I'm doing a bit of testing right now. Hopefully you will not notice any problems, however, if you do please send an error report directly to:
Zaluzec-at-ANLEMC.MSD.ANL.GOV
I'm in the process of slowly upgrading the listserver software abit at a time to get rid of some of the annoying problems that have been creeping into the system. and the subsequent headaches for you and me. This process will likely take awhile, and I will be switching periodically between old and new links. Most of the installation was done over the holidays however, I've still got a load of reconfigurations which need to be handled "gently" to minimize any crashes. However, as Murphy has it I expect some problems. I'll keep you all informed as I make any major changes, but expect some hickups.
Subject: Time:2:17 PM OFFICE MEMO Resolved-Focusing on JEOL 1010 Date:1/3/94 Just a short note to thank everyone who responded to my request for help in rectifying our focusing problem with our the JEOL 1010. As I indicated in an earlier message, we learned a lot from the many comments and suggestions by the members of this list. It ends up that the gun alignment was changing when the shutter closed, which caused beam sensitive specimens like we use, i.e. slotted grids, to charge. Evidently, this is not a serious problem with mesh grids as are used in calibration. JEOL indicated that an early design innovation of the 1010 which was intended to blank the beam prior to exposure was later found to be less than satisfactory and abandoned this method in favor of the traditional system. Unfortunately, in our scope some remnants of this circuitry were not eliminated, thus contributing to our problem. They were able to track this problem down after considerable, and diligent effort, and corrected it by modifying the deflector lens amperage and by removing circuit elements linking the shutter and gun alignment. I am not sure what all this means, however, all or photographs since this correction have been in perfect focus. JEOL has been very cooperative and attentive to our frustrations. We are thankful that they did not take the approach of one of the members of this list who indicated that: "This is really simple! If anyone can take sharp pictures using standard specimens then the machine is working fine!" Hopefully, this information will be of use to other users of the JEOL 1010 which may encounter similar focusing problems with beam sensitive specimens.
Subject: Time:2:17 PM OFFICE MEMO Resolved-Focusing on JEOL 1010 Date:1/3/94 Just a short note to thank everyone who responded to my request for help in rectifying our focusing problem with our the JEOL 1010. As I indicated in an earlier message, we learned a lot from the many comments and suggestions by the members of this list. It ends up that the gun alignment was changing when the shutter closed, which caused beam sensitive specimens like we use, i.e. slotted grids, to charge. Evidently, this is not a serious problem with mesh grids as are used in calibration. JEOL indicated that an early design innovation of the 1010 which was intended to blank the beam prior to exposure was later found to be less than satisfactory and abandoned this method in favor of the traditional system. Unfortunately, in our scope some remnants of this circuitry were not eliminated, thus contributing to our problem. They were able to track this problem down after considerable, and diligent effort, and corrected it by modifying the deflector lens amperage and by removing circuit elements linking the shutter and gun alignment. I am not sure what all this means, however, all or photographs since this correction have been in perfect focus. JEOL has been very cooperative and attentive to our frustrations. We are thankful that they did not take the approach of one of the members of this list who indicated that: "This is really simple! If anyone can take sharp pictures using standard specimens then the machine is working fine!" Hopefully, this information will be of use to other users of the JEOL 1010 which may encounter similar focusing problems with beam sensitive specimens.
This newsgroup passed its vote by a significant margin. Barring SERIOUS controversy, it will be created in five days.
Newsgroups line:
sci.techniques.microscopy The field of microscopy.
Voting closed at 23:59:59 UTC, 2 January 1994.
After this result appears on news.announce.newgroups it will be sent to the following mailing lists (minus the individual ACK lists), with the permission of their respective maintainers:
microscopy-at-anlemc.msd.anl.gov {Microscopy Mailing List} maintained by Nestor J. Zaluzec {zaluzec-at-anlemc.msd.anl.gov}
nih-image-at-soils.umn.edu {NIH Image Mailing List} maintained by John Ladwig {jladwig-at-soils.umn.edu}
This vote was being conducted by a neutral third party. For voting questions only, contact pschleck-at-unomaha.edu. For questions about the new group, contact John F. Mansfield {John.F.Mansfield-at-umich.edu} .
(Vote-takers Note: I apologize for the lack of a 2nd CFV. It was my intention to send one out halfway through the voting period, on December 19th. However, I wasn't paying close attention to the news.announce.newgroups submission deadline before the holidays, which came up earlier this year, on December 18th. The extremely decisive results, combined with the usual lull in activity over the holidays, makes it almost impossible that this accidental oversight affected the the outcome of the vote, anyway.)
CHARTER
The main aim of sci.techniques.microscopy is to provide an open forum for the discussion of microscopy and related fields on the Internet.
PURPOSE
The purpose of sci.techniques.microscopy is to provide an open discussion forum for the microscopy community on the Internet. The newsgroups allow the rapid and timely discussion of opinions and information that would take months or years (or not at all) on conventional paper journals. It is hoped that this newsgroup will eventually be linked with the microscopy-at-anlemc.msd.anl.gov mailing list and archived at the same site. Technical suggestions as to the best way to accomplish this are welcome, and may be directed to either John F. Mansfield or Nestor J. Zaluzec via their respective E-mail addresses above.
Please note that this proposed newsgroup is intended to be an open forum for discussion of microscopy. Thus relevant topics for this newsgroup should only be limited to what the participants in this proposed newsgroup regard as microscopy.
TOPICS FOR DISCUSSION
Optical Microscopy Confocal Microscopy Scanning Probe/Force Microscopy (formerly Atomic Force Microscopy) - SPM, SFM, AFM Scanning Tunnelling Microscopy - STM Scanning Electron Microscopy - SEM Transmission Electron Microscopy - TEM High Resolution Electron Microscopy - HREM Analytical Electron Microscopy - AEM Scanning Transmission Electron Microscopy - STEM High Voltage Electron Microscopy - HVEM X-ray Energy Dispersive Spectroscopy - XEDS Electron Energy Loss Spectroscopy - EELS Electron Microprobe Analysis (EMPA) Wavelength Dispersive X-ray Spectroscopy (WDS) Diffraction Contrast Imaging Phase Contrast Imaging Selected Area Electron Diffraction - SAED or SAD Convergent Beam Electron Diffraction - CBED Image Filtering Field Ion Microscopy Electron Holography X-ray Microscopy Scanning Acoustic Microscopy Ultrasonic Imaging Specimen Preparation (Electropolishing, Ion Milling, Ultramicrotoming, etc.) 3D reconstruction Image Processing Software Data formats Databases Hardware/Equipment - specs, opinions, etc. Applications Announcements/reviews of papers/conferences. Preparation techniques. Non-ambient techniques General Discussion/opinions/questions. Positions vacant
As well as anything else that is relevant to microscopy in general.
[sci.techniques.microscopy group vote Final Vote Ack deleted to avoid overloading the mailing lists. The full results may be obtained via anonymous FTP from ftp.uu.net under /usenet/news.announce.newgroups/ sci/sci.techniques.microscopy.]
Hello, I just started working with magnetic steel specimens in the TEM and find that the beam is just "bent" everywhere. Has anyone worked with steel before and/or does anyone have any pointers they picked up along the way? Thanks..
Return-Path: {P.V.Hatton-at-sheffield.ac.uk} Received: from oxmail.ox.ac.uk by vax.ox.ac.uk (MX V3.3 VAX) with SMTP; Tue, 04 Jan 1994 14:43:36 +0000 Received: from sheffield.ac.uk by oxmail.ox.ac.uk via JANET with NIFTP (PP) id {17715-0-at-oxmail.ox.ac.uk} ; Tue, 4 Jan 1994 14:43:26 +0000 Received: from pp2.shef.ac.uk by pp.shef.ac.uk with SMTP (PP) id {04547-4-at-pp.shef.ac.uk} ; Tue, 4 Jan 1994 14:41:54 +0000 Received: from gorgon.shef.ac.uk by pp2.shef.ac.uk with SMTP (PP); Tue, 4 Jan 1994 14:33:35 +0000 Received: From LADYBOWER/GORGON_WORKQ by gorgon.shef.ac.uk via Charon-4.0A-VROOM with IPX id 100.940104143216.2368; 04 Jan 94 14:33:35 +0000 Message-ID: {MAILQUEUE-101.940104142755.448-at-UNDERBANK.shef.ac.uk} To: rms-at-vax.ox.ac.uk
I would be grateful if you could circulate the following info:
"X-Ray Microanalysis - Advances and Applications" The Spring Meeting of the X-ray Microanalysis Group will be held at the School of Clinical Dentistry, University of Sheffield on Thursday 7th April 1994. Enquiries can be directed to Dr Paul Hatton, tel. 0742 670444 ext. 3051, fax 0742 797050
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 1--8.
The observation of large magnetite crystals from magnetotactic bacteria by electron and atomic force microscopy
by MARCOS FARINA,* BECHARA KACHAR,"" ULYSSES LINS,* RAYMOND BRODERICK~~ & HENRIQUE LINS DE BARROS##,
*Instituto de Biofisica Carlos Chagas Filho - CCS - Bloco G -Universidade Federal do Rio de Janeiro, 21949-900, Rio de Janeiro, Brazil. ""Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, Bethesda, Maryland, U.S.A. ~~Department of Pharmacology and Experimental Therapeutics, University of Maryland at Baltimore, School of Medicine, Maryland, U.S.A. ##Museu de Astronomia e Ciencias Afins, Rio de Janeiro, Brazil
Summary Magnetite crystals inside coccoid magnetotactic bacteria found in lagoons near Rio de Janeiro city were examined by electron microscopy (EM) and atomic force microscopy (AFM). For AFM, ultrathin sections of bacteria embedded in Epon resin were etched with an ethanolic NaOH solution and observed both in the height and in the force modes. Comparative electron microscope images were useful for identifying crystalline reliefs in the etched sections. Different situations representing particular arrangements of crystal chains were observed by AFM. The majority of the bacteria examined presented unusually large magnetite crystals which remained strongly attached in linear chains even after the laboratory procedures for their isolation. This behaviour is different from all other biogenic magnetite crystals isolated so far. It is suggested that this attachment is due to the strong field between individual crystals as well as to the contact areas, which are the largest observed until now. The correct identification of a particular topography by AFM as a crystal relief may be critical when crystals are not aligned in chains; in these cases the linear dimensions and the presence of well-defined edges and faces are important features to be taken into account. Characterization of the crystal faces is important for the study of magnetotactic micro-organisms since the crystalline habits seem to be species-specific. Observation of etched sections proved to be a helpful approach for crystal relief observation, especially when small amounts of bacteria were available.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 9--25.
Development, quantitative performance and applications of a parallel electron energy-loss spectrum imaging system
by G. BOTTON* & G. L'ESPERANCE,
Centre for Characterization and Microscopy of Materials, Departement de Metallurgie et Genie des Materiaux, Ecole Polytechnique de Montreal, C.P. 6079, Succ. ``A'' Montreal, (Quebec), Canada
Summary The development of a parallel electron energy-loss spectrum imaging system is presented. The analytical performance of the imaging technique was investigated and the system applied to materials science problems. The system, which allows acquisition and storage of a parallel electron energy-loss spectrum at each pixel of an image, was developed by interfacing a multichannel analyser and a microscope to a computer workstation. In the experimental conditions used for imaging, detection limits and quantification errors were large and varied as a function of spatial resolution and the range of chemical elements of interest in the image. Applications of this imaging technique in materials science showed that quantitative chemical information is provided by the system and that the use of relative thickness maps and detailed statistical analysis of the spectrum-image allowed an unbiased interpretation of the images. As energy-loss spectra are available after processing, spectroscopic information about the analysed material can be used to provide supplementary information.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 27--38.
Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold-labelled riboprobes in breast cancer cell lines and histological specimens
by G. LINARES-CRUZ,* J. P. RIGAUT,"" J. VASSY,"" T. C. DE OLIVEIRA,"" P. DE CREMOUX,* B. OLOFSSON~~ & F. CALVO* ,
*Laboratoire de Pharmacologie Experimentale, Institut de Genetique Moleculaire, Hopital Saint-Louis, Universite Paris 7, 27 rue Juliette Dodu, F-75010 Paris, France. ""Laboratoire d'Analyse d'Images en Pathologie Cellulaire, U. 263 - I.N.S.E.R.M., Universite Paris 7, 2 place Jussieu, F-75251 Paris Cedex 05, France . ~~U. 248 - I.N.S.E.R.M., Faculte de Medecine Lariboisiere Saint-Louis, 10 avenue de Verdun, F-75010 Paris, France
Summary A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 39--51.
Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts
by J. S. BURMEISTER, G. A. TRUSKEY & W. M. REICHERT* ,
Department of Biomedical Engineering, Duke University, Durham, NC 27708, U.S.A.
Summary Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degrees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 (plus or minus 13) nm. Focal contact areas estimated by TIRFM (1.3 plus or minus 0.7 square micrometres) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 plus or minus 0.3 square micrometres).
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 53--66.
Semiautomated methods for cancellous bone histomorphometry using a general-purpose video image analysis system
by W. E. HUFFER,* P. RUEGG,* J.-M. ZHU"" & R. B. LEPOFF* ,
*Department of Pathology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado, U.S.A.
Summary Semiautomated methods are used to measure elongated, curved and complex branching profiles and isolated perimeter segments in monochrome video images with a general-purpose analysis system. These methods are used to make the major primary measurements of bone histomorphometry. Accuracy and reproducibility of the image acquisition, processing and measurement system is documented by measuring a semicircular standard of known dimensions. Semiautomated applications of the Ar/Le method for measuring areas and perimeters, and calculating lengths and widths of osteoid seams, lengths of mineralization labels and mineral apposition rate, wall width, indirect measurements of eroded, osteoclastic and osteoblastic perimeters without tracing, and measurement of mineralized or total cancellous bone area and perimeter gave values comparable to measurements of the same parameters by tracing or grid counting techniques with equal or better reproducibility and much greater efficiency. Intraindividual variation in measuring multiple bone biopsies was comparable to that reported with current standard methods. Major sources of variability for semiautomated methods were image magnification and selection of profile edges by thresholding, and sources of variability for manual methods are image magnification, numbers of orthogonal intercepts, tracing speed and accuracy of the algorithm used to measure traced pixels. Semiautomated methods are accurate, reproducible and rapid methods suitable for bone histomorphometry.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 67--72.
Some remarks on the accuracy of surface area estimation using the spatial grid
by K. SANDAU* & U. HAHN , Institut fur Angewandte Mathematik und Statistik, Universitat Hohenheim, D-70593 Stuttgart, Germany
Summary A set of three line grids in three orthogonal directions is called a spatial grid. This spatial grid can be used for surface area estimation by counting the number of intersection points of a surface with the grid lines. If direction and localization of the spatial grid are suitably randomized, the expectation of this number is proportional to the surface area of interest. The method was especially developed for cases where the surface to be measured is embedded in a medium, which is the usual case in microscopical applications, and where a stack of serial optical sections of the surface is available. The paper presents an improvement of an earlier version of the counting rule for intersection points. Furthermore, if the direction of sectioning is not uniform random, a bias results. This bias is calculated for a disc as a perfectly anisotropic object. A generalization of the estimator is considered by introducing a weighted mean instead of the usual arithmetic mean. The variance due to the randomized direction is investigated depending on the weights, and the minimum of this variance is derived. The relationship between the covariogram and the variance of the surface area estimated with the spatial grid is considered.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 73--78.
Volume-weighted mean particle volume estimation using different measurement methods
by E. ARTACHO-PERULA & R. ROLDAN-VILLALOBOS,
Department of Morphological Sciences, School of Medicine, University of Cordoba, Avda. Menendez Pidal s/n, 14071 Cordoba, Spain
Summary The use of the `point-sampled intercepts' method on histopathological material is evaluated for the main purpose of comparing different methods of intercept length measurements. The volume-weighted mean nuclear volume of the carcinoma of the ampulla of Vater is calculated using three methods for measuring intercept lengths: a semiautomatic image analyser, an equidistant ruler and a logarithmic ruler. Rulers of several classes and lengths are used, and the results of the volume-weighted mean nuclear volume estimations are compared. The equidistant ruler is more accurate than the logarithmic ruler. With a greater number of ruler classes and with adjustment of ruler length to the greatest nuclear intercept lengths, the systematic deviation from the true value of the volume-weighted mean nuclear volume is smaller. The volume-weighted mean nuclear volume parameter has great power to differentiate intestinal carcinoma from normal tissue.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 79--82.
A simple calibration for routine section thickness measurements using current density ratios
by Y. M. HENG,* F. P. OTTENSMEYER,"" A. L. ARSENAULT* & G. T. SIMON*,
*Electron Microscopy Facility, McMaster University Medical Centre, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada
Summary A method to calibrate current density ratios for the determination of specimen thickness is presented. This method uses a tilt series from a single noncrystalline specimen to create different thicknesses; these are used to generate data points to establish the relationship between specimen thickness and current density ratio. The actual specimen thickness at 08 tilt was determined to an accuracy of 5nm by a parallax method. From the calibration curves obtained, we observed that the current density ratio was sensitive to relative thickness changes on the same section of less than 1nm when a 50-micrometre objective aperture was used.
Journal of Microscopy, Vol. 173, Pt. 1, January 1994, pp. 83--86.
A simple filter system for processing small or transparent specimens
by B. CRIBB & J. ZHU ,
Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Qld 4072, Australia
Summary A novel method of attaching a fine mesh filter to the end of a disposable plastic pipette is described. Such a pipette filter can be used to exclude specimen uptake during specimen preparation procedures, particularly when processing small or transparent materials. The pipette filter-tip does not interfere with fluid exchange and is non-reactive with normal processing fluids.
THE JOURNAL OF MICROSCOPY IS AVAILABLE AT A REDUCED RATE FOR MEMBERS OF THE ROYAL MICROSCOPICAL SOCIETY, THE MICROSCOPY SOCIETY OF AMERICA AND THE INTERNATIONAL SOCIETY FOR STEREOLOGY. PLEASE CONTACT THE SECRETARY OF EACH SOCIETY FOR DETAILS.
INSTRUCTIONS FOR AUTHORS AND DISK SUBMISSION FORMS CAN BE OBTAINED FROM DR GILLIAN WILSON, THE JOURNAL OF MICROSCOPY, 37/38 ST CLEMENTS, OXFORD OX4 1AJ, UNITED KINGDOM. TELEPHONE +44 (0)865 248768, FAX +44 (0)865 791237, EMAIL RMS-at-UK.AC.OX.VAX.
I would like to thank everyone for their help in my decalcification problem. Several techniques were suggested and I am sure one of them will work for my needs. Again, many thanks!
Subject: Time:2:31 PM OFFICE MEMO TEM: Mag Specs Date:1/4/94 TEM work on steel specimens can be very difficult, because they are almost always magnetized. You may be able to reduce the magnetic inhomogenieties a bit by passing the specimens through a strong AC field, a process industrially known as 'degaussing'. I believe degaussing coils can be purchased from most electronics supply stores that deal in the television market. In use, you insert the specimen into the center of the coil and withdraw it very slowly, with the AC current flowing through the coil.
Besides doing what Robert Keller suggests, you should note the objective lens current for a non-magnetic sample and set the lens to the same value. Then you can bring the specimen to the eucentric height by noting when the image is at the minimum contrast condition.
Russell Cook Electron Microscopy Center for Materials Research Argonne Natonal Laboratory Argonne, IL
Automatic print processing is a great benefit whether or not you also have an automatic exposure system. Indeed, we had a Kodak Royalprinter for many years before we aquired our LogEtronics (now Egoltronics) EM55 automatic dodging enlarger. Even now some of our users (not many) prefer to use our old Durst enlarger although they have only two simple aids to exposure determination: a Kodak Projection Print Scale (cat. no. 1557248) which cost about $10 and an Ilford EM10 Exposure Monitor which cost about $25.
Russell Cook Electron Microscopy Center for Materials Research Argonne Natonal Laboratory Argonne, IL
Subject: Time: 2:17 PM OFFICE MEMO TEM Analysis of Diffusion Couples Date: 1/4/94 We have considered using a TEM to study diffusion couples, but preparation of thin foils is a major stumbling block. Is anyone familiar with a possible technique that could leave the diffusion structure intact at the interface between two unlike materials in a diffusion couple? Is anyone aware of reported work relating to TEM analysis of diffusion couples?
There were several articles/abstracts in the Proceedings of the XIIth International Congress for Electron Microscopy (1990 EMSA Proceedings) on this subject, mine among them. If memory serves me, there are articles in the EMSA or MSA proceedings for years prior to and after 1990. My primary sample preparation technique, and that of many otherr researchers, is to ion-mill cross-sectioned samples.
Russell Cook Electron Microscopy Center for Materials Research Argonne Natonal Laboratory Argonne, IL
Eaton Publishing invites submission of papers for peer review in consideration for publication in the new journal "Cell Vision - Journal of Analytical Morphology." The first issue of Cell Vision is scheduled for publication in May/June 1994.
Cell Vision is edited for those scientists and physicians that analyze morphology as a means of diagnosis or research. It is also intended for those who are interested in advances in immunocytochemistry, confocal microscopy, image analysis and more recent developments such as in situ polymerase chain reaction and probe scanning microsopy.
Cell Vision focuses on these novel analytical methods in morphology and their applications in biomedical research and diagnostics. Developments reported in this journal will benefit any scientist who visualizes and analyzes chemical components against the background of tissue structure.
Cell Vision will have an international circulation and will publish articles contributed by multinational authors. All articles will be rigorously peer reviewed and promise to be of very high quality. The international Editorial Board of Cell Vision, led by Dr. Jiang Gu of the Deborah Research Institute, includes many top scientists in modern morphology.
General information about Cell Vision (including instructions for authors and subscription information) is available by contacting Eaton Publishing, 154 East Central Street, Suite 201, Natick, MA 01760. You may also fax your requests to 508-655-9910 or submit electronic mail requests to internet address: fweaton-at-biotechnet.com.
Thanks for your comments. I'm headed off to look at my samples this afternoon and see how the suggestions work. For those who are wondering, I prepare my samples by cutting with a wire saw to 100microns and slowly (as possible) jet polish with perchloric until there is a hole.
We have just taken on a project which involves cutting "thin" sections of 35 um fenestrated glass beads. I am using tungsten coated glass knives broken via the "balanced-break" method with , as expected, less than desireable results. Has anyone had any experience with anything like this? Incidentaly, the beads are fixed and embedded in Spurr's resin. Any ideas for better sections would be appreciated.
Subject: Time:1:13 PM OFFICE MEMO Re} EM Atlas Date:1/5/94 {I am looking for EM atlases (atli?) for both "normal" and pathologic {tissues. {I work almost exclusively with mouse tissue, but any good atlas should {be enough of a guide. I'd appreciate any infomation or suggestions. Margaret, You did not mention the type of tissue you are looking at. If you are interested in nervous system tissue, the definitive "atlas" is, The Fine Structure of the Nervous System, by Peters, Palay and Webster. The most recent edition (3rd) is published by Oxford. Mike Mike_Schwartz-at-qm.yale.edu
} Date: Wed, 5 Jan 94 12:15:01 EST } From: meh-at-jax.org (Margaret E. Hogan) } To: microscopy-at-anlemc.msd.anl.gov } Subject: EM Atlas
} I am looking for EM atlases (atli?) for both "normal" and pathologic tissues. } I work almost exclusively with mouse tissue, but any good atlas should be } enough of a guide. I'd appreciate any infomation or suggestions. Thanks!!!! } } Peggy Hogan } The Jackson Laboratory } meh-at-aretha.jax.org
Another atlas, not the usual normal or pathological atlas, but very useful: Artifacts in Biological Electron Microscopy R.E.F. Crang and K.L. Klomparens, eds. Plenum Press, NY 1988
Organisers: Dr A Entwistle Dr C V Howard Dr H Gundlach
The Digital Imaging Light Microscopy Summer School is aimed primarily at scientists in biological and related disciplines and will comprise of a basic introduction, followed by a selection of modules.
-------------------------------------------------------------- Monday and Tuesday
Common Core - Introduction to Light Microscopy. This will consist of lectures, demonstrations and practical work on the following:
The History of microscopy; Introduction to microscopy; Limitations of the eye; Resolution, Contrast, Magnification; Refraction and lenses, geometrical optics, conjugate planes; Aperture; Illumination of the specimen in transmitted and reflected light; Lens aberrations and their correction; the choice of optical components; Diffraction and its consequences for the microscope image; Generation of contrast; Photomicrography.
Modules - The following five options are available:
Comprehensive Digital Light Microscopy - Stereology and digital imaging, digital image collection, digital image processing, digital image display and digital image storage.
Confocal Microscopy (students must bring their own specimens for study) - Fluorescent staining of samples, imaging of samples (fluorescence, reflectance and DIC techniques), visualization of 2-D and -D data sets and aspects of confocal theory.
Advanced Fluorescence Microscopy - Fluorescent staining of samples, ion ratioing methods, fluorescence decay-time measurements, fluorescence confocal microscopy.
Stereology and Digital Light Microscopy - Basic concepts of systematic random (ie unbiased) sampling in 3-D from histological material, efficient design-based stereological methods for estimating number, surface, length and volume (both manual methods and those employing image analysis systems will be covered) and methods of particle sizing in 3-D.
Techniques in Digital Light Microscopy - Introduction to stereology, introduction to confocal microscopy and introduction to digital imaging and processing.
FOR FURTHER DETAILS CONTACT THE ROYAL MICROSCOPICAL SOCIETY, 37/38 ST CLEMENTS, OXFORD OX4 1AJ, UNITED KINGDOM. TELEPHONE +44 (0)865 248768, FAX +44 (0)865 791237, EMAIL: RMS-at-UK.AC.OX.VAX.
I'm interested in the morphology of Panthera Pardus epidermis. Does anyone know if there is something unusual about the epithelial cells of this animal skin?
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
Charles Bradley at Argonne National Laboratory has been sectioning very small bits of radioactive waste glasses embedded in resin. I believe the size of the glass pieces are about 40 microns or so. I am not sure of the resin he has been using, however. He has been using a diamond knife to get TEM sections from a Reichert-Jung Ultracut E.
Russell Cook Electron Microscopy Center for Materials Research Argonne Natonal Laboratory Argonne, IL
I have come across a structure, many times, that I have identified as a perichromatin granule. A dense body of about 30-40 nm surrounded by a transparent halo an usually associated with dense chromatin. I think this structure was first mentioned by Bernhard 20 or more years ago. Does any one know if this structure has been further characterized? Any leads on this would be greatly appreciated.
A paper by Bottke may be of interest: Bottke W (1976). Chromosome-associated paracrystalline nuclear inclusions in the spermatocytes of a pulmonate snail, Planorbarius corneus L. Chromosoma (Berl.) 55: 273-287.
On Thu, 6 Jan 1994, Greg Erdos ICBR EM Core Lab University of Florida wrote:
} Cell Biologists: } } I have come across a structure, many times, that I have identified } as a perichromatin granule. A dense body of about 30-40 nm surrounded by a } transparent halo an usually associated with dense chromatin. I think this } structure was first mentioned by Bernhard 20 or more years ago. Does any } one know if this structure has been further characterized? } Any leads on this would be greatly appreciated. } } ***************************** } * Greg Erdos * } * Director, ICBR EMCL * } * University of Florida * } * Gainesville, FL 32611 * } * gwerdos-at-gnv.ifas.ufl.edu * } * 904-392-1295 * } ***************************** }
With encouragement from a number of you folks out there, I tried sectioning these thing with an old diamond knive. Much to my amazement, it worked beautifully. As a biologist, I never would have thought that this was possible. We have good sections with minimal obvious damage to the knife. We have saved a great deal of time and many headaches. Thanks to all who responded and HOORAH for the system.
We are looking for a used but recent vintage electron microprobe to replace a very old ARL system. Preferences are for a 4-channel system with EDS, something like a Cameca SX-50. We are aware of Don Lesher's operation in northern Ohio and have talked with him about acquiring a rebuilt ARL-SEMQ. This may be our eventual route, but if there is a good recent vintage machine sitting out there somewhere we would be very interested in knowing about it. Thanks. Warren D. Huff Dept. of Geology University of Cincinnati Cincinnati, OH 45221-0013 phone (513) 556-3731 fax (513) 556-6931 e-mail huff-at-ucbeh.san.uc.edu
Due to the continuing recession in California and especially the hardships the UC system is undergoing we are forced to begin a recharge for our microscopy services. I would very much like to know what current recharges are for biological TEM use and prep services. If anyone has a freeze fracture device (we have a Balzers BAF 400T) what are the going rates? How about for SEM prep?
Thank you in advance. Rick A. Harris Electron Microscopy Evolution and Ecology Univ. of Calif. Davis, CA 916 752 2914
None Re: Recent request for help on volume calculations -
"Our laboratory has expressed interest in calculating volumes on microscopic data and I am looking for software that can perform the following tasks:
2: 3-D reconstruction (including EM image alignment) 5: Wire-frame generation and simple volume rendering 3: Volume calculation 1: Run on a Mac or PC 4: File I/O from a Mac or PC format (preferrably using MacDraw objects of known dimensions)
The non-proprietary file format support is neccessary to allow cross-platform data manipulation. My U*NIX access is limited, so ability to un on a PC is important.
I keep reading how people did 3-D reconstruction, but their programs are hard to find. Any referrals to sources of this software is appreciated. It WOULD be neat if it was available in C by anonymous FTP, but perhaps this is too narrow a requirement. Any leads would help."
Reply - There are simple methods for estimating volume densities that do not require complicated computer systems or sophisticated software. If your aims are to estimate the volumes of subcellular structures and you can directly measure at least one reference volume then you might like to try cross latice overlays as described by Weibel as far back as 1979 (Stereological methods vol 1. practical methods for biological morphometry Academic Press NY). More recent reviews can be found in APMIS (Gundersen et al 1988 vol 96 379-394), J. Microsc (Cruz-Orive 1982 vol 125 89-102) Am. J. Physiol (Cruz-Orive & Weibel 1990) vol 258 148-156) and TICB (Luquoc 1993 some time this fall). Another source is a recently published book where the final chapter covers all these methods (Griffiths 1993 Fine Structure Immunocytochemistry, Springer Verlag, Heidelberg). Best of all is to take a course on stereology (there is one in Banff, Canada in May 1994 and one here at Yale in August 1994). If you still want to do 3-D reconstructions, which will only give you information on the structures you reconstruct, then the best software I have seen up to now are the VoxelView programs. We run them on a Silicon Graphics workstation which never seems to have enough memory. These programs allow you to reconstruct sections, rotate and manipulate the images as well as measure the parameters of the reconstructed structures.
Immunocytochemistry and Cryosections Practical Course 22 - 27 August 1994. An intensive practical course mixed with theoretical sessions where you can learn how to produce cryosections as well as immunolabeling, colloidal gold production and much more.
Additional we are offerring a three day practical workshop on Stereological methods. This will be on 18 -20 August 1994, prior to the cryosectioning course.
A team of instructors headed by Hans Gundersen will take you through the theoretical and practical details of modern stereological methods. These will include volume and surface densities, the fractionator, the nucleator, the disector and much much more. Address for further details on both courses;
Paul Webster, Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510.
Re} Stereology Re: Recent request for help on volume calculations -
"Our laboratory has expressed interest in calculating volumes on microscopic data and I am looking for software that can perform the following tasks:
2: 3-D reconstruction (including EM image alignment) 5: Wire-frame generation and simple volume rendering 3: Volume calculation 1: Run on a Mac or PC 4: File I/O from a Mac or PC format (preferrably using MacDraw objects of known dimensions)
The non-proprietary file format support is neccessary to allow cross-platform data manipulation. My U*NIX access is limited, so ability to un on a PC is important.
I keep reading how people did 3-D reconstruction, but their programs are hard to find. Any referrals to sources of this software is appreciated. It WOULD be neat if it was available in C by anonymous FTP, but perhaps this is too narrow a requirement. Any leads would help."
Reply - There are simple methods for estimating volume densities that do not require complicated computer systems or sophisticated software. If your aims are to estimate the volumes of subcellular structures and you can directly measure at least one reference volume then you might like to try cross latice overlays as described by Weibel as far back as 1979 (Stereological methods vol 1. practical methods for biological morphometry Academic Press NY). More recent reviews can be found in APMIS (Gundersen et al 1988 vol 96 379-394), J. Microsc (Cruz-Orive 1982 vol 125 89-102) Am. J. Physiol (Cruz-Orive & Weibel 1990) vol 258 148-156) and TICB (Luquoc 1993 some time this fall). Another source is a recently published book where the final chapter covers all these methods (Griffiths 1993 Fine Structure Immunocytochemistry, Springer Verlag, Heidelberg). Best of all is to take a course on stereology (there is one in Banff, Canada in May 1994 and one here at Yale in August 1994). If you still want to do 3-D reconstructions, which will only give you information on the structures you reconstruct, then the best software I have seen up to now are the VoxelView programs. We run them on a Silicon Graphic workstation which never seems to have enough memory. These programs allow you to reconstruct sections, rotate and manipulate the images as well as measure the parameters of the reconstructed structures.
There is a tutorial on quantitative morphometry written by Dr. Robert Bolender, Dept. Biological Structure at Univ. Washington. It also provides templates for point and intersect counts and for QM computations. It is available from the Health Sciences Center for Educational Resources, University of Washington SB-56, Seattle, Washington, 98195 (206) 685-1156.
Dr. Bolender teaches a course on QM in even years.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Subscribers who might find it difficult to attend the stereology courses in Banff or Yale should note that the RMS will be running a Digital Imaging and Stereology course at Liverpool University, UK, in July 1994, coordinated by the current President of the International Society for Stereology, Dr Vyvyan Howard. Contact RMS-at-UK.AC.OX.VAX with your full name and address to obtain details!
From Paul Sheppard, Tree-Ring Lab, Univ. of Arizona
To microscopy forum members:
I am having slight, but definite, difficulty in attaining repeatable focus of my binocular common main objective microscope. In my attempt to apply image- analysis techniques to tree-ring science, my focus problem has led to systemat- ic differences in values as measured by different technicians. I am amazed at how little the focus differences must be before we see differences in our data. Can anyone suggest ways to ensure repeatable focus?
I have inquired into adding on autofocus hard- and software, but my first estimate on that was $7,000, which is prohibitive at this time. I have also heard about a dual-light focus aid, where two spot beams are projected onto the subject to intersect exactly at the focal distance of the objective lens; if the subject is above or below that distance, then the spot will be elongated or even split into two. Has anyone tried this? I have also tried focussing at high magnification and then working at my usual lower magnification. This re- quires parfocal optics, which I have, but this process is cumbersome and other- wise prone to error.
Thanks in advance for any and all suggestions,
Paul Sheppard Laboratory of Tree-Ring Research University of Arizona GRAD12-at-CCIT.ARIZONA.EDU
My work is in the materials sciences where for reflected light microscopy specien preparation usually begins with a grinding and polishing sequence in order to obtain an optically smooth surface. I am now faced with a material I do not wish to grind/polish, I passed by a reference that suggested that if the specimen surface is not optically smooth I can use glass coverslips and an index matching fluid - this sounds vaguely familar from highschool biology. Is there anyone out there with experience in Light Microscopy who could point toward the correct products and describe for me some of the considerations involved.
} Rick Harris asked about charges for EM facilities..... } --------------------------------------------------- } } Sandy Silvers of the South Eastern EM Society has a fairly } comprehensive database on EM facilities in the US. You } can contact her at
** some stuff deleted **
I just got off the phone with Sandy Silvers and told her that, since she doesn't currently have Internet access, I'd pass this information along. She has moved from Florida. Her current address and phone number are:
Sandra Silvers EM Complex USDA, ARS, RRC PO Box 5677 Athens GA 30613-6199 (706) 546-3471
Her database has listings, which include contact people, for about 200 facilities. Information about usage charges is included for most of them. She is looking for more input and will send a questionaire on request. Printed copies of the database are available for $8 (US). She does all this personally now, so needs to recoup printing costs.
She's intrigued with the possibility of having the information available more widely, perhaps through FTP.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Subject: Time:4:14 PM OFFICE MEMO LM???-Reply Date:1/11/94 I do not know the nature of your specimens, but I seriously dobut that you would gain much useful information by using a cover slide and oil to overcome the usual polishing process. Polishing is usually necessary because optical microscopes have such limited depth of field (Typically about 10 microns for a 10X objective, 1 micron for a 40X obj, and 0.1 or 0.2 microns for a 100X obj) that they are not at all suited for looking at rough surfaces. Placing a layer of oil and a cover glass over a rough surface will not solve this problem. Furthermore, the objective lenses of metallographic microscopes are not designed to work through cover glasses - you'll probably need to go to the biology or mineralogy dept to find a microscope that is. I would suggest that you try examining the specimen with a stereo binocular microscope as a start. A good instrument of this kind should give magnifications up to about 75X. If that is not sufficient, then the next step would be to try SEM - although contrast may be a problem, depending on the nature of the specimens. microscope will not work with a cover glass,
The previously posted information about Sandy Silvers address was incorrect. She has since moved, her new address is apparently.... Thanks to L. Melsen for the correction....
Rick Harris asked about charges for EM facilities..... ---------------------------------------------------
Sandy Silvers of the South Eastern EM Society has a fairly comprehensive database on EM facilities in the US. You can contact her at
SANDY H. SILVERS EM COMPLEX RUSSELL RESEARCH CENTERS USDA, BOX 5677 ATHENS, GA 30613-6199 (706) 546 3471 FAX (706) 546 3452
Subject: Time:5:15 PM OFFICE MEMO Stereographic Calcs Date:1/11/94 We have obtained optical goniometric measurements of angles between the faces of about 75 macro crystals. These angles (commonly called phi and rho) were measured relative to the axes of the optical goniometer. Now, we want to convert them to angles that are relative to principal planes of the crystals. Does anyone have , or know of, a readily available computer program for doing this?
We are working on a project that requires us to shadow myosin with Pt and C. We are trying to see the myosin heads and another conjugated molecule. We are using our Balzers 400 to shadow at 15 to 25 degrees for the Pt and then at 90 degrees for the C. We can find the molecules but they are extremely faint. We have used anywhere from 5 to 15 angstroms of Pt. Best results were with higher angles and thinner coats. Then we increase exposure in the TEM to 3 seconds to bump the contrast and shift the s/n ratio. Has anyone a suggestion for making the myosin more visible? The micrographs have little contrast between the molecule and the substrate.
Rick A. Harris Electron Microscopy Evolution and Ecology Univ. of Calif. Davis, CA
Excerpts from mail: 11-Jan-94 LM - ??? by Peter Joyce-at-utxvms.cc.ut } My work is in the materials sciences where for reflected light microscopy } specien preparation usually begins with a grinding and polishing sequence } in order to obtain an optically smooth surface. I am now faced with a } material I do not wish to grind/polish, I passed by a reference that } suggested that if the specimen surface is not optically smooth I can use } glass coverslips and an index matching fluid - this sounds vaguely familar } If I understand your question, I don't think index matching will help. Reflections occur where refractive index changes, if you match the refractive index of your specimen you not see much reflection. I am not a materials scientist, but biologists use reflected light microscopy to generate contrast by interference of the first surface reflection (usually the coverslip/medium interface) with the second surface reflection (usually the medium/specimen interface) in order to see regions where the specimen makes close contact with the coverslip. If your application is based at all on similar effects, index matching will at least attenuate one of the reflections.
As part of a research project, I am looking for -any and all- references that pertain to the staining of mineral species for their characteriza- tion and to make certain minerals more distinguishable under the optical (polarizing) microscope.
I greatly appreciate all info and help in this endeaver.
Thanks, Rob
X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X X Rob Tayloe X MSM Spelunkers Club X X Metallographic Lab. X Missouri Speleological Survey /-v-\ \-v-/ X X Rolla Research Center X Bat Conservation International X X U.S. Bureau of Mines X Missouri Cave & Karst Conservancy \-v-/ X X tayloe-at-rorc.usbm.gov X National Speleological Society #32993 /-v-\ X X (314) 364-3169 x247 X American Cave Conservation Association X X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X
I am wondering if anyone out there has tried incorporating the new Mac AV computers with their video systems? I've got the money for the Mac, but I need to know how the software is for image analysis, and how the final product actually looks.
If anyone has done an EM video with the Mac I would like to see it.
Thanks for the help...Go Giants...long live the VW!
John L. Grazul BBR EM Facility Rutgers University Box 1059 Piscataway, NJ 08854
On the NIH-Image mailing list, many, many postings have gone through with the general concensus being that the AV input on the Macs was not intended for scientific quality. There are a number of high-quality frame grabbers for 640 x 480 pixel pictures (e.g., Scion LG-3 at 301-695-7870, Data Translation QuickCapture at 508-481-3700, Perceptics at 615-966-9200, others) as well as some higher-end systems for cooled CCDs, etc. There are also some slow-scan TEM interfaces for the Mac (Gatan, 4pi Analysis at 919-489-1757 for STEM, others). There has been considerably less discussion about the video output. If this is the issue, someone else should comment.
As for image analysis software, there are a number of packages ranging from classical microscopic image analysis to part inspection on assembly lines. My choices are NIH Image (free from NIH via anonymous FTP to zippy.nimh.nih.gov) for workhorse viewing and automation as well as "simple" particle analysis and PrismView (marketed by Signal Analytics 703-281-3277) for high-end work. NIH Image is quick to learn, readily extensible and is extremely popular with its infinite return-on-investment! There are other systems as well.
These vendor lists are NOT exhaustive. If you can download NIH Image, appendices B,C, and D are a little more exhaustive for Mac imaging vendors.
Bill ========================== Bill Heeschen / Analytical Sciences - Materials Characterization 1897-D Building / The Dow Chemical Company Midland, MI 48667 U.S.A. phone: (517)636-4005 fax: (517)636-5453 Email: waheeschen-at-dow.com ==========================
} } As for image analysis software, there are a number of packages ranging from } classical microscopic image analysis to part inspection on assembly lines. My } choices are NIH Image (free from NIH via anonymous FTP to zippy.nimh.nih.gov) } for workhorse viewing and automation as well as "simple" particle analysis and } PrismView (marketed by Signal Analytics 703-281-3277) for high-end work. NIH } Image is quick to learn, readily extensible and is extremely popular with its } infinite return-on-investment! There are other systems as well. } } These vendor lists are NOT exhaustive. If you can download NIH Image, } appendices B,C, and D are a little more exhaustive for Mac imaging vendors. } } Bill
Another note regarding easy info on NIH Image, subscribe to the mailing list located on nih-image-at-soils.umn.edu. They're alll the time fielding most any question you can think of, including questions regrading PrismView. If it's too much to subscribe, post your question anyway and have replies sent directly to you. Good luck.
I was wondering if there was a software package similar to NIH-Image that will run on the IBM or Silicon Graphics workstation that one can get through ftp.
I need a small sample of Ti203 for some microscopy work I'm doing. It's only a really small amount needed so If someone has some that they can give me I would be most appreciative, failing that does anyone know of a supplier?
Thanks David
-- ---------------------------------------------------------------------- David Bell |E-mail: dcb-at-electron.ph.unimelb.edu.au School of Physics |Phone : +61 3 344 5451 The University of Melbourne |Fax : +61 3 344 4783 Parkville, Victoria, AUST, 3052 |
Here is a supplier for Ti2O3, otherwise known as titanium (III) oxide: AESAR/Johnson Matthey 30 Bond Street P.O. Box 8247 Ward Hill, MA 01835-0747 (800) 343-1990 (508) 521-6300. You should be able to get 50 grams for about $50.
J. Ester asked } I was wondering if there was a software package similar to NIH-Image } that will run on the IBM or Silicon Graphics workstation that one can } get through ftp
There is no equivalent for NIH Image that will run on the PC. The closest thing (and it's not close) is NCSA Image fro the National Center for Supercomputing Applications at the Univ. of Ill. It is available via FTP. Their address is "ftp.ncsa.uiuc.edu". NIH Image as I understand from Wayne Rasband (the author at NIH) will be ported to the PowerPC when he gets a unit. So those of you who are tied to PC will be able to run as long as you run the machine in it's Mac Compatible Mode instead of the PC Compatible Mode.
Department of Anatomy and Neurobiology Colorado State University Fort Collins, CO
July 11-15, 1994
The CSU Electron Microscopy Center in the Department of Anatomy and Neurobiology offers an intensive course in freeze-fracture and freeze-etch techniques for research scientists and senior technicians.
Basic and advanced freeze-fracture and freeze-etch techniques will be taught in five days of concentrated lectures and laboratory sessions (12-15 hr/day). Advanced techniques will include sequential confocal mapping/freeze-fracture examination of identified cells in tissue slices. This course will prepare research scientists and laboratory technicians to use freeze-fracture techniques in cell biology research. No previous freeze-fracture experience is necessary, but the individual must be proficient in transmission electron microscopy.
Participants will prepare high-resolution replicas of their own specimens, become proficient at interpreting the resulting images, and learn to prepare and label their own stereoscopic micrographs. Faculty and participants will discuss the physical and chemical bases for interpreting freeze-etch replicas, the major sources of specimen preparation artifacts, and the unique advantages and limitations of freeze-fracture and freeze-etch techniques. Methods for reducing specimen contamination during the fracturing and replication processes, as well as techniques for identifying, fracturing, and mapping individual cells in tissue slices, will be described.
This course is organized by Drs. John Rash, John Walrond, Robert Lee and John Chandler in collaboration with major instrument manufacturers.
Freeze-fracture/freeze-etch equipment used in the course includes: Balzers BAF-301 and BAF- 400, JEOL JFD-9010-CR and additional rapid freeze and freeze-etch devices supplied by RMC, Inc. and Bal-Tec, Inc. Molecular Dynamics multi-probe 2001 confocal laser scanning microscope will be used.
Registration fee of $1250 includes textbook, laboratory instruction manual, all necessary supplies and implements, noon meals, refreshments, EM negatives and prints, and unlimited use of equipment during the course.
Information concerning this course, hotel accommodations and travel arrangements may be obtained from:
Eileen Diepenbrock Colorado State University Department of Anatomy and Neurobiology Fort Collins, CO 80523 (303) 491-5847 ediepenbrock-at-vines.colostate.edu
Registration closes May 15, 1994
Course registration will be for a minimum of 10 and maximum of 12 participants.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
I've had multiple requests lately for permission to post resume's and job "hunting notices" on this listserver. As we set forth early in the setup of this discussion/information forum that type of posting is not appropriate here. However, I realize that with the current budget situations across the country & world there will be a significant number of highly talented individuals looking for positions in the near term. Allow me then to remind all of you that should you have acess to information concerning research/teaching positions this type of BULLETIN/ANNOUNCEMENT is allowed on the Mailserver and I would encourage you to post it. If you are not sure about any posting feel free to Email it directly to me and I will review it's appropriateness.
Individuals interested in posting (SHORT) electronic resume's are welcome to access the ANLEMC/MSA electronic bulletin board. It can be reached via INTERNET/TELNET link at the address: ANLEMC.MSD.ANL.GOV, login with the username EMCBBS and password EMCBBS then follow directions on the screen. Please note that there is only a single connection from INTERNET to this BBS line and that the line may be in use. Pay attention to any messages on your terminal when you login this will clue you in to what is happening....
I received a copy of your response to John L. Grazul regarding digital TEM. We are using a system consisting of a Gatan wide angle video camera (model 673mkIII ), a Dage video processor (DSP200), Quick Capture board and a MacII running NIH-Image. The combination yields 640x480 images directly from the microscope. With the frame processor we can average frames (2,4,8,16,32) and adjust the gain and offset of the video signal so the Mac receives a full range signal. I have written some macros for Image to permit rapid specimen ID, magnification setting and storage. Folders of images are then sent over the network to the pathologist's desk.
We have found that the images are sufficient for diagnostic purposes and have begun to shift to electronic imaging.
The images are NOT the quality of film, but the cost for the system is low (NIH Image is free and VERY good). My concern was (and is) that with a slow scan system the increased cost doesn't really get you that much more in terms of resolution (maybe twice the resolution for lots more money). The microscope has such excess magnification that it is much cheaper to acquire multiple images at higher magnification (and hence greater resolution) than it is to purchase a slow scan camera and the associated software drivers. My thought is that if you want film-like resolution you should take micrographs and either print them or digitize the negative with a flatbed or drum scanner for image analysis. If all you want to do is some image analysis, it is entirely possible that a 640x480 image will be sufficient.
Feel free to forward this to Grazul and others on whatever mail list or news group you are on.
I would be interested to learn how you are using your system.
Charles Daghlian Rippel E. M. Facility Dartmouth College Hanover, NH 03755 603-650-1337
The list administrator is John Ladwig (jladwig-at-soils.umn.edu), but don't bug him until you've tried the above instructions! 8-)
As many of you list participants are aware, there is movement afoot to set up a NewsGroup which will be a superset of both the nih-image and microscopy lists. As this develops, there will be postings to both lists describing how/when this happens.
Bill ========================== Bill Heeschen / Analytical Sciences - Materials Characterization 1897-D Building / The Dow Chemical Company Midland, MI 48667 U.S.A. phone: (517)636-4005 fax: (517)636-5453 Email: waheeschen-at-dow.com ==========================
* SC3 Ion Projection Lithography Instructor: John C. Wolfe, Univ. of Houston
Resists -------
* SC4 Introduction to Microlithography, Resist Materials and Processing Instructors: Larry F. Thompson, AT&T Bell Labs.; Murrae J. Bowden, Bell Communications Research, Inc.; C. G. Willson, Univ. of Texas/Austin
* SC5 Optical Lithography Modeling Instructors: Chris A. Mack, FINLE Technologies, Inc.; Andrew R. Neureuther, Univ. of California/Berkeley
* SC6 Resist Thickness, Bake, Exposure, and Development Control Instructor: W. Tom Batchelder, Semiconductor Systems, Inc.
* SC7 Resists for Deep-UV Lithography Instructor: C. Grant Willson, Univ. of Texas/Austin
* SC8 Diazonaphthoquinone-Based Resists Instructor: Ralph Dammel, Hoechst Celanese Corp.
Metrology & Process Control ---------------------------
* SC9 IC Critical Dimension Measurement System Instructors: Sadri Khalessi, Metrologix, Inc.; Kevin M. Monahan, Metrologix, Inc.
* SC10 Fundamentals and Pitfalls of Submicrometer Dimensional Metrology Instructor: Terrence E. Zavecz, TEA Systems Corp.
* SC11 The Physics and Simulation of Metrology Instruments Instructor: Mark P. Davidson, Spectel Co.
* SC14 The Use of the Low-Voltage SEM in IC Fabrication Instructor: Michael G. Rosenfield, IBM Thomas J. Watson Research Ctr.
Microlithography ----------------
* SC15 Advanced Topics in Optical Lithography Instructor: Chris A. Mack, FINLE Technologies, Inc.
* SC16 Fundamentals of Cameras and Projectors Instructor: Warren J. Smith, Kaiser Electro-Optics, Inc.
* SC17 Introduction to Optical Lithographic Tools Instructor: Timothy A. Brunner, IBM Thomas J. Watson Research Ctr.
* SC18 Plasma Etching Technology Instructor: Daniel L. Flamm, Univ. of California/Berkeley
* SC19 The Exposure-Defocus Tree and Its Uses in Understanding and Extending Optical Lithography Instructor: Burn J. Lin, Linnovation, Inc.
* SC20 Optimization Methods for Microlithographic Materials and Processes Instructor: Daniel J. Herr, Semiconductor Research Corp.
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Don Grimes of Microscopy Today has sent the enclosed message along to me for approval for posting on the Microscopy Mailserver. I do not have a problem with it as it is a general announcement meant as a service to Microscopists who will be looking for jobs.
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To: Membership
Thanks to many for your interest and comments regarding our newsletter. Several have inquired over the possibility of our assisting in their search for new employment. I have previously not done this as it does not quite meet my objective as "material of interest to a reasonable number of microscopists". However I would like to try to help. If you seek new employment and would like to provide me a summary of either "what" you are or "what" you are looking for, I will publish the summary in my next issue. I only ask that you keep the summary under around 60 words and I MUST have it no later than this coming Friday (21 Jan) to make the issue. And if you wish to keep your name "quiet", we can do the Box # thing - where a number rather than your name is listed, and I will forward any interests direct to you. I expect that my newsletter is received in 99+% of the microscopy labs in the U.S. but can not guarantee that it goes to the right person.
Due to the number of replies to the attached message here is more info. The QM2000 tutorial runs on DOS and Windows. Its development platform is due out for the Mac and Unix, so there may be ports to those operating systems. Dr. Bolender has released a quantitative morphometry dataabase for the nervous system, developed with Sybase, running on a Sparc.
A summer quarter course in quantitative morphometry will probably be taught here this summer.
For more information: Dr. Robert Bolender Dept. Biological Structure SM-20 University of Washington Seattle, WA 98195 rpb-at-u.washington.edu
******old message There is a tutorial on quantitative morphometry written by Dr. Robert Bolender, Dept. Biological Structure at Univ. Washington. It also provides templates for point and intersect counts and for QM computations. It is available from the Health Sciences Center for Educational Resources, University of Washington SB-56, Seattle, Washington, 98195 (206) 685-1156.
Dr. Bolender teaches a course on QM in even years.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
I will am about to start as a postdoctoral scientist working with Prof. Kirz at the State University of New York at Stony Brook/Brookhaven National Laboratory in the field of x-ray microscopy. I am very interested in your EM-course on Immunocytochemistry and Cryosectioning. Please let me know what I need in order to apply. Sincerely, Joerg Maser
The Department of Materials Science and Engineering at Lehigh University has an opening for a senior (associate/full) professor with expertise in electron microscopy and/or microanalysis (SEM, TEM, AEM, etc.). The successful candidate must have a proven record of research in the application of microscopy and/or microanalysis to the solution of materials problems and be able to conduct independent and cooperative research in MS&E. Ability to teach undergraduate and graduate courses in microscopy and materials is essential. Experience in running a microscopy facility will be an advantage. Position available September 1, 1994.
Curriculum vitae and the names of three references should be sent by May 15, 1994 to: Professor David B. Williams Chairman, Search Committee Dept. of Materials Science and Eng. Lehigh University 5 East Packer Avenue Bethlehem, PA 18015-3195
Lehigh University is an equal opportunnity employer and welcomes applications from all qualified candidates.
Thanks to B.Miner for forwarding the useful comments from P.Catinella and B.Edwards on RIE etching on microelectronic devices.
One other question that I would like to direct to them or other interested parties would be the requirement for installing a "scrubber" on the exhaust fumes from the RIE. We are working up a estimate for the possible total cost of purchasing/operating/maintaining a plasma etcher in our laborato- ry. The requirement for a "scrubber" on the exhaust has been mentioned as a possibile environmental requirement. I have spoken to some manufactures and they don't use scubbers due to low flow rates in their machines and small quantities of gas involved.
Thanks again.
Richard Sartore US Army Research LAboratory AMSRL-EP-RA Fort Monmouth, NJ 07703 RSARTORE-at-MONMOUTH-ETDL1.ARMY.MIL
To Membership, International (or local) readers who wish to receive a no cost copy of our newsletter "Microscopy Today" and are having trouble contacting me, as I am on Compuserve, are encouraged to do so by FAX. My number is (608)836-1969. And the several who have requested the newsletter but did not supply their addresses are encouraged to resent their requests.
We have a Nanoscope III here, and would like to study the images off-line on another pc. Does anyone know good image analysing & processing programs for the PC? Basic filtering and measurement functions (cross sections etc) are a must.
I've heard names like Global Lab Image, Mocha, Image Pro Plus. Any experiences on these? Where to take contact: companies, addresses, fax numbers?
Thanks in advance,
Markus Levlin
-- Markus Levlin Laboratory of Physics tel +358 0 451 3144 markus.levlin-at-hut.fi Helsinki University of Technology fax +358 0 451 3116 Otakaari 1 M, 02150 Espoo, Finland
Thanks to B.Miner for forwarding the useful comments from P.Catinella and B.Edwards on RIE etching microelectronic devices.
One other question that I would like to direct to them or other interested parties would be the requirement for installing a "scrubber" on the exhaust fumes from the RIE. We are working up a estimate for the possible total cost of purchasing/operating/maintaining a plasma etcher in our laborato- ry. The requirement for a "scrubber" on the exhaust has been mentioned as a possibile environmental requirement. I have spoken to some manufactures and they don't use scubbers due to low flow rates in their machines and small quantities of gas involved.
Thanks again.
Richard Sartore US Army Research LAboratory AMSRL-EP-RA Fort Monmouth, NJ 07703 RSARTORE-at-MONMOUTH-ETDL1.ARMY.MIL
Dear Tamara Howard, I'm not sure if this is exactly what you are looking for but I have seen people in Jeremy Pickett-Heaps' lab at Melb. Uni. use teflon powder to coat glass slides before embedding between them. They don't actually grow the cells on the slides but just use them for thin layer embedding of algae in Spurr's. I'm not sure where teflon powder comes from - sorry. Can you grow the cells on plastic coverslips that you can section? I too have experienced having coverslips smashing instead of peeling nicely from the resin and actually found patience, practice and the gentle persuasion of a single-edged razor blade angled down between the glass and the resin to yield enough glass-free resin + cells to make it worth the trouble. David Orlovich.
Tamara, Try growing your cells on Permanox plastic dishes. The plastic is resistant to ethanol, acetone and resin and the polymerized resin is easily separated from the petri dish as long as the resin isn't too thick. I usually use a resin layer of about half a mm. If you need to use glass coverslips, you can try pushing the still-warm coverslip against a block of dry ice. The differential contraction rates will sometimes free the resin but it isn't 100% effective.
Rod Kuehn University of Minnesota
On Wed, 19 Jan 1994, Tamara Howard wrote:
} Does anyone have any experience with cell monolayers grown on glass coverslips? } I've seen a method reported where you coat the coverslip with carbon before } adding the cells; this is supposed to allow the resin to be stripped from the } glass for sectioning. Does it work? We thought the regular culture } substrates would allow release, but the glass just breaks when we try to "peel" } the resin away. Any suggestions would be VERY HELPFUL...I'm trying to section } glass, now. } Thanks! } Tamara Howard } Pitt Med/Pathology } Pittsburgh, Pa } tah-at-med.pitt.edu
} One other question that I would like to direct to them or other } interested parties would be the requirement for installing a } "scrubber" on the exhaust fumes from the RIE. } ...The requirement for a "scrubber" on the exhaust has been } mentioned as a possibile environmental requirement. I have spoken } to some manufactures and they don't use scubbers due to low flow } rates in their machines and small quantities of gas involved. } } Thanks again. } } Richard Sartore } US Army Research LAboratory } AMSRL-EP-RA } Fort Monmouth, NJ 07703 } RSARTORE-at-MONMOUTH-ETDL1.ARMY.MIL
The requirement for a scubber is more likely to be a matter of law and agency regs (military regs in your case) than real need, BUT it may also depend on what's coming off of the targets. The gas would be no trouble (so the manufacturers aren't going to worry about it), but if you're etching something like Gallium Arsenide semiconductors, there'll be reactive arsenic ions and the like coming out; that could be a problem. Scrubbing should be easily and cheaply done be bubbling the exhaust through distilled water (with maybe cotton batting in the outlet to make sure), which would be then disposed of by the usual regs for chemical waste. Phil Oshel po-at-parmly1.parmly.luc.edu
Re} TEM biol. cells on glass Tamara, I assume that you must embed the cells as a monolayer so that you can either orientate them or locate specific cells. If so, then you can take any of the blocks that you have already embedded, place them in liquid nitrogen and then warm them up. Repeated cycles of cooling and warming will crack the glass and will often cause it to shoot off the plastic. You must remove all resin from the top side of the block beforehand and be careful that the glass pieces do not get into your eyes. We use this method routinely to locate, and section, single cells that have been microinjected. The cells are grown on a locator slide and the pattern is transferred to the resin. You can help the glass removal by scoring the coverslip with a diamond before cooling and do not worry if the resin breaks. You will usually be able to find what you want amongst the pieces.
Cutting plastic coverslips is not easy, but a viable alternative to consider, if you only want orientation, is to grow the cells on the special filters produced by Costar and Falcon. These embed well and can be sectioned in resin. They are more difficult to cut cryosection from, but even this is possible.
If you only want a pellet of cells then it is better to grow them in plastic dishes. You can remove them, before fixation, by treating them with proteinase K, and after fixation by scraping with either a soft wooden or teflon scraper (not the normal cell scrapers that are available).
Good Luck
Paul Webster Yale School of Medicine (203) 785 5072.
Tamara Howard asks about stripping processed cells off glass coverslips, and suggests using carbon coated coverslips.
I have 3 suggestions:
1. I seem to recall seeing a paper/idea/suggestion once somewhere (???...) that rapid temperature changes at the glass resin interface will help break off the coverslip cleanly. Place a solid (pre-filled with resin and then polymerized) gelatin/Beem capsule over the cells on the coverslip and then polymerize them, attaching the cells to the capsule. Later, rapidly cool the coverslip, twisting the beem capsule. Play around, and let me know what works ! *** ONE ADVANTAGE: You could also first polymerize the cells between 2 coverslips (the one that it was grown on, and the other greased with vaseline so that it can easily be disloged after polym.), so that you could observe the cells clearly under phase contrast, mark the cells of interest, and finally place a solid resin capsule over the marked area, thus selecting the cells of interest.
2. Use propylene oxide (PO). This works like a dream when processing cells in plastic culture dishes: do all your processing for TEM right up to full dehydration with ethanol in the dishes. Monolayers process (fixation and dehydration) very quickly, and so it is a real easy technique to try. Decant the 100% ethanol from the dish, then quickly pour on pure PO, gently tilt the dish once or twice, and as the plastic of the dish starts to dissolve, the whole monolayer "peels off" and can be decanted into a suitable tube or small vial. Replace PO with the first change of resin, and carry on, embedding the mat of cells, perhaps using gentle centrifugation for the pure resin changes. Dave Sanan, now somewhere in California (give me a call, man!) first showed me this trick.
Perhaps the PO could even lift monolayers off the glass ? Would certainly work for plastic coverslips.
3. Hydrofluoric acid. Here's the real McCoy! Ref: J. Microsc. 104: 205- 207. Use hydrofluoric acid to etch away the coverslips, leaving the resin and cells behind. Be real careful as HFA is very corrosive. Work in a polyethylene or polytetrafluoroethylene dish in a fumehood.
Good luck
Ian
*********** ************ Dr Ian S Harper Int. Tel #: 27-21 938 0347 Experimental Biology Programme Int. Fax #: 27-21 938 0456 Medical Research Council Internet: iharper-at-eagle.mrc.ac.za PO Box 19070 Tygerberg, 7505 South Africa *****************************************
Thanks to B.Miner for forwarding the useful comments from P.Catinella and B.Edwards on RIE etching microelectronic devices.
One other question that I would like to direct to them or other interested parties would be the requirement for installing a "scrubber" on the exhaust fumes from the RIE. We are working up a estimate for the possible total cost of purchasing/operating/maintaining a plasma etcher in our laborato- ry. The requirement for a "scrubber" on the exhaust has been mentioned as a possibile environmental requirement. I have spoken to some manufactures and they don't use scubbers due to low flow rates in their machines and small quantities of gas involved.
Thanks again.
Richard Sartore US Army Research LAboratory AMSRL-EP-RA Fort Monmouth, NJ 07703 RSARTORE-at-MONMOUTH-ETDL1.ARMY.MIL
---------------------------------------------------------------------------- ---------------------------------------------------------------------------- --------------------------------------- Richard, If you are only dealing with a low volume of gas and you have a fume hood near by then you can do as we do exhaust fumes through there as most fume hoods, I would imagine, have a scrubber attached.
we only use plasma etching techniques for "deprossessing" integrated circuits for failure analysis so our gas flow is minimal.
For embedding monolayers we have been growing cells on a polymer film in place of glass cover slips. It is called Aclar and is available (in large quantities only) from ProPlastics, Linden NJ. It is optically very clear and separates easily from all embedding resins. It is apparently a Teflon- like substance. I was required to purchase more than a lifetime supply, so anyone who would like to try a sample should contact me. Not all cell lines will grow on the naked stuff. Some require a collagen coat and others never grow at all as they do on polystyrene dishes. or glass.
FROM THE PRESIDENT OF THE ROYAL MICROSCOPICAL SOCIETY
Something for Nothing! Bursaries Bursaries Bursaries ************************************************************************ Over many years it has never ceased to amaze me that RMS bursaries, to help eligible people attend meetings or courses, are not snapped up. It has even been known for them not to be fully taken up by the date of the sponsored event. Although admittedly the value of the bursary may not cover all of the expenses incurred in attending an event, they can be very useful primers in persuading other sponsors to make a contribution. Anyway, we announce here, bursaries for events in 1994.
RMS International Bursaries in Microscopy ************************************************************************ The RMS International Bursaries are intended to help young microscopists working outside Western Europe to attend RMS Courses or Conferences. The awards will be made to help with the registration and accommodation costs but it will not normally be possible to help with the cost of travel to the United Kingdom. The Society will offer up to six Bursaries annually and it is unlikely that any single award will exceed œ250. There are no strict rules or definite age limits but it is likely that they will be made to assist young scientists who would otherwise be unable to attend the Course or Conference. The Bursaries are not limited to Fellows or Student Members of the RMS, but it is unlikely that an award will be made to an Applicant currently working in North America or Japan.
The application for an RMS bursary can be made at any time, but should be made as far in advance of the Course or Conference as possible. The application should include details of the Course or Conference to be attended, a copy of any abstract(s) to be submitted and also a copy of the Applicant's Curriculum Vitae and publication list (if appropriate). The application should be accompanied by a letter of support from the Applicant's Head of Department or from a Fellow of the Royal Microscopical Society.
It is expected that the Applicant will have made efforts to find funding from elsewhere and he/she will be expected to show that such an application has been made - even if it was not successful.
RMS Bursaries in Microscopy ************************************************************************ The RMS bursaries will only be available to Fellows or Student Members of the Society. Non-members of the RMS are not eligible. The awards will be made to help with the registration costs of a Course or Conference, but it will not normally be possible to help with the cost of travel or accommodation. There are no definite age limits, but it is likely that they will be made to assist young microscopists who would otherwise be unable to attend. It is unlikely that any single award will exceed œ250 nor be made to an Applicant currently working in North America or Japan.
The application for an RMS bursary can be made at any time, but should be made as far in advance of the Course or Conference as possible as funding may be limited and to allow for fair refereeing of the applications.
It is expected that the Applicant will have made efforts to find funding from elsewhere and he/she will be expected to show that such an application has been made - even if it was not successful.
Applicants are advised that preferential consideration will be given to DipRMS and TechRMS candidates and those who have no financial support from their employer or other source. Please note that each application must be endorsed by the Applicant's Head of Department or employer.
The names of the successful Applicants will be published in the RMS Proceedings.
Application Forms for RMS Bursaries are available from: The Administrator, Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, United Kingdom. Completed applications must be returned to the Administrator at the same address.
The Summer School in Light Microscopy for 1994 will be organised on a modular basis, as in 1993, to allow flexibility in matching the needs and interests of participants to the subject matter provided, and to make it possible to include minor or specialist topics for which the demand might not justify the provision of separate courses. These topics can be offered in conjunction with others and thereby share the availability of instructors and expensive up-to-date equipment. There will be evening lectures and discussions to obtain the best use of the time available, and the numbers of participants will be strictly limited.
The first three days (Sunday evening to Wednesday evening), the Principles of Light Microscopy module will consist of lectures, demonstrations and practical classes on the fundamental aspects of light microscopy and the various imaging and contrast modes which can be used in the observation and characterisation of biological specimens, polymers, ceramics, minerals and metals. This module will cover the basic concepts of all forms of microscopy, leading on to the functioning and limitations of the light microscope, and introducing techniques for enhancing contrast. Practical work will make use of a variety of types of microscope from the major manufacturers, and the module will provide a practical understanding of the phenomena which lie behind the principles and practice of light microscopy.
From Thursday morning, the course will be divided into three specialist modules from which participants may select. Analytical and Applied Microscopy is an extension of the Principles module, these two together covering approximately the same ground as our former one-week Principles course. It will build on the topics covered in the Principles module and will show how they may be applied in practice. This will assist the development of a systematic and analytical approach to microscopy. A workshop format will enable participants to examine and discuss the images obtained from samples which will be provided and their own specimens, using a wide variety of techniques. The emphasis will be on the correct adjustment of the instruments, the strengths and weaknesses of each technique and the interpretation of images. This module will be especially valuable for microscopists working in industrial laboratories.
The Polarised Light module will address itself to the interpretation of contrast, not only in ceramics and minerals, but also in biological materials in which components of the structure are birefringent. It will attempt to explain by the use of simple diagrams and demonstrations, and with the minimum use of mathematics, the colour changes which are observed, and how contrast may be enhanced by the use of accessories. This module is designed to be of use to biologists, materials scientists and geologists.
In the Image Recording module it will be assumed that participants have a thorough understanding of the techniques of imaging and contrast enhancement. The module will include the principles of photography and video imaging, the transfer of the image from the microscope to the camera, and the design and construction of image-recording equipment. Time will be available for exposing black-and-white and colour film, which will be processed and evaluated before the end of the module.
Participants may register for the whole week (for the Principles module and one of the three specialist modules), for the Principles module alone, or for one of the specialised modules alone. Because the specialised modules are designed to build upon the groundwork presented in the Principles module, we expect that participants in specialised modules will normally either attend the Principles module at the beginning of the week, or have attended a previous RMS Light Microscopy Summer School.
We anticipate the customary extremely generous provision of equipment and materials from the manufacturers, for all parts of the course.
Principles of Light Microscopy Sunday evening 17 July to Wednesday evening 20 July 1994 (a 3 day module)
This module will consist of lectures, demonstrations and practical work on:
The history of microscopy. Introduction to microscopy. Limitations of the eye. Resolution, Contrast, Magnification. Interactions between light and matter. Refraction and lenses, geometrical optics, conjugate planes. Aperture. Illumination of the specimen in transmitted and reflected light.
Lens aberrations and their correction; the choice of optical components. Diffraction and its consequences for the microscope image. Generation of contrast. Introduction to bright-field, dark ground, phase contrast, polarized light, differential interference contrast and fluorescence.
This module will adopt a workshop approach and consist of short informal lectures, demonstrations and practical sessions. It will cover the correct adjustment of the microscope, the strengths and weaknesses of each technique, and the interpretation of images, in both transmitted and reflected light. Participants will have the opportunity to become familiar with techniques of their own choice according to their interests, using where possible, their own specimens. Facilities for specimen preparation will, however, be limited. We expect equipment for the following techniques to be available:
This module is designed to introduce biologists, materials scientists and geologists to the usefulness of polarized light techniques, and will involve the minimum use of mathematics. Lectures, demonstrations and practical work will cover the following topics:
The nature of light and polarization. The interaction of birefringent materials with plane-polarized light. Stress optical effects, the use of accessories and compensating plates. Minerals, ceramics, biological materials and fibres. Reflected polarized light techniques. Introduction to conoscopic techniques.
This module will discuss the recording of images by drawing, by photography and by video techniques. Equipment and materials will be available to enable participants to record images in black and white and colour of their own specimens and to discuss difficulties and results. The following topics will be covered:
Drawing, photography and video methods; an overview. Transferring the microscope image to the film and video camera. Monochrome and colour film; basic principles, use and processing. Negative and reversal films. Printing. 'Instant' monochrome and colour films. Colour temperature, light sources, films and filters. Equipment for photomicrography; focusing and determining exposure. Photomacrography. Video cameras, monitors and printers.
TO OBTAIN FURTHER DETAILS OF THIS COURSE SEND AN EMAIL MESSAGE WITH YOUR NAME AND ADDRESS TO RMS-at-UK.AC.OX.VAX, OR CONTACT THE RMS ON TELEPHONE +44 (0)865 2488768, FAX +44 (0)865 791237.
A range of techniques have been devised for localising antigens at the electron microscope level. One of the difficulties commonly faced is the choice of which method to use with any particular antigen-antibody combination. The aim of the course is to provide a theoretical and practical introduction to the various methods available and covers antigen location, using both transmission and scanning electron microscopy.
The advantages and disadvantages of the various methods available will be discussed, but the emphasis will be on the practical aspects of the techniques and will provide experience of the following methods: pre-embedding labelling for scanning electron microscopy; low temperature embedding in Lowicryl resins; preparation and labelling of both resin sections and thawed cyrosections; rapid freezing by impact and high pressure followed by freeze substitution and low temperature embedding; silver enhancement of colloidal gold for light and electron microscopy; immunolabelling of 1æm resin sections; and epi- polarised light microscopy. Registrants are encouraged to discuss specific problems with the course organisers and if possible, to bring samples with them for processing and labelling during the practical sessions, which will be supported by Leica (UK) Ltd.
The course is primarily aimed at electron microscopists with experience of routine processing methodology who wish to become familiar with ultrastructural immunocytochemical labelling techniques.
The practical nature of the course, means that numbers will be restricted to a maximum of 10 registrants.
FOR FURTHER DETAILS CONTACT THE RMS - TELEPHONE +44 (0)865 248768, FAX +44 (0)865 791237, EMAIL RMS-at-UK.AC.OX.VAX.
The unprecedented upsurge in the application of immunocytochemistry in the life sciences during the past decade continues with increasing vigour. Both light and electron microscopy are important techniques in routine diagnosis and research in medicine and biology. This technology has contributed so much towards our current understanding of the cell, that the modern microscopist must be part immunologist as well as being skilled in microscopy.
The underlying principles of immunocytochemistry apply equally to light and electron microscopy. This five-day course has been specially designed to utilise this overlap and therefore, will be of value to both light and electron microscopists. The course is structured towards a technical appreciation of immunocytochemical techniques, since once they are mastered, they can be applied to any system. Each year this popular course is updated in the light of new developments in immunocytochemistry and is also suitable for participants interested in the plant sciences, as we teach some of the specialist techniques required for the handling of plant cells. The course will be of immense value to any life science microscopist/cell biologist of any background, who intends to use or is starting to use immunocytochemistry for routine purposes or research.
The course counts towards qualification for the Diploma of the Royal Microscopical Society.
The main emphasis of the week will be to give participants sufficient practical experience and knowledge of immunocytochemistry to carry out immunolabelling in their own laboratories. At the same time, a series of lectures will be given by more specialist exponents in various areas of immunocytochemistry.
The three practical days will be led by experts in their particular field: Tony Leathem and Susan Brookes (LM immunocytochemistry), Paul Monaghan (immunogold labelling for EM) and David Hughes (silver enhancement). After an introduction on the biology and production of antibodies, the practicals will be backed up with lectures on the applied aspects of the respective techniques. Of particular interest is the series of specialist lectures which will include, botanical immunocytochemistry (Chris Hawes), in situ hybridisation (John Davies), confocal microscopy (Mark Fricker), immuno-scanning EM and special applications of colloidal gold. An evening workshop will be held to introduce cryo-techniques in immunocytochemistry and to demonstrate some of the latest equipment used in low temperature tissue preparation.
FOR FURTHER DETAILS CONTACT THE RMS - TELEPHONE +44 (0)865 248768, FAX +44 (0)865 791237, EMAIL RMS-at-UK.AC.OX.VAX.
Last year we experimented with a new format for our Flow Cytometry course. Following its success both in attracting students and instructing and informing them, we will continue with the new format. There will be two courses - basic and advanced - run sequentially to give the potential student the choice of attending either or both of the courses. We anticipate that there will be at least three bench top cytometers for use by the students and one machine equipped with two lasers for the advanced course. As usual, we will rely on the generous co- operation of the manufacturers, Becton-Dickinson, Coulter and Ortho, who lend us both the machines and experienced operators to run them.
The basic course will assume little prior experience and take the student through the most important applications. Although it will fill the needs of someone working in a research environment, it will have a slight clinical bias. The majority of bench top flow cytometers are now to be found in clinical laboratories.
The first day will consist of a series of talks describing the basics of flow cytometry. A simple practical (measurement of a DNA histogram from cultured cells) will serve as an introduction to using the bench top flow cytometers.
There will be two practicals on the second day. In the first, students will use antibodies labelled with three different fluorochromes to identify lymphocytes subsets in human peripheral blood. This practical will also demonstrate the importance of using light scatter to separate lymphocytes, monocytes and granulocytes in samples of blood. In the second practical (lead by Richard Camplejohn from St. Thomas's Hospital), nuclei will be extracted from a formalin-fixed, paraffin embedded tumour and the DNA histogram recorded. The rest of the programme will include lectures on the analysis of DNA from clinical samples and on immunophenotyping in a hospital laboratory.
The third and final day will also be the first day of the advanced course. In the practical demonstration, lead by George Wilson (GRC Gray Laboratories), students will investigate the cell cycle kinetics of a mouse tumour which will have been labelled in vivo with a thymidine analogue (bromodeoxyuridine, BrdU). There will be a lecture on applying this technique and its clinical application and on DNA measurement and cell cycle analysis, the principles of cell sorting and the measurement of antigens associated with cell proliferation.
The advanced course will join us for the last day of the basic course (see above). On their second day, they will prepare and analyse chromosomes, using both bivariate and univariate analysis. We will set up a flow cytometer in the lecture theatre and project the computer screen so that it can be seen by the whole class. Jim Watson (MRC, Addenbrookes, Cambridge) will lecture on time as a parameter and then run experiments in the lecture theatre on intracellular enzyme kinetics looking at esterases and glutathione-S-transferase. A lecture on the measurement of intracellular pH and calcium ions will also be followed by a live demonstration. The other lectures will be on chromosome analysis and sorting and on further applications in cell and molecular biology.
The third and final day will consist of lectures on studying apoptosis, measuring multi-drug resistance in tumours and lectures and practical demonstrations on the measurement of cell cycle kinetics using the BrdU-Hoechst/PI method and on the interaction of fluorochromes and DNA.
We expect that anyone attending our basic course will come away with a sound grasp of the principles of flow cytometry, an understanding of the concepts behind data analysis, a feel for some of the problems and the confidence to run the commoner applications. Everything in the course will be relevant to workers in a clinical environment.
The advanced course will be at the forefront of the technology. It should give students a broad understanding of the wide range of applications of flow cytometry in a modern research laboratory. It will help them to establish new techniques and ought to give them ideas for new experiments in their own laboratories .
We hope that many students will take the opportunity to stay the whole week and to benefit from a broad look at flow cytometry - from basics to the most advanced applications. The RMS course is the only comprehensive course on flow cytometry to be run in the UK
FOR FURTHER DETAILS CONTACT THE RMS - TELEPHONE +44 (0)865 248768, FAX +44 (0)865 791237, EMAIL RMS-at-UK.AC.OX.VAX
We have versions of Diffract, DiffractII, and Desktop Microscopist. The best one is the Desktop Microscopist by far. It works much better, doesn't have as many faults as diffract and is more user friendly. It doesn't have that annoying propensity to crash all the time as diffract.
I would like to use freeze substitution for a low magnification TEM study of cell-extracellular matrix relationships in amphibian embryos. Can anyone suggest an appropriate fixative and concentration for adding to the sustitution medium (MeOH) prior to embedding in EPON?
Dave Parichy Section of Evolution and Ecology Univ. of California, Davis 916 752 3634
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: "Rick A. Harris" {szrick-at-bullwinkle.ucdavis.edu}
} I would like to use freeze substitution for a low magnification TEM study } of cell-extracellular matrix relationships in amphibian embryos. Can } anyone suggest an appropriate fixative and concentration for adding to } the sustitution medium (MeOH) prior to embedding in EPON? } } Dave Parichy } Section of Evolution and Ecology } Univ. of California, Davis } 916 752 3634
You might find useful information in:
Allanspach, A. 1993. Ultrastructure of early chick embryos after high pressure freezing and freeze substitution. Micoscr. Res. Techn. 24:369-384.
Hippe-Sanwald, S. 1993. Impact of freeze substitution on bioolgical electron microscopy. Microsc. Res. Techn. 24:400-422.
Hunziker, E.B. 1993. Application of cryotechniques in cartilage tissue preservation and immunoelectron microscopy: potentials and problems. Microsc. Res. Techn. 24:457-464.
McDonald, K. and M. Morphew. 1993. Improved preservation of ultrastructure in difficult-to-fix organisms by high pressure freezing and freeze substitution: I. Drosophila melonagaster and Strongylocentrotus purpuratus embryos. Microsc. Res. Techn. 24:465-473.
I have just had a look in my favourite freeze-substitution book (Cryotechniques in Biological Electron Microscopy, eds: R A Steinbrecht and K Zierold) and they suggest the following freeze-substitution medium for methanol substitution:
Methanol containing 0.5% uranyl acetate, 1% OsO4, 3% glutaraldehyde and 3% water (the water comes from the 50% glutaraldehyde they used).
It actually looks a bit complex to make the solution up - they suggest:
add 9 mL of 50% aq. glutaraldehyde (in a liquid nitrogen precooled flask) to 60 mL methanol, then 3 mL of a 20% (w/v) soln of uranyl acetate in methanol are added; in a second precooled flask 1.5 g osmium tetroxide are dissolved in 75 mL methanol; both flasks are cooled to about 220 K, their contents poured together and vigorously shaken.
They say that the mixture is highly reactive even at 240 K and so should be used in a few hours.
Substitution time was 8 hours each at -95 deg C, -60 deg C and -30 deg C. Then 30 minutes at 0 deg C. Replace the substitution mix with pure acetone (still at 0 deg C), warm to 7 deg C and infiltrate with araldite/Epon.
The original reference to this protocol is: Muller, M., Marti, T., and Kriz, S. (1980). Improved structural preservation by freeze-substitution. In: Brederoo, P., and de Priester, W. (eds). Electron Microscopy 1980, vol.II. Proc. 7th Eur. Congr. Electron Microsc., Leiden, pp. 720-721.
I've always used 2% OsO4 in acetone at -70 deg C for up to 7 days and then embedded in Spurr's resin. The best reference I have for that is:
Howard RJ and O'Donnell KL (1987). Freeze-substitution of fungi for cytological analysis. Exp. Mycol. 11:250-269.
I hope this is of some use.
David Orlovich School of Biological Science University of NSW PO Box 1 Kensington NSW 2033 Australia
Subject: Time:2:18 PM OFFICE MEMO RE} SEM RES STD Date:1/24/94 The method I have used with good success was published on p. 68 of the May 1987 issue of the EMSA Bulletin. If you don't have access to that issue of the Bulletin, send me your FAX address and I'll send a copy to you. Bigelow-at-umich.edu.
I am looking for software (vendor / public domain) that can draw crystal structures and print these diagrams on a laser printer. I would like to be able to enter the crystal system, cell edges, etc.. and have the diagram show the coordination of differing cation sites. Any suggestions?
Message-Id: {MAILQUEUE-101.940125090324.512-at-FS-IAM-1.JRC.NL} To: Microscopy-at-anlemc.msd.anl.gov
I intend to do some internal strain measurements using HOLTZ lines, so could anyone suggest any software which allows the simulation of HOLTZ lines, and where I could find it?
Kodak has discontinued direct duplicating film 2468 in 100 foot rolls. We have used this film successfully for years to make presentation slides from halftone prints. What should we use now as a replacement?
Received: from MAILQUEUE by MICROBIO1 (Mercury 1.11); Tue, 25 Jan 94 11:24:30 EST Return-path: {EMLAB-at-opus.mco.edu} Received: from emoryu1.cc.emory.edu by transporter.microbio.emory.edu (Mercury 1.11); Tue, 25 Jan 94 11:24:27 EST Received: from anlemc.msd.anl.gov by emoryu1.cc.emory.edu (5.65/Emory_cc.3.4.12) via SMTP id AA15050 ; Tue, 25 Jan 94 10:51:32 -0500 Return-Path: EMLAB-at-opus.mco.edu
Kodak has discontinued direct duplicating film 2468 in 100 foot rolls. We have used this film successfully for years to make presentation slides from halftone prints. What should we use now as a replacement?
KODAK OFFERS A FILM CALLED " RAPID PROCESS COPY " (CAT. NO 174 6031 FOR THE 150 ft ROLL) WHICH IS A DIRECT REVERSAL FILM DESIGNED FOR THE PURPOSE YOU DISCRIBE. THIS FILM IS QUITE SLOW : EXPOSURES IN THE 30 TO 45 SECOND RANGE AT f 4.0 ON MY SETUP. ONE NEEDS TO CALIBRATE THEIR COPY STAND SETUP TO THE FILM AND PROCESSING.
THE PROCESSING IS VERY SIMPLE: DK 50 DEVLOPER FOR 10 MINUTES, FOLLOWED BY STOP AND FIXER.
I HAVE BEEN USING THIS FILM SINCE THE MID- EIGHTIES WITH EXTREMELY GOOD RESULTS. ANY QUESTIONS? CALL ME. lmelsen-at-unix.cc.emory.edu
Via: uk.ac.birmingham.computer-centre.ibm3090; Tue, 25 Jan 1994 16:27:15 +0000
On Tue, 25 Jan 1994 MACLAREI-at-ibm3090.computer-centre.birmingham.ac.uk wrote:
} I wonder if anyone has any good ideas on how to electropolish commercial } purity Titanium without getting precipitation of hydride needles. } } I have tried using a variety of acid based solutions, most of which } contained perchloric acid in varying concentration. All of these, however, } result in hydride precipitation. } } The net result of all of this is that I find it very difficult to prepare } decent specimens of Titatium by electropolishing. If anyone has any good } ideas as to how these problems can be overcome I would be very glad to hear } from you. } } Ian MacLaren
Ian,
You may want to try the following, (although I have NOT tried this particular recipe):
Ethanol (96%)................90 ml n-Butyl alcohol..............10 ml Aluminum Chloride.............6 g Zinc Chloride................28 g for 1-6 minutes; 20 -25 V dc; stainless steel cathode; room temp.; need to keep agitated (the solution, NOT the user {grin!} ) to prevent a passivating layer from forming: can use a stirrer, or oscillate the anode quite rapidly at a fixed distance from the cathode (approx. 1 to 2 cm). * remember: nicely TOXIC!!! {ref.: The Electrolytic and Chemical Polishing of Metals; Tegart; 1959; Pergamon Press}
Hope this is helpful, Rob
X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X X Rob Tayloe X MSM Spelunkers Club X X Metallographic Lab. X Missouri Speleological Survey /-v-\ \-v-/ X X Rolla Research Center X Bat Conservation International X X U.S. Bureau of Mines X Missouri Cave & Karst Conservancy \-v-/ X X tayloe-at-rorc.usbm.gov X National Speleological Society #32993 /-v-\ X X (314) 364-3169 x247 X American Cave Conservation Association X X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X\X
I think there is new chemistry for using T MAX films for direct positives. Someone from Kodak should pick up on this message and let the rest of us know about it. ***************************** * Greg Erdos * * Director, ICBR EMCL * * University of Florida * * Gainesville, FL 32611 * * gwerdos-at-gnv.ifas.ufl.edu * * 904-392-1295 * *****************************
I called my supplier of 2468 today. They say that the film has NOT been discontinued. (And this company claims they are the only E. Coast supplier!) What's the truth???
If your prints come from large-format negatives, you can photograph the negatives on a light box with technical pan or t-max. If you need a direct positive film, you can use Ektachrome color slides or use t-max with a direct-positive developing kit.
Rod Kuehn University of Minnestota
On Tue, 25 Jan 1994 EMLAB-at-opus.mco.edu wrote:
} Kodak has discontinued direct duplicating film 2468 in 100 foot rolls. } We have used this film successfully for years to make presentation } slides from halftone prints. What should we use now as a replacement? }
Return-path: {BERGRH-at-MSUVX1.MEMST.EDU} Received: from memstvx1.memst.edu by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id {01H848IIDF408WW4DH-at-gnv.ifas.ufl.edu} ; Tue, 25 Jan 1994 23:26:34 EST Received: from MSUVX1.MEMST.EDU by MSUVX1.MEMST.EDU (PMDF V4.2-14 #5958) id {01H845ET8EPC9BWPOF-at-MSUVX1.MEMST.EDU} ; Tue, 25 Jan 1994 22:28:15 CST
Greg-- I am reading your Microscopy post and, not adept at replying to these public forums, am writing you direct. I used the TMax direct reversal kit for my talk at this summer's MSA meeting and was very pleased with the quality of the slides it produced. I made "superslides" by using (hard to find) 127 film mounts and TMax 100 film size 120. The kit specifies rating the film at ASA 50, half its normal speed. The kit instructions are well written and I got good results from the first roll on. Instructions for extending development with subsequent rolls are clearly written--as I recall the kit will process about 8 or 10 rolls and cost me $35 (?). Significantly more rolls of 35mm would be possible
Direct Pos. Film users: Kodak makes a TMax direct postive film developing kit for making black and white slides. Catalog number 812 1188. Cost: approx. $30. When doing line copy I have also used LPD4 film for continuos tone copy and have had very good results. There's a little trickery involved but it allows you to do line and continuous copy on the same roll of film. If anyone is interested, I'll tell how it is done. Phil Rutledge prutle1-at-gl.umbc.edu
Message-Id: {MAILQUEUE-101.940126090559.480-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
} Date: Wed, 26 Jan 1994 09:26:33 -0500 (EST) } From: rutledge phil {prutle1-at-gl.umbc.edu} } Subject: Tmax } To: microscopy-at-anlemc.msd.anl.gov
} Direct Pos. Film users: } Kodak makes a TMax direct postive film developing kit for making black } and white slides. Catalog number 812 1188. Cost: approx. $30. When doing } line copy I have also used LPD4 film for continuos tone copy and have had } very good results. There's a little trickery involved but it allows you } to do line and continuous copy on the same roll of film. If anyone is } interested, I'll tell how it is done. } Phil Rutledge } prutle1-at-gl.umbc.edu
I'll second this--I've used LPD-4 for making direct-positives of photographs for slides when I only want to mess with developing one type of film. 8 secs. at 1/2-stop intervals from f111/2 to f4 or 41/2 will usually get you a usable frame. This range of f-stops is conservative; actually shouldn't need more than a couple of brackets once you figure out your local conditions. The Direct Positive sold by Ted Pella does do a better job, but.... Phil Oshel
Kodak make a T-Max Reversal Kit for direct positives. The catalogue number is K-8121188. It comes as a 1 quart unit. In Canada it sells for about $35.00.
Kodak make a T-Max Reversal Kit for direct positives. The catalogue number is K-8121188. It comes as a 1 quart unit. In Canada it sells for about $35.00.
} } I have found something that I think works better, but takes a day to } develop - regular color Ectachrome slide film (Tungsten). The results } are very good and having somebody else fool with the wet chemistry is } much better. I think that the development process is C-47 which is the } same for color negatives which means that you can have it done at 1 hour } photo shops. As a reult, I only use the MP 5360 when I have an } emergency. } } } Scott Walck } walcksd-at-ml.wpafb.af.mil } Materials Directorate } Wright Patterson AFB, OH
I have had excellent results with Ektachrome, but find it takes 25-30 minutes to development: process E-6 with the Kodak hobbyist pack, 10-30 minutes to dry, depending on if you have a film drier. Most 1-hr photo-shops that I know of won't do E-6 films. Phil Oshel po-at-parmly1.parmly.luc.edu Parmly Hearing Inst. see c. v. on MSA Bulletin Board. Please!
Slides I can't believe that anyone is satisfied with the 35mm format for projection slides. Besides, copying prints with a camera always results in a loss of quality. The best way is to take the information straight from the negative, although this does not let you label the image with letraset. I print directly onto film, using an enlarger and mount the image in the super-slide frames (40x40mm) loved by electron microscopists and hated by projectionists. The image can be carefully framed and the contrast can be easily manipulated. The film to use is Agfa sheet film, so you will have to find your own supplier, and can be purchased in boxes of either 10x8 or 10x12. Use it as you would paper under an enlarger. For a soft image use "Litex premium camera film 0910P". For a harder finish use "Litex camera halftone film 0811P". These films are normally developed in Gevaline G7C developer, also from Agfa, which will produce high contrast but they can also be developed in D-19 for a softer contrast. I am sure that acutance developers will also work well. The film can only be handled in red light and has to be dish developed but the results are worth it. Try it and compare with camera copies. There is nothing like a big image to make your point. If you are interested and require more details then feel free to contact me. Paul Webster Yale School of Medicine paul_webster-at-quickmail.yale.edu (203) 785 5072.
} Slides } I can't believe that anyone is satisfied with the 35mm format for projection } slides. Price! Money! Grant funds! 35mm is cheap! And everyone has a slide projector that will take 35mm. Phil Oshel
I would appreciate any feedback from users of Digital confocal software packages like MICRTOME from VayTek or comparable systems.
1) Does cost justify capabilities.
2) How it compares to laser systems?
3) Are manus userfriendly?
4) If you used package already would you buy an upgrade or move on to a different system?
Thanks.
Cesar D. Fermin, Ph.D. Tulane University Medical School Department of Pathology 1430 Tulane Ave/SL79 New Orleans, La 70112-2699 (504) 584-2521 Fax 587-7389
"Price! Money! Grant funds! 35mm is cheap! And everyone has a slide projector that will take 35mm. Phil Oshel"
What a rude reply Phil! I don't deserve that.
Most EM labs have a darkroom with an enlarger. A box of 100 sheets of film is cheap and will last for years (I bought my boxes four years ago and still have enough to send to you, Phil Oshel, if you want to try out the system. The 40x40mm slide holders (from GePe) fit into a 35 mm slide projector -they just use up more of the available space. If anything it is all cheaper than buying a copystand and 35 mm camera. Why do I bother?
During my short stay in Sendai, Japan, I learned a good way to make overheads from electron microscope negatives. The material is FUJIGRAPH PROJECTION FILM PT-100, which comes at least in size 21x29,7cm (DIN A4) in 100 sheets packages. This film can be used just like the enlarging paper in the darkroom. The results are far better than with any oldfashioned way. These overheads are good for teaching purposes because you can easily make markings on them.
Regards, J M
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Maki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
} Re} Slides } } "Price! Money! Grant funds! 35mm is cheap! } And everyone has a slide projector that will take 35mm. } Phil Oshel" } } What a rude reply Phil! I don't deserve that.
My apologies...but I repeatedly run into people, usually from medical schools, who don't understand why others don't use this or that, usually expensive, technology, instead of all the old stuff. The litany can get very frustrating. The answer is usually money. And often administrators. Labs and smally schools have photographic equipment & 35mm cameras, 35mm slide mounts, etc.. I have worked in EM labs for 13+ years, and have spent many hours all of the equipment you mention below. I have had much trouble with 40X40 slide holders jamming in 35mm projectors, but haven't tried the brand you recommend. Plus, multiple-image plates & 4"X5" Polaroid images still need to be photographed on the copy stand. And a good 35mm camera is still the cheapest, easiest way to produce the slides. Pros don't use them out of tradition. Personally, I'd prefer to use 120 film, or even 4X5, but....And most light microscopes come with 35mm camera backs. Meaning you need all that stuff anyway if you're a service lab, or anyway, not a dedicated lab where only one photpgraphic system is used (which most are). But what type of film comes in 100 sheet boxes & lasts for years? The film I've seen & used in TEMs & any other microscope--sheet, 35mm, or 120 roll--goes quickly. Printing paper's worse. Especially with students and EM courses! Thank you for the offer to send some materials, but it'd be a loss now--our grant & my job got cut, so it wouldn't be used. Phil Oshel } Most EM labs have a darkroom with an enlarger. A box of 100 sheets of film is } cheap and will last for years (I bought my boxes four years ago and still have } enough to send to you, Phil Oshel, if you want to try out the system. The } 40x40mm slide holders (from GePe) fit into a 35 mm slide projector -they just } use up more of the available space. If anything it is all cheaper than buying } a copystand and 35 mm camera. Why do I bother? } } }
To E'mers wanting EM slides: The easiest way to make EM slides is to put your neg in an enlarger and shoot onto another piece of EM film (type 4489). Develope it as you would normally process em film. This allows you to develope in a tray under a red safelight and allows control of the density of the slide. It gives great contrast in the slide or you can control the contrast by the way you develope the film. I started doing it this way for em slides only over 25 years ago and have always gotten good contrast whether the original negative was on the flat or contrasty side. Phil Rutledge prutle1-at-gl.umbc.edu
As I said in a previous message, I use LPD-4 for continuous tone slides on the same roll of film when I am doing line copy. The way I do this is as follows: I use a Polaroid MP-4 copy stand equipped with a Nikon F3 and a 90mm macro lens.
Line Copy __________
Exposure: 5 seconds/f:6 Develope: D-19 full strength- 2 minutes Fix: 2 minutes in rapid fix with Orbit Bath added. Orbit Bath allows a 2 minute fixing time and a 5 minute wash. It's better than Kodaks Hypo Clearing Agent. Wash: 5 minutes, dry, mount
Continuous Tone ________________
This requires a little playing with to determine your exposure. I use same exposure for the line copy but I add 3-5 seconds more. I pre-fog the film by exposing the copy for 8-10 seconds. At the same time I continuously move a grey scale card under the lens until I get to the 5 second mark for the line copy exposure. Process as above. I have always had good results with this method. You just need a little coordination exposing this way. Phil Rutledge prutle1-at-gl.umbc.edu
This is the text of an advertisement that will appear in the british press next week for a postdoctoral position which will become vacant on the 1st of June this year. We would like to identify an appropriate candidate as soon as possible - hence the short deadline. Applications will be accepted by FAX but NOT BY ELECTRONIC MAIL. Please note that, due to circumstances beyond our control, we will have to give preference to candidates who are citizens of nations in the European Economic Community.
The University of Birmingham School of Metallurgy and Materials
POSTDOCTORAL RESEARCH FELLOWSHIP
Applications are invited for the above post to work on a 2.5 -year SERC-funded project entitled "Mechanical Behaviour of Nb3Al Alloys". The successful applicant will investigate the basic defect structure and deformation mechanisms in these alloys and to explore strategies for enhancing their ductility.
A Ph.D. degree, extensive experience of electron microscopy techniques and familiarity with the physical metallurgy of intermetallic compounds are essential.
Preliminary enquiries should be directed to Dr. M. Aindow - Tel: (44) 21 414 5188, FAX: (44) 21 414 5232, Email M.AINDOW-at-BHAM.AC.UK, or Dr. I.P. Jones - Tel: (44) 21 414 5184.
Application forms and further particulars may be obtained from The Director, Staffing Services, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom or telephone (44) 21 414 6483 (24 hours) and quote reference G10613/94. The closing date for receipt of applications is 18/2/94.
The University of Birmingham is an equal opportunity employer.
Mark Aindow, School of Metallurgy and Materials, Telephone; (021) 414 5188 The University of Birmingham, FAX; (021) 414 5232 Elms Road, Edgbaston, Birmingham, Email; M.AINDOW-at-BHAM.AC.UK GB B15 2TT, United Kingdom.
I have had quite a few requests asking how to access the new sci.techniques.microscopy newsgroup. So, I thought I would post a summary of the whats, wheres and hows of Usenet News, as I understand them, to the NIH-Image and Microscopy mailing list.
What is Usenet News? Usenet News, unlike email, is not distributed to an individuals account, but rather to a server at a particular institution (be it a government lab, university or private company) that can then be accessed by individuals at that institution. Usenet News is a system where messages are posted to newsgroups that are designed to cover a particular topic. Examples of topics are rec.windsurfing (recreational: windsurfing), comp.sys.mac.digest (Computer: Macintosh question and answer digest), sci.materials (Science: Materials Science) and, of course, sci.techniques.microscopy (Science: Microscopy Techniques of all kinds). There are many hundreds of worldwide newsgroups, i.e. groups that are distributed around the world, and then there also are groups that are only distributed locally. Examples of world-wide groups are those I have mentioned above and here at the University of Michigan we have local groups for class disscussion. As an example, for our electron microscopy course (MSE 562) we could have a group umich.eng.mse562 and it would only be available to readers in the umich.edu domain.
Postings to each newsgroup are stored on the institution's local NNTP (sorry guys I dont what this is an accronym for unless it is Network News Transport Protocol!) server and distributed to other sites. If the same kind of system is used now as was used when Usenet was first started, again I am no expert here - just a user, then each news server will exchange messages with a number of other servers geogrpahically close to it and the postings will sort of hop from one system to another and propgate around the world. When Usenet was first started the transfer was by dial-up modem lines, now the how system uses the Internet. If there is someone out there who can describe this process more exactly, please let me know or post a summary.
How do I read Usenet News and post to Newsgroups? If you want to use Usenet News you need access to an NNTP server and news reading/posting software to run on your local computer, workstation or terminal. You should contact your local network adminsitrator and ask them if you have access to a Usenet feed and which net-news software they recommend for the machine you use. If they do not have access, ask them why not! The functionality of each news program is best expalined by a local expert, I cannot possibly go into all of the details of even one news program here. Suffice to say however, if you have access to a news posting and reading program and access to the accompnaying NNTP server, you can post articles, questions and information to be read by people around the world.
Which software should I use? As I say it depends on you machine and what is available to you locally. I use a Mac and my favorite software is NewsWatcher (which is free, a definite bonus). The lastest verion of this software is available by anonymous ftp from ftp.acns.nwu.edu (the same place as Disinfectant, the antiviral utility for the Mac) in the directory /pub/newswatcher. The current version is 2.0d17. Ask around locally to see what you have available.
Why News and not just Email? The advantage of News over email mailing lists is the messages reside on a remote machine and do not clog up your mailbox. Some people have severely restricted mailbox size allocations and the output from a mailing list can swamp them and prevent them from receiving other mail. With Usenet you only download the News you want to read. You can subscribe to a small subset of the total newsgroups and selectively scan through those. You may view all of the subjects of the articles in a group before you read any of them. It is quite a flexible system.
I cant get to the Internet, what do I do? The one major complaint about Net News is that it is not accessible unless you have a connection to the Internet. The mailing lists can be accessed from Compuserve, America On-Line, GEnie, etc. An connection used to be an expensive proposition, however, these days it can be as reasonably priced as Compuserve or the other dial-up network services. For example "The World" (1-617-739-0202) charges $20 per month for 20 hours of connect time to Internet services. Another provider "The WELL" (Whole Earth 'Lectronic Link - 1-415-332-4335) charges $15 per month and $2 per hour for use. For a local provider please call the InterNIC Information Services at 1-800-444-4345.
This is just a brief outline that I have put together on the spur of the moment (as someone recently put it "its a stream of consciousness"!) Hope it helps. Drop me a line if I have made any major mistakes or left anything vital out of this message. Cheers, John Mansfield.
} 2) How it compares to laser systems? One potential problem is that to collect images using a cooled CCD camera the exposure times must be much longer than with a laser scanning confocal microscope. For instance, we did a comparison of a double labeled cultured cells scanned with a cooled CCD camera and with the BioRad MRC 600. With the CCD camera the Cy3 stain looked great (deconvolved with the quick mode of the Vaytek system). However, the FITC bleached before we could collect a few sections. On the BioRad, we were able to collect both signals simultaneously without significant bleaching of the FITC. Also, we looked at the BDS deconvolution system. We were very impressed with the deconvolved images, but here is a major difference between confocal and deconvolution: Using a confocal, investigators from all over the university can come in, scan their samples, immediately see results, get hard copy or send images to other computers instantly, and leave. If they were using deconvolution they would have to post-process every optical section before getting results. Personally, I would like to see how the BioRad confocal images would look processed with one of the deconvolution packages. -Michael Cammer
The problem "jjerome-at-isnet.is.wfu.edu (Jay Jerome)" mentioned as spectral effects when he mounted slides in glass mounts may be Newton rings. This problem may be eliminated by using anti-Newton glass in the mounts. Companies such as Wess or Gepe market this kind of glass for slide mounts. Contact your local photo shop or look in one of the photography magazines for advertisements.
} With regard to suggestion that our problem of spectral effects, we do use } Gepe anti-newton glass slides. However, the effect is reminiscent of the } effects one gets if you do not use anti-newton glass. It does not occur } however when we use thinner based film for making our slides. Only when we } use EM negative film such as 4489. Any other suggestions?
If you're not doing so already, you might try using a mask between the backing side of the film and the glass. This will eliminate the contact between the two flat surfaces that can produce Newton rings.
Way back when, we used to use commercially available masks cut from black paper. I've also used aluminum ones from EMDE Products, Inc. I don't even know whether they are still in business. The address in the literature I have from them doesn't even have a zip code. The address given is 2040 Stoner Ave., Los Angeles, CA.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
} Return-Path: {Vitor-at-dsif.fee.unicamp.br} } From: Vitor-at-dsif.fee.unicamp.br } Date: Wed, 26 Jan 94 09:26:00 EDT } To: venema-at-dutentb.et.tudelft.nl, Snitka-at-dsif.fee.unicamp.br, } tprohas-at-email.tuwien.ac.at } Subject: CALL FOR PAPERS } } } } } ----- Begin Included Message ----- } } } From PHOTOEXE%ILCTEHOL-at-VMS.HUJI.AC.IL Sun Jan 23 10:06:12 1994 } Date: Sun, 23 Jan 94 09:59 GMT } From: INTL BULLETIN ON PROCESSES AND APPLICATIONS } {PHOTOEXE%ILCTEHOL-at-VMS.HUJI.AC.IL} } Subject: CALL FOR PAPERS } To: VITOR-at-dsif.fee.unicamp.br } Content-Length: 1791 } } FRONTIERS IN SCIENCE AND TECHNOLOGY } AT NANO-MICRO SCALE } } July, 28-29 - 1994 } Guaruja - Sao Paulo State - Brazil } } Topics: } (not limited to) } } Scaling Laws - Theory } Scanning Probe Microscopies (STM, AFM,....) } Sensors } Piezoelectric Drivers } Machining (Beam, Plasma, Molecular, ...) } New Materials (Diamond-like, Porous Silicon....) } Synthesis and Structures (Biology, Microelectronics....) } Computation } ..... } } Abstract Deadline March, 20, 1994 (two printed A-4 pages) } Manuscripts at the Conference Site } } Organizing Comitttee } ==================== } } } Prof. Vitor Baranauskas - State University of Campinas - Brazil (chairperson) } Prof. Valentinas Snitka - Vibrotecnika - Lithuania } Prof. Ioshiaki Doi - State University of Campinas - Brazil } Prof. Aaron Peled - CTEH - Israel } Dr. Vladimir J. Trava-Airoldi - Instituto Nacional de Pesquisas Espaciais -Brazil } Prof. Gilberto Mattos Gualberto - State University of Campinas - Brazil } } } Organized by: } ============ } Sociedade Brasileira de Vacuo - Brazilian Vacuum Society } } This Conference will be a satellite of the Annual Brazilian Vacuum Conference, to be held in Sao Carlos City - 24-27 July, 1994 } } Guaruja is a pleasant island-city on the South Atlantic Sea. The Conference will be held in a Hotel on the beach. Proceedings will be refereed and published by an International Journal. } } For further information or abstracts submission contact : } } Prof.V.Baranauskas } Faculdade de Engenharia Eletrica } Universidade Estadual de Campinas UNICAMP } 13083-970 - Campinas - SP - Brasil } } FAX: +55-192-391395 } e.mail vitor-at-dsif.fee.unicamp.br } } ============================================================================= } ----- End Message ----- } } } } ----- End Included Message ----- } } }
Via: uk.ac.birmingham.computer-centre.ibm3090; Fri, 28 Jan 1994 11:37:44 +0000
Images from Nanoscope III can be saved as TIF files and studied on a PC using the VISILOG Software. This software is distributed (english language) by NOESIS Vision Inc 6800 Cote de Liesse Suite 200 Ville St Laurent, Quebec, Canada H4T 2A7 Tel (514) 345-1400 Fax (514) 345-1575
Fellow Microscopists: Here in the SEM lab, we scan a wide range of specimens (Insects, rodent teeth & skulls, fossil vertebrates & invertebrates) This is just to name afew. We use gold-palladium coating on these specimens. Is there anyone out there who has a sure fire method to remove coatings without damage to the specimen?
Thanks in advance, Peling Fong Melville
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
Does anyone out there have a number for Sony for color printer tech support? The loony I got in the DC area told me that I couldn't do half of the things that were printed right in the manual.
Jay: I have always made my EM slides using EM film and the only time I use glass is when I am making a composite slide. EM film mounts quite nicely in cardboard mounts, if your not doing any taping of the slides as you might do in making composite slides, why use glass? I have alwas had problems when I had to use Gepe glass. The best glass to use is by EMDE. They make an ultra thin glass (cat.# 135-NRT) slide making kit for the prevention of Newton Rings. Normal thickness glass can kill you every time with EM film. Don't know if EMDE is still in business. My box says Los Angeles 25, California. It's a few years old. You can call information in Los Angeles and see if they still have a phone # or check one of the big industrial photo supply houses or photo shop. Good Luck! Phil Rutledge prutle1-at-gl.umbc.edu
Further to Ron Anderson's suggestion: Bernie Kestel has written a paper "Non-Acid Electrolyte Thins Many Materials for TEM Without Causing Hydride Formation" (Ultramicroscopy 19 (1986) pp 205-212. I have a copy of the paper and would be pleased to FAX it to you and/or mail you a copy. You may contact me via CompuServe or as follows:
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673 USA
TEL: 714-492-2600 FAX: 714-492-1499
You may also reach Bernie Kestel at: TEL: 708-252-4945 FAX: 708-252-4798
I also have a bibliography of over 40 papers dealing primarily with TEM sample preparation. I would be pleased to send you a copy and can subsequently provide reprints at no charge.
Bernie Kestel, Argonne National Laboratory's expert at electropolishing, suggests the following for Ti alloys:
13% HCl and 87% methanol temperature = -50 C 90 Volts and 35 mA in a single jet electropolishing machine
or
60ml Percholoric Acid 590 ml Methanol 350 ml 2-butoxy ethanol (butyl cellosolve) temperature = 0 C 40-50 Volts and 50 mA in a single jet electropolishing machine
or
5.30 g LiCl 11.16 g Mg(ClO4)2 500 ml methanol 100 ml 2-butoxy ethanol (butyl cellosolve) temperature = -40 or -50 C 150 to 250 volts and 30 mA flow speed = medium single jet electropolishing machine (South Bay) The flow restriction of the Tenupol holder may require less of the 2-butoxy ethanol which increases the viscosity of the solution. Results seem less satisfactory as the temperature is raised. You may need another power supply for the higher voltage. Greater or lesser amounts may be tried of the LiCl and Mg(ClO4)2. Add the powders to the methanol with simultaneous stirring. Do not reverse the voltage polarity: this introduces hydrogen into the specimen. Keep the specimen at a positive voltage. This approach worked well on compacted Ti nanocrystalline wafers.
Message-Id: {9401282054.AA22451-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Electropolishing of Titanium Orig-Author: {MACLAREI-at-ibm3090.computer-centre.birmingham.ac.uk}:ddn:wpafb ----------------------------------------------------------- Dear all,
Thank you to the two of you (Rob Tayloe and Cameron Begg) who replied to my earlier message concerning the electropolishing of Titanium for TEM specimens. However I can't believe that there is no one out there that has personal experience of preparing specimens of Ti or dilute alpha-Ti alloys for the TEM. If there is anyone out there who has done some TEM on such materials I would be very grateful if you could describe the specimen preparation route that you used.
Message-Id: {9401281628.AA21291-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Electropolishing of Titanium Orig-Author: {MACLAREI-at-ibm3090.computer-centre.birmingham.ac.uk}:ddn:wpafb ----------------------------------------------------------- Dear all,
Thank you to the two of you (Rob Tayloe and Cameron Begg) who replied to my earlier message concerning the electropolishing of Titanium for TEM specimens. However I can't believe that there is no one out there that has personal experience of preparing specimens of Ti or dilute alpha-Ti alloys for the TEM. If there is anyone out there who has done some TEM on such materials I would be very grateful if you could describe the specimen preparation route that you used.
Message-Id: {MAILQUEUE-101.940128134052.576-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
} Fellow Microscopists: } Here in the SEM lab, we scan a wide range of specimens (Insects, } rodent teeth & skulls, fossil vertebrates & invertebrates) This is just to } name afew. We use gold-palladium coating on these specimens. Is there } anyone out there who has a sure fire method to remove coatings without } damage to the specimen? } } Thanks in advance, } Peling Fong Melville } } -------------------------------------------------------------- } Peling Melville peling-at-amnh.org } Interdepartmental Laboratories } American Museum of Natural History
I have never heard of such a method, and do not believe that one exists. If I'm wrong, I would be VERY interested in hearing of it. But why bother? A light coat of Au/Pd won't hurt specimens, and coated SEM specimens can be embedded for TEM or LM. If there is some compelling reason not to have a coat on your specimen, the only choice is to examine them uncoated at low kV. This is pretty simple for bones & teeth, and many inverts. It is also doable with arthropods, even allowing for their setae, but you may need to go down to 300-500 V accelerrating voltage. Phil Oshel po-at-parmly1.parmly.luc.edu
Peling: Trying to remove gold-palladium coatings can be done on specimens such as yours but it can be a pain. I have used 20-50% acetone in water and sonicated the specimens. On some specimens this has worked and on others it hasn't. I know of no sure fire method to do this. What you can use instead of gold-palladium, is silver. I have removed silver off of hard and soft tissue samples rather easily. Sometimes I need to look at cells or whole tissue by SEM and then using the same sample for TEM. I use silver most (95%) of the time. At high mags in the SEM silver has a much smaller grain size than gold or gold-palladium and I get a better signal. All it takes to remove the silver is a basic darkroom chemical called Farmers Reducing Agent. This can be bought at just about any photo shop. You just soak your sample in this solution (full strength) until the silver is removed, then do what you need to do to the sample. If I am processing for TEM, I wash the specimen 3 x 15 minutes in distilled water first, 3 x 15 minutes in buffer and start my normal processing routine for TEM starting with the dehydration cycle. If you have any questions call me or Email me. Phil Rutledge, Director Center for Electron Microscopy UMBC Bio. Dept. Phone: (410) 455-3852 FAX: (410) 455-3875 Email: prutle1-at-gl.umbc.edu
Greetings, I am looking for a postdoc to join my lab to work on the relationship between cellulose synthesis and morphogenesis. Experience with either polarized light or electron microscopy, or with spatial measurements of growth is desirable. I encourage persons of all sexes, sexual orientations, colors, nationalities, cultures, sizes, shapes and physicalities to apply. Please send me your cv, names and contact #'s (or email handles) of several references, and if available, a statement of your "world view". If you have further q's, please ask. A brief description of the project, as funded, follows.
What is the role of cellulose deposition in controlling the degree of growth anisotropy? Although it is well known that highly anisotropic growth can be rendered isotropic by the randomization of the direction of deposition of cellulose microfibrils, it is not known whether the degree of growth anisotropy is also governed by the alignment of cellulose microfibrils. The major approach taken in this project is to measure growth anisotropy at a cellular scale and then quantify the alignment of cellulose microfibrils. Cells growing at various degrees of growth anisotropy will be obtained both by taking advantage of natural variations and through use of low concentrations of microtubule inhibitors. Cellulose alignment will be quantified throughout the whole wall with polarized light microscopy and at the innermost layer in metal-carbon replicas viewed in em. Experimental material will be roots of Arabidopsis and elongating tissue culture cells. There is support to work with Prof Andrew Staehelin at Boulder to use rapid-freezing deep etch techniques to make replicas without previous fixation. The project will also focus on several root morphology mutants in arabidopsis. These appear to have tissue-specific defects in cellulose deposition, and the relation between their processing of cellulose and their aberrant morphology will be studied.
Thanks for your interest,
Tobias Baskin
******************************** *************** Tobias I. Baskin 109 Tucker Hall /~~~\ Biol. Sci's * Univ. of Missouri c|o o\ Columbia, MO 65211 USA \ = / Tel:314-882-0173 FAX 314 - 882 - 0123 """ email: baskin-at-biosci.mbp.missouri.edu
Years ago we used to study cultured hepatocytes with SEM. These cell were coated with gold/palladium. Following SEM studies we would embed these specimens in epon and section the same cells for TEM. This worked beautifully and gave the cells an interesting electron dense outline. Our SEM fix, of course, was a good glutaraldehyde/osmium fix so that the TEM fine stucture was good.
Greetings, Does anyone have any comments on the new(ish) 35 degree diamond knives that are sold by Diatome (and others?)? I am interested in comments about "ambient temperature" knives, not "cryo" knives. The Tech Staff at Diatome say that the 35 degree knives reduce compression in some samples but not all. They further said that there were NO disadvantages to the 35 degree knives. My application is with growing plant tissues, such as roots.
I am a newcomer to electron microscopy and I appreciate help from the many wise folks who are a part of this list.
Thanks in advance, Tobias Baskin
******************************** *************** Tobias I. Baskin 109 Tucker Hall /~~~\ Biol. Sci's * Univ. of Missouri c|o o\ Columbia, MO 65211 USA \ = / Tel:314-882-0173 FAX 314 - 882 - 0123 """ email: baskin-at-biosci.mbp.missouri.edu
I am investigating methods of laser illumination for light microscopy. Specifically, I would like recommendations on methods of get uniform laser illumination free of laser speckle and noise. I am aware of one reference,
Ellis, G. W., "A fiber-optic phase-randomizer for microscope illumination by laser" _J. Cell. Bio._, vol 83, p303a.
but it is rather old, 1979, and I wonder if there are better ways of doing it nowadays. It was suggested to me that a phase randomized fiber bundle might do the trick. I was wondering if anyone has tried this? Although the spatial phases of the light will then vary across the fiber bundle area, I wonder if you will still get a well defined interference pattern, which will require vibration to average away and obtain uniform illumination?
Any ideas, experiences, or references will be appreciated. And I will summarize to the list the information I receive.
Thanks, eer
Erik Reuter, e-reuter-at-uiuc.edu, 217-244-6378(W), 359-4683(H), 244-6375 fax
We are in the market to purchase some microscope objectives to operate in the near infra-red, wavelength range of 700nm to 1100nm, to be used in a lab-built microscope. We would like to achieve the highest resolution allowed by diffraction at these wavelengths (~ 1 micron?) with dry objectives.
The microscope consists of a coaxial laser illuminator, a dichroic beam splitter, an objective lens, and a eyepiece/C-mount adapter/video camera. We are examining III/V semiconductor samples.
I would like to hear your experiences and recommendations on suppliers or manufacturers of infra-red microscope objectives. Are reflecting objectives our best bet? Who makes the best reflecting objectives? In order to obtain the best performance, should we purchase the objectives together with eyepiece/C-mount adapter or camera adapters, since the objectives are probably made for a specific correction?
Thanks, eer
Erik Reuter, e-reuter-at-uiuc.edu, 217-244-6378(W), 359-4683(H), 244-6375 fax
You can get excellent illumination by using a single-mode, polarization preserving fiber coupled to an output beam expander and collimator to achieve the desired beam size for input into a CLSM for example (6 mm). BY using an adjustable collimator the gaussian beam shape is preserved at any size beam and can be adjusted to the proper back focal plane size of your objective.
All multi-mode fibers will give speckles and you have to do something to scramble this at the output (vibration, optical scramblers). See the literature on fiber optics to see why this is always going to happen to your original gaussian beam.
We have a nice fiber and input coupler from Point Source in Winchester, England FAX 44-703-602470 and are using a Spindler & Hoyer output coupler, Goettingen, FRG FAX 49-551-69-35-166.
On Mon, 31 Jan 1994 16:49:17 +0200, { JOHNA-at-SCI.WFEB.EDU} wrote:
} } Years ago we used to study cultured hepatocytes with SEM. These cell were } coated with gold/palladium. Following SEM studies we would embed these } specimens in epon and section the same cells for TEM. This worked } beautifully and gave the cells an interesting electron dense outline. Our } SEM fix, of course, was a good glutaraldehyde/osmium fix so that the TEM } fine stucture was good. } } John Aghajanian.........JOHNA-at-sci.wfeb.edu
I do not understand why people want to get rid of the coating. It does not harm the TEM-specimen preparation. It does not harm the images. So what's the big need for removal. Actually, if I see a report stating that the same specimen has been first examined in a SEM with a metal coating and then prepared for TEM and there is no evidence of metal coating, bells would ring in my head and I would doubt wether the same samples are in question or not.
/jm
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Maki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
ABSTRACTS FOR THE JOURNAL OF MICROSCOPY - FEBRUARY 1994
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 87--101.
ESTIMATION OF THE DIRECTIONAL DISTRIBUTION OF SPATIAL FIBRE PROCESSES USING STEREOLOGY AND CONFOCAL SCANNING LASER MICROSCOPY
T. MATTFELDT,* A. CLARKE"" & G. ARCHENHOLD"", *Department of Pathology, University of Ulm, Oberer Eselsberg M23, D-89081 Ulm, Germany. ""Molecular Physics and Instrumentation Group, Department of Physics, University of Leeds, Leeds LS2 9JT, U.K.
Fibrous structures like polymers, glass fibres, muscle fibres and capillaries are important components of materials and tissues. A spatial fibre process is the union of smoothly curved or linear one-dimensional features of finite length, arranged in an unbounded three-dimensional reference space according to some random mechanism. Design-based stereology was combined with confocal scanning laser microscopy to study samples of fibre-reinforced composites, which were considered as realizations of not necessarily isotropic fibre processes. The methods enable the unbiased estimation of the intensity and of the directional distribution of spatial fibre processes from arbitrarily directed pairs of registered parallel optical sections a known distance apart. The directions of fibres sampled by a frame of observation on the reference plane are estimated from the coordinates of the intersection points of the fibres with both optical planes using confocal scanning laser microscopy. The true directional distribution of the fibre process is estimated by weighting each measured direction by the reciprocal of its chance of being sampled, which can be inferred from the data. The concept of complete directional randomness for uniformly and independently distributed spatial directions is introduced. The cumulative distribution function of the angular distances between different directions and other exact relations are derived for complete randomness of vectorial and axial directions. A Monte Carlo method is constructed to test spatial fibre processes, whose fibres have negligibly small curvature, for complete directional randomness. Confocal scanning laser microscopy was used to study the angular distribution of glass fibres in a polymer composite which was subjected to increasing hydrostatic extrusion. The hypothesis of complete directional randomness had to be rejected for all samples with 1% probability of error. The directional distribution was of the bipolar type, with the principal axis directed parallel to the axis of extrusion. Progressive stretching of the material increased the degree of anisotropy of the glass fibres. Although presented for an application in polymer physics, the methods are general and may also be applied in biological investigations.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 103--114.
AUTOMATED TRACING AND VOLUME MEASUREMENTS OF NEURONS FROM 3-D CONFOCAL FLUORESCENCE MICROSCOPY DATA
A. R. COHEN,* B. ROYSAM* & J. N. TURNER*"", *ECSE Department, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, U.S.A. "" Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509, U.S.A.
Three-dimensional (3-D) image analysis algorithms and experimental results that demonstrate the feasibility of fully automated tracing of neurons from fluorescence confocal microscopy data are presented. The input to the automated analysis is a set of successive optical slices that have been acquired using a confocal scanning laser microscope. The output of the system is a labelled graph representation of the neuronal topology that is spatially aligned with the 3-D image data. A variety of topological and metric analyses can be carried out using this representation. For instance, precise measurements of volumes, lengths, diameters and tortuosities can be made over specific portions of the neuron that are specified in terms of the graph representation. The effectiveness of the method is demonstrated for a set of sample fields featuring selectively stained neurons. Additional work will be needed to refine the method for unsupervised use with complex data involving multiple intertwined neurons and extremely fine dendritic structures.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 115--126.
ALGORITHMS FOR AUTOMATED CHARACTERIZATION OF CELL POPULATIONS IN THICK SPECIMENS FROM 3-D CONFOCAL FLUORESCENCE MICROSCOPY DATA
B. ROYSAM,* H. ANCIN,* A. K. BHATTACHARJYA,* M. A. CHISTI,"" R. SEEGAL"" & J. N. TURNER"", *Rensselaer Polytechnic Institute, Troy, NY 12180-3590, U.S.A. "" Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509, U.S.A.
Methods are presented for the automated, quantitative and three-dimensional (3-D) analysis of cell populations in thick, essentially intact tissue sections while maintaining intercell spatial relationships. This analysis replaces current manual methods which are tedious and subjective. the thick sample is imaged in three dimensions using a confocal scanning laser microscope. The stack of optical slices is processed by a 3-D segmentation algorithm that separates touching and overlapping structures using localization constraints. Adaptive data reduction is used to achieve computational efficiency. A hierarchical cluster analysis algorithm is used automatically to characterize the cell population by a variety of cell features. It allows automatic detection and characterization of patterns such as the 3-D spatial clustering of cells, and the relative distributions of cells of various sizes. It also permits the detection of structures that are much smaller, larger, brighter, darker, or differently shaped than the rest of the population. The overall method is demonstrated for a set of rat brain tissue sections that were labelled for tyrosine hydroxylase using fluorescein-conjugated antibodies. The automated system was verified by comparison with computer-assisted manual counts from the same image fields.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 127--141.
MEASUREMENT OF ABSOLUTE TRACER CONCENTRATIONS IN TISSUE SECTIONS BY USING DIGITAL IMAGING FLUORESCENCE MICROSCOPY. APPLICATION TO THE STUDY OF PLASMA PROTEIN UPTAKE BY THE ARTERIAL WALL
P. D. Weinberg,* C. P. Winlove"" & K. H. Parker"", *Department of Biochemistry and Physiology, University of Reading, Whiteknights, PO Box 228, Reading RG6 2AJ, U.K. "" Centre for Biological and Medical Systems, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BY, U.K.
Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to other studies employing DIFM. Nonlinearities were found to arise from offsets in the video digitizers, from background fluorescence and stray light within the microscope and from the transfer characteristics of the intensified CCD camera. Camera gain controls showed complex behaviour. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in response were caused by the geometry of the microscope optics and by the image intensifier. However, the ratios between areas were not affected by light intensity or camera gain settings. Measured intensities were independent of the depthwise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not concentration dependent. Methods used to correct these effects and obtain a uniform, linear and constant relationship between concentration and grey level are described. Using the system and appropriate corrections, in vivo uptake of sulphorhodamine-B-labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean uptake of tracer and its local variation were quantitatively similar to those previously obtained with nonmicroscopic methods.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 143--147.
A CRYOGLUE TO MOUNT VITREOUS BIOLOGICAL SPECIMENS FOR CRYOULTRAMICROTOMY
K. RICHTER, Laboratoire d'Analyse Ultrastructurale, CH-1015 Lausanne, Switzerland
Mixtures of ethanol, 2-propanol and 2-butanol can be used as a cryoglue to mount vitrified biological specimens for ultrathin cryosectioning. Brought directly from room temperature to a cutting support at 140 K in the cold chamber of a cryoultramicrotome, these alcohols stiffen to a viscous and gluey consistency allowing the attachment or embedding of a vitreous biological sample. The mass hardens at lower temperatures fixing the sample well for microtomy. With ethanol: 2-propanol (2:3), samples are applied at 140 K and ultrathin cutting can be done at 115 K.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 149--154.
THE USE OF THE STABLE ISOTOPE 44Ca IN STUDIES OF CALCIUM INCORPORATION INTO DENTIN
T. LUNDGREN, E. U. ENGSTROM,* R. LEVI-SETTI+, A. LINDE & J. G. NOREN++, Departments of Oral Biochemistry and ++ Pedodontics, Faculty of Odontology, University of Goteborg, Sweden. * Department of Physics, Chalmers University of Technology, Sweden. + Enrico Fermi Institute and Department of Physics, University of Chicago, U.S.A.
The incorporation into rat incisor dentin of two calcium isotopes, the stable 44Ca and the radioactive 45Ca, was studied using secondary ion mass spectrometry (SIMS) step-scanning and imaging, and autoradiography, respectively. The results demonstrated a time-dependent incorporation of the calcium isotopes into the mineral phase of dentin. With the SIMS step-scanning, detecting 44Ca, the ion yield was high in the odontoblasts 2 min after intravenous injection. After 10 min a marked increase in signal intensity was found at the dentin mineralization front. This result was consistent with those obtained by 45Ca autoradiography; a peak of incorporation occurred 10 min after injection of the isotope. Likewise, localization of 44Ca to the mineralization front could be demonstrated 10 min after injection by SIMS imaging. In images obtained at earlier intervals, no such increase in ion yield could be detected. The results show that the nonradioactive, stable isotope 44Ca can be used as a marker for biomineralization in a similar way to radioactive 45Ca.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 155--164.
STRUCTURAL FEATURES OF CELL WALLS FROM TOMATO CELLS ADAPTED TO GROW ON THE HERBICIDE 2,6-DICHLOROBENZONITRILE
B. WELLS,* M. C. McCANN,* E. SHEDLETZKY,"" D. DELMER"" & K. ROBERTS*, * Department of Cell Biology, John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K. "" Department of Botany, Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel
Evidence from high-resolution images of primary cell walls suggests that the cell wall is constructed from at least two independent yet coextensive fibrous networks, one based on cellulose/hemicellulose and the other on pectin. The ability to analyse the structure of each of these networks in isolation has been hampered by a lack of suitable biological material such as mutants. However, the recent use of the cellulose-synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) that prevents the formation of the cellulose--xyloglucan network while allowing the pectin network to form a functional wall offers the unique opportunity of studying at least the pectin network independently. A range of electron microscopy techniques and a novel spectroscopy method are used to study the walls from tomato suspension cells adapted to growth on DCB. Measurements of the minimum cell wall thickness derived from thin sections of dehydrated walls show that the marked reduction in level of the cellulose/hemicellulose network affects neither the thickness of the wall formed, nor the apparent spacing of pectin molecules. However, images obtained by the fast-freeze, deep-etch, rotary-shadowed (FDR) replica technique show that the three-dimensional architecture of these pectin-rich walls is very different from that of nonadapted walls. Fourier transform infrared (FTIR) microspectroscopy data and immunogold-labelling studies provide additional evidence that supports the previous biochemical data.
Journal of Microscopy, Vol. 173, Pt. 2, February 1994, pp. 165--172.
A PARTICLE SEGMENTATION METHOD BASED ON NUCLEATION AND GROWTH OF THE BACKGROUND
XIN ZHENG & MAX KOLB"", Laboratoire de Chimie Theorique, Ecole Normale Superieure de Lyon, 46, Allee d'Italie, 69364 Lyon Cedex 07, France. Institut de Recherches sur la Catalyse, CNRS, 2, Ave. A. Einstein, 69626 Villeurbanne Cedex, France
A new algorithm for image segmentation is proposed, which is capable of extracting particles in the presence of noise and background fluctuations. It begins with the detection of small regions belonging to the background, called background nuclei, and then lets these nuclei grow to become the entire background. Edge information and region information of the image are used simultaneously.
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Message-Id: {MAILQUEUE-101.940201080121.320-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
I second Jouko Maki's opinion below. I've never noticed that the metal coating on the specimen causes any damage (or other problems) to the knife in sectioning, & it can be cut with a glass knife. So, the presence of the coating could be considered required to demonstrate that the section was taken from a SEM specimen. Phil Oshel
} From: "Jouko M{ki" {jokamaki-at-utu.fi} } Subject: Re: removing SEM specimen coatings ... } } Years ago we used to study cultured hepatocytes with SEM. These cell were } } coated with gold/palladium. Following SEM studies we would embed these } } specimens in epon and section the same cells for TEM. This worked } } beautifully and gave the cells an interesting electron dense outline. Our } } SEM fix, of course, was a good glutaraldehyde/osmium fix so that the TEM } } fine stucture was good. } } } } John Aghajanian.........JOHNA-at-sci.wfeb.edu ... } I do not understand why people want to get rid of the coating. It does not } harm the TEM-specimen preparation. It does not harm the images. So what's } the big need for removal. } Actually, if I see a report stating that the same specimen has been first } examined in a SEM with a metal coating and then prepared for TEM and there } is no evidence of metal coating, bells would ring in my head and I would } doubt wether the same samples are in question or not. } } /jm } } - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - } Jouko K. Maki Navigare necesse est... } Laboratory Manager, Ph.D. } Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND } University of Turku Tel.: + 358 21 633 7318 } INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380 }
In my efforts to produce even better sections of these 35 um glass beads, I spoke with the folks at Diatome last week. They advised me to try a new 45 degree diamond knife (I had asked her about a 55 degree knife as someone had suggested on this bulletin board) and then to try a 35 degree knife. They predicted that we would get our best sections with the 35 knife. Sectioning glass beads with a 35 knife, of course, would cause Gib Ahlstrand to cringe. But we're going to try it to see how it goes. I'll keep you abreast of our progress. The prediction is: good sections and a very short edge life.
Message-Id: {9402011548.AA10683-at-umail.UMD.EDU} To: microscopy-at-anlemc.msd.anl.gov
************************************************************************ The University of Maryland Short Course
PRACTICAL ASPECTS OF SCANNING ELECTRON MICROSCOPY
Session I: March 14 - March 18, 1994
Session II : March 21 - March 25, 1994
This intensive four an one-half day course provides participants with a thorough coverage of the basic theory and practice of scanning electron microscopy and related techniques, including X-ray microanalysis. The course employs easy to understand lectures coupled with supervised laboratory exercises to provide practical instruction in the operation of scanning electron microscopes equipped with modern accessories. Class size is limited assuring those attending of the opportunity for extensive hands on experience on the instrumentation available. Attendees in the past have represented a broad range of disciplines (materials, semiconductors, failure analysis, pharmaceuticals, food science, forensics, biology, etc.).
For more information about the course, contact: Tim Maugel University of Maryland Department of Zoology College Park, MD 20742 Phone : (301)405-6898 Fax : (301)314-9358 E-Mail : maugel-at-zool.umd.edu *************************************************************************** Tim Maugel University of Maryland Laboratory for Biological Ultrastructure Department of Zoology College Park, MD 20742 Phone: (301)405-6898 Fax: (301)314-9358 EMail: tm11-at-umail.umd.edu
We are using Unicryl as embedding medium for immunoEM experiments. One of the problems is that you cannot use Os before embedding. Does anyone have experience with on grid Os after immuno. Which grids? Which protocol?
We are at the moment trying to use a Vidas system from Kontron to do some ploidy analysis using Feulgen. We have to calibrate the system using well known 2n, 3n, 4n , .... cells. Does anyone have an idea were to get such celllines or slides for calibration.
Thanks to everyone for the many useful comments on scrubbers used for RIE etching. There seems to be a consensus that some sort of scrubbers are required before exhausting into atmosphere. I have checked some of the chemical supply house and point of use scrub- bers are available for modest cost as suggested. If a scrubber is not already attached to main exhaust in the laborstory, the point of use scrubber would be more than adequate for low or moderate users of the RIE system.
Chlorine gas for etching aluminium presents a more costly problem due to its toxicity and the safety requirements for using in an enclosed laboratory. Does anyone have experience/recommendation for alternative gases for the aluminium etch that would not have toxicity requirments for handling and operation and as a conse- quence be less costly to handle/use.
Thanks again.
Richard Sartore US Army Research LAboratory AMSRL-EP-RA Fort Monmouth, NJ 07703 RSARTORE-at-MONMOUTH-ETDL1.ARMY.MIL
John Mansfield North Campus Electron Microbeam Analysis Lab University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 4:49 PM Date:2/2/94 NC EMAL
Applications of Environmental Scanning Electron Microscopy
Dear Fellow ESEM, Wet SEM & Low Vacuum SEM User(s):
Many of the electron microscope manufacturers now offer an "environmental", "low-vacuum" or "wet" SEM as part of their product lines. These instruments have the unique capability of forming secondary or back-scattered electron images while the sample is in what would typically be considered an extremely poor vacuum. While novel, these instruments have not yet been taken too seriously by the microscopy community.
To highlight the flexibility and functionality of these low-vacuum systems, this year's joint Microscopy Society of America and Microbeam Analysis Society meeting in New Orleans will feature a symposium sponsored by MAS entitled "Applications of Environmental or Low-Vacuum Scanning Electron Microscopy".
This is an invitation to you and your colleagues to contribute papers to this symposium.
This symposium will be broadly based and not material, instrument or technique specific. Submissions covering novel in-situ experiments; advances in hot-stage, cold-stage or deformation microscopies and studies on non-conducting materials, ceramics, polymers etc., liquids and emulsions are encouraged. More "conventional" SEM experiments performed in an environmental SEM are also appropriate
Organizers: Roger Bolon General Electric Corporate R&D Building K-1 Schenectady NY 12309 Tel: (518) 387-7783 email: bolonrb-at-crd.ge.com
Stuart McKernan University of Minnesota High-Resolution Microscopy Center 100 Union St. S.E. Minneapolis MN 55455-0153 Tel.: (612) 624-6590 e-mail: stuartm-at-maroon.tc.umn.edu
John Mansfield University of Michigan North Campus EMAL 413 SRB 2455 Hayward Ann Arbor MI 48109-2143 Tel: (313) 936-3552 email: John.F.Mansfield-at-umich.edu
Abstract deadline is 15th March Please submit TWO PAGE manuscripts (plus three copies) to: Meeting Office, PO Box EM, Woods Hole MA 02543.
} From: lherault-at-acs.bu.edu (Ronald LHerault) } Date: Wed, 2 Feb 94 12:33:32 -0500 } To: microscopy-at-anlemc.msd.anl.gov } Subject: Removing coatings.
} We often use replication on specimens that can't be changed by coating. } Since this is a dental school, we have ready access to polyvynil } oops, polyvinyl siloxane impression materials. The negatives produced } are filled with Spurr low viscosity embedding material and oven cured. } We then mount and coat the specimen for SEM. The only time I ever } had a problem was when I took an impression of a sand dollar. The } impression material stuck in the many pores o the saample.
I am not familiar with this material, other than having it used on me--is it anything like silicone bathtub chaulk? I've used that to fill the lateral line canals of fish, and have gotten very nice impressions of the sensory neuromasts, sensory strip and all (including possible pits of hair cells!?) Phil Oshel please see c.v. on MSA bulletin board--job cut
A few spaces remain in the upcoming school on EELS imaging and analysis techniques to be held at the Gatan R&D facility in Pleasanton, California during 1-4 March 1994. The school covers the basic theory and practice of electron energy loss spectroscopy and energy filtered imaging in transmission electron microscopy and provides opportunities for hands-on experience with these techniques, both in data acquisition directly at the microscope and in data analysis at computer work stations. Registration is open to all interested parties. The school is run at cost, the registration fee of US $750 covering tuition, course materials, and lunches (travel, hotel, and other meals are not included). For further information, please contact
Mike Kundmann Gatan EELS Software R&D 6040 Washington Street Downers Grove, IL 60516-1978 USA (708) 964-2153 (24-hour voice message and/or fax)
Spaces remain in the upcoming school on Digital Microscopy to be held at the Gatan R&D facility in Pleasanton, California during 22-25 February 1994. The school covers digital image capture and analysis techniques and their application to computer-assisted operation of scanning and (fixed-beam) transmission electron microscopes. The school also provides opportunities for hands-on experience with these techniques, both in image capture directly at the SEM or TEM and in data analysis at computer work stations. Registration is open to all interested parties. The school is run at cost, the registration fee of US $750 covering tuition, course materials, and lunches (travel, hotel, and other meals are not included). For further information, please contact
Jacob Wilbrink Gatan R&D 6678 Owens Drive Pleasanton, CA 94588-3334 USA Phone: (510) 224-7316 (24-hour voice mail) Fax: (510) 463-0204
I'm interested in the morphology of Panthera Pardus epidermis. Does anyone know if there is something unusual about the epithelial cells of this animal skin?
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
We are attempting to set up some image processing capabilities on our SEM. Does anyone have any information on systems that can do image analysis (particle size, phases, etc) on a pc or Mac. Any comments on likes and dislikes would be extremely helpful, as would neighborhood prices.
} We are attempting to set up some image processing capabilities on our SEM. } Does anyone have any information on systems that can do image analysis } (particle size, phases, etc) on a pc or Mac. Any comments on likes and } dislikes would be extremely helpful, as would neighborhood prices. } } Thanks!!
A little more information would be helpful. Do you want to do image capture and then analysis of those images? Is your SEM analog or digital? Do you want EDS, too?
One excellent option to look at is the system from 4pi Analysis, Inc. Their system can interface with digital or analog scopes. They capture into NIH-Image, the public domain program from NIH that runs on the Mac. They can also add the capability to do EDS, but that's not required. Their prices are very reasonable. Their image capture board, which resides in a NuBus slot takes control of the beam for slow scanned images.
I saw one of their units in a busy, high volume, high quality lab that could buy pretty much what they want. They have the 4pi and tell me they're very pleased with it.
Dapple also has a system for image capture. Theirs is passive, and takes the image the scope gives it. It's also much more expensive.
4pi Analysis, Inc. 3500 Westgate Drive, Suite 403 Durham, NC 27707 (919) 489-1757
Dapple Systems, Inc. 355 W Olive Ave. #100 Sunnyvale, CA 94086 (408) 733-3283
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
We've simply cabled a framegrabber board from a mac running NIH Image to the SEM video port. This means manually synching the grab to the scan sweep, but its cheap and effective. You could probably do the same with any PC based image system. I've read about the 4pi system and that would prbably be our choice when our needs for SEM digital capture expand. Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
I want to take the image from a ccd camera on my optical microscope, combine it with just a few parameters (temperature, sample positioning, etc) output from a pc and combine them for recoding on a video recorder. Anyone got any ideas?
I am not a subscriber to the list. Please post to me personally. If requested, I will summarize responses as best I can.
A friend is looking for a computer program written in C or Pascal (or any language compatible with the hardware) that would enable her to use the arrow keys on the PC keyboard to move a mechanical microscope stage in the x and y directions via a PC21 Indexer card and compumotor stepper motor. We are looking for program or possible source for program or better place to post this request. Thanks for your help.
Keith Hallam writes: } I just want to take the image from a ccd camera... a few parameters ...
Just thought I would offer you the following:
My company makes a series of small devices called video marking and measuring systems. They hook up in series with the video to your monitor and give you all kinds of capabilities. For example: Add labels to the image. You choose the size and style. Add markers. Choose among 16 styles, up to 16 different ones at once Use a stopwatch Count things by marking them. Tally shows on screen. Measure distances, areas, pathlengths, radius, lots of options. Draw anything at all freehand. Put up grids, targets, reference ruler. Other stuff that I can't think of right now. Oh.. Send data to a printer or pc.
This may not be what you were looking for, but these systems were designed for microscope use, and are being used by quite a few people at this time. They have only been out for about 6 months.
The problem, I think, is that you wanted to take the data from your pc automatically. This system can't do that, but it does offer a lot of things that you may find useful.
The marking and measurement system is called the XR-2000 series, and comes in a few flavors, depending on what type of video you have.
We also make a stand alone real time video processor called the OMNEX which does many of these things and also video averaging, integration, memory stuff, low light camera control, and all kinds of other things.
The XR-2000 marking and measurement systems cost around $1700 depending on the type of video. The OMNEX processor is about $5350.
Deadline for advance registration and submission of papers for the ICEM13 in Paris is March 1, 1994. Before March 1st the registration fee is 2000FFR and after March 1st it increases to 2200FFR. Student registration is 1100 FFR at all times, including at the meeting. Due to problems with the mail, the organizing committee will be reasonably flexible about accepting the "before 1st March" rate. If registrations are post marked by the 1st of March, they will be accepted at the advance registration rate.
As for abstracts, they can also be flexible. Everyone must send a copy of their contribution to the ICEM13 before March 1st, using either fax or email for the text. These can then be sent to the symposia chair for review. HOWEVER, the printed copies with complete illustrations must reach France before the contributions go to the printers. If the fully-illustrated printed contributions are not received in time, they may be accepted as talks or posters, but will not appear in the proceedings.
To receive registration information, etc. (the 3rd Circular) for the Paris ICEM13, contact Dr. Bernard Jouffrey directly by email at: jouffrey-at-mssmat.ecp.fr or by fax at: 33 1 41 13 14 30. Send your name, address, fax no., and email no.
Another question, totally unrelated to the last one. Has anyone any experience, or references, to the use of image analysis to differentiate between different types of blood cells in an optical microscopy image?
To: Anyone interested From: Cesar D. Fermin, Ph.D. Tulane Pathology.
life. Excellent condition. Water chiller included.
Available free, but:
1) you must pay for packing and shipment to your destination,
2) You must arrange for packing at time of dismantling,#000# sometimes in April.
3) Respond via e-mail, but acceptance must be arranged in writing or via fax to me.
Cesar D. Fermin, Ph.D. Tulane University Medical School Department of Pathology 1430 Tulane Ave/SL79 New Orleans, La 70112-2699 (504) 584-2521 Fax 587-7389
Via: uk.ac.birmingham.computer-centre.ibm3090; Tue, 8 Feb 1994 11:46:36 +0000
We are considering purcahsing a Quadra 840AV for image capture and manipulation of phase, DIC and fluorescence microscopy of biological material, including quick-time - in particular of Platyhelminths. I would appreciate contact or advice from anyone with experience with this machine for similar applications. I understand that the image grabber board is probably inadequate for such applications and would need to be replaced by a higher quality card. What remaining advantages are there (apart from the audio which we are not interested in) for the AV machine over an 800 with the addition of the appropriate card?
There has probably been considerable discussion on this issue in the past in this group and in the NIH Image group and I would also appreciate instructions on where to look for this in an FAQ site.
Dr N.A. Watson Department of Zoology University of New England Armidale, NSW, 2351 AUSTRALIA Fax: 067 711 869 Phone: 067 732181
Tom Gore from Universtiy of Victoria asked about the Argus 10 processor. My company (Imagen Inc) makes the main competitor to the Argus 10, the OMNEX. We consider it to be an improvement on the Argus 10 in many ways.
The OMNEX is easy to use. It uses a mouse and on-screen menus. There are no manual controls at all.
The OMNEX can do real time averaging, integration, memory operations (subtraction, addition, etc.), take a gradient and lots more. It has digital contrast enhancement with 20 storage macros and a custom lut that you can draw with the mouse. It has distance, area, path, event, radius measurements with four calibrations. It does psuedocolor and can store 4 images and cycle them automatically (like a slide show).
It can control long exposure cameras for low-light work. There is a built-in help menu system, although most people don't need it because the menus are pretty straightforward.
There are quite a few other things that it does that I can't think of at the moment, but if you like, I can drop a brochure in the mail (the REAL mail). We have distributors all over US, Canada, and are now getting into Japan and Europe.
We also have a marking and measuring system that is quite nice. I can send you that info also.
Hope this tells you maybe what you were looking for. Of course, my opinion is absolutely politically unreliable, but the device stands for itself. People seem to enjoy using it. (It even plays breakout.)
We used Cr2O3 standard for Cr2O3 in microprobe analyses. Some time ago standard was changed to metallic Cr. When analysing Cr2O3 with new metallic standard we noticed that it gives analyses which are 4 wt% below. It is obvious that the ghange to metallic Cr standard caused the problem. Why Cr2O3 and metallic Cr as standards give different values ? Peak positions are same in both standards. Best wishes,
Jussi Liipo Department of Geology University of Oulu SF-90570 Oulu FINLAND
I teach SEM to undergraduate Biology students and would like to provide them with a brief introduction to specimen preparation techniques used by material scientists. Does anyone know of a review article or basic text on the subject that would be useful for this purpose? Thanks for any references you can provide!!!!
Kenneth M. Bart Director, Electron Microscopy Facility Hamilton College Clinton, New York 13323 USA kbart-at-hamilton.edu (315) 859-4715
I have the following surplus equipment in my lab that I would like to sell:
1. LKB 8800 Ultramicrotome
2. EmScope CPD 750 Critical Point Drier
Both instruments are in good working order, but have not been used for a few years. If anyone is interested please contact me directly via e-mail , do not reply on this Microscopy listserver.
Kenneth M. Bart Director, Electron Microscopy Facility Hamilton College Clinton, New York 13323 USA kbart-at-hamilton.edu (315) 859-4715
I need to obtain citations and/or procedures for the use of rutheniun red in the staining of mucopolysaccharides in thin and semi-thin sections.*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
JOURNAL OF MICROSCOPY, VOLUME 173 PART 3, MARCH 1994 **************************************************************
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 173--181.
ATOMIC FORCE MICROSCOPY OF FREEZE-FRACTURE REPLICAS OF RAT ATRIAL TISSUE
by L. KORDYLEWSKI, D. SANER & R. LAL, University of Chicago, Department of Medicine/Cardiology, 5841 S. Maryland Ave., Chicago, IL 60637, U.S.A.
Summary
Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 183--197.
TIP ARTEFACTS IN SCANNING FORCE MICROSCOPY
by U. D. SCHWARZ,* H. HAEFKE, P. REIMANN & H.-J. GšNTHERODT, Institute of Physics, University of Basel, Klingelbergstrasse 82, CH-4056 Basel, Switzerland
Summary
Since its invention in 1986, scanning force microscopy (SFM) has experienced great success as a characterization method for topography on small scales. In spite of the enormous potential of the method, it is limited by the quality of the tip used for probing the surface topography. Convolutions of non-ideal tip shapes with the real topography and tip bending, flexing and jumping effects produce artefacts in the resulting images. A brief description of the preparation and characteristics of the most commonly used SFM tips is given. A variety of different artefacts originating from tip properties is presented and illustrated with selected scanning force micrographs. Methods to minimize tip artefacts in SFM images are described.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 199--210.
CALIBRATION OF THE SCANNING (ATOMIC) FORCE MICROSCOPE WITH GOLD PARTICLES
by S. XU & M. F. ARNSDORF, Department of Medicine, The University of Chicago, Chicago, IL 60637, U.S.A.
Summary
Scanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 211--218.
CONTRIBUTION OF ENERGY-FILTERING TEM TO THE DETECTION OF CALCIUM: APPLICATION TO MAST CELLS
by M. HOROYAN,* M. SOLER,* J. M. MARTIN,""A.-M. BENOLIEL,* M. FRATERNO,~~ M. PASSEREL,## E. KATCHBURIAN, P. BONGRAND* & C. FOA*, *INSERM U 387, Laboratoire d'Immunologie, H“pital Ste Marguerite, 13277 Marseille Cedex 9, France. ""Ecole Centrale de Lyon, Departement de Technologie des Surfaces, URA CNRS 855, 36 Avenue Guy de Collongue, BP 163, 69131 Ecully Cedex, France. ~~Service commun de Microscopie ‚lectronique, Facult‚ de M‚decine, Bd Jean Moulin, 13005 Marseille, France. ##Centre de Microscopie ‚lectronique et de Microanalyse, Facult‚ des Sciences et Techniques de St J‚rome, avenue Escadrille Normandie-Niemen (Case 151) 13397 Marseille Cedex 13, France. Department of Histology, Federal University of Sao Paulo, Sao Paulo CEP-0402,3, Brazil
Summary
The ultrastructural distribution and quantification of calcium in mast cells prepared by anhydrous processing was investigated by energy-filtering transmission electron microscopy (EFTEM) using a Zeiss 902 electron microscope. Optimal conditions for calcium detection were determined using inorganic (calcium phosphate) and organic (calcium-loaded chelex beads) standards with known amounts of calcium. Electron energy-loss spectroscopy (EELS) revealed calcium at the L-2,3 edge and also at the M-2,3 edge for all specimens examined. Comparison with X-ray microanalysis confirmed the results obtained with EELS. Electron spectroscopic imaging (ESI) was applied for mapping calcium both in standards and in cells and we showed that mast cell granules were the main site of calcium localization. Although, results have shown that a combination of analytical techniques is required to obtain reliable results.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 219--225.
CONTRAST IN THE TRANSMISSION MODE OF A LOW-VOLTAGE SCANNING ELECTRON MICROSCOPE
by U. GOLLA, B. SCHINDLER & L. REIMER, Physikalisches Institut, Universita„t uMnster, Wilhelm-Klemm Str. 10, 4400u Mnster, Germany
Summary
The contrast thicknesses (x_k) of thin carbon and platinum films have been measured in the transmission mode of a low-voltage scanning electron microscope for apertures of 40 and 100mrad and electron energies (E) between 1 and 30keV. The measured values overlap with those previously measured for E (more than or equal to 17keV) in a transmission electron micro-scope. Differences in the decrease of x_k with decreasing E between carbon and platinum agree with Wentzel--Kramer--Brillouin calculations of the elastic cross-sections. Knowing the value of x_k allows the exponential decrease in transmission with increasing mass-thickness of the specimen and the increasing gain of contrast for stained biological sections with decreasing electron energy to be calculated for brightfield and darkfield modes.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 227--237.
MINIMIZING SAMPLE EVAPORATION IN THE ENVIRONMENTAL SCANNING ELECTRON MICROSCOPE
by R. E. CAMERON & A. M. DONALD, Polymers and Colloids Group, Cavendish Laboratory, Madingley Road, Cambridge CB3 0HE, U.K.
Summary
The ElectroScan environmental scanning electron microscope (ESEM) enables wet samples to be observed by eliminating air but allowing water vapour into the sample chamber. However, evaporation from, and condensation on, the sample may occur during the pumpdown sequence used to reach this state, which means that the sample may not be in its natural state when viewed if due care is not taken. In this paper, the pumping system of the ESEM is described mathematically and expressions are derived for the evaporation and condensation. This treatment is then used to calculate the optimum pumpdown sequence. The importance of using the optimized procedure is illustrated by micrographs of fat emulsions.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 239--243.
MORPHOMETRIC ASSESSMENT OF SURFACE CONFORMATION AS AN ESTIMATE OF ROUGHNESS
by D. A. SILAGE & G. R. BARAN, Departments of Electrical Engineering and Operative Dentistry, Temple University, Philadelphia, PA 19122 U.S.A.
Summary
One of the standard methods for assessing the roughness of a material subjected to wear uses the surface arithmetic means and root-mean-square deviation. However, these parameters often do not provide a qualitative assessment of the difference in materials worn under the same conditions of load and elapsed time. The profile and surface roughness parameters are frequently inconsistent. Such measurements are also required to determine the wear characteristics of various materials under different conditions. A morphometric assessment of wear characteristics, based on the surface area fraction of localized deviations in the surface texture and stress fractures, is provided, and clearly indicates the observed difference.
Journal of Microscopy, Vol. 173, Pt. 3, March 1994, pp. 245--256.
VOLUME AND SURFACE AREA MEASUREMENT OF VIABLE CHONDROCYTES IN SITU USING GEOMETRIC MODELLING OF SERIAL CONFOCAL SECTIONS
by F. GUILAK, Musculo-Skeletal Research Laboratory, Department of Orthopaedics, Health Sciences Center T18-030, State University of New York at Stony Brook, Stony Brook, NY 11794-8181, U.S.A.
Summary
This study describes a technique for noninvasive determination of the surface area and volume of chondrocytes using the confocal scanning laser microscope, and the fundamental limitations associated with its application. Using geometric modelling principles, an isointensity surface contour was formed from a series of optical sections recorded with the confocal microscope. Using a combined surface- and volume-based algorithm, the surface area, volume and other morphometric descriptions were calculated from a polygonal description of the cell surface. The high image contrast required for repeatable identification of the cell border was achieved through the use of a fluorescent dye, which was excluded from cells by an intact membrane. Calibration results indicated that the theoretical modelling algorithm is relatively precise when applied to simulated convex (ellipsoidal) cells, with overall errors of less than 0.5% in surface area and volume measurements. When applied to low-noise, high-contrast volume data recorded on the confocal microscope, typical coefficients of variation of 2--4% were determined for length measurements, 2--5% for volume measurements and 3--6% for surface area measurements either for latex microspheres or for chondrocytes. While the precision of the method is comparable to standard histological techniques, its accuracy is difficult to assess, as systematic errors are unpredictable and may be introduced from several sources.
FOR DETAILED INSTRUCTIONS TO AUTHORS, PLEASE CONTACT DR GILLIAN WILSON, THE JOURNAL OF MICROSCOPY, 37/38 ST CLEMENTS, OXFORD OX4 1AJ, UNITED KINGDOM. TELEPHONE +44 865 248768, FAX +44 865 791237, EMAIL RMS-at-UK.AC.OX.VAX.
NB. PAPERS FROM NORTH AMERICA AND CANADA MAY ALSO BE SUBMITTED TO PROFESSOR DAVID WILLIAMS, MATERIALS SCIENCE & ENGINEERING, LEHIGH UNIVERSITY, BETHLEHEM PA 18015-3195, USA. FAX +1 215 758 4244, EMAIL DBW1-at-LEHIGH.EDU.
Yes, I've thought since being made aware of MOSAIC that a server with images would be a wellcome addition to the net. I'd be willing to get involved. J.T. Stanley Oregon Graduate Institute Portland OR
On Wed, 16 Feb 1994 WALCKSD-at-ml.wpafb.af.mil wrote:
} This is an addenum to my previous suggestion. } } One of our computer gurus here at the lab suggested that we already have } the capability of sending digital images over the microscopy server, } just uuencode the graphic image and put a header in it saying what it } is. } } Nestor, will this work? Can we standardize such a transmission of an } image? Can it be tacked on to the end of the Email text? } } } -Scott Walck } } Dear Dr. Walck: The question you should also ask is what resolution will be available if you send images. In North Carolina we will soon be connected to a network that will allow high resolution images to be relayed around the state.
If you get further information about this and the cost of doing it , I would be very interested. Nina Allen, wake forest university
Reply_ RE} } Digital micrographs on I Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu The anonymous ftp server at the University of Michigan could be a good site for this. I have a directory on feebie.engin.umich.edu for storing MSA & MAS related stuff, it is /pub/MSA+MAS. You can upload stuff to /pub/incoming and then email to me so I can move it to the MSA+MAS directory. If it is left in incoming it is liable to be deleted if the system is rebooted. (incoming is essentially a /tmp directory). If we got a large number of images I would archive them to optical disk or tape and then make them available on an MSA+MAS CD ROM. I was thinking of also making available on this disc archived messages sent to this list and the sci..techniques.microscopy newsgroup. I have been working with some peopl here to make the freebie ftp server accessible from Gopher and Mosaic. It seems that there may be some security questions, however. I am still working on it. Takes time and I dont seem to have enough hours in the day lately!! Cheers Jfm.
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This has been planned for the microscopy server, however, we have been waiting for our "new" computer to arrive, be installed & tested. The old system which we are currently running on is not worth spending any more $$ on and will be sacrificed to the silicon god in the near future. I expect the new system to be up, running and debugged within 2 months time.. Hopefully the transition will be seemless, but expect a few hickups. Just bear with the current setup a little bit longer.
Scott W asked about Emailing images over the server using uuencode.
The answer is yes it will work, however......
DONOT UNDER ANY CIRCUMSTANCES DO IT!!!!
It will jam up/fill all subscribers Email tremendously. Their mail boxes will fill and they (and I) will scream bloody murder. If someone wants a copy of an image they should contact you directly for it. If you want to make a particuliar image available to the subsribers for comment then wait until the new system is up an running and I will see how to most apprropriately (and simply) make this option avaiable to those who want to see the data. There are literally hundreds of subscribers who would not be interested in seeing your data, while maybe only a few dozen would be interested.
Also uuencode tends not to be universally accepted. Lots of users of this server donot have the capability to decode uuencoded data. That is a UNIX based system encoding many PC and Mac and Vax's which access this system will not have the appropriate translators (I note that they do exist however). Thus if you arbitrarily chose to send the data via a format that many users can't even read then it would be a waste of everyones time.
BTW this system is a VAX and not a UNIX machine and although we can translate uuencoded data we prefer alternatives which preserve more information, particuliarly if the data comes originally from a Mac or PC.
Message-Id: {MAILQUEUE-101.940217072125.480-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
} From: "Nestor J. Zaluzec (708)-252-5075, -4964" } Scott W asked about Emailing images over the server using } uuencode. } } The answer is yes it will work, however...... } } DONOT UNDER ANY CIRCUMSTANCES DO IT!!!! } ... An ignorant question, for when the new server is up: Is it possible to have mail sublists, say neuron.microscopy-at-..., that everyone on micrscopy-at-... could have access to, but not necessarily automatically get, as we do from this list? Thumbnails of images could be posted to the sublists for anyone to browse, and then contact the source of the thumbnail for the full image. This might make images available to the largest number of people, without overloading the server (?) or people's mailboxes. Phil Oshel
The University of New Mexico has an opening for a full-time position of Research Technologist V in the Transmission Electron Microscopy Laboratory in the Department of Earth and Planetary Sciences. The laboratory have a JEM 2000FX scanning transmission electron microscope with Noran EDS attachment and a newly installed JEM 2010 high resolution microscope with a Link ultra-thin window EDS and advanced image processing systems (including both Gatan live-rate TV system and slow-scan camera as well as IBM PC compatible and Macintosh computers). The laboratory also has a darkroom and full TEM sample preparation equipment. The responsibility of the technologist (under the supervision of the laboratory manager) includes but not limited to: routine maintenance of the laboratory and all the equipment (electron microscopes, ion mill, carbon coater, dimpler, diamond saw, microtome and darkroom equipment, etc.); supervise service engineers; train students, research staff and faculty on the use of microscopes and laboratory equipment; purchase consumable parts and supplies for the laboratory; participate in the research projects by preparing TEM samples, performing TEM/AEM analysis, preparing micrographs, slides and technical reports. The qualified candidate should have at least a BachelorÕs degree in science and engineering (Master's degree desirable) and four years of experience in a TEM laboratory (two years with MasterÕs degree). Experience on TEM maintenance and research projects using analytical and high resolution electron microscopy, knowledge with both IBM PC and Macintosh computers are desirable. The salary range is from $22,400 to $30,800/year depending on the qualifications. Application with a cover letter and resume must be received by Dr. Lumin Wang, Department of Earth and Planetary Sciences, University of New Mexico, Albuquerque, NM 87131-1116, by February 25, 1994.
The answer is maybe. We will also be getting the newgroup running and hopefully digest mode (?). It's just too early to say yes or no. The Newgroup function will effectively act as a sublist.. Please hold off on questions about the newsgroups etc.. for awhile, I've just gotten back from a meeting and have a bunch of catchup work to do, I will post details and information as new options are added to the server system. Again it will be ~ 2months before things are likely running...
A new professor in our department wants to purchase a nice used dissecting/operating microscope . I need names of places where one can purchase used microscopes.
Thanks for any help, nina allen, biology, wake forest university
Nina Allen asks were to buy a nice used micrscope. I am afraid I can't help with that. However, I am involved in getting some wonderful scopes from China. We are getting all types, from low power inspection, to phase contrast and darkfield 2000X trinoculars.
In general, the prices are lower than used Nikon/Zeiss/Olympus, etc. The quality is great. Most of these types have never been exported.
I am not ready to supply anything, but will have a sheet of sorts in about a week. If you would like to know more, send me a note. I am excited about the qualtiy/price of these devices.
Excuse the misspelled "where" in the first line herein. Slipped an "h".
Here is a summary of the recent discussion concerning magnetic specimens in a TEM:
TEM work on steel specimens can be very difficult, because they are almost always magnetized. You may be able to reduce the magnetic inhomogenieties a bit by passing the specimens through a strong AC field, a process industrially known as 'degaussing'. I believe degaussing coils can be purchased from most electronics supply stores that deal in the television market. In use, you insert the specimen into the center of the coil and withdraw it very slowly, with the AC current flowing through the coil.
Besides doing what Robert Keller suggests, you should note the objective lens current for a non-magnetic sample and set the lens to the same value. Then you can bring the specimen to the eucentric height by noting when the image is at the minimum contrast condition.
Russell Cook Electron Microscopy Center for Materials Research Argonne Natonal Laboratory Argonne, IL
Dear Colleagues,
Does anybody know an accurate value for the lattice parameter of GaAs at 120K (or 100K) ?
What are people using as a standard for determining the point spread function when the image is visualized with bright field (transmitted) light at high (1.3-1.4) numerical aperture? All of the articles I've seen involve fluorescence microscopy. Thanks for any leads.
Message-Id: {9402221505.AA29320-at-mogate.sps.mot.com} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Imaging Magnetic Materials Orig-Author: {finger Displays information about a user {bandaru-at-gandalf.rutgers.edu} }:ddn:wpafb ----------------------------------------------------------- A request for suggestions from those having experience with the Imaging of Magnetic Materials( Fe based). Any ideas on how to correct for the OL astigmatism , or good references to the above will be greatly appreciated. Please mail your requests to bandaru-at-gandalf.rutgers.edu Thanks in advance, Prabhakar R. Bandaru
The address for the company that manufactures the automatic dodging enlarger is: Logetronics Corporation, 7001 Loisdale Road, Springfield, VA 22150 (703) 971-1400
The company that repaired our enlarger recently is: Egoltronics Corporation, 7036 Tech Circle, Manassas, VA 22110 (703) 751-5522
LogEtronics has been bought out by another company (I believe) and has changed the name to Egoltronics Corp. Address is: 7036 Tech Circle Manassas, Virginia 22110 703-335-1501 (fax) 703-335-1234
I need to attach a 3-8 nm colloidal gold [preferably 5nm] particle directly to the primary antibody. Does anyone know of a company or companies that will do this with client's antibody?
Message-Id: {9402222328.AA20467-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Imaging Magnetic Materials Orig-Author: {finger Displays information about a user {bandaru-at-gandalf.rutgers.edu} }:ddn:wpafb ----------------------------------------------------------- A request for suggestions from those having experience with the Imaging of Magnetic Materials( Fe based). Any ideas on how to correct for the OL astigmatism , or good references to the above will be greatly appreciated. Please mail your requests to bandaru-at-gandalf.rutgers.edu Thanks in advance, Prabhakar R. Bandaru
You might try E-Y Laboratories. They specialize in colloidal gold conjugates (tracers, antibodies, etc), and I think (!) I remember that they would conjugate your reagent for you. Anyway, it might be worth checking:
E-Y Laboratories, Inc. 107 N. Amphlett Blvd. San Mateo, CA 94401 415-342-3296
Good luck
David Morilak morilak-at-cmgm.stanford.edu -------
Message-Id: {9402231439.AA22563-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Lacquer as protective coating Orig-Author: {"Joel Hoehn" {Hoehn-at-maroon.tc.umn.edu} }:ddn:wpafb ----------------------------------------------------------- I am trying to protect the front side of some Fe-3%Si while polishing from the back side in a tenupol using 95% acetic / 5% perchloric solution. I have heard of a sort of lacquer used for the procedure and would like to know if anyone could tell me of a supplier of such a compound. From what I understand, the usual microscopy type suppliers (i.e. ted pella, spi, etc.) do not carry this sort of product, but some industrial application type companies do.
Any leads would be helpful.
Joel Hoehn e-mail: Hoehn-at-maroon.tc.umn.edu Dept. of Chem. Engg & Materials Science phone#: (612) 625-1018 University of Minnesota 421 Washington Ave. S.E.
Message-Id: {9402231451.AA22632-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Magnetic specimens in TEM Orig-Author: {jrmicha-at-saix367.sandia.gov (MICHAEL, JOSEPH)}:ddn:wpafb ----------------------------------------------------------- I spent 6 years at BEthlehem Steel's research department using TEM and STEM to study steel microstructures. The most important thing that can be done to minimize the difficulty of magnetic specimens is to make the specimen blank as thin as possible. Normally, we would electrochemically polish the specimen blanks to a thickness of about 50 microns before we would jet polish them to perforation. This minimizes the amount of magnetic material within the field of the lens.
We tried de,agnetizing the specimens, but found that the fields generated within the coil usually destroyed the thin area of the specimen.
} } We have a Kevex 8000 PDP 11 DEC EDS computer with horrible 8 inch } Bernoullis. They are expensive, unreliable. We have two questions. } } 1) Has anyone successfully used kermit to communicate with a pc from this } type of computer? } } 2) Is there a file translation utility that would transfer the intensity } vs. energy to an ascii } format? } We have a similiar system with some of the same needs. We replaced the 8 inch Bernoullis with twin Bernoullie 44MB drives and improved things considerably. If you do this you will have to replace a chip on one of the boards in the computer. This may be the only item you have to purchase from Kevex.
As for kermit, we have placed kermit on the system with success. We had a couple of problems doing it though. The first was getting kermit on a proper media. The second was adding it to the menu. We purchased a program called TIFMKR from Kevex and had them load Kermit on the same disk. TIFFIT/TIFMKR converts Images from Advanced Digital Imaging, Screen Images, and EDS images to TIFF format files that can be transfered from the DEC to your PC using Kermit. Unfortunately the conversion is fairly low resolution and not practical for the Advanced Imaging images. Kevex also wants a lot of money for this package but they will negotiate. Check with Kevex for specific information.
There is a red lacquer MICROSTOP used for 'the window pane' method and to protect tweezers, etc. while chemically thinning samples. The lacquer was thinned with acetone. We had it at North Carolina State. It had been in the lab of Professor Ray Benson, at (919) 515-2706.
You could also try photoresist. It should be very easy to obtain at U of Minn, is chemically resistant, and removable with acetone or oxygen plasma. Some types of photoresist clean up from samples easier than others.
Just a bit of information for those of you that are interested in details. The Microscopy Listserver just recorded its 750th subscription request. Seems like it was a reasonable idea after all. ;-)
If you add up the total number of Email transmissions that the system has handled over the last 5 months we have sent out over 250,000 individual messages. No wonder my computer is getting tired!
Honors & a beer (or whatever he would like when I see him) for being the 750th subscriber go to:
Mike Bench Dept. of Chem Eng & Mat. Sci Univ. of Minnesota
============================= Nestor J. Zaluzec ANL EM Center
I am passing on the following message for discussion:
Electron Microscope Center Mississippi State UniversitySubject: Formvar films cast on glass
Question: Our laboratory staff has tried various methods over the years to get the formvar film to release from glass slides on a regular, consistent basis while producing formvar-coated TEM grids.(Problem: the films sometimes do not release at all, sometimes release too wrinkled to use, etc.) We use formvar prepared in chloroform, and make use of an Effa Film Caster Apparatus (Ernest F. Fullam, Inc.). I would appreciate any suggestions which might help alleviate this dilemma.
Can anyone tell me the mechanism of UAc fixation and staining of wood components, specifically lignin, for EM? I am interested in learning about the chemical groups of the sample that may be involved. I am also interested in learning about the structure and chemical groups of lignin and have not been able to find any recent information.
Like many people we have been using double distilled water for washing etc in immuno gold protocols. We will soon have much easier access to an ultrapure (type 1, 18 M ohm) water system. Does anyone know if this will cause any difficulties or is it essentially the same.
Also some people manage to get away with "straight" distilled water, any comments?
Ian Hallett EM Facility HortResearch Auckland New Zealand
Regarding the request about water purity and immuno staining.
I think it very much depends on your local water source. We have used normal distilled water without any problems at all and have in fact obtained very good localizations. Some of the researchers here use the ultra pure water but that is mainly for in situ hybridization, although light microscopy immuno work has been carried out with the ultra pure water without any hassles as well.
Peter RichardsPeter D. G. Richards Dept Anatomy and Cell Biology UCT Medical School Observatory 7925 RSA Tel: 021-406 6285.
Message-ID: {MAILQUEUE-101.940224084456.800-at-anat.uct.ac.za} To: microscopy-at-anlemc.msd.anl.gov
Regarding the recent query about formvar coating passed on from Bill Munroe.
I do all my coating in a similar way but without the film caster. I find that the glass slides I use for the film have to be clean but not ultra clean. If the glass is to clean then there are problems with removal of the film on to the water surface, as the formvar sticks well to the glass, as Bill has obviously found.
When the glass slide is to dirty then obviously a poor film results, however with a slight "greasyiness" the films come off well and evenly with no wrinkling.
Peter RichardsPeter D. G. Richards Dept Anatomy and Cell Biology UCT Medical School Observatory 7925 RSA Tel: 021-406 6285.
Bill, I coat glass slides with Formvar on a regular basis and have had similar trouble in the past. I have solved it completely, however, by a very simple means. Just wipe a finger across your forehead and lightly coat the side of the slide on which the released coating will be (works best towards the end of the day, obviously). Then with a teatowel or the corner of your lab coat, buff the slide moderately clean. ie. leave a microscopic layer of body grease on the surface. This ensures that the Formvar will release if you have scored all around the slide edges with a sharp razor blade. No need for fancy equipment - just dip the slide slowly, at an angle so one corner releases first, into a wide mouthed vessel of distilled water. The cast film with its grids is then very easily picked up with Parafilm rolled across the film and lifted.
Good luck
Nikki Watson
Dr N.A. Watson Department of Zoology University of New England Armidale, NSW, 2351 AUSTRALIA Fax: 067 711 869 Phone: 067 732181
Do Kontron still exist? Are they now part of a bigger company? Do they still make inage analysis systems? Does somebody have a US phone number? Alwyn Eades Center for Microanalysis of Materials University of Illino
Our trick for getting consistent release ofFormvar films is to spray the slide with Fantastic or 409 spray cleaner and polish the slide dry without rinsing. The residual detergent film allows easy release of the film and it is generally of good quality. In our humid climate we also store the solution and the posdre in a sealed container with silica gel. ***************************** * Greg Erdos * * Director, ICBR EMCL * * University of Florida * * Gainesville, FL 32611 * * gwerdos-at-gnv.ifas.ufl.edu * * 904-392-1295 * *****************************
Do Kontron still exist? Are they now part of a bigger company? Do they still make inage analysis systems? Does somebody have a US phone number? Alwyn Eades Center for Microanalysis of Materials University of Illino
Message-ID: {MAILQUEUE-101.940224165025.288-at-anat.uct.ac.za} To: microscopy-at-anlemc.msd.anl.gov
Further to the requests about water and immuno staining.
Some of the local proponents of immuno work out here are using only local tap water without any purification. Certainly the results are good and there does not appear to be any background problems as a result. Peter D. G. Richards Dept Anatomy and Cell Biology UCT Medical School Observatory 7925 RSA Tel: 021-406 6285.
When all else fails: we use nose grease rather then forehead grease (of course, if you are a woman or a man wearing makeup this doesn't work at all ). If nose grease doesn't work, we score the slide normally and place it face side down over a 65mm plastic culture dish containing a few drops of hydrofluoric acid (be careful! this is bad stuff) for a few seconds. Then we exhale on the slide as usual and generally the films strip easily. Too long of an exposure to HF acid results in poor films so you have to practice with it depend upon humidity, barometric pressure, how the stars are aligned, and so on.... If all this doesn't work, we try again tomorrow ! Good luck.
We use ethylene dichloride as the solvent. The slides are cleaned first in ethanol until the grease-streaks disappear. After drying the slide for a minute or two, we score the bottom and side edges (45 degree angle to face of slide) with a razor blade, then we also score the face of the slide on all four sides with a razor blade. I score several times along the bottom face. If one set of score marks fails, the other is likely to succeed. We usually use Gold Seal Microslides but have have used other brands as well. Problems are rare.
Rod
On Wed, 23 Feb 1994, Richard F. Kuklinski wrote:
} I am passing on the following message for discussion: } } From: Bill Monroe } Electron Microscope Center } Mississippi State UniversitySubject: Formvar films cast on glass } } Question: Our laboratory staff has tried various methods } over the years to get the formvar film to release from } glass slides on a regular, consistent basis while producing } formvar-coated TEM grids.(Problem: the films sometimes do } not release at all, sometimes release too wrinkled to use, } etc.) We use formvar prepared in chloroform, and make use } of an Effa Film Caster Apparatus (Ernest F. Fullam, Inc.). } I would appreciate any suggestions which might help } alleviate this dilemma.
The ultrapure water water systems clean out particles, organics and ions--- they do not, however, degas the water. Small bubbles can be a problem in photography (pin holes in emulsion) or in putting grids underwater on filter paper(small bubbles form on the grids and make them hard to manipulate) in preparation to picking up carbon films. We do not consider any of these problems serious and use ultrapure water all the time!
In Center for HREM, Arizona State University (ASU), we read all EDS and EELS data in a Vax system by "RT 11". Then we can convert these binary data using a program "convascii" that was written by our computer manager Mr. Paul R. Perkes, into a ascii files. Then read these files either from Mac or PC.
If you are interested in this "convascii" program, you have to contact Mr. Paul R. Perkes email:smtp%"perkes-at-asuhrm.la.asu.edu"
Lacquer as protective coating In our lab we use a product called Miccroshield 1 ( Miccroshield 2 is its solvent ) as a protective coating during etching of silicon crystals in 5% HF + 95% HNO3. It's a lacquer that is brush on, drys quickly and is easily removed. From the MSDS here's the manufacturer info:
TOLBER DIVISION/PYRAMID PLASTICS INC. 220 WEST 5TH STREET HOPE, ARKANSAS 71801 # (501) 777-5759
The Microshield Lacquer has been used for many years with our Model 550 Electropolisher and Model 550 Chemical Jet Polisher. It is acetone soluble and is available in an 8 ounce kit. Please reference P/N 02-01890-01.
We use formvar dissolved in Dichloroethane (0.6%). We cast onto slides which have been polished in a drop of liquid detergent, leaving a very thin film over the slide and dried 60 C for 10 minutes or so. After dipping the slide in the formvar solution we leave to dry for 10 minutes or so befor stripping, this gives much more consistant release.
Ian Hallett EM Facility HortResearch Auckland New Zealand EM Facility
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: Microscopy-at-anlemc.msd.anl.gov
} Question: Our laboratory staff has tried various methods } over the years to get the formvar film to release from } glass slides on a regular, consistent basis while producing } formvar-coated TEM grids.......
From David Bentley:
When we have trouble with the film not releasing, we briefly 'polish' the slides with a Kimwipe (which may actually end up depositing a thin film of grease from our fingers on the slide); if this does not work, we use Kimwipes to clean the slides with Windex, which seems to leave a 'parting layer' of some kind on the glass. We use Gold Seal slides from Clay Adams (cat. # 3052). The films generally part well when the slides are _fresh_ (e.g. less than three months after the seal of the _inner_ mylar bag of the shipping container is broken). The slides than begin to exhibit patchy 'frosting' or 'fogging' which is visible when a slide is held up to the light; the film seems to stick to the fogged areas. This fog eventually covers the entire surface of the slide, and can be removed by acid (5% HCl/95% ethanol) cleaning followed by Windex.
In the last mail alwyn eades said: } } } The general view is that the best lacquer is "Lacomit", a bright red } lacquer. } It is resistant to almost all polishing solutions but is removed easily by } normal solvents, for example acetone. The red colour helps to see whether } it has been cleanly removed. } Unfortunately, it is not avaiable in the USA. As far as I know it is still } avaiable in England. If you get someone to bring you a large bottle, it } will last until there are no more electron microscopes.
If you do get someone to do this, either get them to bring a bottle of 'lacomit thinner' as well - over time the undiluted stuff tends to turn into a jelly which needs occasional thinning. Also, when you remove it, (I usually use acetone), make sure that the wash is completely clear with no trace of pink; if this is not done, a residue is left which is almost impossible to remove and sits all over your thin area. 8).
Richard Beanland, Dept. of Mat. Sci. and Eng., University of Liverpool, P.O. Box 147, Liverpool, England.
I'm not sure what my options are regarding the microscopy list. I previously tried to unsubscribe by sending a message (UNSUBSCRIBE MICROSCOPY wacb-at-aplcomm.jhuapl.edu) to listserver-at-anlemc.msd.anl.gov, but still I get swarms of email from the list. Perhaps this (MICROSCOPY) was the wrong name to use for the list. Now, in a more circumspect and appreciative mood, I would like to see what other options I have for achieving a more managable flow to me from the list. Some lists have a "digest" mode that collects all of the messages to the list on a single day into a single mailing to the list members, and this would probably be appropriate for me here.
And so, I ask, how and where do I send a message that will elicit a list all of the commands and options that exist for list members? What is the command syntax that will do this for me? "help" or "index" etc. are frequently used in some lists to get the kind of response I have in mind.
Sorry to send this directly to you, but I don't know how else to address this sort of bootstrapping (or maybe I should say bootstripping) problem.
Message-Id: {MAILQUEUE-101.940224155128.448-at-ahabs.wisc.edu} To: microscopy-at-anlemc.msd.anl.gov
While greasing the slide this way works great, anyone who subsequently grows cells on the grid may need to be concerned about the grease transferring to the formvar. I have found that just polishing the slide with a kimwipe (not too much or the formvar will stick) is usually enough to get it to release.
Bill, I coat glass slides with Formvar on a regular basis and have had similar trouble in the past. I have solved it completely, however, by a very simple means. Just wipe a finger across your forehead and lightly coat the side of the slide on which the released coating will be (works best towards the end of the day, obviously). Then with a teatowel or the corner of your lab coat, buff the slide moderately clean. ie. leave a microscopic layer of body grease on the surface. This ensures that the Formvar will release if you have scored all around the slide edges with a sharp razor blade. No need for fancy equipment - just dip the slide slowly, at an angle so one corner releases first, into a wide mouthed vessel of distilled water. The cast film with its grids is then very easily picked up with Parafilm rolled across the film and lifted.
Good luck
Nikki Watson
Dr N.A. Watson Department of Zoology University of New England Armidale, NSW, 2351 AUSTRALIA Fax: 067 711 869 Phone: 067 732181
} I am passing on the following message for discussion: } } From: Bill Monroe } Electron Microscope Center } Mississippi State UniversitySubject: Formvar films cast on glass } } Question: Our laboratory staff has tried various methods } over the years to get the formvar film to release from } glass slides on a regular, consistent basis while producing } formvar-coated TEM grids.(Problem: the films sometimes do } not release at all, sometimes release too wrinkled to use, } etc.) We use formvar prepared in chloroform, and make use } of an Effa Film Caster Apparatus (Ernest F. Fullam, Inc.). } I would appreciate any suggestions which might help } alleviate this dilemma. }
This always works for me:
I use formvar in ehthylene dichloride and if it is over a month old I discard it. I have real problems with Gold Seal micro slides but no problems with Fisher pre-cleaned micro slides, cat no. 12-552. I wipe the slide with a kimwipe but I don't over do it. I don't have a fancy film caster, I just dunk the slide into .25 to .4% w/v soln. for a few seconds and then let it air dry for a minute. I then run the side of my forceps lightly around the edge of the slide, not on top of the slide but on the angle made by the top and side of the slide. Next, I breath on the slide to form a moist layer (I have always done this, don't know why). Then I gently dip slide straight down while holding it at an angle of about 45 degrees. Works every time, never wrinkles. Gold Seal slides would not release formvar. Some people like to use glass strips from the microtome but I have not tried this.
Rick A. Harris Supervisor, Microsopy Facility Evolution and Ecology, Storer Hall Univer. of Calif., Davis
I have read this discussion with interest. There seem to be extremely many methods with all the same principle.
The main idea seems to be that you have to make the surface of the object glas hydrophilic with some method. The usual way is to coat the glas with a "monolayer" of some polaric molecules, e.g. detergent, as many of you have suggested. This layer then attracts the water molecules and they are slipping in between the glas and the formwar thus removing the formwar- layer. There are many different hydrophilic commercially awailable solutions with which you can even adjust the strength of hydrophilicity - as well as the opposite = hydrophobicity.
This monomolecular layer is also good against microscopic pits in the glas surface because it covers the pit to some extent.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Maki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
Plant cell walls become highly fluorescent after fixation with glutaraldehyde. A student here used that method to estimate total cell wall by morphometrics. I think he left them in 10% glut overnight. ***************************** * Greg Erdos * * Director, ICBR EMCL * * University of Florida * * Gainesville, FL 32611 * * gwerdos-at-gnv.ifas.ufl.edu * * 904-392-1295 * *****************************
In reply to your deconvolution questions: 1) The maximum entropy sol- ution is not always the smoothest; in particular, the m.e. solution for dif- fraction gives peaks which get sharper as the phase set is extended. Since, for your example, the maximum resolution is limited by the probe diameter, this effect may not occur; however, if the solution tree gives maximum likelihood for a set of structures which are not smooth, there is nothing which would guide the solution to a smoother structure. That is, suppose there are two structures the first of which is smooth and nearly correct and the second of which is rough at a scale smaller than the probe diameter and whose envelope is even more nearly correct. M.e. would follow the path of the rougher structure, and would not necessarily smooth it out. 2) Leaving aside the question of whether atoms are delta functions--especially when they are at the sum of the Van der Walls radii apart--maximum entropy can find such a structure and will find a structure whose envelope follows the contact points of the atoms with the probe. The often-published STM images of a silicon surface demonstrate what one might expect. It is not a bad idea, however, to include atomicity or other constraints explicitly, and, I believe, this can be done easily in a m.e. calculation. I hope this helps.
IgGs & Gold Dear Dr Ruben, I tried to send this message out on the bulletin board but I seem to be having problems with the connection. I hope that this will answer some of your questions. Feel free to call me if you want more information etc.
There are many reviews out in the literature covering the different methods for producing colloidal gold probes and then coupling them to proteins, including IgGs (look under Jan De Mey, Jan Leunissen, Ralph Albrechts). The methods are easy to follow and easy to do. If you would prefer someone else to do this then I think the lab in Utrecht, Holland provides this service - the contact is George Postuma, Dept of Cell Biol. University of Utrecht, The Netherlands (phone number 011 31 30 507 654) - this is the lab where Jan Slot works, by the way. Coupling immunoglobulins to gold requires lots of protein so make sure you have large supplies of your antibodies, especially if you want to do double label experiments and need IgGs coupled to more than one gold size. If you only have limited supplies of antibody and are planning simple immunocytochemical experiments then it will be simpler and less expensive to use protein A-gold. This will bind to rabbit antibodies and a few other IgGs. However, mouse monoclonals, in general have poor or no affinity for protein A. In this case, buy an unconjugated rabbit anti-mouse and use this as a bridge (ie apply first antibody; rabbit anti-mouse or anti-rat etc; apply protein A gold). Contact me if you want to use the antibodies for double labeling. These are simple methods using bridging antibodies and protein A-gold. Paul Webster Yale School of Medicine (203) 785 5072 Good Luck.
In reply to your deconvolution questions: 1) The maximum entropy sol- ution is not always the smoothest; in particular, the m.e. solution for dif- fraction gives peaks which get sharper as the phase set is extended. Since, for your example, the maximum resolution is limited by the probe diameter, this effect may not occur; however, if the solution tree gives maximum likelihood for a set of structures which are not smooth, there is nothing which would guide the solution to a smoother structure. That is, suppose there are two structures the first of which is smooth and nearly correct and the second of which is rough at a scale smaller than the probe diameter and whose envelope is even more nearly correct. M.e. would follow the path of the rougher structure, and would not necessarily smooth it out. 2) Leaving aside the question of whether atoms are delta functions--especially when they are at the sum of the Van der Walls radii apart--maximum entropy can find such a structure and will find a structure whose envelope follows the contact points of the atoms with the probe. The often-published STM images of a silicon surface demonstrate what one might expect. It is not a bad idea, however, to include atomicity or other constraints explicitly, and, I believe, this can be done easily in a m.e. calculation. I hope this helps.
At the ANLEMC we also use Process Software's FTP & TCP/IP software on the Dec PDP 11's. We use this on both normal PDP 11's and also EDAX PV9900 systems (PDP 11/73 based). All systems transfer both images and spectra back and forth between Dec, Mac, and PC systems. To transfer images we send data in raw (binary) format and then use a variety of programs to import and read the data. For example we can read EDAX Image files directly into the NIH Image program for fast viewing and sorting. We can also transfer spectra both in binary (Edax) format or alternatively translate the spectra on the EDAX system into the MSA (Microscopy Society of America) format, which is a simple ASCII file. Once the data is in the MSA format it can be directly transported into any data analysis/spreadsheet progran on your favorite platform. The MSA format is in the public domain and can be downloaded from the EMMPDL.
I've been off line for the last 2 days, however, I did recieve your collective messages that some of you were having problems. the system should be fixed by now, but please keep me informed of problems whenever they occur. The lastest problem was relatively simple the disk was full and hence Email was either being rejected or sent out with some or all of the text of messages missing. Sorry about that.... :-(
As for the other questions about creating a sublists, digest modes, etc..... These have always been possiblities and I'm always open to the suggestions for improvements (no offense will be taken on anyones comments/suggestions for improvements). Basically these items are on my list of things to do, but as I mentioned earlier this month (or was it last month?) , I'm trying to procur a new system, install it debug and generally get it up and running and then decommission this computer (all in my spare time). Until I have these all completed, I'm going to have to preserve the status quo.
*************************************************************************** CALL FOR PAPERS ***************************************************************************
We are organizing a focused workshop entitled:
"INTERFACE FORMATION AND DYNAMICS IN LAYERED STRUTURES"
This workshop will be part of the Scanning Microscopy 1994 Meeting to be held in Toronto, Canada, 9-11 May, 1994.
The objective of this workshop is to bring together academic and industrial researchers working in the field of epitaxial heterostructures. Topics will include: -surface and interface energetics of defect formation -sensitivity of materials properties to interface imperfections -characterization techniques for interface studies -epitaxial growth modes -phase separation processes -point and line defect engineering -applications of mismatched materials.
The workshop has been organized on the same lines as the Gordon and European Science Foundation Research Conference format. Accordingly there will be much time for discussion. Moreover there will be a large number of invited speakers including:
G.C. Aers (NRC-Ottawa) J.-M. Baribeau (NRC-Ottawa) E. Bauer (U. Clausthal) D.K. Biegelsen (Xerox) D. Cherns (U. Bristol) A.G. Cullis (DRA-Malvern) C.B. Duke (Xerox) K. Eberl (Max Planck Inst.) L.C. Feldman (AT&T) E.A. Fitzgerald (AT&T) J.M. Gibson (U. Illinois) M. Grinfeld (Rutgers U.) D.E. Jesson (ORNL) B.A. Joyce (Imperial College) K.L. Kavanagh (UCSD) R.E. Mallard (BNR-Ottawa) B. Meyerson (IBM) B. Orr (U. Michigan) C.J. Palmstrom (Bellcore) H.E. Ruda (U. Toronto) M. Saran (Northern Telecom) L.J. Schowalter (Rensselaer) T. Tiedje (UBC) P.W. Voorhees (Northwestern U.) G.C. Weatherly (McMaster U.) Y.-N. Yang (U. Maryland) A. Zangwill (Georgia Tech)
The deadline for submission of contributed papers is 15 April, 1994.
For further information about the workshop please contact:
Doug D. Perovic Department of Metallurgy and Materials Science, University of Toronto, Toronto, M5S 1A4 Canada Tel: (416) 978-5635 Fax: (416) 978-4155 Email: perovic-at-ecf.utoronto.ca
To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Problems & Question with the Email Orig-Author: {"Nestor J. Zaluzec (708)-252-5075, -4964" {ZALUZEC-at-anlemc.msd.anl.gov} }:ddn:wpafb ----------------------------------------------------------- Friday 2/25/94 ~ 11 pm
All Subscribers
I've been off line for the last 2 days, however, I did recieve your collective messages that some of you were having problems. the system should be fixed by now, but please keep me informed of problems whenever they occur. The lastest problem was relatively simple the disk was full and hence Email was either being rejected or sent out with some or all of the text of messages missing. Sorry about that.... :-(
As for the other questions about creating a sublists, digest modes, etc..... These have always been possiblities and I'm always open to the suggestions for improvements (no offense will be taken on anyones comments/suggestions for improvements). Basically these items are on my list of things to do, but as I mentioned earlier this month (or was it last month?) , I'm trying to procur a new system, install it debug and generally get it up and running and then decommission this computer (all in my spare time). Until I have these all completed, I'm going to have to preserve the status quo.
I am looking for microscopically orientated people who may be visiting South Africa in the next 6-12 months.
I have recently submitted and had accepted the DipRMS thesis, but am trying to assist the Royal Microscopical Society in finding a microscopist who would be able to convene a local viva board. The person would have to meet the Royal Microscopical Society's approval, as being a person able to perform such a function.
If you know of anyone or if you are able to assist yourself could you please contact me directly at
RETEP-at-ANAT.UCT.AC.ZA
or phone/write to me at the telephone number/address below.
PLEASE DO NOT respond via the Mailserver.
Peter D. G. Richards Dept Anatomy and Cell Biology UCT Medical School Observatory 7925 RSA Tel: 021-406 6285.
*************************************************************************** CALL FOR PAPERS ***************************************************************************
We are organizing a focused workshop entitled:
"INTERFACE FORMATION AND DYNAMICS IN LAYERED STRUTURES"
This workshop will be part of the Scanning Microscopy 1994 Meeting to be held in Toronto, Canada, 9-11 May, 1994.
The objective of this workshop is to bring together academic and industrial researchers working in the field of epitaxial heterostructures. Topics will include: -surface and interface energetics of defect formation -sensitivity of materials properties to interface imperfections -characterization techniques for interface studies -epitaxial growth modes -phase separation processes -point and line defect engineering -applications of mismatched materials.
The workshop has been organized on the same lines as the Gordon and European Science Foundation Research Conference format. Accordingly there will be much time for discussion. Moreover there will be a large number of invited speakers including:
G.C. Aers (NRC-Ottawa) J.-M. Baribeau (NRC-Ottawa) E. Bauer (U. Clausthal) D.K. Biegelsen (Xerox) D. Cherns (U. Bristol) A.G. Cullis (DRA-Malvern) C.B. Duke (Xerox) K. Eberl (Max Planck Inst.) L.C. Feldman (AT&T) E.A. Fitzgerald (AT&T) J.M. Gibson (U. Illinois) M. Grinfeld (Rutgers U.) D.E. Jesson (ORNL) B.A. Joyce (Imperial College) K.L. Kavanagh (UCSD) R.E. Mallard (BNR-Ottawa) B. Meyerson (IBM) B. Orr (U. Michigan) C.J. Palmstrom (Bellcore) H.E. Ruda (U. Toronto) M. Saran (Northern Telecom) L.J. Schowalter (Rensselaer) T. Tiedje (UBC) P.W. Voorhees (Northwestern U.) G.C. Weatherly (McMaster U.) Y.-N. Yang (U. Maryland) A. Zangwill (Georgia Tech)
The deadline for submission of contributed papers is 15 April, 1994.
For further information about the workshop please contact:
Doug D. Perovic Department of Metallurgy and Materials Science, University of Toronto, Toronto, M5S 1A4 Canada Tel: (416) 978-5635 Fax: (416) 978-4155 Email: perovic-at-ecf.utoronto.ca
I have always used gelatin capsules for embedding material in LR-White and I was wondering if anyone has tried using Beem capsules. If I dry the Beem capsules overnight in a 50 C vacuum oven, would this help? I'm currently doing immuno-labeling on Euplodes and would like to be able to spin the specimens down into a conical Beem capsule. Any suggestions? I appreciate any info on this method. Thanks, Phil Rutledge prutle1-at-gl.umbc.edu
This is a request for help. A zoology faculty member wants to count fiber types (muscle fiber cross sections, stained for I, IIA and IIB) and to obtain cross section areas for the fibers. If I understand the needs correctly, we will need software that will allow "sliding" two images and aligniment of the two for fiber type identification. Then, a more usual morphorometry of doing the X-sec. Defining the boundry by hand for the area determination is acceptable. Is there anyone who is doing this or has experience with muscle firber analysis that can recommend software for either the PC or Mac platform? I can capture the image into a PC system in my lab for them, save it in a standard format .tga or tiff etc., but have no microcomputer level analytical software. Any suggestions, vendors etc will be appreciated. Pardon the typo, live time VAX. Thanks David P. Campbell
We also ended up with a big mess when heat polymerizing LR White in polyethylene BEEM caps. However a polypropylene micro-fuge tube will work just fine. Only problem is that one must saw out the specimen or use doggie toenail clippers to cut off the end of the tube wherein lies the specimen and then tease it out. LR White does not bind to the polypropylene but it doesn't slip right out either. If you use the clippers be sure to do this inside your closed fist so that the specimen is not launched across the room never to be seen again. The clipped off tip must then be remounted on something appropriate for sectioning using super glue. Works best if you file done the cut surface so that it is smooth and makes good contact with the plastic stub such as those sold by Pella.
} Phil Rutledge asked about polymerizing LR-white in Beem capsules. We tried } drying at 60 C for 4 hours (haven't tried overnight). Results were } just as bad as with undried capsules. Poor polymerization of the resin. } We ended up with a real funky mess. } -Jay Jerome } jjerome-at-isnet.is.wfu.edu
I've never used LR-white, but I've talked with one of the distributors about its notorious polymerization properties. They told me that they think BEEM capsules are not suitable for polymerizing LR-white. The reason is that the capsules are too porous and permeable to water. Any water will interfere with polymerization. They have never had any trouble polymerizing, as long as they use gelatin capsules.
As I said, I have no experience (yet), but this might help others. I'd be interested in comments from those who use it successfully.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Message-Id: {m0pbe6h-0000PNC-at-stjohns.ohsu.edu} Message-Version: 2 } To: microscopy-at-anlemc.msd.anl.gov
------------------------------------------------------------------------------ POSITION AVAILABLE ------------------------------------------------------------------------------
Research Associate postition open at the Department of Pathlogy in research related to cytoskeletal components of megakaryoscytes and the process of platelet fromation. Successful applicant will have a M.S. or equivalent, experience in electron microscopy and immunology techniques. Experience in standard biochemical and molecular biology techniques preferred.
Contact by phone or regular Mail:
Dr. Paula Stenberg, E.M. Director Department of Pathlogy, L113 Oregon Health Sciences University Portland, OR 97201
Message-Id: {m0pbeRw-0000P0C-at-stjohns.ohsu.edu} Message-Version: 2 } To: microscopy-at-anlemc.msd.anl.gov
Evan's Blue and Blood Brain Barrier
A general question for light level resolution. We use Evan's Blue circulated in vivo for 30 minutes to see if there has been a breakdown of the blood brain barrier. Animals are then perfused (4% para.) the brains sectioned with a vibratome (100 microns). Question is: If we dehydrate and mount in permount will the Evan's blue stay put or get leached out? Any experience with similar situation would be helpful.
Bob Kayton C.R.O.E.T. L606 Oregon Health Sciences Universtiy Portland, OR. 97007 503 494 2504 E-mail kayton-at-ohsu.edu
Message-ID: {MAILQUEUE-101.940302153004.576-at-anat.uct.ac.za} To: microscopy-at-anlemc.msd.anl.gov
Regarding the query about the azo vital dye Evans Blue.
I have not had any personal experiance with the dye but there should be no problem with dehydrating and mounting. Be sure to dehydrate in alcohols and xylene or equivelant.
Peter D. G. Richards Dept Anatomy and Cell Biology UCT Medical School Observatory 7925 RSA Tel: 021-406 6285.
Phil: our lab's experience with yeast, LR White and capsules: two ways: 1. small sample size: Place suspension of sample in LR White in a BEEM capsule, seal and centrifuge ( inside a tube, whose bottom is padded with cotton). Seal the BEEM INSIDE a large gelatin capsule. Available through drugstore or EM supply house. If you can't find a large enough gelatin capsule; use the OOO. Simply "trim" the beem capsule(that extra ridge where the cap goes on), so it will slide in.Then carefully seal the BEEM capsule inside two OOO gelatin capsule bottoms, that have been filled with LR White. A bit sloppy, but very effective. Works for us 100%. You just need to wear gloves and put down extra paper to catch any LR white drops, while you are filling and sealing 2. large sample size: leave material in OO capsules. Place these capsules inside a BEEM capsule, that has been cut to accomodate the capsules (The top half is cut away). Clinical centrifuge, as above.
hope this helps Louisa. Howard-at-dartmouth.edu
P.S. If you have access to a vacuum oven, you can use the BEEM capsules alone. This avoids the problems associated with porous BEEM capsules and the presence of oxygen.
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: MICROSCOPY-at-ANLEMC.MSD.ANL.GOV
} Is there anyone who is doing this or has experience with muscle firber analysis } that can recommend software for either the PC or Mac platform?
NIH-Image, a freeware image processing program for the Macintosh available from the National Institutes of Health at zippy.nimh.nih.gov, directory /pub/image, has facilities for image alignment (in several modes), area determination (including thresholding/density slicing to segment the regions of interest, and automatic counting, analysis and labelling of these regions), and much more.
----- Transcript of session follows ----- 421 nw.oirtorm.net.kiae.su (tcpld)... Deferred: Connection timed out during user open with oirtorm.net.kiae.su
----- Unsent message follows ----- Received: from anlemc.msd.anl.gov by cpuv1.net.kiae.su with SMTP id AA11761 (5.65.kiae-1 for {alekseev-at-nw.oirtorm.net.kiae.su} ); Fri, 25 Feb 1994 02:43:27 +0300
In response to requests for suppliers of protective lacquer for electropolishing I would like to inform you that Microshield Lacquer is available from:
South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673 USA
The Microshield Lacquer has been used for many years with our Model 550 Electropolisher and Model 550 Chemical Jet Polisher. It is acetone soluble and is available in an 8 ounce kit. Please reference P/N 02-01890-01.
} } Is there anyone who is doing this or has experience with muscle firber analysis } } that can recommend software for either the PC or Mac platform? } } NIH-Image, a freeware image processing program for the Macintosh } available from the National Institutes of Health at zippy.nimh.nih.gov, } directory /pub/image, has facilities for image alignment (in several } modes), area determination (including thresholding/density slicing to } segment the regions of interest, and automatic counting, analysis and } labelling of these regions), and much more.
Here's the README file from Image:
NIH Image --------- NIH Image is a public domain program for the Macintosh for doing digital image processing and analysis. It can acquire, display, edit, enhance, analyze, print, and animate grayscale and color images. It reads and writes TIFF, PICT, PICS and MacPaint files It features multiple windows, MacPaint-like editing and 8 levels of magnification. It supports Data Translation and Scion frame grabber cards. Image requires at least 4MB of RAM and 8-bit video. The following files in the directory /pub/image contain NIH Image, documentation, source code, and example images.
nih-image1xx_fpu.hqx NIH Image 1.xx application. Requires FPU. nih-image1xx_nonfpu.hqx Version that does not require floating-point nih-image1xx_docs.hqx Documentation in Word 5.0 format nih-image1xx_source.hqx Think Pascal 4.0 source images Directory with images in TIFF and PICT format stacks Example stacks(3D images and movies) (directory) nih-image_spinoffs Contains variants of NIH Image(directory) programs Directory containing miscellaneous related programs
File Formats ------------ Files are in one of three formats. Those with a ".hqx" suffix are BinHex encoded Mac binary files, those with a ".bin" suffix are MacBinary encoded Mac binary files, and those with a ".txt" suffix are a plain text files. The BinHex and MacBinary formats represent the two parts of a Mac file(the data fork and the resource fork) as a single file. They permit storage of a complete Mac file on a non-Mac system, such as this server.
Most files were compressed using the Mac utility Stuffit 1.5.1 and uploaded using Fetch, which does the BinHex or MacBinary encoding. Both utilities can be found in the util directory. The best way to retrieve files is to use Fetch, which automatically does Binhex (or MacBinary) decoding and file decompression. Unfortunately, Fetch requires a Mac directly connected to the Internet. If this is not the case, use an FTP (File Transfer Protocol) utility to transfer the files to a local host and then transfer them to a Mac via modem.
For Macs not directly on the Internet, BinHex files must be transferred to a Mac using "ascii" mode and then decoded and decompressed using Stuffit or some other Mac compression utility, such as Compact Pro. MacBinary files must be transferred to an intermediate host using FTP in "binary" mode, then transferred to a Mac in "MacBinary" mode and decompressed using Stuffit or Compact Pro. A copy of Stuffit 1.5.1 is in the directory /pub/util in MacBinary format. The document "ftp-primer.txt" in the documents directory provides more information on file formats and FTP.
NIH Image Mailing List ---------------------- There is an NIH Image mailing list. It was set up by a group in the Soil Science Department at the University of Minnesota. To subscribe, send a message containing the line "subscribe nih-image {your name} " to soils.umn.edu.
DepthGauge ---------- DepthGauge is a control panel that allows rapid switching between monitor depths settings(e.g. 8-bit color and 24-bit color).
Fetch(/pub/util) ---------------- Fetch is a slick utility that allows networked Macs to transfer files over the Internet using the File Transfer Protocol(FTP). It does BinHex decoding and file decompression as the files are transferred.
Giffer(/pub/image/peograms) ----------------------- Giffer is a shareware program useful for converting from GIF to Pict format, and vis-versa.
ImageFFT(/pub/image/image_spinoffs) ----------------------------------- ImageFFT is an extension to NIH Image to support frequency domain (power spectrum) display and editing. It can do a 512x512 FFT in 18 seconds on a Mac IIfx.
ImageFractal(/pub/image/image_spinoffs) --------------------------------------- ImageFractal is a version of Image modified to compute the Fractal Index of objects by the Richardson Plot or the Tile-amalgamation methods.
Image/MG(/pub/image/image_spinoffs) ----------------------------------- Image/MG is an extension of Image supporting quantitative evaluation of cerebral blood flow, glucose metabolism, and protein synthesis.
NCSA PalEdit(/pub/image/programs) ----------------------------- NCSA PalEdit is a public domain program from the National Center for Supercomputing Applications for creating and customizing color palettes. With PalEdit, you can modify the whole palette or individual entries in the palette, to create a set of colors tailored to your needs. PalEdit can save palettes in a format compatible with NIH Image.
MockWrite --------- MockWrite is a simple DA text editor that is convenient for editing macros written in Image's Pascal-like macro programming language.
Projectionist(/pub/image/programs) ------------------------------ Projectionist is a utility that allows you to animate stacks created by Image and saved in PICS format. Because all the frames in the stack do not need to be loaded into RAM, Projectionist requires less memory than image. This preliminary version uses the standard system palette, so stacks created with Image may lose some of their colors when animated with Projectionist.
There are a couple of solutions to this problem that we've used. First, capture one image, then print it on mylar with a laser printer (or to paper and photocopy onto mylar if your printer doesn't do mylar). then capture the image to be overlaid and hold the mylar transparency over the computer monitor. This usually works best with a high contrast image on the mylar- otherwise the gray in the background details will obscure the lookup image on the monitor. this works well for physical disectors. The second image can be grabbed during the long print time.
Second, NIH Image, a public domain image analysis program for the Mac, allows alignment of two overlaid images. You can capture the first image, duplicate it, and then capture the second image and perform a live paste which superimposes the live image over the captured image. Live paste allows moving the stage until the live image aligns. - This sounds easy, but usually quite tedious in practice.
The simplest way to align images in Image is to capture your images and then run one of the alignment macros that either come with Image or are avaialble from the User_macros folder from Zippy.nimh.nih.gove (NIH Image ftp source). One alignment macro allows drawing lines on images in a stack, then all members of the stack are automatically aligned. Another macro, which I wrote, works on pairs of images. Draw a line between two landmarks common to the two images, then the macro will rotate one of the images to rotationally align the two lines and transparently paste it over the other image. You tweak the XY registration by hand.
Most PC software with macro capabiblity should allow creating the same type of macros. 24 bit software may allow putting the two images to be aligned into different color planes, then aligning. - Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu -
We just tried Evan's blue for the same purpose. It was somewhat visible in wet tissue coverslipped with glycerol. Dehydrating through ethanols and clearing in xylene, coverslipping with DPX (our standard protocol for paraffin and vibratome sections) dramatically improved the fluorescence. Nearly one week later there is no noticeable fading or diffusion. Sure is pretty.
Could you email your labelling method - concentrations, route of administration and survival times? I'll get the grad. student who did this to make her method available to send to you.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
I have read on this e-mail system about polymerization of LR WHITE RESIN in a vacuum oven. Do you close the lids of the BEEM capsules when in the oven or leave them open?
I`m interested in the morphology of Panthera sp. epidermis. If anyone knows something about it, please, contact me. Thank you.
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
I`m interested in the morphology of Panthera sp. epidermis. If anyone knows something about it, please, contact me. Thank you.
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
I have obtained fixed (2.5% glutaraldehyde in 0.1M sodium cacodylate buffer) lung (rabbit) tissue which has been embedded in LR White and Spurr resin. However, they have NOT been stained with osmium tetroxide prior to embedding. Could anyone suggest possible methods of osmium staining post sectioning? Thankyou.
Microscopists: I am interested in imaging from my TEM directly. For starters, let's say at 1Kx1Kx8bit. Is anyone out there putting the moral equivalent of a CCD in the column? (I suspect that you'd cook an actual CCD in a hurry, not to mention that you don't need response to photons). I see problems with file size, but what are the physical reasons that we don't capture images this way? Julian Smith III Dept. of Biology Winthrop University Rock Hill, SC 29733 Vox 803-323-2111 Fax 803-323-2246
subject:-TEM-PREPARATION OF TEM SAMPLES(PURE COPPER)
I would want to know if it is possible to indicate and to know the initial macro shear direction on a photo of a 3mm TEM disk sample submitted to an shear deformation by extrusion.Moreover, can we prepare and observe square samples instead of conventional disk samples for TEM.I know that some studies have been made in Russia using square samples and showing the direction of initial shear on the pictures. Thanks a lot by advance for any help. Stephane Ferrasse Email address:S0F6296-at-ZEUS.TAMU.EDU
I am studying pure copper samples with the Phillips EM400 and I have problems because my samples seem to be too thick to observe grains,subgrain boundaries and dislocations that I want to observe. My preparation is the following: 1-rough polishing with 600 grit abrasive powder to produce 100-150 micrometers thick parallel sided sheets by using a simple jig. 2-use of a conventional punch device to obtain a 3mm disk 3-use of the Tenupol automatic double jet electropolisher The electrolyte is:20% nitric acid-80% methanol (conditions:methanol cooled in dry ice). QUESTIONS: 1-I don't know if the voltage that I use is good(8-10V);the same for the value of my flow rate (it takes me about 2-4mn to thin my specimen under these conditions) 2-Is it better to have small (those I obtained are about 40-100 micrometer in diameter) or big holes for TEM observations ? 3-For observing grains and subgrains do we have to use diffraction patterns or Kikuchi lines (as it is the case for dislocation observations) to find a better orientation ? More generally, is orientation important to see grain boundaries? 4-I use the highest voltage (120 kV) .Is it correct? 5-Is there something wrong in my initial preparation ( electrolyte,..)? Thanks a lot by advance for any help, Yours Faithfully. Stephane Ferrasse University of Texas A&M phone:409-845-1790 (USA) Email address:S0F6296-at-ZEUS.TAMU.EDU
Message-ID: {MAILQUEUE-101.940307083235.256-at-anat.uct.ac.za} To: microscopy-at-anlemc.msd.anl.gov
I am passing on a query from Peter Linder (Linder-at-botany.uct.ac.za) regarding clearing agents for Botanical Specimens.
He is working with pollen and was wondering if anyone knew of a clearing agent(s) that dealt effectively with tannins. The ones he has used tend to make a mess of the tannins so that they form obnoxious lumps.
Any suggestions?Peter D. G. Richards Dept Anatomy and Cell Biology UCT Medical School Observatory 7925 RSA Tel: 021-406 6285.
Does anyone know the manufacturer of gelatin capsules or if a conical gelatin capsule is available? Since LR-White doesn't seem to work with BEEM capsules, has anyone tried UniCryl with BEEM capsules? I'm doing a lot of cell suspensions for immuno and a conical capsule is ideal. I appreciate all info on this subject. Thanks, Phil Rutledge prutle1-at-gl.umbc.edu
Sponsored by Japan Chapter of SPIE, Co-sponsored by BACUS and SPIE
22 April 1994 Kanagawa Science Park Kawasaki City, Kanagawa Japan ============================================================
Contents ========
1.0 Symposium on Photomask and X-Ray Mask Technology 2.0 Hotel Accommodations 3.0 Registration Information 4.0 General Information 5.0 How to Receive More Information
1.0 PHOTOMASK AND X-RAY MASK TECHNOLOGY =======================================
Symposium Chair: Touru Yoshizawa, SPIE Japan Chapter Steering Committee Chair: Yoshio Tanaka, Oki Electric Industry Co., Ltd. (Japan)
Steering Committee Members: Naoaki Aizaki, NEC Corp. (Japan); Hideaki Hamada, ETEC Systems Japan Ltd. (Japan); Naoya Hayashi, Dai Nippon Printing Co., Ltd. (Japan); M. Ohtaki, Toppan Printing Co., Ltd. (Japan); K. Nakashima, Lasertec Corp. (Japan); Norio Saitou, Hitachi, Ltd. (Japan); Kazumasa Shigematsu, Fujitsu Ltd. (Japan); Yoshiki Suzuki, KLA Japan Ltd.; Yoichi Takehana, HOYA Corp. (Japan); Tadahiro Takigawa, Toshiba Corp. (Japan); Yoshihiro Todokoro, Matsushita Electronics Corp. (Japan); Yaichirou Watakabe, Mitsubishi Electric Corp. (Japan); Hideo Yoshihara, NTT Advanced Technology Corp. (Japan); James A. Reynolds, Reynolds Consulting (U.S.)
2.0 HOTEL ACCOMMODATIONS ========================
The Japan Travel Bureau, Inc. (JTB) has been appointed as the official travel agent for the symposium and will handle reservations of hotel accommodations. Inquiries and applications should be addressed to:
Japan Travel Bureau, Inc. International Travel Division Convention Center (Ref. CD100748-014) 1-13-1 Nihombashi, Chuo-ku, Tokyo 103 Japan
Hotel KSP (Symposium site) 3-2-1 Sakado, Takatsu-ku Kawasaki, Kanagawa 213 Phone: +81-44-819-2211 Y12,650 single w/bath Y24,200 single use of twin room Y25,300 twin w/bath
Hotel Sunroute Den-En Takatsu (15-minute walk to the conference site) 508 Futako, Takatsu-ku Kawasaki, Kanagawa 213 Phone: +81-44-814-3122 Y8,500 single w/bath Y18,000 twin w/bath
Note:
1. The room rates do not include meals. 2. 3 or 6% tax and 10% service charge will be added to the bill when checking out. 3. Should you fail to arrive at the hotel on your scheduled check-in date, your reservation will be automatically canceled.
Application and Payment -----------------------
Participants wishing to reserve hotel accommodations should contact the JTB no later than 20 March 1994. Application for hotel accommodations should be accompanied by remittance of the hotel deposit (one-night room charge) plus a handling fee of Y500 and sent to JTB. No reservation will be confirmed in the absence of this payment. Personal checks are not accepted. All payments must be in Japanese yen. Payment should be in the form of:
* a bank transfer to the Japan Travel Bureau, Inc., account at the Bank of Tokyo, Marunouchi Office, 1-4-2 Marunouchi, Chiyoda-ku, Tokyo 100 Japan (Account number 1025740/Ref. CD100748-014)
* bank draft payable to the order of the Japan Travel Bureau, Inc.
* The following credit cards may be used for payment of hotel deposit: Master Card, Diners Club, Visa Card, AMEX.
Cancellation ------------
In the event of hotel reservations which must be canceled, written notification should be sent to JTB. The following cancellation fees will be deducted before refunding:
Up to 9 days before the first night of stay: Y2,000 Up to 2 to 8 days before: 20% of daily room charge (minimum Y2,000) Less than 2 days before, or no notice given: 100% of daily room charge
3.0 REGISTRATION INFORMATION ============================
To preregister for Photomask Japan '94 contact:
Secretariat-Photomask Japan '94 c/o Business Center for Academic Societies Japan Conference Department 5-16-9 Honkomagome, Bunkyo-ku, Tokyo 113 Japan
Phone: +81-3-5814-5800 Fax: +81-3-5814-5823
* Registration Fees:
SPIE Member . . . . . . . . . .Y25,500 Working Group Member (e.g., BACUS) . . . . . . . Y28,500 Nonmember . . . . . . . . . . .Y30,000
* Cancellation Policy:
No refunds will be made for cancellations received after 16 April 1994.
* Method of Payment (choose one):
All payment must be made in Japanese yen.
* A bank draft for total fee payable to the order of Photomask Japan.
* Bank transfer to the account of Photomask Japan, A/C No. 075-1834926, Daiichi Kangyo Bank, Hongo Branch, Tokyo.
* Credit card (Visa, Diners Club, MasterCard, or American Express)
On-site registration will be accepted at the conference site on 22 April. If you must register on site, please plan to arrive 30 minutes before the program begins. Registration badges are required for admittance to the conference. The technical conference will begin at 8.30.
4.0 GENERAL INFORMATION =======================
* Registration and Information Hours
Registration takes place in front of KSP Hall, Third Floor
Friday 22 April 8.00 to 16.00
* Speakers'/Chairs' Registration Desk
Located in front of KSP Hall, Third Floor
Friday 22 April 8.00 to 16.00
* Location/Travel Information
Photomask Japan '94 will be held at the Conference Hall of Kanagawa Science Park (KSP Hall), 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa, Japan. Phone: +81-44-819-2211
* Climate
The temperature in April ranges between 10 degrees C and 19 degrees C and the average humidity is 65%.
* Visa Requirements
Participants from countries which require a visa to enter Japan should apply at the nearest Japanese embassy well in advance of departure. If you have any questions or requests to obtain a visa, please contact the Secretariat-Photomask Japan ;94 as soon as possible.
Secretariat-Photomask Japan '94 c/o Business Center for Academic Societies Japan Conference Department 5-16-9 Honkomagome, Bunkyo-ku, Tokyo 113 Japan
Phone: +81-3-5814-5800 Fax: +81-3-5814-5823
5.0 HOW TO RECEIVE MORE INFORMATION ===================================
The complete text of the printed advance technical program for Photomask Japan '94 is available via anonymous FTP at:
It is also available through SPIE's automated e-mail server:
Send an e-mail message to,
info-optolink-request-at-mom.spie.org
with the following text in the message body:
send [optolink.meetings.programs]FILENAME.txt
To request a printed advance technical program via e-mail contact:
spie-at-mom.spie.org
For information regarding this meeting or other SPIE symposia or publications, contact SPIE at:
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This advance technical program is based on commitments received up to the time of publication and is subject to change without notice.
---------------------------------------------------------------- SPIE is a nonprofit society dedicated to advancing engineering and scientific applications of optical, electro-optical, and optoelectronic instrumentation, systems and technology. Its members are scientists, engineers, and users interested in the reduction to practice of these technologies. SPIE provides the means for communicating new developments and applications to the scientific, engineering, and user communities through its publications, symposia, and short courses.
SPIE is dedicated to bringing you quality electronic media and online services.
********************************************************************** Biological X-ray microanalysis - applications and recent developments **********************************************************************
The biological XRMA group of the Royal Microscopical Society are holding their Spring Meeting at the University of Sheffield on April 7th 1994. Papers will be presented on a variety of subjects including specimen preparation, elemental mapping, analysis of plant tissue extracts and biological-material interactions. Registration fees are 25 pounds for RMS members and 35 pounds for others.
Further details are available from:
Dr Paul Hatton, School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield S10 2TA, UK
Sender: kayton-at-ohsu.edu (Robert Kayton) Message-Version: 2 } To: microscopy-at-anlemc.msd.anl.gov
We just tried Evan's blue for the same purpose. It was somewhat visible in wet tissue coverslipped with glycerol. Dehydrating through ethanols and clearing in xylene, coverslipping with DPX (our standard protocol for paraffin and vibratome sections) dramatically improved the fluorescence. Nearly one week later there is no noticeable fading or diffusion. Sure is pretty.
Could you email your labelling method - concentrations, route of administration and survival times? I'll get the grad. student who did this to make her method available to send to you.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
There is an immediate opening for a Scanning Electron Microscopy (SEM) Technician at Exxon Research and Engineering Company's Corporate Research Laboratory in Clinton, New Jersey. As a member of the Microcharacterization Team at Corporate Research, this position will involve the operation of a JEOL SEM with associated Energy Dispersive and Wavelength Dispersive Spectrometers. The position will involve the creative application of high resolution SEM imaging and elemental characterization of samples related to a wide range of projects at Exxon's Corporate Research Laboratory. The position will also involve general laboratory operations, sample preparation, SEM maintenance and computer analysis of the SEM data (both image analysis and spectral analysis).
Successful candidates should have experience in chemistry, physics or material science with a Baccalaureate degree or equivalent experience. Previous SEM experience and experience with high vacuum systems and computers (DOS, Unix, and VMS) is highly desirable. Since a number of projects are simultaneously in progress, it is essential for the SEM technician to be very organized and to pay attention to detail and accuracy in reporting results.
Qualified candidates please send resume to:
William Lamberti Associate Research Physicist Exxon Research and Engineering Company Clinton Township Route 22 East Annandale, New Jersey 08801
Equal Opportunity Employer M/F/H/V
All resumes must be received by April 8, 1994.
William A. Lamberti Office LA-196 Exxon Research and Engineering Company Route 22 East Annandale, NJ 08801 Email: walambe-at-erenj.com
TEM-PREPARATION OF PURE COPPER SAMPLES Thank you first to all those who have given me useful information about preparation of pure copper samples. I would want to know however something else about specimen washing and more precisely about storage of copper.I have read (Eddington-Practical electron microscopy) that Cu can not be stored because of oxydation.Then must we use Cu immediatly after jet polishing or can we keep it in a dessicator?If it is the case, for how long? Thanks in advance for any help. Regards. Stephane Ferrasse University of Texas A&M--email:S0F6296&ZEUS.TAMU.EDU
I realize that the Microscopy Listserv is predominantly used for those microscopies involving light or electrons, however, for those interested in "ions" this may be of interest!
(page 1)
- FIRST NOTICE -
SEVENTH ANNUAL WORKSHOP ON SECONDARY ION MASS SPECTROMETRY
Microelectronics Center of North Carolina (MCNC) and Holiday Inn - RTP
Research Triangle Park, NC
May 10-12, 1994
This workshop is an informal gathering of scientists for discussions relating to fundamental aspects, instrumentation and applications of Secondary Ion Mass Spectrometry.
PROGRAM
On Tuesday evening, May 10, 1994, the Seventh Annual Workshop on SIMS will commence with a registration/mixer from 7 - 9 pm. On Wednesday morning, May 11, a special topics session will focus on fundamentals, application and techniques related to secondary ion imaging. Other topics of interest will be accepted for inclusion in follow-on sessions or the third day's program. Following Wednesday's sessions there will be an evening cocktail hour, dinner, and vendor presentations. The third day will be devoted to user's group meetings and contributed presentations.
The format for this workshop will be informal, with topics of interest to both the novice and experienced SIMS user. You are encouraged to prepare an oral or poster presentation on any aspect of SIMS that may be of interest to your fellow workshop attendees. Open forum user's groups are planned for magnetic sector, time-of-flight, and quadrupole-based mass spectrometer instruments. Technical representatives from the instrumentation companies have been invited to address issues involving instrumentation, modifications, methodologies, and new equipment.
______________________ (page 2)
PRESENTATIONS Please indicate on the registration form provided if you are interested in presenting an oral or poster presentation on a topic which may be of interest to other workshop attendees. An abstract must be submitted to the organizing committee by April 15, 1994. As a special option this year, you are invited to submit a full length manuscript for a special issue of the new journal - Microbeam Analysis .
REGISTRATION Please note: the registration fee is $50.00 if received before April 25, 1994; a late registration fee of $100 will apply after this date. The SIMS Workshop is now being held under the auspices of the Microbeam Analysis Society. Checks should be made payable to: Microbeam Analysis Society and sent directly to Dr. Steven Hues with the completed registration form below - to be received no later than April 25, 1994. Note: The registration fee is waved for full-time student presenters only!
LOCAL ACCOMMODATIONS A block of rooms has been reserved at the Holiday Inn - Research Triangle Park, NC [919-941-6000], at a reduced rate of $72/night + tax (mention the SIMS Workshop). Rooms must be reserved before April 19, 1994 to be eligible for this reduced rate. All inquires pertaining to reserving rooms or details on the facilities, as well as payment for your room, should be made directly to the Holiday Inn - Research Triangle Park, NC. The closest airport with free Holiday Inn Shuttle connection (~10 min.) is the Raleigh-Durham International. Bus transportation to and from the hotel and MCNC is also provided.
____ Yes, I will submit a manuscript for publication in a special issue of Microbeam Analysis.
_______________________ (page 3)
ORGANIZING COMMITTEE:
Steven M. Hues Naval Research Laboratory (202) 767-2671
Greg Gillen National Institute of Standards and Technology (301) 975-2190
Richard T. Lareau Army Research Laboratory (908) 532-0119
IMPORTANT: Registration form and the $50 registration fee must be received no later than April 25, 1994 !!!
Please complete the registration form provided (see pg. 2), cut at dotted line, and return with registration fee enclosed (made payable to the Microbeam Analysis Society) to:
Steven M. Hues Naval Research Laboratory Code 6170 4555 Overlook Ave., S.W. Washington, D. C. 20375-5342
____________
Best regards,
Richard T. Lareau, Ph.D. US Army Research Laboratory Electronics and Power Sources Directorate AMSRL-EP-EC-M, Myer Research Center Fort Monmouth, New Jersey 07703-5601
We are considering the purchase of a stage heater/cooler for the Nikon Diaphot. Our main concern is cooling, e.g. stable tempertaure at approx. 22 degrees C. Does anybody have a recommendation of a particularly good stage temperature controller. Right now we have a water cooled system but problems include 1.) vibration and 2.) too narrow for access with high N.A. objectives. Thanks- Michael Cammer
Message-Id: {MAILQUEUE-101.940310095650.288-at-redhot.hut.fi} To: MICROSCOPY-at-ANLEMC.MSD.ANL.GOV
The European Microbeam Analysis Society (EMAS) organises
The 1st EMAS Regional Workshop on June 15-17, 1994, in Helsinki, Finland:
Electron probe microanalysis of materials today - practical aspects
The workshop is intended as a comprehensive introduction for those who are involved with SEM+EDS or EPMA based microanalysis applied to inorganic materials. The emphasis will be on the possibilities and limitations of the methods, and to give enough guidelines, procedures and examples in order to keep the discussion, for which there will be ample time, on a practical and basic level. The participants are encouraged to bring in their own problems for debate. The workshop language is English.
Main subjects:
* Electron-specimen interaction: X-ray generation & detection, spatial resolution * Analytical electron microscopy (AEM): possibilities and limitations * Correction procedures in microanalysis * Practical problems with EDS analysis & practical aspects of WDS analysis * Characterisation of EDS detectors * Standardless vs. quantitative EDS analysis: what can you expect? * SEM + EDS or EPMA with WDS? * Thin surface layer (1 nm - 1 m) analysis with EPMA or SEM + EDS * AEM as a tool for practical problems * How accurate are your results? * Strategy for applying microanalytical techniques
The main lecturers are: Dr. Peter Karduck (Gemeinschaftslabor fuer Elektronenmikroskopie, RWTH Aachen), Dr. W. A. Patrick Nicholson, Dept. of Physics & Astronomy, Univ. of Glasgow) and Dr. Peter Willich (Fraunhofer Institut fuer Schicht- und Oberflaechentechnik, Hamburg).
Posters are invited. Interpretation of the word "regional" can be done in a broad sense. For additional information please see below.
Erkki Heikinheimo ************************************************************* Erkki Heikinheimo e-mail: eheikin-at-redhot.hut.fi Helsinki Univ. of Technology Lab. of Metallurgy SF-02150 Espoo phone +358-0-4512759 FINLAND fax +358-0-4512798 *************************************************************
Has anyone had any experience in processing bacteria grown on 3mm glass beads for SEM? The cells are grown in suspension and the positioning of the beads is irrelevant. Want to look at surface for adhesion properties study. I want to try normal SEM processing procedures but would appreciate any info on an easy method. Thanks, Phil Rutledge prutle1-at-gl.umbc.edu
We are working on an immunoEM project localizing different epitopes in S. mansoni worms. Some of the life cycle stages are fairly small. We are embedding in Unicryl but we have some problems with the small specimens. I tried to put them in gelatine but this gives a problem with the sectioning properties of Unicryl. Does anyone have a better solution. We thought of putting them in agar, but I don't like the temperature this requires (immunoreactivity).
Greetings, We use LOW gelling temperature agar for our tiny specimens. Sigma sells a variety of these, and we use type VII. I forget exactly what the melting T is, but it melts readily on the low setting of a hot plate. We fix, rinse, and then put a few microliters (5-15?) of molten agar around the sample. It sets up "instantly". We use a 2% agar/water solution. Then we dehydrate and embedd as usual. We also throw in a bit of fast green at 100% ethanol, for ease of finding the samples. I have taken this stuff into several kinds of resin and never had any prboblems sectioning, light or em level. Also, we routinely do immunocytochem localizing microtubules, a fairly senstive antigen, so I don't the heat is a big problem. I hope this is useful for you. Please reply if you have further q's. Good Luck, Tobias Baskin
} Hi, } } We are working on an immunoEM project localizing different epitopes in S. } mansoni worms. Some of the life cycle stages are fairly small. We are } embedding in Unicryl but we have some problems with the small specimens. } I tried to put them in gelatine but this gives a problem with the } sectioning properties of Unicryl. Does anyone have a better solution. We } thought of putting them in agar, but I don't like the temperature this } requires (immunoreactivity). } } Any help would be appreciated. } } -------------------------------------------------------------------- } Universitaire Instelling Antwerpen (UIA) } Lab Pathology } 2610 Wilrijk } BELGIUM } } Tel: 32.3.820.25.34 } Fax: 32.3.820.22.48 } E-mail: anapat-at-uia.ac.be } ------------------------------------------------------------------- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Tobias I. Baskin Baskin-at-biosci.mbp.missouri.edu University of Missouri * Division of Biological Sciences * 109 Tucker Hall Columbia, MO 65211 USA voice: 314-882-0173 fax: 314-882-0123 ___ ____ ^ ____ _____ / \ / / \ / \ / / | / / \ / / /___ / /__ /_____\ / /__ / / / \ ( / / / / \ \ / / /____ / \ \____/ /_____
Does anyone know a reference with a derivation of the brightness equation relating brightness to accelerating voltage? Shimoyama, etal, 1972, seem to pull it from the air, and then everyone else seems to just reference them. Shimoyama has B=IeV/pikT where I is emission current density, V is relativistic voltage, T is filament temp. This is stated to be the relativistically corrected expression of the Langmuir theoretical value. Langmuir's equation is I=I(0) (eV/kT) sin2(phi) where I is the max current density in a focused spot, I(0) is the current density at the cathode, T is temp, and phi is the half angle of focused spot.
So does anyone know of a derivation of the brightness vs voltage equation that can be believed??
Subject: Time:11:24 AM OFFICE MEMO Bright vs kV Date:3/11/94 Roseanne: I think you can find the matter of the variation of brightness and accelerating voltage treated in a very understandable manner in Hall's 2nd Ed of "Introd. to Electron Micros.". One of the best general explanations of the characteristics of self-biased guns with tungsten filaments is contained in Chs. VI and VIII of the book "The Electron Microscope" by M. E. Haine, InterSci Pubs. I ran off some calcs of the variation of brightness vs both temp and kV for an illustration in class one time - if you send me your FAX number, I'll be glad to send you a copy. BIGELOW-at-UMICH.EDU FAX: 313-763-4788
Subject: Time:11:37 AM OFFICE MEMO Add'n on Bright. vs kV Date:3/11/94 Sorry, I neglected to mention that Hall's discussion of brightness vs kV appears on p. 158.
I don't do EM, and I've never worked with worms, but I have embedded small pieces of rat spinal cord in agar for vibratome sectioning and it didn't hurt immunoreactivity any for a number of peptides, 5-HT, or tyrosine hydroxylase. The agar stays molten at about 50-55 ¡C, and your tissue is exposed to that for only a very short time. It may even help to cool (not freeze!) your samples slightly before embedding - just a thought. All I did was to attach the blocked pieces to a glass coverslip with SuperGlue, and then squirt the molten agar over and around them. It really isn't embedding in the sense that I'm sure the agar doesn't permeate the tissue at all, but it gave sufficient support to allow sectioning in a consistent plane. You might also consider Low Melting Point agarose (I believe it's available from BRL?) which would keep temperature to a minimum, but it's expensive. Hope this helps...
David Morilak Dept Psychiatry Stanford University morilak-at-cmgm.stanford.edu
RE: Getting a small hole while electropolishing Another "trick"
Another method of setting the automatice cutoff for light sensor s in electropolishers is to temporarily replace your specimen with a conventional TEM aperture. Use an aperture with about a 10-20 micron size hole (it can be an old used one out of your scope) and then turn on the entire electropolishing unit (except the voltage) get the flow of liquid running and adjust the "sensitivity" until your instrument just detects the hole. This is a bit more systematic than just arbitrarily setting the sensor without a calibration point.
You could try embedding in alginate. This can be done at room temperature (or cooler). We have used a method based on Tamponnet et al, Stain Technology 1988, v 63, 155-158. for ultrastructure of free cells and protoplasts.
Mix the cells with a 1-2% sodium alginate solution in buffer (0.1M cacodylate in the original). Extrude the solution through a narrow pippet/syringe into a solution containing 50mM Calcium chloride (or let drops fall into this, or spread a thin film on a slide and dip into this) where the alginate coagulates and holds the cells together.
The only problem we have had is with different batchs of alginate. Some coagulate well and some fail to.
} Do you have } reason to think that the epidermis in the cat family is different in some } special way from the epidermis of other mammals?
I wish thank you for your answer. I will give some explanation. I was in my lab when a engineer asked to speak to me. He was very excited about something he had discovered about the way that the epithelial cells of Panthera pardus epidermis are disposed. He wanted some information or a slide (histological section) of this animal to confirm his ideas, but he did not explained what ideas or what was his intention. He alleged professional secret. That sounds strange, isn t? I am a public employee so I must aid people who ask me aid, they pay my salary. I did a search in Biological Abstract and found nothing. I will do a search in Zoological Abstract, maybe I will be luckier. Thank you.
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
I need to convert TIFF files generated in a PC program using specification 5 to TIFF files following specification 6 for use on a Silicon Graphics machine that requires specification 6. Does anyone know of a program that can do this conversion? Thanks for any pointers.
-- ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Nancy L Desmond, Ph.D. Department of Neurosurgery University of Virginia Health Sciences Center, Box 420 Charlottesville, VA 22908
Message-Id: {MAILQUEUE-101.940314085545.384-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
} From: rutledge phil {prutle1-at-gl.umbc.edu}
} Has anyone had any experience in processing bacteria grown on 3mm glass } beads for SEM? The cells are grown in suspension and the positioning of } the beads is irrelevant. Want to look at surface for adhesion properties } study. I want to try normal SEM processing procedures but would } appreciate any info on an easy method. } Thanks, } Phil Rutledge } prutle1-at-gl.umbc.edu } Probably the quickest way is to pour your bacteria/beads solution into a funnel with a 0.22 or 0.45 micron nucleur-pore membrane filter, then run your fixatives/ OsO4/ dehydration etc. solutions through the same funnel, maybe 5 min. each by volume sample. Dry by CPD or hexamethyldisilizane at 60 C or Peldri. Pour out onto stub with something sticky & conductive on it, like conductive tape or a THIN coat partially dried silver paste (not paint). Phil Oshel
} Do you have } reason to think that the epidermis in the cat family is different in some } special way from the epidermis of other mammals?
I wish thank you for your answer. I will give some explanation. I was in my lab when a engineer asked to speak to me. He was very excited about something he had discovered about the way that the epithelial cells of Panthera pardus epidermis are disposed. He wanted some information or a slide (histological section) of this animal to confirm his ideas, but he did not explained what ideas or what was his intention. He alleged professional secret. That sounds strange, isn t? I am a public employee so I must aid people who ask me aid, they pay my salary. I did a search in Biological Abstract and found nothing. I will do a search in Zoological Abstract, maybe I will be luckier. Thank you.
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
Sharath Yes you did miss something. Look in the EMMPDL subdirectory called CBED. There is a program there by John Porter which plots Stereograhic projections &CBED patterns. By adjusting the convergence angle you will get a conventional SAED pattern. I'm not sure if the UMICH library mirror has the master copy, however I would be very suprized if it doesnot since John Mansfield is very careful on backups and copies. I've appended a copy of the program abstract to this message.
Nestor Zaluzec ANLEMC --------------- Title :STERPROJ_IBM_V4 Keywords :Stereographic Projection, CBED, SAD, TEM, AEM, HOLZ Computer :IBM PC/XT/AT or compatible Operating System :PC-DOS Programming Language :QUICKBASIC 4.5 Hardware Requirements :Hewlett Packard HP7470A Plotter required. Author(s) :John R. Porter Correspondence Address :Rockwell International Science Center, :Thousand Oaks, CA 91360. Abstract:
This QuickBASIC program plots stereographic projections, CBED and HOLZ line simulations, and performs axis/angle pair calculations. Output is to the screen or a Hewlett Packard HP 7470A Plotter. Stereographic projections are drawn to scale for subsequent manipulations with a standard 18cm. Wulff net and can be plotted for cubic, hexagonal, tetragonal and monoclinic crystal systems in any orientation. Plotted poles can be restricted to those allowed by structure factor considerations (for certain structures) or restricted to those in certain Laue zones. The program leads the user through menus to determine the structure and orientation for the projection, which is then plotted accordingly. Version 4.00 is significantly enhanced compared to earlier versions.
EDITORS NOTE: THIS IS A NEW UPDATED VERSION OF STERPROJ- IBM (FEB.1990) -----------------------------------------------------------------------------
Subject: Time:5:16 PM OFFICE MEMO Bright. & Acc. Voltg. Date:3/14/94 My previous comment in response to Roseann Csencsits did not did not actually answer her question concerning the origin of the equation commonly used for brightness vs acc. voltage, because Hall doesn't take into account relativistic effects. A better source is the book "Transmission Electron Microscopy" by Reimer (Springer-Verlag 1984). The underlying equation is Eq. 4.10 on p. 90, which Reimer derives in a fairly understandable manner. This equation can be recast in terms of accelerating voltage U (in Reimer's terminology) by substituting eU for E and mc^2 for Eo (see table 2.1, p. 21), whereupon the terms for the 'relativistic voltage' Ur= U + (e/2mc^2)U^2 can be formed, and the equation becomes b = j/pi + (j/pi)(e/kT)Ur. Assuming the (j/pi) term is small compared to the rest of the equation, and can be neglected, this reduces to the approximation Roseann asked about. For a good definition of the 'relativistic voltage' see p. 30 of Spence. The experimental measurement of brightness is discussed by Spence in Sect. 7.2. Reimer also discusses this the brightness equation in Sect. 2.1 of his book "Scanning Electron Microscopy".
This is my second attempt to get some feedback on my request to get off the microscopy list. I've also sent the proper (but nonfunctional) message to listserver. Still I am swamped with msgs from the list.
Please remove my name from the list, and please let me know if there's a problem. I'm not the only person with this problem - there are enoguh of us that we are considering forming a new list: those_who_can't_unsubscribe_from_the _microscopy_list :}
This is my second attempt to get some feedback on my request to get off the microscopy list. I've also sent the proper (but nonfunctional) message to listserver. Still I am deluged with msgs from the list.
Please remove my name from the list, and please let me know if there's a problem. I'm not the only person with this problem - there are enoguh of us that we are considering forming a new list: those_who_can't_unsubscribe_from_the _microscopy_list :}
To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Thinning Cerium Oxide Orig-Author: {del-at-sol1.lrsm.upenn.edu (David E. Luzzi)}:ddn:wpafb ----------------------------------------------------------- We need to thin a single crystal of Cerium Oxide (CeO2) to electron transparency but are having major difficulties with brittleness. Does anyone have experience with this, or analogous materials? Thanks in advance.
David E. Luzzi Dept. of Materials Science University of Pennsylvania 3231 Walnut Street Philadelphia, PA 19104-6272
I am looking for two second-hand reflective optical microscopes. One should be something like Bausch&Lomb Stereo Zoom 7, the other should have well calibrated focal depth. Can anyone tell me where to look? Thanks.
Lumin Wang TEM Lab., Univ. of New Mexico Bitnet: zircon-at-unmb
Lumin Wang wrote: } I am looking for two second-hand reflective optical microscopes. One should } something like Bausch&Lomb Stereo Zoom 7, the other should have well } calibrated focal depth...
I have no information on used microscopes. Maybe a dealer could help. I am, however, involved in importing new microscopes of all types. In fact, we have a 10X-160X zoom that is priced very low.
There have been a few problems during the last fe months with users trying to unsubscribe from the list. For the most part the problems were related to how they tried to unsubscribe, the MOST common problem was related to mail forwarding! The basic problem was that the particuliar user would subscribe from one address then have that computer forward mail to a different second address on a differnet computer system.
Upon attempting to Unsubscribe, the user would supply his/her second address to the server, rather than the original subscription address. Since the "second" address was unknown to the server nothing happened and they continued to receive their mail! This has happend at least a dozen times in the last few months so please remember to Unsubscribe you must inform the server of your original subscription address, not the one to which you have your mail forwarded. This is particuliarly important for sites/users that heavily use aliases and forwarding, so try to give me a break and remember from whence you came!.
The other problem which requires less investigative work, but is still a headache is that you must unsubscribe to the Listserver not to Microscopy List. (See below)
Also I've had a few comments come back saying that people are not sure if their messages have been posted. If your message is posted you WILL receive a copy back of your original message. The most common problem here is some subscribers simply "REPLY" to a message. This is fine AS LONG AS your Email system recognizes that the message came from the MICROSCOPY-at-ANLEMC.MSD.ANL.GOV, then there is no problem Some Email systems reply to the originator of the message (i.e. the person who started the question/comment). If this is the case then the members of this listserver will not see your reply! Please check your Email header before you send out a REPLY and insure that the message is going to MICROSCOPY-at-ANLEMC.MSD.ANL.GOV. If you do not receive a copy of the message you posted, then it is 99+% likely that your message was not posted to the subscription list....
Just a reminder to Unsubscribe you should send a message to:
Listserver-at-anlemc.msd.anl.gov
and include the following line in your mail message
Unsubscribe Microscopy Username-at-HostAddress
where Username = User Name that you originally subscribed with HostAddress = Address of the host you originally subscribed from ====================================================================
Alot of traffic goes through the computer now adays, and I'm pretty much swamped with things to do.... Yes, the new Alpha system has finally arrived, however, haven't had time to setup the software so it will still be awhile before anything is upgraded. We are now approaching the 900 subscriber mark so business is booming, but the Gov. has cut back our funds & people.
The never-ending-battle continues.........
Nestor J. Zaluzec ANL EM Center & Proprietor of the "Hotel California" Microscopy Listserver :-)
On the question of brightness vs voltage, I REPLYed, which sent my response only to the sender. Others may be interested in Principles of Electron Optics by P.W. Hawkes and E. Kasper, Academic Press, a good two-volume set with more than I want to remember about electron microscopy.
The 46th Annual Meeting of the Scandinavian Society for Electron Microscopy, June 13-15, 1994, Kuopio, Finland
INVITATION AND CALL FOR CONTRIBUTIONS
SCIENTIFIC PROGRAM
The aim of the symposium is to focus interest broadly in the recent developments of microscopy, not only emphasizing the importance of electron microscopy but paying attention also to the development of e.g. confocal microscopy, immunocytochemistry, image processing and image analysis.
The scientic program is designed to have two plenary sessions and parallel sessions for biological and materials scientists. The following sessions are included in the program: - High resolution microscopy - Confocal microscopy - Image processing, analysis and quantitation (plenary) - Electron microscopy in molecular biology - Scanning probe microscopies (plenary) - Electron beam assisted microanalysis - Immuno electron microscopy - Environmental cell pathology of plants - Electron microscopy in clinical medicine - New techniques and products
Invited speakers are amongst others A. Bardal (Norway), C.-H. von Bonsdorff (Finland), F. Cuisinier (France), S. Enestršm (Sweden), H.E. Gaub (Germany), H. Gundersen (Denmark), M.S. Gunthardt-Goerg (Switzerland), T. Holopainen (Finland), S. Huttunen (Finland), L. Kanerva (Finland), I. Kottke (Germany), T. Lepistš (Finland), A.B. Maunsbach (Denmark), M. Mšrgelin (Sweden), J. Paranko (Finland), J.C. Russ (USA), M. Ruhle (Germany), E. Soini (Finland), P. Willich (Germany).
LANGUAGE
The congress language is English
ABSTRACTS
Papers are invited on all aspects of electron microscopy and related techniques. The extended abstracts will be published as a separate proceedings book of the meeting. The abstracts, including photos and references, may contain two pages. The first page should contain text only, the second page may be used for text, figures and tables. The abstract must neve exceed two pages. Use a word-processor with TIMES 12 font and italics for taxonomic terms. The text and figures must fit inside a rectangle measuring 160 mm x 240 mm (width x height). One camera-ready original with one photocopy should be sent before March 31, 1994 to: Dr. Raija Tammi, Department of Anatomy, University of Kuopio, P.O.Box 1627, FI-70211, Kuopio, Finland
POSTERS
At the poster exhibition, a short time will be reserved to each participant for oral presentation. An area of 1100 x 1300 mm (width x height) is allocated for the poster.
SOCIAL PROGRAM
A Get-Together Party will take place on June 12, at the Snellmania Building of the University of Kuopio. On June 13, we take off for a cruise on Lake Kallavesi and continue with the smoke sauna and supper at JatkŠn- kŠmppŠ (Lumberjacks' Lodgings). On June 14, there is reception at the City Hall and after this the conference dinner at the Hotel Arctia
REGISTRATION
The registration fee is 800 FIM for members, 950 FIM for non-members (if you join the society you will get advantage of membreship immediately), and 650 FIM for students and technicians. The fee includes the Get- Together Party, daily lunches and coffees and one copy of the published abstracts. Please contact SCANDEM 94 Secretariat, c/o Finnish Medical Society Duodecim, Savilahdentie 6, FI-70210 Kuopio, Finland (fax. Int.+ 358-71-240361). Deadline is April 15, 1994. Surcharge for registration is 150 FIM.
DEADLINES
March 31, 1994 Submission of abstracts April 15, 1994 Registration
LOCATION
Kuopio is located in the centre of the Lake-district of Finland about 400 km northeast of Helsinki. Connections to Kuopio by air, train or bus are good. The venue site of the meeting is the Snellmania Building of the University of Kuopio close to the centre of Kuopio.
David, I also routinely make TEM samples from GaAs and InP, which are reasonably brittle (but not as bad as YBCO). I simply mount the samples in thermoplastic wax on a glass slide about 20 mm x 20mm and polish down to about 100 microns using the finest grit paper I can find - 'worn out' 1200 grit paper is fine. I then mount it in Lacomit on a PTFE or nylon stub and chemically polish it to transparency using Cl in methanol jet from a wash bottle chopped in half and held upside down in a clamp. I can make 30 samples in a morning and the kit costs virtually nothing to make! The only thing to be careful of is to make sure that the wax ('Crystalbond' in my case) lies all around the sample so that no corners stick out, and to go slowly. If your material is as bad as 1-2-3 superconductor, you may need to be a bit more high-tech; I haven't done it myself, but work done here used a Dimpler to very slowly polish down to about 5 microns before ion milling to transparency. The samples took about 2 days to make and when they broke (about 1 in 5 samples) it was heart rending. I can give you the Email address of the person who did this, if you want - he's moved on now.
Regards,
Richard Beanland,
Department of Materials Science and Engineering, University of Liverpool, P.O. Box 147, Liverpool L69, 3BX, England.
} From: Francisco Javier H Blazquez {fjhblazq-at-fox.cce.usp.br} } } Do you have } } reason to think that the epidermis in the cat family is different in some } } special way from the epidermis of other mammals? } Try doing a literature search of "Science" and "Nature" for the last 5 or so years, using "self-organizing" and "Turing" in your keywords along with "leopard" & "jaguar". The title may have been something along the lines of "How the Leopard got his spots". Also, it may have been in the research news section and not an article. There was a short piece (which I copied & can't find), about some group discovering that the spots on a leopard's (or jaguar's) where a self-organizing phenomenon similar to some chemical reactions (the ones that swirl in a petri-dish?); the principle involved had been proposed orginally by Alan Turing (of math & comupter fame). Your engineer may have stumbled across this. ? Then again, it may be more mundane, like something to do with intercellular junctions. Phil Oshel (312) 274-6348
I just subscribed, got my welcome to microscopy notice and then sent out 2 messages regarding LM on the same day (about a week ago). I never saw either one or heard any response - although I have been catching up on the EM discussion of thinning CeO, etc. Just checking did my messages get out? Is there somewhere I can browse old messages from the list - an ftp site?
P. Joyce
Peter Joyce GRA Materials Science, University of Texas Phone: (512) 471-5723
I have two questions. We have a Leica MeF3 inverted metallograph we just purchased about a year ago. The bulb in the main lamphouse burned out and it was not easy to replace. The manual calls it a 12V/100W LV halogen bulb. When removed all it says on it is Philips. I tried to get Leica to tell me what it's called and who to get it from apart from them (they only had one in stock in Houston) and they were no help at all - although they did try to help me, sort of. The supplier they gave me as their source didn't know anything about being an OEM soure for Leica or Reichert or whoever they are??? What I'm looking for is anyone who has been this or as similar cycle with Leica equipment, or the name of a GOOD specialty bulb store that's been tried and tested on microscope equipment. I tried one really poor one in FLA and another not so bad in NY with little success.
Second question is related to an earlier post of mine from a few months back. I inquired about the use of index matching fluid to help observe my specimens. The response raised more questios than answers, thanks just the same. Experiments have shown that witha thin layer of oil and no coverslip (coverslip helps keep my optics safe from contamination, esp. inverted scope!) the difference in my specimens is night and day. I get really good clarity. The facts are - I'm looking at rough composite prepreg (carbon fibers in polysulfone) and the surface is much too diffuse to see anything. I went to our Plant Biology dept. who gave samples of permount oil, Zeiss immersion oil, mineral oil, and clove oil and some coverslips. The coverslips had little or no effect on optical performance with the oils. Also the Zeiss immersion oil was not very fruitful. The other 3 oils, in particular the clove oil gave excellent clarity at about 200x in darkfield. I went back to the Plant Biology guys for data on the clove oil, specifically the refractive index - they couldn't help me. I called the chemical supplier Sigma Chemical they haven't done that test. I checked the library, there no such data on clove oil and no mention at all of permount oil or mineral oil in the Chemistry CRC and the like. Is there anyone out there using the immersion technique who can comment on the use of such oils and the source of such data? Anyone ever studied clove oil? We're also observing a strange side effect, when the oil is applied tiny white lines, (maybe cracks in the surface of the material) become visible. We're not sure if they're there before the application of the oil because before the oil is applied we can't see well enough. The available data wouldn't suggest this type of oil should be attacking the polysulfone. Perhaps this effect actually arises from the oil getting in pre-existing cracks and highlighting them. Any comments.
P. Joyce
Peter Joyce GRA Materials Science, University of Texas Phone: (512) 471-5723
} I have two questions. We have a Leica MeF3 inverted metallograph we just } purchased about a year ago. The bulb in the main lamphouse burned out and } it was not easy to replace. The manual calls it a 12V/100W LV halogen } bulb. When removed all it says on it is Philips. I tried to get Leica to } tell me what it's called and who to get it from apart from them (they only } had one in stock in Houston) and they were no help at all - although they } did try to help me, sort of. The supplier they gave me as their source } didn't know anything about being an OEM soure for Leica or Reichert or } whoever they are??? What I'm looking for is anyone who has been this or as } similar cycle with Leica equipment, or the name of a GOOD specialty bulb } store that's been tried and tested on microscope equipment. I tried one } really poor one in FLA and another not so bad in NY with little success.
I just happen to have my local independent light microscope service person in the lab today doing routine maintenance. He tells me that, unless you're using a specialty housing, you should do fine with any standard 12V 100W lamp of the ANSI code FCR. You can get these from OSRAM, Philips or Ushio. There should not be any real difference between them. This lamp is rated at 50 hours lamp life at 12V.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
There were a couple of questions concerning LM bulbs and where to purchase them. I quit going to the manufacturers of LMs unless there was no alternative. I have been buying my bulbs from Bulbman,Inc. in Reno, Nevada for over 10 years. They seem to be pretty knowledgable about the bulbs and they have always been able to provide the bulbs or tell me where they may be purchased other than the manufacturer. Their toll free number is 1-800-648-1163. They also take PO's for small amounts of bulbs. You don't have to order more than you need to get a good price if the bulb you want is available. Hope this info helps. Phil Rutledge
I am seeking contact addresses/faxes/e-mail for distributors and manufacturers of CCD video cameras for biological light microscopy (currently brightfield, darkfield, phase and DIC) to be used in conjunction with a VCR and Macintosh Quadra 650 and image grabbing and manipulating facilities.
A colleague in this department uses a Pulnix TM-765 for similar purposes and this camera would be ideal. I have faxed the President of Motion Analysis, Inc at Eugene, OR, which was the the agent when he purchased his camera a couple of years back, but have had no reply. Can anyone give me other contacts or agents for Pulnix, and/or information on other manufacturers/agents for similar systems. We need a camera control system also, and hoped to pay only in the $2500-3000 range.
Any information on Australian distributors would also be welcome.
Thanks
Nikki Watson
Dr N.A. Watson Department of Zoology University of New England Armidale, NSW, 2351 AUSTRALIA Fax: 067 711 869 Phone: 067 732181
For additional support you can cut the conical tip from a BEEM capsule, place your sample insidek (i did this for rat spinal cord) and fill the capsule with agar. Then take the cap off, supergl;ue the thing to the slide after trimming down the open end of the BEEM capsule to expose a couple of millimeters of sample.
a thin walled polyethylened vial cap also works, if you can find an appropriate size, and will be easier to retirm if you need to keep sectioning farther down.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Fri, 11 Mar 1994, David Morilak wrote:
} I don't do EM, and I've never worked with worms, but I have embedded small } pieces of rat spinal cord in agar for vibratome sectioning and it didn't hurt } immunoreactivity any for a number of peptides, 5-HT, or tyrosine hydroxylase. } The agar stays molten at about 50-55 !C, and your tissue is exposed to that } for only a very short time. It may even help to cool (not freeze!) your } samples slightly before embedding - just a thought. All I did was to attach } the blocked pieces to a glass coverslip with SuperGlue, and then squirt the } molten agar over and around them. It really isn't embedding in the sense that } I'm sure the agar doesn't permeate the tissue at all, but it gave sufficient } support to allow sectioning in a consistent plane. You might also consider Low } Melting Point agarose (I believe it's available from BRL?) which would keep } temperature to a minimum, but it's expensive. Hope this helps... } } David Morilak } Dept Psychiatry } Stanford University } morilak-at-cmgm.stanford.edu } } ------- }
I would suggest buying refractive oils from Cargille Labs in Cedar Grove New Jersey, 201-239-6633. They sell liquids certified for refractive index from about 1.25 to 2.0 (this is from memory, and therefore mostly suspect). I have used refractive index kits containing 80 or so liquids that cover a range, but if you are just looking for something for immersion microscope objectives that is more consistant than clove oil they can supply one quarter ounce bottles.
} I am seeking contact addresses/faxes/e-mail for distributors and } manufacturers of CCD video cameras for biological light microscopy } (currently brightfield, darkfield, phase and DIC) to be used in conjunction } with a VCR and Macintosh Quadra 650 and image grabbing and manipulating } facilities. } } A colleague in this department uses a Pulnix TM-765 for similar purposes } and this camera would be ideal. I have faxed the President of Motion } Analysis, Inc at Eugene, OR, which was the the agent when he purchased his } camera a couple of years back, but have had no reply. Can anyone give me } other contacts or agents for Pulnix, and/or information on other } manufacturers/agents for similar systems. We need a camera control system } also, and hoped to pay only in the $2500-3000 range. } } Any information on Australian distributors would also be welcome. } } Thanks } } Nikki Watson } } Dr N.A. Watson } Department of Zoology } University of New England } Armidale, NSW, 2351 } AUSTRALIA } Fax: 067 711 869 } Phone: 067 732181 } } (using 'Eudora' on a Macintosh)
I have looked around in my files and have a few suggestions and corrections.
In my lab we use a couple of Hamamatsu C2400 CCD-video cameras, one with an intensifier coupled to it. I could not find the price for just the CCD camera + controller, but you can contact
Robert A. Wick, Ph.D. Photonic Microscopy, Inc. 2625 Butterfield Road, 103 West Oakbrook, IL 60521 Tel 312-571-1241 Fax 312-571-1244
Other addresses+numbers (form Laser Focus World, Buyers' Guide, 1994)
PULNIX America, Inc. 1330 Orleans Dr. Sunnyvale, CA 94089
Tel 408-747-0300 Fax 408-747-0880 ___________________________________
Dage-MTI Inc 701 N. Roeske Ave. Michigan City, IN 46360
Tel 219-872-5514 Fax 219-872-5559 ___________________________________
I am studying pure copper and copper-niobium samples with SEM.I would want to observe microstructural morphology of the specimen.Is some specific preparation of the specimen needed (for example special cleaning,etching...)?I would want to avoid any kind of preparation involving high temperatures. Thanks in advance for any help. Stephane Ferrasse Texas A&M University_E-mail:S0F6296&ZEUS.TAMU.EDU
A laboratory in our department is seeking a buyer/barterer for an Axiomat with good Nomarski (the machine was used for microtubule motility assays etc.) and other optics. If you are interested, please send us a message. Thanks- Michael Cammer cammer-at-aecom.yu.edu
Greetings, Has anyone tried dehydration in acetonitrile instead of ethanol, as suggested by Edwards et al Micr. Res. Tech 21:39-50 (1992)??? Experience with plant tissue would be particularly relevant, but any comments will be welcomed.
In Canada we have a vendor that sells Sony CCD video cameras, the address is: Soquelec, 5757 Cavendish blvd, suite 101 Montreal, Quebec, Canada, H4W 2W8 Tel:514 482-6427 fax:514 482 1929
Hoping that it will be of some help. Diane Montpetit Food Research Center, Agriculture Canada Quebec, Canada.
In Canada we have a vendor selling Sony CCD cameras: Soquelec 5757 Cavendish blvd, suite 101, Montreal, Quebec, Canada, H4W 2W8 tel:514-482-6427 fax:514-482-1929 Hoping that it will be of some help Diane Montpetit Food Research Center, Agriculture Canada, Quebec, Canada
Introduction to the structure of computer networks Local networks and network protocols; Larger networks; The Internet; Network structure at Tulane Medical Center
Local network functions Controlling network connections with the Chooser; File sharing; E-mail; Networked scheduling; Lab system access; Remote access (connecting from home); Software tools: Alias Manager; Microsoft Mail; Eudora; Meeting Maker; Termy; Apple Remote Access; ARA Commander.
Using the Internet IP addresses and MacTCP; E-mail and internet addresses; Listserv groups; Telnet and File Transfer Protocol (FTP); Transfer formats: binhexing and compression; Special communication protocols: the World Wide Web and Gopher; USENET news groups; Internet resources in the biomedical sciences; Software tools: MacTCP, NCSA Telnet, Fetch, Versaterm Link, Binhex 4.0, Stuffit, Compactor, Mosaic, TurboGopher, ph.
Reference searching and management Using Grateful Med for Medline searching; Current Contents on diskette; Reference management with EndNote; Software tools: Grateful Med 2.0, EndNote Plus, Microsoft Word
Procedures for connecting to the network at Tulane
In Canada we have a vendor selling Sony CCd cameras: Soquelec 5757 CAVENDISH BLVD, SUITE 101, MONTREAL, QUEBEC, CANADA, H4W 2W8 TEL:514 482-6427 FAX:514 482-1929
HOPING THAT IT WILL BE SOME HELP,, dIANE MONTPETIT, FOOD RESEARCH CENTER, QUEBEC, CANADA ,MONTPETITD-at-QCRSSH.AGR.CA
I am looking for suggestions on thin sectioning polymeric materials (polypropylene/synthetic rubber) for TEM analysis. I am having difficulty transfering sections (~80 nm) from the knife to grids. Common problems with the sections are curling and flying away. I was wondering if anyone has had any success embedding these types of materials. *************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
Plastic sectioning I am looking for suggestions on thin sectioning polymeric materials (polypropylene/synthetic rubber) for TEM analysis. I am having difficulty transfering sections (~80 nm) from the knife to grids. Common problems with the sections are curling and flying away. I was wondering if anyone has had any success embedding these types of materials. *************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
If you are sectioning on a dry knife you might like to try the trick that Tokuyasu used for recovering cryosections. Pick them up on the surface of a drop of sucrose or other inert solution held in a small wire loop. The liquid will stop your problems with static and the surface tension should flatten out the sections. Once the sections are recovered, place the liquid drop onto a coated grid. The recovery solution can be removed by floating the grid specimen face down on drops of water. I have seen this method used successfully for rubberized samples in Arizona. It will not be of much use to you if you cannot wet your sample.
Message in response to questions asked by Stephane Ferrasse, Texas A&M.
I assume you are interested in the morphology and distribution of niobium precipitates within a copper matrix. I have worked on rapidly solidified Cu-Nb microcomposites at Iowa State a couple of years ago and have published a technique which has given me excellent results in the SEM. Detailed procedures may be found in ASTM STP 1165, Metallography: Past, Present and Future, George Vander Voort et al., ASTM, 1993, article written by myself and collegues from Iowa State and Penn State.
Briefly, the technique involves an attack-polish method to remove some of the cumatrix. After a standard grind polish finishing on 0.25um diamond with kerosene as the lubricant, the sample was first immersed in a 20%HF, 20%H2SO4, 20%HNO3, 40%H2O solution to remove the smeared Nb created during polishing. Then the samples are immersed in a 30%H3PO4, 15%CH3COOH, 10%HNO3, 45% Ethanol solution to remove some of the copper surrounding the Nb ppts. To aide in this process, this step was performed in an ultrasonic cleaner - promoted homogeneous removal of copper. Final rinsing was done in the ultrasonic cleaner using fresh, 111 trichlorotrifluoroethane. The copper removal and final cleaningstep was repeated as necessary until the desired results were obtained.
Kevin L. Zeik U.S.Steel Technical Center Monroeville, PA 15146 EMail_Zeik-at-USS.COM
To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: Image Archiving Forwarded: Message from DRStadden:R_D:Armstrong of 3-23-94 ------------------------------------------------------------------ SEE FOLLOWING
--------------------- Forwarded Message Body --------------------- To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: Image Archiving ------------------------------------------------------------------ I'm curious about whether anyone is routinely electronically archiving images from SEM or LM, and/or sending them over a PC network. If so, what sort of hardware is required; particularly, what is retrofittable to a digital imaging SEM or a PLM, and what does it cost? I have one quotation for an SEM-interfaceable unit that is around $20K.
Thanks for any leads,
Dave Stadden Testing and Analysis Lab Armstrong World Industries, Inc.
When grabbing the image with CCD camera I a often getting problem with the very high signal which when looking at preview is almost white. I use f=22 but the signal is still to strong. The only solution besides a pice of hardware is to deminish the light source but it is a bad solution. Is there any software for Mac which can solve my problem ?
Michel Deschuyteneer suggested CD-ROM as a method for archiving images. This is something we have been trying to do for the last few months. If you plan to purchase a CD writer, you need to be aware that you may also need to buy a high speed hard disk from which the images will be downloaded to the CD. Look carefully at the input specifications of the CD writer before you buy the high speed disk.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
------------------ RFC822 Header Follows ------------------ Received: by qmgate.anl.gov with SMTP;25 Mar 1994 09:41:29 U Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
Ron Wroblewski asked about software for the Mac to help his intensity saturation problem....
The simple answer is you need a hardware fix not a software one. If the CCD is saturating then there is no software solution. You must decrease the signal. Some CCD camera's have autoIris lenses which attempt to adjust the input to the CCD by closing down the iris via a motorized mechanism.. Alternatively you could put in some type of neutral density filter to reduce the signal that is hitting your CCD.
Yes, we have just (today!) purchased the above printer for producing STEM/XRAY images primarily. For effectiveness/cost ratio it is nothing short of superb. But it does have some limitations: can be SLOW for continuous tone color, does not support Postscript (yet - they "tell me" that a 3rd. party is working on this), fonts can be a problem but not if you store them as image files when used as text on micrographs, line drawings can get excessive cases of the "jaggies" from the mechanical drive ( only shows up on images if you magnify } 5X or so. There is a very objective review of this printer in the August 1993 edition of "Advanced Imaging" Vol 8 No 8 pp 37 - 39. P.S. If you use a Mac you'll need to purchase a $199 interface.
Peter Ingram ingram-at-rcc.rti.org =============================== 3)
Some time ago we had a demo of the Fargo Primera (wax transfer) printer and compared it to the Tektronix Phaser IIID which we have on site. The system demoed only had a three colour system so the blacks were not great. A 3 colour + black system is available. The ouput was not quite as good as the Tek. but when you consider that the Fargo was around a quarter of the price (in Australia) it was pretty good value. I have seen the dye sub. output but not had a chance to do direct comparisions.
I think that Byte reviewd the printer not long ago (or it may have been another computer journal).
I hope this is of some use.
Colin V.
John Fournelle Electron Microprobe Lab Internet:johnf-at-geology.wisc.edu Dept of Geology & Geophysics Telephone: (608) 262-7964 University of Wisconsin Fax: (608) 262-0693 1215 West Dayton Street Amateur radio: WA3BTA/9 Madison, WI 53706 (14.030, 21.030 mHz)
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 1 - 14.
STANDARD PERFORMANCE CRITERIA FOR ANALYTICAL ELECTRON MICROSCOPY
by S. M. ZEMYAN & D. B. WILLIAMS , Department of Materials Science & Engineering, Lehigh University, Whitaker Laboratory, . 5 E. Packer Ave., Bethlehem, PA 18015, U.S.A.
Summary Users of analytical electron microscopy lack easy-to-use standards for assessing the consistency and quality of analytical performance. We propose using a Cr thin film of known thickness to measure three important characteristics related to performance: the Cr K alpha peak-to-background (P/B) ratio, the X-ray spectrometer relative efficiency, and the spectrometer energy resolution. We used a Cr specimen to determine the instrumental factors which influence the P/B ratio, finding that the highest P/B ratios are achieved in scanning transmission mode at the highest available accelerating voltage. We present values of the P/B ratio, and the detector relative efficiency and energy resolution which can be used for comparison in other laboratories using the standard film.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 15 - 22.
ATOMIC FORCE MICROSCOPY IN THE PHOTOCHEMISTRY OF CHALCONES
by G. KAUPP , Universitaet Oldenburg, FB 9 - Organische Chemie I, Postfach 2503, D-26111 Oldenburg, Germany
Summary The application of atomic force microscopy (AFM) to photodimerization of crystalline chalcones provides new insights into the detailed mechanisms of solid-state reactions on the molecular level. Well-directed long-range transport phenomena are found which reach far beyond the crystal lattice distances. Reactions occur in the surface region where the light is absorbed. Characteristic features are built up that depend on crystal structure and crystal face. This could not be foreseen by previous theories based solely on a topochemical postulate/principle. There is now a much more intimate correlation of crystal structure with solid-state reactivity and this is directly studied and proven experimentally by AFM. Even solid-state reactions which are in opposition to topochemistry can be studied and understood on a molecular basis. The three-dimensional resolution of undisturbed insulating surfaces which is obtained by AFM is not available by any other technique.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 23 - 30.
REGRESSION METHODS FOR AUTOMATED COLOUR IMAGE CLASSIFICATION AND THRESHOLDING
by E. J. BREEN , CSIRO, Division of Mathematics and Statistics, Institute of Information Science and Engineering, Locked Bag 17, North Ryde NSW 2113 Australia
Summary Regression methods are used to perform automated image thresholding and colour pixel classification. This is done by considering threshold levels and pixel classification labels as pattern attributes. A regression equation that performs a mapping from the J dimensional feature-pattern space to the K dimensional attribute space is derived. The approach is non-parametric and deterministic, hence no assumptions about the statistical properties of the input patterns or images need be made. Initially a known set of input patterns with associated attributes are used to constitute a training set. A mapping function is then determined from the training patterns and used for estimating attribute values from unknown input patterns, such as images.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 31 - 38.
LIP-MODEL-BASED THREE-DIMENSIONAL RECONSTRUCTION AND VISUALIZATION OF HIV-INFECTED ENTIRE CELLS
by P. GREMILLET,* M. JOURLIN + & J.-C. PINOLI ++ , *Misis Image, BP 711, 42950 Saint-Etienne Cedex 9, France. + Laboratoire Lyonnais des Signaux et Systemes, Ecole Superieure de Chimie, Physique et Electronique, 31 Place Bellecour, 69288 Lyon Cedex 02, France, and Laboratoire de Traitement du Signal, Universite de Saint-Etienne, 23 Rue Paul Michelon, 42023 Saint-Etienne Cedex 02, France. ++Pechiney, Centre de Recherches, BP 27, 38340 Voreppe, France
Summary This paper presents a global solution from acquisition to visualization for the three-dimensional reconstruction of cell sections. Original techniques are proposed for the correct handling of the geometrical section distortions, and a new interpretation based on the logarithmic image processing (LIP) model is applied in order to create normalized grey-level sections where these are missing. Finally, a new method for generating a mesh of triangles to describe the envelope of the reconstructed cell is proposed, as well as a visualization mixing image synthesis and grey-level information. The product allows the user to explore the reconstructed cellular block in any desired direction, by showing user-defined grey-level sections inside the block mixed to a synthetic view of the cell envelope.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 39 - 46.
SURFACE ULTRASTRUCTURE OF OLFACTORY RECEPTOR SENSE HAIRS IN THE SILKMOTH, ANTHERAEA PERNYI
by V. I. POPOV, A. A. NIKONOV, N. K. AGAFONOVA & E. E. FESENKO , Institute of Cell Biophysics, Russian Accademy of Sciences, Pushchino, Moscow Region, 142292, Russia
Summary The fine structure of silkmoth sense hair surfaces has been investigated by freeze etching with Pt/C rotary shadowing. To do this, the hydrophobic layer in the cuticle was removed using 20 - 25% methanol or ethanol. Freeze-etch patterns of sense hair surfaces, as well as the pore structure and pore distribution, are shown. The hair surface has a nonhelical `band' structure, in which every `band' lies at an oblique angle with respect to the axis of the hair. Each `band' is separated from its neighbour by a 30-nm step. The average density of pores is 11.3 plus or minus 2.4 pores per square micrometre. Freeze-etch patterns of the single and multiple pore tubules are shown. Evidence for direct contact between the pore tubule and dendrite membrane of an olfactory receptor neuron is presented.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 47 - 50.
AGAR STANDARDS FOR QUANTITATIVE X-RAY MICROANALYSIS OF RESIN-EMBEDDED PLANT TISSUES
by E. FRITZ & G. JENTSCHKE , Institute of Forest Botany, University of Gottingen, Busgenweg 2, 37077 Gottingen, Germany
Summary Calibration standards for quantitative X-ray microanalysis of resin-embedded plant tissue were prepared by adding 6 - 600 mm KC1 to 5% agar. Agar blocks with an edge length of 1 - 2 mm were rapidly frozen, freeze-dried and embedded in styrene-methacrylate. Dry sections 1 micrometre thick were mounted on adhesive-coated grids. Apart from fine-scale inhomogeneities caused by ice crystal formation, the KC1 is evenly distributed in the agar blocks. The peak-to-continuum values of K and Cl were highly linearly correlated to the K and Cl contents over the whole concentration range.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 51 - 54.
A SIMPLE METHOD FOR PROCESSING INDIVIDUAL OOCYTES AND EMBRYOS FOR ELECTRON MICROSCOPY
by C. NOGUES, M. MARTI, M. BOADA & M. PONSA , Departament Biologia Cel.lular i Fisiologia, Facultat Ciencies, Universitat Autonoma de Barcelona, 08193-Bellaterra (Barcelona), Spain
Summary A simple method for handling individual specimens that must be processed either for scanning or transmission electron microscopy studies is described. For scanning microscope processing, dehydration is carried out with samples enclosed in small cages made from TAAB capsules in which top and bottom are substituted by plankton nets, and for transmission electron microscopy, samples are pre-embedded in agarose. This procedure significantly reduces mouth pipetting, dissecting microscope observations, is less labour intensive and, most importantly, reduces sample loss.
Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. 55 - 58.
ENHANCED RETENTION OF MAGNETIC PARTICLES (E.G. MICROTOMED SECTIONS) IN A TEM
by W. A. FURDANOWICZ & K. E. DOWNEY , Homer Research Labs, Bethlehem Steel Corporation, Bethlehem, PA 18016-7699, U.S.A.
Summary An electron-transparent layer of adhesive is used to restrain magnetic particles from being stripped off the support film by the magnetic field of an objective lens in a TEM.
ÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ RAPID PUBLICATION Journal of Microscopy, Vol. 174, Pt. 1, April 1994, pp. RP1 - RP 2.
MONOMERIC COLLAGEN IMAGED BY CRYOGENIC FORCE MICROSCOPY
by M. B. SHATTUCK, M. G. L. GUSTAFSSON, K. A. FISHER, K. C. YANAGIMOTO, A. VEIS, R. S. BHATNAGAR & J. CLARKE
IF YOU ARE INTERESTED IN SUBMITTING A MANUSCRIPT TO THE JOURNAL OF MICROSCOPY, OR HAVE ANY OTHER QUESTIONS ABOUT THE JOURNAL, PLEASE CONTACT DR GILLIAN WILSON, JOURNAL OF MICROSCOPY EDITORIAL OFFICE, 37/38 ST CLEMENTS, OXFORD, OX4 1AJ, UNITED KINGDOM. TELEPHONE +44 865 248768, FAX +44 865 791237, EMAIL RMS-at-VAX.OX.AC.UK.
AA Happy EA H ÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
A belated reply to the person asking about cat skin. In felis domesticus the skin has all of the same layers as found in human skin. However, the skin is thinner because there are only a few layers of dermins (2-4 layers) as compared to the {10 layers found in most areas of human skin.
We also have been considering CD-ROM for archival and image transfer. I have been told that only recently some of the authoring drives allow reading a partially filled platter, and then only on the authoring drive, not on a standard reading-only drive. I was hoping to use CD-ROM as an alternative to buying magneto-optical drives for portable data, as oppposed to setting up a server.
What has anyone experienced or read about this?
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Fri, 25 Mar 1994, Russell E. Cook wrote:
} Michel Deschuyteneer suggested CD-ROM as a method for archiving images. } This is something we have been trying to do for the last few months. If } you plan to purchase a CD writer, you need to be aware that you may also } need to buy a high speed hard disk from which the images will be downloaded } to the CD. Look carefully at the input specifications of the CD writer } before you buy the high speed disk. } } } Russell E. Cook } Electron Microscopy Center for Materials Research } Materials Science Division } Argonne National Laboratory } Argonne, Illinois 60439 } USA } } }
If all people are talking about is archiving, then I would strongly suggest that DAT tapes are considered. The prices of the hardware for CD-ROM and DAT are comparable, but the price of a single tape is $16 and it can hold 2-16 Gbytes of data. The only reason in my mind for using CD-ROM is if you want additional disc space which is less expensive than true hard disks; for archiving it may well be a waste of money.
As Robin Wright says, CDs are forever. Also, they allow random access and it is extremely cheap to buy an internal CD-ROM player with new computers
Glen
On Mon, 28 Mar 1994, L. D. Marks wrote:
} If all people are talking about is archiving, then I } would strongly suggest that DAT tapes are considered. The } prices of the hardware for CD-ROM and DAT are comparable, } but the price of a single tape is $16 and it can hold } 2-16 Gbytes of data. The only reason in my mind for using } CD-ROM is if you want additional disc space which is less } expensive than true hard disks; for archiving it may well } be a waste of money. } } Laurie Marks, NU }
Message-Id: {MAILQUEUE-101.940329082400.480-at-FS-IAM-1.JRC.NL} To: microscopy-at-anlemc.msd.anl.gov
I agree that DAT tape is not ideal for archiving due to eventual degradation etc, but what about these new Phase-Change optical drives? I don't know much about them, except that they are re-writeable, cost about half that of CD-ROM writers (at least in Europe) and can store approx. 1Gb. I think that they are sold by Panasonic over here. Maybe somebody else knows more about them. As far as I can see they appear to have significant advantages, and are based on Sony's mini-disk technology, so the prices should drop fairly soon if that is a success.
Doug Arrell Mechanical Performance and Joining Institute for Advanced Materials 1755 ZG Petten Netherlands
Subject: Time:8:16 AM OFFICE MEMO CD archiving Date:3/29/94 Just my two cents worth on archiving on optical discs, CDs or DAT. We have been looking into CD archiving and feel that it has several advantages over the other two formats. 1) CDs allow relatively fast and easy acess to the images/files. So if you are planning to manipulate these images (of course you will have to work on copys placed on your hard disk) or require frequent acess to them this method is about equal to opticals, but superior to DAT, 2) Both CDs & opticals will have a longer expected life then DAT before media breakdown, and 3) the most important consideration for us is that CD readers are cheap, going down in price and are incorporated into the hardware of an increasing number of computer systems. This is important since every potential user is already equipped or makes only a small investment ( {$300 for many readers). The larger number of potential users will make the system more cost effective and may partially offset the initially higher cost of a CD writer. A further consideration is that both Mac and IBM clone users need acess. The formatting for opticals is usually specific to one or the other of these standards (unless you plan to go with Apple PowerPCs) and will thus be more limited. Photo CDs can be read on drivers of either machine format with no conversions or translations required. We think that for these reasons, CDs are the way to go.
I have been requested to pass on the following message for discussion:
From: Bill Monroe Electron Microscope Center Mississippi State University
His Question: Occasionally our laboratory staff has experienced problems with thin sections coming off the grid during immuno-gold labeling steps. The tissue sections are from L.R. White embedded material and are mounted on 200 mesh formvar-coated nickel grids.
The Question: Are there any suggestions to remedy this problem of sections coming off the grid?
Richard Kuklinski, rfk2-at-ra.msstate.edu Electron Microscope Center, Mississippi State University
How do I persuade Image to measure the number of intercepts on a line or to measure the length of individual intercepts? Both of these measurements are useful in determining the grain size of metals, for example, see ASTM standard E112 and E1181.
We originally posted this to the NIH-Image list but received no responses, so if anyone can help, we'd appreciate it. Thanks.
You can reply to my e-mail address smiths-at-mlc.lib.mi.us OR directly to my patron at the following address:
Sam Purdy National Steel Corp. Technical Research Center 1745 Fritz Drive Trenton, MI 48183 313/676-2682 or FAX:313/676-2030
I am looking for the name and address of the manufacture of crystalbond. We had purchased in the past sticks about 3/4" in diameter at a very reasonable price (much below Gatan's price) but can't locate the source now.
Serial Sections? An easy way to keep serial sections attached to one another is to coat the block with a sticky substance. Previously, I used something called "Takki-wax". Does anyone know of a supplier for this wonderful stuff, we just ran out?
We are interested in collecting RHEED and LEED images with a video camera. Stuff like line profiles, spot intensity fluctuations. I checked into this several years ago and found a system by Staib Instruments It was a lot of money. I then thought of buying a system with a Sony XC-77 camera, Fuji CF50L lens, DT2851 Framegrabber, Halo 88, and Image Pro software. This sytem would have cost about $7K. Several years have past and now we have a Mac Quadra 840AV. Someone here thinks he can do the same thing with the Mac AV inputs that these other systems could do with dedicated boards. Any comments from people running such systems would be helpful.
} } Serial Sections? } An easy way to keep serial sections attached to one another is to coat the } block with a sticky substance. Previously, I used something called } "Takki-wax". Does anyone know of a supplier for this wonderful stuff, we just } ran out? } ================================================================
I have also heard of using diluted rubber cement for keeping serial sections together. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
We've been using the Becton-Dickinson mouse monoclonal antibody againsty bromodeoxyuridine (BrdU) for a few years for a variety of samples and fixatives. We'vew been using at 1/4000 successfully for ABC-peroxidase and fluorescently labelled secondary antibodies.
The BD is very pricey, $245 per bottle. Has anyone tried other brands of antibodies or fluorescently conjugated anti-BrdU antibodies? What dilutions did you use, how sensitive did you find them (especially the conjugated Ab)?
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Message-Id: {9403291926.AA22920-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Crystalbond Orig-Author: {"M. J. Kramer" {MJKRAMER-at-vaxld.ameslab.gov} }:ddn:wpafb ----------------------------------------------------------- I am looking for the name and address of the manufacture of crystalbond. We had purchased in the past sticks about 3/4" in diameter at a very reasonable price (much below Gatan's price) but can't locate the source now.
Re} I-G-labeled sections falling off Are the sections falling off or is it the formvar that is comming away from the grids? I've never had any problem with sections comming detached from formvar-carbon films.
} } I have been requested to pass on the following message for discussion: } } } From: Bill Monroe } Electron Microscope Center } Mississippi State University } } His Question: Occasionally our laboratory staff has experienced } problems with thin sections coming off the grid during immuno-gold } labeling steps. The tissue sections are from L.R. White embedded } material and are mounted on 200 mesh formvar-coated nickel grids. } } The Question: Are there any suggestions to remedy this problem of } sections coming off the grid? } } } Richard Kuklinski, rfk2-at-ra.msstate.edu } Electron Microscope Center, Mississippi State University ========================================================== REPLY
We have never experienced this problem. We do however pick up sections from above rather than from below thereby eliminating the possibility of trapping water between the section and the formvar film. I don't know if this would make any difference. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Crystal bond is supplied and made by Universal Photonics 1-800-645-7173. Universal Photonics is a supplier of materials for lens grinding and polishing (used to be Universal Optics) and developed Crystal bond for holding prisms and crystals to the work tool as they are being lapped. Their catalog has a lot of interesting stuff of potential use to microscopists, but for years was out of print. I think you can get one now, however.
South Bay Technology produces 4 standard mounting waxes designed for mounting samples for precision lapping, polishing and cutting. SBT makes a wax which is essentially identical to Crystalbond 509. The only difference is that QuickStick 135 is very clear and does not have the amber color normally associated with Crystalbond.
QuickStick 135 is acetone soluble and has a flow point of 135 degrees C. QuickStick 135 is used extensively in TEM sample preparation operations and is supplied with all South Bay Technology TEM sample preparation equipment. Quick Stick 135 is packaged in a plastic tray consisting of 20 3" x .25" x .25" unwrapped sticks (Approx. 360 grams) for $40. It can also be supplied in the 7" long cardboard tubes as is Crystalbond or in a variety of other forms.
For information on our other mounting waxes, free wax samples or information on any of our sample preparation products, please contact me as follows:
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
TEL: 714-492-2600 Toll-free in USA: 800-728-2233 FAX: 714-492-1499
e-mail: via CompuServe at: David Henriks, 73531,1344
Bill Monroe asks about sections falling off of grids during immunostaining. I use uncoated gold grids (no grid induced astigmatism) with LR White or Spurr's resin. I section blocks, pick up sections and let dry at least one day before immunostaining. Once in a while a few sections might fall off but this is not a major problem. I also immerse the grids in all solutions on parafilm. Hope this helps.
Anybody out there have suggestions for measurement of particles from the TEM? The particles are thought to measure about 80 angstroms. We are assuming using 80-200,000x magnification and need very precise measurements. The commercial standards are only good up to 50,000x.
I appriciate any help!
Kathy Walters Central Electron Microscopy Researh Facility University of Iowa
Kathy, How about using carbon graphite at 3.4 Angstrom lattice spacing or there is a type of asbestos that gives 9 Angstrom lattice spacing. These could be used at 100KX or above and the mag accuracy of your TEM could then be calculated. Cal
Message-Id: {9404011318.AA00750-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
On Fri, 1 Apr 1994 dennis%odin-at-odin.morph.med.umich.edu wrote: Snip } My lab is looking for a citation in the literature about the stability of } specimens over time in polymerized Epn blocks. I've checked the literature } but have not had much luck. If anyone knows offhand where this can be find, I } would appreciate the reference.
Hi, I've recently been reading a book called _Artifacts iin Biological Electron Microscopy_ by R.F.E. Crang and K.L. Klomparens, Plenum Press, 1988, and have found much of the info to be very applicable to some of my general problems. There is a section by H.H. Mollenhauer that discusses dehydration and epoxy embedding that might have the info you're looking for. Good luck.
Dwight U. Beebe beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
Picking up on Cal's idea, would it be possible for you to prepare your particles right on top of the carbon graphite, on Formvar coated grids for support. That way you could photograph them together in the same micrograph and get the highest accuracy measurement of the particle sizes by direct comparison to the carbon lattice image.
The question I have, is can one compare the particles directly to the image of the crystal lattice spacings, or is there a calculation that must be done to correctly use that spacing information? Anyone? Gib Ahlstrtand.
------------ Forwarded Message begins here ------------
Kathy, How about using carbon graphite at 3.4 Angstrom lattice spacing or there is a type of asbestos that gives 9 Angstrom lattice spacing. These could be used at 100KX or above and the mag accuracy of your TEM could then be calculated. Cal ---------- Forwarded Message ends here ------------
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
In reply to Kathy Walters query about resolution measurements, try using a colloidal gold preperation, say a 5nm gold. The makers of the probes send along info which contains a distribution curve of the size of the gold. Hope this helps.
After finding a suitable calibration standard, you could use one of the video measurement systems on the market to do the actual measurements. The standard is used to calibrate the measurement system only, and does not have to be closely related to the size of your sample. This would still give very accurate results, down to a fraction of the size of the standard.
My company makes such a device, called the XR-2000. It has a very large array of measurements, like distance, area, angle, pathlength, etc, and lots of other functions. It is very inexpensive and easy to use.
Your system must use standard video (like Y/C or RGB) for this to work.
If you would like to know more about the XR-2000, private Email to me.
Message-Id: {9404041513.AA02301-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
Greetings! I will teach a comprehensive, semester-long TEM course next Fall and am trying to find a good text. My choice at the moment is: Electron Microscopy. Principles and Techniques for Biologists by J.J. Bozzola and L.D. Russell. Does anyone have other favorite texts? The course will be small (6 graduate students) and will include both theory and practical sessions. Both plants (my specialty) and animals will be used as material. I would also be very interested in hearing directly from people who have taught such a course or one similar to it, for tips and "what works, what doesn't" info. We will also use the RMS Handbook: The Operation of Transmission and Scanning Electron Microscopes by D. Chescoe and P.J. Goodhew, as we have a JEOL 100S for the course and this book has a nice description of alignment procedures which match our instrument. Thanks in advance, any help will be greatly appreciated.
Dwight U. Beebe beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
} In reply to Kathy Walters query about resolution measurements, try } using } a colloidal gold preperation, say a 5nm gold. The makers of the } probes } send along info which contains a distribution curve of the size of } the gold. Hope this helps.
Colloidal gold is easy to see and measure, but is not all that uniform. As Ed says, the manufacturers send along a size distribution curve. The sizes - diameters and distribution - will vary from batch to batch. One would also want to know how the manufacturer's size distribution was determined. They presumably would also have to measure the sizes microscopically, and the data would not need to be really accurate to suit their purposes.
The Journal of Microscopy has another few new books for review. Interested parties should contact Gillian Wilson at RMS-at-VAX.OX.AC.UK.
The books are:
1. Introduction to Scanning Tunnelling Microscopy. By C Julian Chen. OUP, 1994. 412 pages.
2. Polymerase Chain Reaction. By C R Newton & A Graham. BIOS in association with the Biochemical Society, 1994. 161 pages.
3.Rapid Methods and Automation in Microbiology and Immunology. Edited by R C Spencer, E P Wright & S W B Newsom. Intercept Ltd, France, 1994. 502 pages.
Subject: Time:2:05 PM OFFICE MEMO Re:EM Text Date:4/5/94 Another good text to consider might be "The Principles & Practice of Electron Microscopy" by Ian M. Watt, Cambridge Univ. Press. It is very well written and covers a good range of subjects for a one-term course.
Subject: Time:2:09 PM OFFICE MEMO Re: SEM Screen color Date:4/5/94 I think one justification for using green phosphors is that the human eye has maximum sensitivity in the green range of the visible spectrum.
Additional note: to avoid to thick a formvar which can remain between grid bars, use a quick blast of compressed air directed the grid to vaporize formvar between the grid bars.
On Fri, 1 Apr 1994 wrightr-at-zoology.washington.edu wrote:
} My very first immunolabeling experiment ended when every section } floated off the 25 grids that I was labeling at the time. Since } then, I use a method shown to me by Bonnie Chojnacki. Specifically I } use grids that have been dipped in a dilute formvar solution (about } 0.1% in chloroform) and then dried on filter paper. The grids can } either be dipped individually into the formvar and then placed on } filter paper, or a whole canister can be dumped into the formvar and } each grid retrieved individually onto filter paper. } } } This procedure produces a sticky coat on the grid bars and sections } (epoxy, LR-white, etc) really stay attached throughout all of the } incubations. } } } } } Robin Wright }
The answerto Your questionK about the same information after 30 year depends on what information you want. I wouldnt trust immunocytochemistry of material embedded that long, since we see a difference even after a month (post embed stain of etched epon). However, the structures have not changed and quantitation done will be the same. Do you need a reference if the experience of a great many microscopists says there is no difference?
On Mon, 4 Apr 1994 dennis%odin-at-odin.morph.med.umich.edu wrote:
} Thanks to everyone who responded the my question. However, most of the } citations sent had to do with the stability of sections under beam current, } heat, etc... } } The situation is that a drug company that has provided a grant to us would } like to know for certain that the tissue that has been embedded in epon can be } pulled out from storage thirty years and will still be good. Will the tissue } be sectionable and will it still give the same data as before? } } As I said, most of the people here still have blocks that they did their } undergraduate work in back in the 1960s, and they are still as sectionable as } the day they were pulled out of the oven. In fact, I understand that the } quality of epon actually improves over time because it will have longer to } fully polymerize. } } So I am then simply looking for a citation on something that we already seem } to know, but still have not been able to find it. } } Thanks again, } Dennis Shubitowski } dennis-at-odin.morph.med.umich.edu }
Green phosphor has a greater luminous efficiency, and so can be made much brighter than white phosphor with equivalent electron optics. This makes a green TEM screen useful, but does not make a good argument for a green monitor in a darkened lab. The SE M manufacturer probably chooses a phosphor based on decay time. The green P20 phosphor has a decay time similar to that of the human eye, and so "integrates" the noisy signal optimally.
regards Mark W. Lund, Director MOXTEK, Inc. Orem UT
} I'm looking for a good inexpensive seconhand ultramicrotome similar } or identical to RMC MT-7000 ULTRA or REICHERT ULTRACUT S.
I don't have an Ultracut S., but I do have a Reichert Jung Histocut II that has never been used, and we are now considering selling it. It was purchased for a project that never materialized, and it has never actually cut anything.
As Nestor indicated several times before, and also in the introductory welcome material sent to new subscribers, is important including in your message the suject header. It allows determining nature of message without having to read the content. Given the increased usage of the service, inclusion of the subject header is a nice gesture toward other users. In fact, many more will probably not unsubscribe.
Cesar D. Fermin, Ph.D Tulane Medical School Pathology/SL79 New Orleans, La 70112
Hi, I have not used Image, but have tried to use another image analyser to measure the number of intercepts on a line and the intercept lengths in the past. The general procedure was as follows: 1) Use grey level to differentiate your grains from the grain boundaries and create a bit map of the grains. 2) Decrease your active measurement field until you have only a single rastor line across the screen. 3) If only the "detected" portion of your image is overlayed with a colored "bitmap", then you should have a colored broken line across the screen at this point. 4) Count the objects detected to give you a measure the number of intercepts. 5) Take the maximum dimension of the objects detected to give you a measure of the intercept length. 6) By rotating the image and repeating the procedure, information may be collected in relation to variation with orientation.
Hope this is of assistance. ______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
I am trying to locate an e-mail address for Dr. Mark E. Welland -at- Cambridge University, Engineering Dept., St. John's College. Last I knew, he was working in Scanning, Tunneling Microscopy. If anyone knows of his address (or, if you're on this list, Mark) please respond directly to my e-mail address. Thanks very much!
Susan K. Smith National Steel Corp. smiths-at-mlc.lib.mi.us
Hi, Sorry to broadcast this, but I deleted the message before I realized I might have an answer. A request was made about a source for a OM-3 motor. I checked with a company I buy equiment from and was told they have several on hand. The company is Marivac, Ltd., 5821 Russell St., Halifax, Nova Scotia, B3K 1X5 Canada. 800-565-5821, FAX 902-455-4007. I have purchased equipment from these folks (Balzers MED 010) and have a service contract with them for my Ultracut. The person I deal with is Chris Cathcart; he operates out of Toronto (416-495-0389), but can be reached via the 800 number. I've been pleased with the quality of the service I get. I have no connection with this company other than as a satisfied customer, standard disclaimers apply, blah, blah, blah. Good luck!
Dwight U. Beebe beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
Anybody have a good fixative for cross-linking polysaccahridases? I'm trying to do SEM on a "gliding" bacteria strain and find my bacteria being lifted off the agar. I've tried vapor fixation using osmium and then fixing overnight in 2% glut. One colony strain will lift off the agar surface and the other will remain. Any Suggestions?
I have used 50 X 9 mm culture dishes with friction fit lids manufactured by Simport Plastics, Beloeil, Quebec, Canada (514) 464- 1723. The catalogue number was D210-14.
I would be interested in a source of smaller diameter dishes with a larger internal clearance for storing my metallographic samples.
Hope this is of assistance. ______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
Falcon 35x10mm dishes (available through Fischer) work for embedding cultured cells.
Another possibilty is to grow the cells on culture inserts (available from Millipore, Falcon, and other companies) which are placed into 24 well plates. After fixation (or post-fixation if you prefer), the filter on the bottom of the insert can be removed with a razor blade, and the filters dehydrated with ethanol and embedded. This uses even less reagents that the 35x10mm dishes, and the 24 well plate is easier to manipulate than several round dishes. I've used this technique for culturing fetal kidneys and for endothelial cells.
Hello, Our laboratory is in the process of investigating ion mills. It appears that Baltec, Fischione and Gatan are the principle manufacturers of this piece of equipment. Any comments, suggestions, experiences or thoughts about ion mills would be appreciated. Thanks, John
In answer to Phil Rutledge's query about fixation of "polysaccahridases": The closest thing I know to a fixative for polysaccharides is Alcian Blue. This works reasonably well in most cases where bacterial capsules need to be insolubilised. Try the following refs: Tuffery - 1969, J gen Microbiol 57:41-50 Goldberg & Septier - 1986, Anat Record 216:181-190 Scott - 1972, Histochemie 32:191- (Primary reference) Trachtenberg - 1986, J Ultrastruct Mol Res 97:80-102 Steedman - 1950, Quart. J Microsc Sci 91:477-479 Powell et al - 1992, J Fish Biol 41:813-824 (useful ref) Dellorbo et al - 1992, J Electr Microsc 41:475-479.
Hope this is of some use.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Message-Id: {9404111414.AA06580-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: ion mills Orig-Author: {PHELPS-at-ENH.NIST.GOV}:ddn:wpafb ----------------------------------------------------------- Hello, Our laboratory is in the process of investigating ion mills. It appears that Baltec, Fischione and Gatan are the principle manufacturers of this piece of equipment. Any comments, suggestions, experiences or thoughts about ion mills would be appreciated. Thanks, John
Phil Rutledge writes: } Anybody have a good fixative for cross-linking polysaccahridases? I'm } trying to do SEM on a "gliding" bacteria strain and find my bacteria being } lifted off the agar. I've tried vapor fixation using osmium and then } fixing overnight in 2% glut. One colony strain will lift off the agar } surface and the other will remain. Any Suggestions? } Phil,
Here is a well worn ruthenium red staining method for mucopolysaccharides from M.A. Hayat: "Positive Staining for Electron Microscopy", p. 169-170. Whether this will fix polsaccahridASES, or prevent them from sliding off the agar, I cannot say.
A few preliminary notes from Hayat: 1. Maximum contrast obtained when ruthenium red and osmium tetroxide applied together(refering to TEM prep here). 2. Stain can be applied by adding it to fixative solution, water, or buffer solution. 3. Phosphate buffers are not recommended for ruthenium red-osmium tetroxide procedures. Chloride-free cacodylate buffer considered most ideal one to use.
For general purposes, the following method of fixation and staining for acid mucopolysaccharides recommended:
Solutions: A. Aqueous glutaraldehyde (4%) 5 ml 0.2 M Cacodylate buffer (pH 7.3) 5 ml Ruthenium red stock solution 5 ml (1,500 ppm in water)
B. Aqueous osmium tetroxide (5%) 5 ml 0.2 M Cacodylate buffer (pH 7.3) 5 ml Ruthenium red stock solution 5 ml (1,500 ppm in water)
Mix solution B just prior to use. 1,500 ppm is 30 mg ruthenium red in 20 ml water. Fix and stain samples in solution A for 1 hr. at room temperature. Rinse briefly with buffer, then post-fix and stain with solution B for 3 hr at room temperature. Rinse briefly with buffer, then dehydrate and embedd, or critical point dry(in your case) according to standard procedures.
Ruthenium red stain is available from most EM or chemical suppliers.
The above is sort of the mother of all ruthenium staining techniques for EM. Others have since modified its use and here are more recent references:
1. B. Giammara and J. Hanker, Ruthenium Red-osmium bridging with TCH: New techniques to stain biological specimens for light and TEM and to coat them for SEM, Proceedings of the 46th Annual Meeting of the Electron Microscopy Society of America, 1988, pp20-21.
2. M. Jacques and L. Graham, Improved preservation of bacterial capsule for electron microscopy, Journal of Electron Microscopoy Technique 11:167-169 (1989). This paper compares ruthenium-glutaraldehyde staining to glutaraldehyde-amine, which they found to be superior.
The following papers are on non-ruthenium red methods, and involve TCH (thiocarbohydrazide) and tannic acid procedures:
3. R.O. Kelley, R. Dekker and J. Bluemink, Ligand-mediated osmium binding: Its application in coating biological specimens for SEM, J. Ultrastructural Research, 45:254-258 (1973). (The classic reference in applying TCH for SEM)
4. J. Murphy, Non-coating techniques to render biological specimens conductive/1980 update, Scanning Electron Microscopy/1980/I, pp209-220.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
This Master Class is intended for novices, experienced specimen preparers and scientists involved in the examination of transmission electron microscopy specimens. The course will present the latest materials, tools, and methods for preparing high-quality specimens.
Description
While most aspects of specimen preparation will be covered, emphasis will be placed on preparing cross section specimens of complex composite samples, such as those found in the semiconductor industry; mechanical polishing to transparency; and ion milling. The course will review a variety of modern physical science specimen preparation methods, discussing the advantages, disadvantages, and limitations of each. Experience has shown that a wide range of bulk specimen configurations may be successfully prepared. Discussion will include the preparation of metals, ceramics, thin films and coatings, powders, wires, and difficult (small volume, radioactive, etc.) specimens. Laboratory demonstrations are planned.
Instructors
Ron Anderson; Senior Physicist at the IBM East Fishkill Facility. Vince Carlino; President of VCR Group. George Lane; Materials Product Manager for Bal-Tec, Inc. Jo Ellen Tyson; Applications Specialist for Mager Scientific.
Registration and Attendance
To register, call or e mail Kim Knudtson at 612 626 7594 (kimberly-at-maroon.tc.umn.edu). Enrollment is limited; please respond early to guarantee a space. Disability accommodations will be provided upon request. This publication and material is available in alternative formats upon request.
Tuition
The fee will cover course materials, refreshments, and box lunches. For members of the University community, the charge is $70; for all others, the fee will be $250. University of Minnesota attendees should provide a CUFS number, others should provide information so that they may be billed after the course.
Program Sponsor
The Center for Interfacial Engineering (CIE) is an NSF Engineering Research Center supported by the NSF, the University of Minnesota, and the corporate members of CIE. The CIE Characterization Facility and High-Resolution Microscopy Center provide instrumentation and personnel to facilitate the physical and chemical study of interfaces. These facilities are available to all members of the University community as well as external users. Tours of the labs will be available to interested attendees.
Accommodations
The University of Minnesota is located 10 miles from the Minneapolis-St. Paul International Airport. The Radisson Metrodome Hotel is conveniently located within two blocks of the lecture and laboratory sessions. The telephone number is 612-379-8888. A Days Inn is a 20-minute walk from the University; the telephone number there is 612 623 3999.
Schedule
Day 1, April 28
Registration (8:00 am), 210 Amundson Laboratories held in the High-Resolution Microscopy Center, Shepherd Laboratories
Lectures: Ron Anderson ¥ Strategic plan Initial processing from bulk samples Sawing and grinding, dimpling ¥ Final processing the thin specimen Ion milling/FIB methods Mechanical microthinning
Vince Carlino ¥ Specimen Prep by Dimpling and Ion Milling
George Lane ¥ High Performance Ion Beam Thinning of Solid State Materials
Day 2, April 29
Lectures: Ron Anderson ¥ Other Methods: Microtomy, Cleavage, Replication ¥ Cross-sectional specimen preparation The IBM East Fishkill Method
Demonstrations
¥ Ron Anderson: Tripod polisher
¥ Vince Carlino: VCR Dimpler Ion Beam Sputterer Model IBS/TM200S Ion Milling System
¥ George Lane: RES 010 Rapid Etching System MED 020 Modular High Vacuum Coating System
¥ Jo Ellen Tyson: Reichert Ultracut S/FCS cryo-ultramicrotome Attendees may bring their own samples.
Stuart McKernan stuartm-at-maroon.tc.umn.edu Chemical Engineering and Materials Science OR High Resolution Microscopy Center University of Minnesota, Minneapolis, MN 55455
we charge based on what it costs us: embed.....$6 thick sections (glass slide)....$16 Thin sections........$60 TEM time.............$36/hr TEM negative..........0.65 8X10 print............4.10 Publication quality print.....$5.50 Technician time if needed (microscope operation).......$25/hr
equipment costs included depreciation and service contract
hope this is helpful
On Mon, 11 Apr 1994, Tamara Howard wrote:
} A while back someone asked about the standard charges for a routine TEM } workup on a biological sample...I don't recall ever seeing any answers to that, } and now I am looking for similar information. What is considered to be a } reasonable charge for say one sample, plain old fix, embed, a few grids, and } maybe 2 hours max scope time plus prints? At an academic level, that is - I kno0w } clinical charges can be astronomical. } You can answer by e-mail to tah-at-med.pitt.edu if you do not want to } go through the usegroup on this. } I would really appreciate some help on this one! } Tamara Howard } U. Pittsburgh School of Medicine } Pittsburgh, PA }
I haven't had the chance to actually do it, but according to info. I received at the Bio-Rad Confocal course last month, it should be possible. The relative brightness of Cy3 may mean titrating its molar ratio relative to Cy5 and/or careful selection of filter blocks and PMT filters to prevent Cy3's long trailing slope from comming through the Cy5 signal. Take a look at Brelje et al, Chapter 4, Methods in Cell Biology, vol. 38, edited by Brian Matsumoto.
On Mon, 11 Apr 1994, Michael Cammer wrote:
} If you have a Kr/Ar laser, you can triple label with FITC, Cy3, and Cy5. } } }
} ratio relative to Cy5 and/or careful selection of filter blocks and PMT } filters to prevent Cy3's long trailing slope from comming through the Cy5 } On Mon, 11 Apr 1994, Michael Cammer wrote: } } If you have a Kr/Ar laser, you can triple label with FITC, Cy3, and Cy5.
We have not had a problem with Cy3 being imaged in the Cy5 channel (we image Cy5 only while exciting with the red laser line). However, we have had problems when imaging bright propidium iodide or Texas red with the standard long pass rhodamine filter block; some Cy5 appears to be excited.
We have generous institutional support and I am told that our charges are quite low. We do not have to recover any salary and about half our operational cost are from institutional support.
THESE ARE OUR IN HOUSE RATES. For outside clients I, at least triple the rates
The following charges are in effect at the EM Core .
Standard TEM or SEM sample $65.
Additional similar samples sub- mitted at the same time $55.
Immunolabeling or other cyto- chemical reactions per sample $75.
Negative stain per sample $35.
Partial preparation is also available with price appropriate to our input for example:
Fix and embed only $20/sample
Thin section only $20/block
Use of the microscopes by qualified users is $20 per hour and $1 per photograph. If you want Polaroid you must also supply the film.
For the benefit of the curious Materials Scientist like me, would someone please post a description (short) of what CY3/CY5 is and what it is used for.
I have been having problems with Quetol 651 resin for the past year, after using it successfully in my lab for 2 years. Has anyone else experienced difficulties? Of what sort? I'd like to correspond with someone with a background/knowledge of resin che mistry to possibly diagnose the defect.
Thanks to everyone who gave suggestions to the problem I am having with the fixation of the bacteria and having the cells float off of the agar. Now I'll have to sort through the suggestions and see what works best. Again, thanx to all, Phil Rutledge, Director Center for EM UMBC
Greetings, We just received 176 grams (ampules) of osmium tetroxide. The ampules, we are told, were bought some time back in the 1960's from Fisher, Englehard (in individual wooden cases!), National Lead, and MCB. The integrity of the ampules seems good as evidenced by the translucent greenish color of the osmium. However, the osmium seems to have coalesced into a single mass. Does anyone have any comments, warnings or advice? Considering that this gift is worth over $6K, I'd like to be able to use it.
************************************************* * * * * * Charles J. Butterick * * Electron Microscopy Center * * Department of Cell Biology and Anatomy * * Texas Tech University Health Sciences Center * * Lubbock, Texas 79430 * * USA * * Phone 806 743-2706 voice * * 806 743-2707 fax * * Email hecub-at-ttacs.ttu.edu * * * * * *************************************************
SOMEONE WROTE: In message {199404121522.IAA18262-at-ucsd.edu} writes: } I have been having problems with Quetol 651 resin for the past year, after } using it successfully in my lab for 2 years. Has anyone else experienced } difficulties? Of what sort? I'd like to correspond with someone with a } background/knowledge of resin che } mistry to possibly diagnose the defect. } } Hello,
We've used Quetol for years without trouble. You do not mention what problems you are having so I have no idea what to tell you. Put out another message on the MICROSCOPOY net describing what used to work well for you and what kind of trouble you are having now. That way I think people with experience will be able to zero in on what might work for you, and I will reply then. Thanks.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Greetings, We just received 176 grams (ampules) of osmium tetroxide. The ampules, we are told, were bought some time back in the 1960's from Fisher, Englehard (in individual wooden cases!), National Lead, and MCB. The integrity of the ampules seems good as evidenced by the translucent greenish color of the osmium. However, the osmium seems to have coalesced into a single mass in each of the ampules. Does anyone have any comments, warnings or advice? Considering that this gift is worth over $6K, I'd like to be able to use it.
************************************************* * * * * * Charles J. Butterick * * Electron Microscopy Center * * Department of Cell Biology and Anatomy * * Texas Tech University Health Sciences Center * * Lubbock, Texas 79430 * * USA * * Phone 806 743-2706 voice * * 806 743-2707 fax * * Email hecub-at-ttacs.ttu.edu * * * * * *************************************************
Message-ID: {MAILQUEUE-101.940412174341.320-at-bmg.bhs.uab.edu} To: Microscopy-at-anlemc.msd.anl.gov
Does anyone know if there is a User's Group for the Macintosh-based image analysis software IP-LabSpectrum from Signal Analytics Corp.? I would be interested in linking up with users of that particular software platform to exchange ideas, information, scripts ect.
Thanks Kevin McCarthy Assistant Professor Department of Cell Biology University of Alabama at Birmingham Birmingham, Alabama 35294 Phone 205-934-9923/9924 Fax 205-934-7029
I notice from the literature that people setting up microscope-based ion measurement systems to quantitfy Na, Ca or H by fluorescence ratio microfluorimetry usually use a Xenon lamp for excitation of the fluoprophores. We are putting together a system for measuring Ca using Indo on an Olympus IMT-2 but lack a fluorescence source. The mercury lamp is half the price of the xenon, and certainly excites at the wavelengths we require (Indo exc. at 365 =B1 10nm), so we are thinking of going that way rather than the very expensive Xe.
Is the main advatage of Xe then the more constant output over the whole UV range so that dual excitation is more reliable (both excitation intensities the same, whereas with mercury one may fall over a peak and the other not) ? I'd appreciate comments from anyone who has experience in this area. Thanks, Ian Harper iharper-at-eagle.mrc.ac.za
I notice from the literature that people setting up microscope-based ion measurement systems to quantitfy Na, Ca or H by fluorescence ratio microfluorimetry usually use a Xenon lamp for excitation of the fluoprophores. We are putting together a system for measuring Ca using Indo on an Olympus IMT-2, but still lack a fluorescence source. The mercury lamp (100W) is half the price of the xenon, and certainly excites at the wavelengths we require (Indo exc. at 365+/- 10nm), so we are thinking of going that way rather than the very expensive Xe lamp.
Is the main advatage of Xe then the more constant output over the whole UV range so that dual excitation is more reliable (both excitation intensities the same, whereas with mercury one may exc wavelength may fall over/near one of the mercury peaks, whereas the other may not) ? I'd appreciate comments from anyone who has experience in this area. Thanks, Ian Harper iharper-at-eagle.mrc.ac.za
Message-Id: {MAILQUEUE-101.940413083221.320-at-parmly1.parmly.luc.edu} To: microscopy-at-anlemc.msd.anl.gov
} In answer to Phil Rutledge's query about fixation of "polysaccahridases": } The closest thing I know to a fixative for polysaccharides is Alcian Blue. } Jan Coetzee } Ruthenium red is used to demonstrate polysaccarhide coats of cyanobacteria. I don't have a reference to hand, but I believe a receipe is in Dykstra's & Bozzola & Russell's books? Phil Oshel
Hi Ian Mercury sources tend to be much less stable, and are not white enough to provide both wavelengths at comparable intensities for many dual excitation combinations. They can be used for some dual emmission work. We use the same instrument you are considering, and have added the OSP-3 illuminator and photmeter system for rapid spot measurements of ion [] and pH. The system works well, exposes cells to minimal photo-oxidizing irradiation during the measurements, and can be driven from pc-based software. Let me know if I can help
Message-Id: {199404122000543157-at-qmgate.secrc.abb.se} X-Mailer: InterCon Dispatcher/SMTP for QuickMail X-Priority: 4
Looking for someone that has been working with oxidation, hydriding and wear properties of surface coated or surface modified zirconium base alloys (like Zircaloy 2 or 4). The environment is water or steam at ]300!C. I am interested in all topics related to the subject; coating or surface modification methods ( how to get a coating without defects going through the coating?), corrosion tests, investigation of tested samples (LOM, SEM, TEM, SAM, XRD, electrochemical impedance spectroscopy.
Bengt Stridh ABB Corporate Research S-721 78 Vasteras Sweden E-mail: best-at-secrc.abb.se Fax: +46-21-13 41 00 Phone: +46-21-32 30 67
REGARDING Cerius Software Vendor According to a brochure picked up at the latest MRS meeting, the Cerius software is sold through: Molecular Simulations, 16 New England Executive Park, Burlington, MA 01803 ph: (617)-229-9800.
In reply to Charles Butterick's question about old osmium - try calling a vendor of osmium to ask their "proffesional" advice, such as EMS, or Stevens Metallurgical corp.
} Does anyone maintain a list of software suppliers for TEM } applications? I am interested in programs such as MacTempas, } Crystalkit, Desktop Microscopist, etc. Addresses, phone numbers, ftp } site information would all be very useful.
MacTempas and Crystalkit Total Resolution 20 Florida Street Berkeley, CA 94707 Attn: Roar Kilaas 510-527-9100 Fax 510-527-9151
Desktop microscopist Virtual Laboratories 37 Highland Court Ukiah, CA 95482 Attn: Eric Schlienger 707-462-8037
Crisp and ELD Calidris Manhemsvagen 4 S-191 45 Sollentuna Sweden Attn: Sven Hovmoller 46 8 625 00 41
Prism Signal Analytics 440 Maple Ave East, Suite 201 Vienna, VA 22180 Attn: Brian Culhane 703-281-2509
DigitalMicrograph Gatan Inc. 6678 Owens Drive Pleasanton, CA 94588 Attn: Sheri Kurland 510-463-0200
NCEMSS and Interface Tool Telnet from ORAC.LBL.GOV
TCBED from ASU
Image from MSA EMMPDL
That's my list of software sources.
Roseann Csencsits Electron Microscopy Center Argonne National Lab
} In reply to Charles Butterick's question about old osmium -- What exactly could go wrong with a sealed vial of the metal? If the vial seal had been breached, wouldn't the osmium vaporize? I know em makes a person really paranoid about materials, but is there any other reason not to use the stuff?
Tobias Baskin * TEMPORARILY C/O Peter Hepler * Biology Department University of Massachussetts * Amherst * Mass 01003 Tel: 413 - 545 - 2698 FAX 413 - 545 - 3243
In message Charles J. Butterick writes: } Greetings, } We just received 176 grams (ampules) of osmium tetroxide. The } ampules, we are told, were bought some time back in the 1960's from Fisher, } Englehard (in individual wooden cases!), National Lead, and MCB. The } integrity of the ampules seems good as evidenced by the translucent } greenish color of the osmium. However, the osmium seems to have coalesced } into a single mass. } Does anyone have any comments, warnings or advice? Considering that } this gift is worth over $6K, I'd like to be able to use it. } Charles,
We have heard your plea for help and are delighted to be able to respond with our assistance. In our lab, we have over 25 years experience of working with osmium tetroxide and biological systems. Just send us about 25 ampoules(grams), we will test the lot against biological systems we are currently working with, and report the results back to you.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Sounds like the osmium has melted at some time and then reformed. After quizzing another EM veteran here, learned that such ampules should still be fine as long as they show that bright green color. Avoid using any vials showing black flecks in the coalesced osmium, as that's a sign of degradation. Similarly, when put into solution, good vials will give a bright yellow-green solution as usual; vials with black specks will probably give solutions with no color or the off-gray color of an old solution.
David Hall Dept. Neuroscience Albert Einstein Col. Med. Bronx, NY
We consider to purchase sample cutters for bulk materials and 3mm slices to prepare transverse TEM samples for HREM. I appreciate for your advice. Welcome companies to send us the price.
Sandy Burany Physics Department Simon Fraser University Burnaby, B.C. V5A 1S6 (604)291-4082 Fax (604) 291-3592 Email burany-at-sfu.ca
Message-Id: {MAILQUEUE-101.940414095946.705-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
We are attempting to demonstrate peroxidase/myeloperoxidase activity in neutrophils in cytospin preparations by using DAB. We find it works well on fresh cytospins, but if cytospins are left for a few days we get no reaction. Any suggestions as to how best to maintain enzyme activity in stored cytospins? Mark Elliott
Re} Peroxidase & Cytospins Why not try fixing the cytospins with aldehyde to retain the peroxidase activity? Neither formaldehyde of gluteraldehyde will affect the enzyme activity but may inhibit proteolysis. The aldehydes will work best on cells that have not been allowed to dry out and will only continue to work if the cells remain hydrated. Leave them in buffer or, if formaldehyde-fixed, in formaldehyde. Glutaraldehyde fixation is only useful if you are using DAB. It will not be useful for fluorescent studies unless the autofluorescence is quenched with sodium borohydride.
Does anyone have experience with a good commercially available Monoclonal or polyclonal antibody against collagen I. Our experiments are performed with rabbits so a polyclonal made in rabbit is useless, or should be conjugated to something (biotin, dig.) Thanks in advance.
with likely the addition of a Hitachi in the next year. each is "Best" for us for what it does and the application for which it is used for.
Let's see what everyone has to say on this one. It should be interesting. I'll just sit back and "listen" for abit.
Remember try to be objective if you are going to respond. This is intentionally not a moderated list and I want to keep it that way.
I'm sure that the EM manufacturers who are also subscribers to this list will be interested in the responses. In that regard, I'll ask them (i.e. any manufacturers) not to respond to this question. If I see something which is not a fair discussion or an objective reply. I will cut the discussion off. If any manufacturer wants to complain please address all mail to me personnally and I will formulate any appropriate notice(s) to the listserver....
Nestor J. Zaluzec ANL EMCenter zaluzec-at-anlemc.msd.anl.gov
For reliability I have been most happy with my Hitachi H-7000 and S-4000. They serve about 99% of the needs we have in a multi-user facility, for mostly biological EM. There are a few times when I wish I had something else for very speialized work, but on the who;e these serve us well. My suspicion is that one could say the same for most of the major manufacturerers' products on the market today.
I agree with Nestor, the best microscope is dependent upon what you wish to do. Even in cameras, the Nikon is not ideal for all applications. However, to join in the foray: We love our Philips 400, 515, and CM-30s.
On Fri, 15 Apr 1994, Larry Hawkey wrote:
} } I have tried to send this message twice but is keeps getting bounced back } to me. } } Some one is asking me about my recommendations on which EM to } buy. So my question to all of you is this: } } Is there in the EM community a sense that there is one } Electron Microscope that is the best that money can buy? } } If some one was to ask about 35mm cameras for instance. I } think that the general consensus is that Nikon is ahead of } the pack. Minolta and Cannon are good and may be a good deal } for the price but if money is not the first concern Nikon is } the first pick. } } I use the JEOL 1200EX II. It is great. I have also used } several of the 100 incarnations. I used the Ziess EM 10 } about ten years ago but I have really only used JEOL for the } past ten years. In addition there seems to be a heavy } concentration of JEOLS in the Duke University community so I } feel that my impression that Ziess is nice but JEOL is best } may only reflect my small world. } } Try to put your biases aside and send me your thoughts. } } Larry Hawkey } Hawkey-at-neuro.duke.edu }
Some one is asking me about my recommendations on which EM to buy. So my question to all of you is this:
Is there in the EM community a sense that there is one Electron Microscope that is the best that money can buy...
Larry Hawkey Hawkey-at-neuro.duke.edu
I will start by saying I am not a TEM user and my answer will support this. I am a microprobe operator and an SEM operator. However, I have supervisory responsibility for support and operation of a lab that has a JEOL 100CX STEM, a JEOL 4000FX 400kV STEM, a Philips EM301 TEM in addition to 2 JEOL 35C SEM's, a JEOL 6400 SEM and a JEOL JXA-8600 EMPA so I am not insensitive to the needs of a TEM user. When we bought our most recent STEM, the 4000FX, we came down to a choice between Philips and JEOL. When we bought our most recent SEM, the 6400, we chose between Philips, CamScan, and JEOL. I would advise any one that in buying a piece of EM equipment there are three considerations: Performance, Cost, and Service. Of the three, cost is the lowest priority. Obviously, if you have limited funds, cost is a consideration up front (i.e. you can't get something for nothing). Once you purchase, what you paid is meaningless. The two remaining criteria will be with you for the life of the instrument. I will let the rest of the EM community steer you on the performance subject but I want to stress that efficent, prompt, compitent service is in my opion as important as performance. The best instrument, when inoperable, is not very useful. Waiting weeks for service is not pleasant, especially when deadlines come and go. Service personel that are not equipped or trained can lead to frustration. Personally, I will sacrifice a liitle performance if the competitor provides better service. Admitedly, I can do this because I do little work that "pushes the envelope". Mosty of my work is routine and not instrument limited. If you are beating back the frontiers of the science, then weigh performance heavily. But always keep in mind that the instrument must be well maintained if you expect to achieve optimum performance on demand. This takes good service.
Message-Id: {9404151852.AA01866-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: Jay Jerome {jjerome-at-isnet.is.wfu.edu} Cc: Larry Hawkey {hawkey-at-neuro.duke.edu} , Microscopy-at-anlemc.msd.anl.gov, dennis-at-med.umich.edu {Pine.3.89.9404151432.A9297-0100000-at-isnet.is.wfu.edu} "Larry Hawkey" {hawkey-at-neuro.duke.edu} , microscopy-at-anlemc.msd.anl.gov
Reply to: RE} } Who makes the best TEM? Just a note of clarification as to what the person who is looking for a TEM is going to use it for;
The person I am talking to is only interested in Routine Biological TEM, looking at ultra-structure (yes that is still done). Things like counting synapses or looking to see if the Mitochondria are screwed up. Also by best I am interested in compairing The preception that one company just carries a slightly but consistantly better line of EMs.
--------------------------------------
The problem with your question "Who makes the best TEM" is that you haven't specified what you wany to use it for. The best microscope for ultra-high resolution imaging is not necessarily the best for general teaching of students or the best for high performance microanalysis. You need also to factor in the service available. For example, how many engineers does a company have in your area, how many microscopes like the one you are thinking of buying does each engineer have responsibility for (put another way, how much experience does the engineer have on your instrument - a person can be very good, but if yours is the only TEM in his area he will have only limited experience with it). What kind of microscopy do you need to do - fast turn-round screening of biological samples or extensive analysis of materials samples, and so forth. There are good and bad points about all the major manufacturers, and probably each instrument from each maker is the best in its own niche. The analogy with the 35mm camera is a little over simplified.
On Fri, 15 Apr 1994, Nestor J. Zaluzec-ANL EMCenter wrote:
} } Talk about a loaded question. Which microscope is best? } } To state the obvious which I'm sure will be repeated at least } twice or three times in the repies to this question. } } ***It depends on what your trying to do!*** } } CTEM, STEM, AEM, CBED, XEDS, EELS, AES..... } and the list continues, choose your poison. } } At the ANL EMCenter we have: } } JEOL (100cx & 4000EXII) } Philips (EM420T & CM30T) } AEI (EM-7) } VG (603Z) } } with likely the addition of a Hitachi in the next year. } each is "Best" for us for what it does and the application } for which it is used for. } } Let's see what everyone has to say on this one. It should } be interesting. I'll just sit back and "listen" for abit. }
Well, I'm close to retirement so I'll stick my neck out. For biological applications I have used RCA, Siemens, Hitachi, Jeol, Zeiss, and Philips. For ease of use, quality of construction, resolution, and reliability I'll go with the Philips. My work has always been conventional biological TEM.
No, we have not tried cutting the fibers in to disks and perfroming a 3-D reconstruction. I have not heard of this previously applied to composite microstructures, have you? Can you tell me more about this methodology? Can you point me to specific papers in the literature? Is this similar to the type of 3-D reconstructions we read about being used in medical imaging? If so don't I need some features in the image to key off of?
Thanks for your help,
P. Joyce
Dear Peter,
If you consider biological systems to be composite microstructures, then, yes we have a lot of experience with this technique. As applied to your carbon-fiber system, there are several things to consider: 1) Is there enough contrast between the fibers and the matrix? 2) If not, is there a way to add contrast, such as by infiltrating the matrix? 3) Is the thickness of each slice limited by transmissivity or by overlap of features--in the HVEM a spec- imen of several microns can be penetrated, but after about one micron, typical biological specimens have so many features that these can no longer be untang- led? Another possibility if there is low contrast is to find an element in the matrix which is not found in the carbon, and to do element mapping--this is in- herently much lower resolution than imaging--or energy-filtered imaging looking at an edge or the low-loss region. In any event, I presume you have settled on appropriate conditions and obtained images which have then been scanned and converted into digital files. If the sections were thin enough so that the carbon fiber position and orienta- tion do not change appreciably from section to section, you can just stack the images from successive sections and process the resulting file as if it arose from a confocal microscope series or some such. There are many image proces- sing packages which will allow you to view the image volume from various angles with various features highlighted--you do have to tell the program how to iden- tify the features, but at worst, you can do this pixel-by-pixel if necessary. We use SEMPER on our confocal microscope to do this, and some in-house programs in combination with, I believe, MOVIE.BYU to do this for electron micrographs. If the sections can be thicker--e.g. if there are rather few carbon fibers distributed in a non-carbon-containing matrix and if energy-filtered imaging sees the carbon as dark while the matrix is transparent--you should either take stereo pairs and use a pointer to outline the features of interest within the section volume while viewing the stereo pairs, or take a large ser- ies of images at various tilts and do tomographic reconstruction. This may be more work than thin sections, but it has the advantage that features in the middles of sections are not perturbed by the cutting process--which can be a problem with thin sections. For reconstruction using stereo pairs, we use an in-house program, STERECON, and for tomagraphy, we use SPIDER, also an in-house program. SPIDER does reconstructions very much like medical imaging using either Fourier tech- niques, back-projection or projection onto convex sets. Fourier techniques are an old tried-and-true (mostly) method which allows filtration to smooth out noise, and which is well-understood, but which also gives artefacts due to the inevitable missing wedge of information. Back-projection just models the three dimensional object which would give rise to the observed projected views. Fin- ally, projection onto convex sets is a recent method incorporating constraints which, among other things, is used for de-blurring photographs. I don't under- stand this method well enough to try to explain it. Dr. Joachim Frank at our lab is in charge of the image processing end of things, Drs. Bruce McEwen and Michael Radermacher have experience with both programming and using SPIDER, Mr Mike Marko is our resident expert on STERECON and Mr. Ardean Lieth knows about all the programs. A letter (or e-letter) to any of these people should get you more information, and would be better than reading the papers cited for using these programs. I'm sure there are very good write-ups of serial-section techniques, but I am not familiar enough with the literature to reccommend any in particular. All of the programs developed here are available (I'm told the price is reasonable). Dr. Frank is the correct person to tell you about this aspect. I have probably told you more than you wanted to know, but I'd be happy to try to answer any other questions I can. The address for any of the people mentioned is Wadsworth Center for Labs. & Research Empire State Plaza, P.O. Box 509 Albany NY 12201-0509 and the e-mail addresses are username-at-tethys.ph.albany.edu, however, I don't know every one's usernames--try lastname, initial+lastname or initials as gues- ses. Good luck.
Your 'old' osmium should be ok! Concerning the fact that it coalesced into a mass on the bottom of the tube, this is not important: I remember a recipe (by S.Palay) recommending heating the tube (in a water bath) in order to collect all the osmium tetroxide in a single mass at one end of the tube. It worked ok (by the way, there is no point in doing so, if you carefully wash the outside of the ampulla before breaking it ;-)) Cheers! John
*********************************************************** * Jean C. Desclin (John), Associate Prof. of Histology * * Laboratory of Histology - Faculty of Medicine * * Brussels Free University (U.L.B.) * * e-mail: desclinj-at-ulb.ac.be (internet) * * snail mail: route de Lennik 808 * * B - 1070 Brussels Belgium * ***********************************************************
Reply_ RE} } General: Which Scope is Best? Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu IMHO (In My Humble Opinion):
For quality of construction and reliability I would say Philips. In addition they have always been the best scope for CBED.
The one major thing that I dont like about ALL of the newer microscopes is the manufacturer's insistance in making the tilting and translation functions motorized. With these instruments you have no "feel" for the specimen position or tilt and when performing diffraction experiments I like to be able to "tweak" the tilts and sample position manually.
Our lab currently has 2 JEOLs, a 200cx and a 6100. Both instruments are under service contracts with JEOL, and I cannot imagine life otherwise. The contracts may seem expensive at first glance, but when a problem arises the service technicians have always fixed them much more quickley than we could have. The routine maintenance provided by the contracts also helps keep the instruments in good working order even with multiple users. If you have the option, life is much better witha service contract from the manufacturer.
John Phelps NIST, Boulder PHELPS-at-ENH.NIST.GOV
P.S. I promise that I wasn't paid by the service people for the response.
At Argonne National Laboratory, we have maintenance contracts on all of our TEM's, including an old JEOL 100CXII. We simply don't have the time to devote to repair work while the field service techs have the resources to take care of most of the problems that arise. We do some of the repairs ourselves, if they can be done quickly. For instance, we don't ask for help in changing filaments or in fixing a camera if the film gets jammed. We know our limits. One recent repair on our Philips EM420 took a week, and we could not have done it ourselves. Since we have a large base of users, 4 TEMs, 1 STEM, and only 3 staff members, time is very valuable to us.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, IL 60439, USA
Dr. Bogers I talked with our ECM expert for advice on ab's and he suggested a company in Germany called HEYL Goerzallee 253, D-1000, Berlin 37 phone 030-8-17-60 52 FAX 030 8 17 40 49
He also mentionned a company called Collagen Corporation in Palo Alto, California USA, and one in Birmingham Alabama USA which also deals specifically with collagen antibodies. There is also a Developmental Biology Hybridoma Bank in the US which deals with ECM antibodies. He said that any of these may be able to help you and should be fairly good.
IMHO I would not suggest buying a JEOL 100B. This is the microscope that I never could qualify on at ASU, it took a lot of maintenance, and is 20 years obsolete. :)
I would also like to point out that if you want to unsubscribe you must send the message to the listserver, not the list:
listserver-at-anlemc.msd.anl.gov
We should all keep the information that was sent us when we first logged onto this list so that we know how to log off without annoying 1200 listmembers .
regards Mark W. Lund, PhD Director MOXTEK, Inc. Orem UT
I know we are a special case--we have a 1.2 MV HVEM from AEI--but we get along very well without a service contract (} 80% uptime). Our secret is that no ser- vice contracts being available on our beastie, we have had to learn to do it all ourselves. The service people in-house are VERY good--a must. Since we do a lot of development and improvement, we need a person who knows the machine very well, and those people also can service the microscope. If you have a fac-ility which just uses the microscope--as opposed to redesigning it--it is prob- ably just as well to have a service contract, but if you need a machine person or two on hand anyway, you can get along without a contract.
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I am sending this again because I am not sure if it was received properly the first time.
Dr. Bogers: I talked with our ECM expert for advice on ab's and he suggested the following companies as good sources. HEYL, Goerzallee 253, D-1000, Berlin 37, Germany Phone 030 8 17 60 52 FAX 030 8 17 40 49
also, a company called Collagen Corporation in Palo Alto California, USA and one in Birmingham, Alabama USA which also deals specifically with collagen antibodies. There is also a Developmental Biology Hybridoma Bank in the US which deals with ECM antibodies . He said that any of these may be able to help you and should be fairly good.
Message-Id: {1994Apr18.101055.761680489-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy list)
Dear fellow microscopists, At San Joaquin Delta College, Dept. of Microscopy, Stockton, CA we have an intensive 2 yr hands-on program to train microscopists. We issue certificates in both biological and materials area. We will be graduating 17 students from the program May 28, 1994. If you need an entry level person with 2 yrs intensive experience in TEM, SEM, EDS, OLM, all preps, routine maintenance, report writing, etc. etc. please contact Judy Murphy (murphy-at-ms.sjdccd.cc.ca.us, phone 209/474-5284; fax 209/474-5649) If you want more info about the program or a copy of the Delta Microscopy Newsletter, also contact as above. Our program has been in existence for 24 yrs. and we have graduates all over the United States and several abroad. Hope to hear from those interested. Judy Murphy, San Joaquin Delta College, Dept. of Microscopy, 5151 Pacific Ave., Stockton, CA 95207
JEOL 1200EX Purchased 1989, S/N EM157085-528 Under service contract since purchase Has Tracor Northern EDS (don't know model) Available immediately, shipping negotiable
For more information, contact: melilly-at-aol.com directly, not this list.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Interesting and topical question. In my opinion unless inhouse expertise includes a trained microscope service engineer, you are playing russian roulette without a service contract. sooner or later the chamber is going to come up loaded. Over the long term it is not cost effective. However, I have often thought that facilities with good general in house expertise (we fix a lot of minor problems ourself) should be able to negotiate a different kind of contract (with different cost) than a facility that needs a service engineer for every minor problem. Perhaps we need a lobby with the microscope companies.
Having said that, and persuant to Larry Harkey's queries about microscopes,.o Our Philips service engineer in North Carolina is the best I have ever encountered and Philips in general has always provided superior service. However, with any company their are regional differences in the quality of service.
On Mon, 18 Apr 1994, Griffin, Robin wrote:
} } How are the majority of EM labs maintaining their electron microscopes? Are } they using maintenance contracts, in-house repair or something else? I } know how much maintenance contracts cost (EEK!). How well does it work } without maintenance contracts? I would appreciate any opinions about this } very expensive issue. }
How are the majority of EM labs maintaining their electron microscopes? Are they using maintenance contracts, in-house repair or something else? I know how much maintenance contracts cost (EEK!). How well does it work without maintenance contracts? I would appreciate any opinions about this very expensive issue.
How are the majority of EM labs maintaining their electron microscopes? Are they using maintenance contracts, in-house repair or something else? I know how much maintenance contracts cost (EEK!). How well does it work without maintenance contracts? I would appreciate any opinions about this very expensive issue.
Support person for the Electron Microscopy Core Laboratory of the Interdisciplinary Center for Biotechnology Research at the University of Florida.
DUTIES: Preparation and examination of biological specimens for transmission and scanning electron microscopy in a facility that serves the entire university community on a fee for service basis. Collaborative research possible as part of the program. Incumbent is expected to deal directly with the clients on experimental design, execution of the project and preparation of the data for publication, in consultation with the Scientific Director of the laboratory.
QUALIFICATIONS: B.S. or M.S. in a biological science with proven experience in most aspects of biological electron microscopy. Good communication skills, both oral and written. Ability to deal on a one to one basis with investigators from a wide variety of disciplines and backgrounds.
STARTING DATE: July 1, 1994. (Possibly sooner)
APPLICATION DEADLINE: Open until position is filled.
RANK: Appropriate to qualifications (EM Tech., Sr. EM Tech, EM Lab Manager)
SALARY: $17,873.-$22,633.
Inquiries by E-mail or full resumes to the address below
We currently have a Kodak slow-scan CCD system mounted on a 2010; this is a side-mount systems as opposed to the more common Gatan bottom-mount. Does anyone have any comments comparing the technical capabilities of these systems? How much does the placement affect usage, etc...?
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
I'm trying for the first time to make some preparation of DNA for TEM. Grids and plates, with some initial problem, seem to be promising. My problem, now, is the elaboration of these pictures. I have seen that I can obtain good digital images with a scanner from the positives, and save them as TIFF files in a PC. I would like to find some PD program (possibly DOS, Windows or Amiga) to elaborate these files: the program should recognize DNA in the image and make some kind of elaboration: I don't know exactly what kind: I think (I hope!) I should be able to do something with these digitalised pictures.
Thank you for your help.
Nanni Iazzetti Dept. Genetics, General and Molecular Biology University of Naples Italy
Re} Best Microscopes I didn't see much use in joining the debate about "best microscopes" until the new data arrived. Buying a microscope for the US is a relatively easy choice and the discussion has covered all points. However, I lived and worked in Nairobi, Kenya for 7 years where we had a Zeiss EM 10A which worked well (and is still working). I think that this was the best choice for an EM in Africa. Although we only saw a service engineer once a year, the microscope was constructed for ease of service and repair. The machine was only used by a few people so a talented local electronics engineer and myself were able to keep this microscope running continually with relatively little effort. The long range support from Zeiss was exceptional and any serious problems were overcome with a few phone calls and faxes. Currently, I work on a Phillips 410 and Jeol 100cx, neither of which I feel capable of fixing when they go wrong. I could fix a Peugeot in Africa but have no idea on where to start with the computerized, fuel-injected cars over here.
Paul Webster Yale University School of Medicine 333 Cedar Street, New Haven, CT 06510.
Even more important, is how much knowledge the use (s) has. For stardard biological observations upt 50K even an old Zeiss S2 wil do the job. Of course, electron coherence, astigmatic correction, and vacum cleanness could drive you crazy, not to say much about frequent aligments. Thus, check your packet book, test your technicians, and determine what you need answered, then have all manufacturers to view and examine the same grid with the problem and make decision.
Cesar D. Fermin, Ph.D Tulane Medical School Pathology/SL79 New Orleans, La 70112
I agree with Nestor, the best microscope is dependent upon what you wish to do. Even in cameras, the Nikon is not ideal for all applications. However, to join in the foray: We love our Philips 400, 515, and CM-30s.
On Fri, 15 Apr 1994, Larry Hawkey wrote:
} } I have tried to send this message twice but is keeps getting bounced back } to me. } } Some one is asking me about my recommendations on which EM to } buy. So my question to all of you is this: } } Is there in the EM community a sense that there is one } Electron Microscope that is the best that money can buy? } } If some one was to ask about 35mm cameras for instance. I } think that the general consensus is that Nikon is ahead of } the pack. Minolta and Cannon are good and may be a good deal } for the price but if money is not the first concern Nikon is } the first pick. } } I use the JEOL 1200EX II. It is great. I have also used } several of the 100 incarnations. I used the Ziess EM 10 } about ten years ago but I have really only used JEOL for the } past ten years. In addition there seems to be a heavy } concentration of JEOLS in the Duke University community so I } feel that my impression that Ziess is nice but JEOL is best } may only reflect my small world. } } Try to put your biases aside and send me your thoughts. } } Larry Hawkey } Hawkey-at-neuro.duke.edu }
There are several short courses offered (1 week) through the Marine Biological Laboratory in Woods Hole, Massachusetts. For information call 508-548-3705 . The next one is in May. Regards, Nina Allen
On Tue, 19 Apr 1994 IDESOUSA-at-BRMVM1.VNET.IBM.COM wrote:
} Is anybody aware of a good short course on optical microscopy? } It is such a vast subject (polarized , UV,IR, confocal,filters, } image analysis, interferometry, ellipsometry etc.)
Might try the following:
1. McCrone Research Institute 2820 S. Michigan Ave. Chicago, IL 60616-3292 (312) 842-7100 [phone] 842-1078 [fax]
*some of their offerings: photomicrography, polarized light microscopy, hotstage microscopy, SEM, TEM, pharmaceutical, forensic, asbestos id, hair and fiber id, wood and pollen id, anb many others, including custom planned on-site courses. I've taken the Applied Polarized Light Microscopy course there, and found it -very- good; although not enough emphasis for materials type. Courses are spread out through the year. Prices for 1994: $1050 (ouch!) for almost all, w/ TEM being higher.
2. Institute for Microstructural Analysis c/o Buehler Ltd. P.O. Box 1 Lake Bluff, IL 60044 (702) 295-4659 [phone]
*courses offered include: image analysis, petrographic prep., basic metallography, advanced materials, ceramics, etc. Prices vary from $700 for 2-day to $1095 (again, ouch!) for 5-day, and higher prices at their Irvine, CA locale. I myself have not been there, but have heard good reports.
3. ASM International Materials Park, OH 44073 (216) 338-5151 [phone]
*courses include: metallographic interpretation, met. techniques, advanced tech. in optical (& electron) microscopy, failure analysis, optical microscopy of ceramics, quantitative metallography, etc... I've taken one course there, and have had several home-study classes. Good all around training. They offer what's called Materials Engineering Institute Extension diploma, and a three-tiered Center for Applied Metallography levels. Prices are similar to Buehler's.
} Could someone suggest books or reference material which is neither } trivial or too technically involved in the physics. (level of a good } metallurgical technician)
Some of my favorites include:
Metallography: Principles and Practice...........Vander Voort, George McGraw-Hill, 1984 (wish it would be updated tho) Polarized Light Microscopy.......................McCrone & Delly Ann Arbor Science Publishers, 1978 (likewise...) -and- many of the Microstructural Science volumes from the proceedings of the annual technical meetings of the International Metallographic Society. -and- many of the ore microscopy books have very detailed and highly useful information.
In addition, the other large met. supply houses besides Buehler, LECO [(616) 983-5531; St. Joseph, MI] and Struers [(216) 871-0071, Westlake, OH] offer tech. tips and misc. publications that are -very- handy and helpful. LECO also offers some training.
} One particular item I would find very handy is a compilation of } uv fluorescence wavelength of common substances. } } Thanks to all!
I'd be interested in such also...
Hope the above is helpful; glad to see another met online! -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /-v-\ | | Metallographic Lab. | Missouri Speleological Survey \-v-/ | | Rolla Research Center | Bat Conservation International \-v-/ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /-v-\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
On Tue, 19 Apr 1994 IDESOUSA-at-BRMVM1.VNET.IBM.COM wrote:
} Is anybody aware of a good short course on optical microscopy? } It is such a vast subject (polarized , UV,IR, confocal,filters, } image analysis, interferometry, ellipsometry etc.)
Might try the following:
1. McCrone Research Institute 2820 S. Michigan Ave. Chicago, IL 60616-3292 (312) 842-7100 [phone] 842-1078 [fax]
*some of their offerings: photomicrography, polarized light microscopy, hotstage microscopy, SEM, TEM, pharmaceutical, forensic, asbestos id, hair and fiber id, wood and pollen id, anb many others, including custom planned on-site courses. I've taken the Applied Polarized Light Microscopy course there, and found it -very- good; although not enough emphasis for materials type. Courses are spread out through the year. Prices for 1994: $1050 (ouch!) for almost all, w/ TEM being higher.
2. Institute for Microstructural Analysis c/o Buehler Ltd. P.O. Box 1 Lake Bluff, IL 60044 (702) 295-4659 [phone]
*courses offered include: image analysis, petrographic prep., basic metallography, advanced materials, ceramics, etc. Prices vary from $700 for 2-day to $1095 (again, ouch!) for 5-day, and higher prices at their Irvine, CA locale. I myself have not been there, but have heard good reports.
3. ASM International Materials Park, OH 44073 (216) 338-5151 [phone]
*courses include: metallographic interpretation, met. techniques, advanced tech. in optical (& electron) microscopy, failure analysis, optical microscopy of ceramics, quantitative metallography, etc... I've taken one course there, and have had several home-study classes. Good all around training. They offer what's called Materials Engineering Institute Extension diploma, and a three-tiered Center for Applied Metallography levels. Prices are similar to Buehler's.
} Could someone suggest books or reference material which is neither } trivial or too technically involved in the physics. (level of a good } metallurgical technician)
Some of my favorites include:
Metallography: Principles and Practice...........Vander Voort, George McGraw-Hill, 1984 (wish it would be updated tho) Polarized Light Microscopy.......................McCrone & Delly Ann Arbor Science Publishers, 1978 (likewise...) -and- many of the Microstructural Science volumes from the proceedings of the annual technical meetings of the International Metallographic Society. -and- many of the ore microscopy books have very detailed and highly useful information.
In addition, the other large met. supply houses besides Buehler, LECO [(616) 983-5531; St. Joseph, MI] and Struers [(216) 871-0071, Westlake, OH] offer tech. tips and misc. publications that are -very- handy and helpful. LECO also offers some training.
} One particular item I would find very handy is a compilation of } uv fluorescence wavelength of common substances. } } Thanks to all!
I'd be interested in such also...
Hope the above is helpful; glad to see another met online! -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /-v-\ | | Metallographic Lab. | Missouri Speleological Survey \-v-/ | | Rolla Research Center | Bat Conservation International \-v-/ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /-v-\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
the McCrone Institute in chicago has a number of short courses on various aspects of light microscopy - including things that would probably be of interest to you such as polarization, dispersion staining, etc
there are several good, simple books around that are probably at the level you want - check out the Oxford series - I know thee is an flouresence & think there may also be a polarization book - they present the basic theory and are inexpensive - there is also a very practical-oriented book with a yellow and white paper cover whose exact and author escape me ( it is siiting on my bookshelf at home)
if you are not satisfied with your other responses send me a message & I will try to get back to you
We are thinking of purchasing a sapphire knife for our Vibratome so that we can cut high-quality sections. The knives are about $700. Being somewhat poor, we were wondering if the knife is a good investment. What has been people's experience with these knives? Do they cut reliable high-quality sections? Any problems with them? Thanks for any opinions.
-- ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Nancy L Desmond, Ph.D. Department of Neurosurgery University of Virginia Health Sciences Center, Box 420 Charlottesville, VA 22908
We (actually Ed Lachica) have found it an absolute requirement for cutting unfixed chick embryos. Fixed post-hatch brains didn't seem to cut any better. But sectioning unfixed brain, or lightly fixed embryonic brain is helped tremendously.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 20 Apr 1994, Nancy L. Desmond wrote:
} We are thinking of purchasing a sapphire knife for our Vibratome } so that we can cut high-quality sections. The knives are about } $700. Being somewhat poor, we were wondering if the knife is a } good investment. What has been people's experience with these } knives? Do they cut reliable high-quality sections? Any } problems with them? Thanks for any opinions. } } -- } ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ } Nancy L Desmond, Ph.D. } Department of Neurosurgery } University of Virginia } Health Sciences Center, Box 420 } Charlottesville, VA 22908 } } 804.924.5607 (voice) } 804.982.3829 (fax) } ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ }
I am studying Aluminium alloys samples with TEM and especially two kinds of them: Al 3003 (0.12%Cu-1.2%Mn) AL 6061 (0.6%Si-0.27%Cu-1%Mg-0.2%Cr) I will use a double jet electropolisher (Tenupol 2) and I would want to know, if possible: 1-what are the best electropolishing solutions for these alloys (the books are not enough specific ) ? 2-what are the value of current,voltage and what is the temperature used for the solution ? 3-I suppose that we use the classical technique for cleaning but there must be some problems to prevent samples from oxydation which is an important problem for aluminium.Is there a special technique to avoid oxydation ?
NOTE:if no answer is possible, what are at least the best solution and electropolishing conditions for PURE aluminium.
It took nearly three months for BioRad to replace our laser when it failed after only 100 hours of use. So much for 24 hr. service. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Does anybody know of a good procedure on the infiltration of yeast cells with LR-White? I dehydrate the pellet normally, starting with 35%ETOH for 10 minutes, 50%ETOH 10 minutes, 75%ETOH 10 minutes, 2 x 100%ETOH 10 minutes, 1:1- 100%ETOH/LR-white 1hour, 1:2- 100%ETOH/LR-White 1hour, 1:3- 100%ETOH/LR-white 1hour, 2x pure LR-white 1hour, bake at 50C for 72 hours. I am still ending up with holes around the cells and some cells aren't being infiltrated with the resin. I have never had problems with infiltration of any type of tissue, bacteria or anything else I've had to embed. This is getting to be pretty frustrating (Jack Daniels-black, here I come). Thanks Phil
Does anybody know the name of the journal that replaced "The Journal of Histochemistry and Cytochemistry" ? I was interested in getting a subscription to it and when I called Elsevier I was told it was discontinued 9 years ago. Someone said it was replaced by another outfit and they weren't sure who the publisher was. Anybody know? Thanks, Phil Rutledge
Message-Id: {MAILQUEUE-101.940422072110.367-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
Phil Rutledge Our library carries a journal by the title of Journal of Histochemistry and Cytochemistry published by the Histochemical Society. It is printed by Williams and Wilkens in Baltimore. Probably is the same one you are looking for. In addition, concerning LR white and yeast infiltration, it was suggested by someone here that you might try using a mixture of osmium and potassium permanganate to fix the tissue, or possibly ruthenium red. You need to permeabilize the cell wall to let the LR white in. Try also to increase infiltration times, or put it under vacuum. Was also suggested you look up papers by C. Bracker on fixation of fungi. Hope this info helps.
I did the studies about Al3003 by TEM. You can find my paper "ATEM study of precipitates in an Al-Mn-Si alloy" publisned on METALLOGRAPHY 21:293-315 (1988). I used 20 HClO4 + 20 HCl + 80 ethyl alcohol, around 25 mA and temperature aroun -25 C. As you can see from my paper I have get lattice image. I am the first author of the paper XIANYING MENG-BURANY. Sandy X. Burany
Apologies to all concerned if this query is a bit dumb! After trawling the literature on HREM imaging theory (particularly the application of the wave-optical Abbe theory of image formation) I am confused as to whether the Fraunhofer diffraction pattern is:
i)a Fourier transform of the object exit wavefunction (being a product of the incident wave amplitude and a transmission function)
OR
ii)as some authors simply state, "the transmission function"
The J Histochem Cytochem is published by the Histochemical Society. The address is: Mount Sinai School of Medicine, 1 Gusatave L. Levy Place, Box 1045, New York NY 10029 Ph: 212-362-1801. You should find this journal in most university libraries. If your library does not get it, talk to your acquisition librarian. It is still the leading histochemical journal.
Denis G. Baskin, Ph.D. University of Washington
On Fri, 22 Apr 1994, rutledge phil wrote:
} Does anybody know the name of the journal that replaced "The Journal of } Histochemistry and Cytochemistry" ? I was interested in getting a } subscription to it and when I called Elsevier I was told it was } discontinued 9 years ago. Someone said it was replaced by another outfit } and they weren't sure who the publisher was. Anybody know? } Thanks, } Phil Rutledge }
} One particular item I would find very handy is a compilation of } uv fluorescence wavelength of common substances.
For ultraviolet(365nm) and blue-violet(400nm) fluorescence of over 600 materials see:
The Particle Atlas, Edition Two, Volume IV, McCrone and Delly (1979). [Out of Print]
The entire six-volume, electronic edition of the Particle Atlas is now available (PAE^2) on CD-ROM from McRI or:
Microdataware 2894 Tribune Ave Hayward CA 94542 TEL 510.582.6624 FAX 510.582.8295
McCrone Research Institute is offering the PAE^2 Version 1.0 for Windows with an educational discount of $550.00, which reduces the price to students to $950.00 if purchased within 90 days of course completion.
For a basic library of microscopy:
"A Basic Microscopy Library", John G. Delly, The Microscope, vol. 34, pp. 11-25 (1986).
This article contains a list and description of books on microscopy for the fields including biomedicine, mineralogy, petrology, petrography, metallography, chemical microscopy, and, of course, general works.
100%ETOH/LR-white 1hour, 2x pure LR-white 1hour, bake at 50C for 72 hours. I am still ending up with holes around the cells and some cells aren't
being infiltrated with the resin. I have never had problems with
infiltration of any type of tissue, bacteria or anything else I've had to
embed. This is getting to be pretty frustrating (Jack Daniels-black,
here I come). Thanks Phil
} } } } } } } } } } } } } } } } } } } } } } } } }
We routinely use LR White for preparing EM sections of yeast in immunolocalization experiments. The critical factor for getting good infiltration is a brief treatment of the fixed, washed cells with 1% sodium metaperiodate. This treatment alters the cell wall so that the resin infiltrates perfectly. After treatment with metaperiodate, a relatively standard dehydration/infiltration schedule can be followed. We also use a temp block (like the ones normally used for restriction digests) to precisely control temperature during polymerization. It seemed to be important to keep the temperature low during polymerization to minimize shrinkage (we used 45-50C for 24 hr).
For details of the procedure see:
Wright and Rine, Methods in Cell Biology 31:473-512.
van Tuinen and Riezman, J. Histochem. Cytochem. 35:327.
Phil wrote: ""Does anybody know of a good procedure on the infiltration of yeast cells with LR-White? I dehydrate the pellet normally, starting with 35%ETOH for 10 minutes, 50%ETOH 10 minutes, 75%ETOH 10 minutes, 2 x 100%ETOH 10 minutes, 1:1- 100%ETOH/LR-white 1hour, 1:2- 100%ETOH/LR-White 1hour, 1:3- 100%ETOH/LR-white 1hour, 2x pure LR-white 1hour, bake at 50C for 72 hours. I am still ending up with holes around the cells and some cells aren't being infiltrated with the resin.""
If you want to try and look at some of your poorly infiltrated yeats. If you have a 200KV instrument, you can cut 1/4 micron sections(maybe the yeast will stay in the plastic and not fall out!); stain with aqueous UA for 45'-1 hr. at 45 degrees, LC 30' and use the 200kv. We do this alot when people send us blocks that are bad.
This is what we do in our facility, with success on yeast: we use LR white, both medium and hard grades; for regular ultrastructure(medium grade)- if we have problems with infiltration: we increase the changes in LR white and make sure each change is accompanied by time on the rotator( -at- 4 RPM)(called rotamix in protocol below) we leave the sample in LR white overnight, on a rotator at 4 degrees. If we have trouble with air bubbles, we reduce stirring to a minimum and only use the rotator for mixing; we polymerize at 55 degrees for only 24 hours. For IEM, we use hard grade -if we have problems with infiltration: we increase the changes in LR white first and accept some yeast cells will not be infiltrated well, in some samples; we always have plenty in each section that are fine, even if we leave the cell wall intact. Note that there are two procedures; if your label is O.K. with procedure b, you'll get better infiltration; but either way gives use plenty of well-infiltrated yeasts cells in each section. If we have trouble with air bubbles, we reduce stirring to a minimum; we polymerize Hard grade at 50 degrees for only 24 hours.
Phil: here's the last half of our protocols; please call or send me e-mail anytime. It should work. Regular ultrastructure 6. Dehydrate : 30% - 5 mins 50% - 5mins 70% - 5mins 100% - three 5min. washes.
7. LR White:100% ETOH 1:1 - 30-mins RT on a gentle Rotamix*** .
8. LR White:100% ETOH 3:1 - 30-mins RT on a gentle Rotamix*** .
9. LR White - 2-3 changes 30-mins each, RT on a gentle Rotamix***
10. Overnight in fridge (-at-4oC), on a gentle Rotamix.
11. LR white - 1 change 1 hr (Warm LR White to RT before change)
12. Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle for 1 hour;
NOTE: if you need to centrifuge material and you have a small sample size, place cells in trimmed beam capsule, seal and centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge; then carefully seal beam capsule inside two 000 gelatin bottoms, that have been filled with LR White. Make sure you seal well with LR White. Use regular mold holders to hold capsules. OR if you need to centrifuge material and you have a large sample size, leave cells in original 00 gelatin capsule(i.e.complete step 12. Then place 00 capsule inside a BEEM capsule that has been cut to accomodate 00 gelatin capsule(i.e. top half is removed); centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge, until sample is spun down.
13. Polymerize at 55-60oC for 24 hours.
*** Use glass vials ; keep out of direct light; Use a rotomix, asLR White needs to be GENTLY stirred. KEEP Oxygen out of LR White; avoid excess air bubbles in resin. The dehydration and embedding are fairly straightforward. USE LR White Medium GRADE Open gelatin capsules to stop all polymerization.
For section stain, start with 2%UA aqueous for 20-45 mins. and LC 2-5 mins. try UA-45'/LC 2' first
FOR IEM Dehydrate : 50% - 30 mins 70% - 2 changes, 30 mins. each
LR White:70%ETOH - 2:1 2 changes 30 mins each ***
a. LR White - 3 changes 30-60 mins each, on a gentle Rotamix. b. LR White - 4-6 changes over three hours, on a gentle Rotamix.
a.Overnight in fridge (-at-4oC), on a gentle Rotamix. b. Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle. SEE NOTE step 8. Polymerize at 50oC for 24 hours.
a.LR white - 1 change 1 hour (Warm LR White to RT before change)
a.Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle for 1 hour;
NOTE: if you need to centrifuge material and you have a small sample size, place cells in trimmed beam capsule, seal and centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge; then carefully seal beam capsule inside two 000 gelatin bottoms, that have been filled with LR White. Make sure you seal well with LR White. Use regular mold holders to hold capsules. OR if you need to centrifuge material and you have a large sample size, leave cells in original 00 gelatin capsule(i.e. complete step 8). Then place 00 capsule inside a BEEM capsule that has been cut to accomodate 00 gelatin capsule(i.e. top half is removed); centrifuge inside a 12-15ml cenrifuge tube{with a cotton bottom pad}, using a clinical centrifuge, until sample is spun down.
9. a.Polymerize at 50oC for 24 hours.
TEST one block for sectioning quality. Polymerize another hour, if necessary.
*** Use glass vials to mix; mix solns. at RT; keep out of direct light; Use a rotomix, as solution is not very miscible & needs to be GENTLY stirred. KEEP Oxygen out of LR White; avoid excess air bubbles in resin. The dehydration and embedding are fairly straightforward.
LrWhite blocks should be stored at -20oC to preserve antigenicity indefinitely. Remove the gelatin and expose block to air in order to stop polymerization.
For section stain, start with 2%UA aqueous for 20-45 mins. and LC 2-5 mins. try UA-20' with no LC first, so you can locate the gold particles easily; adjust stain, as needed for ultrasruture.
JOURNAL OF MICROSCOPY - MAY 1994 ISSUE - VOLUME 174 PART 2
PUBLICATION DATE - 20 MAY 1994
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 61-68
ACCURATE ALIGNMENT OF SETS OF IMAGES
By W. O. SAXTON Department of Materials Science and Metallurgy, Pembroke Street, Cambridge CB2 3QZ, U.K.
Summary Two modifications are described to the conventional procedure of cross-correlation, widely used for establishing the relative alignment of the members of a set of images from which a higher resolution or more interpretable restoration is sought. Both achieve a high and sharp peak in circumstances where the conventional peak is too ill defined to be recognisable; neither involves significant additional computational time. The more general method requires a rough knowledge of the imaging conditions, but a variant applicable to images with axial resolution has no such requirement. In addition, a least-squares procedure is presented for achieving an optimum compromise between many pair-wise displacement measurements, preventing the accumulation of alignment errors across a set of images.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 69-73
RESOLUTION IN NONLINEAR LASER SCANNING MICROSCOPY
By J. DEITCHE, M. KEMPE & W. RUDOLPH The University of New Mexico, Department of Physics and Astronomy, Albuquerque, NM 87131, U.S.A.
Summary The lateral and depth resolution of nonlinear microscopy was studied systematically. Nonlinear microscopy can be classified into several categories depending on the coherence properties of the process that generates the imaging signal from the illuminating light, on whether a single- or a two-beam geometry is used and whether the optical setup is Type I or Type II. An evaluation of the imaging equations shows that (i) lateral and depth resolution improve with increasing nonlinearity, (ii) the differences between coherent and incoherent imaging diminish, and (iii) nonlinear imaging allows depth discrimination in Type I microscopy.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 75-84
CRYO AUTOMATED ELECTRON TOMOGRAPHY: TOWARDS HIGH-RESOLUTION RECONSTRUCTION OF PLASTIC-EMBEDDED STRUCTURES
By M. B. BRAUNFELD, A. J. KOSTER, J. W. SEDAT & D. A. AGARD The Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143-0448, U.S.A.
Summary The use of fully automated data collection methods for electron beam tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self-consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post-cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium. Surprisingly, the substantial dose reduction made possible by automated data collection did not result in a significant decrease in specimen shrinkage even for samples stabilized by pre-irradiation. The accelerated shrinkage is a direct consequence of the stroboscopic illumination patterns inherent to automated data collection. Furthermore neither the choice of embedding resin nor microwave post-curing greatly affected shrinkage. Finally, cryogenic data collection was investigated as a means to minimize the effects of secondary radiation damage. Minimal pre-irradiation coupled with low-temperature automated data collection greatly reduces shrinkage and should result in high-quality data for three-dimensional reconstructions.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 85-92
A NOVEL METHOD FOR MEAN CELL VOLUME ESTIMATION
By P. WEBSTER* & G. GRIFFITHS+ *Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT 06510, U.S.A. +European Molecular Biology Laboratory, Postfach 10.22.09, 69012 Heidelberg, Germany
Summary A novel method is described for the estimation of the mean cell volume of cell populations grown in suspension. The cells are filtered on to a nitrocellulose filter to form a cylindrical pellet which is embedded in epoxy resin. Using estimates of pellet height and radius, the number of cells in the pellet and the volume density of cells in the pellet, it is possible to produce an unbiased estimate of the mean cell volume. This method is compared, using cell suspensions of the blood parasite Trypanosoma brucei, with other published methods for mean cell volume estimation. A Coulter channelizer was also used to compare the mean cell volume of living trypanosomes with that of aldehyde-fixed populations, and the values obtained were compared with those obtained with the new method. The estimated mean cell volume of a T. brucei clone was used to derive values from volume densities obtained by point and intersection counts for the absolute volumes of the flagellar pocket, the nucleus and the endocytic organelles containing internalized horseradish peroxidase and transferrin-gold after 30-min incubations at 310K. Estimated values for the cell were also obtained. From known data on the total amount of variant surface glycoprotein molecules per cell and the known packing density of membrane proteins, it is estimated that approximately 80% of the molecules must reside in intracellular compartments. It was estimated that the equivalent of 5% of the surface membrane may be internalized per minute, an amount which is almost the size of the entire flagellar pocket membrane.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 93-100
THE INFLUENCE OF TISSUE PROCESSING ON QUANTITATIVE HISTOPATHOLOGY IN BREAST CANCER
By M. LADEKARL Stereological Research Laboratory, University Institute of Pathology and Second University Clinic of Internal Medicine, Institute of Clinical Experimental Research, University of Aarhus, Denmark.
Summary Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume-weighted mean volume of cancer call nuclei was estimated using the method of point-sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, the nuclear volume fraction, the nuclear profile density and the mitotic profile frequency were obtained. The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5.0, 6.0, 7.0, 7.4 and 8.0 respectively. Similarly, no significant correlations between estimates and the period of formalin fixation (24h, 3 days and 3 months) were found in samples from five other tumours. However, the volume-weighted mean nuclear volume of cancer cell nuclei was 13% larger (2p=0.004) and the mean nuclear profile density was 17% smaller (2p=0.04) in hydroxyethyl- methacrylate-embedded samples from 17 tumours as compared to paraffin-embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 101-110
METHOD FOR THE STUDY OF THE THREE-DIMENSIONAL ORIENTATION OF THE NUCLEI OF MYOCARDIAL CELLS IN FETAL HUMAN HEART BY MEANS IF CONFOCAL SCANNING LASER MICROSCOPY
By Y. USSON,* F. PARAZZA, * P.-S. JOUK + & G. MICHALOWICZ+ *Dynamique de l'organisation du genome, Universite Joseph Fourier, BP53, 38041 Grenoble cedex 9, France +Medecine Neonatale, Centre Hospitalier Regional Universitaire, Grenoble, France
A series of three-dimensional image analysis tools are used to measure the three-dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 micrometre inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 111-119
A NOVEL APPLICATION OF MICROSPHERE PERFUSION AND SCANNING ELECTRON MICROSCOPY TO THE IDENTIFICATION OF PULMONARY ARTERIOLES IN GUINEA PIG AND RABBIT LUNGS
By D. C. WALKER, S. HOSFORD & A. MACKENZIE Christmas Seals Electron Microscopy Laboratory of the Pulmonary Research Laboratory, U.B.C. Pathology, St Paul's Hospital, Vancouver, B.C., Canada V6Z 1Y6
Summary In arterioles of the lung the intravascular blood pressures are lower than in comparable vessels in the systemic circulation and the arteriole walls are thinner. Therefore, it is very difficult to distinguish between arterioles and venules of the same size using scanning electron microscopy. This study describes a novel application of latex microsphere perfusion and scanning electron microscopy which distinguishes between pulmonary arterioles and venules on the basis of endothelial cell morphology. Microspheres, 90 and 45 micrometres in diameter, were perfused into the arterial side of the pulmonary circulation of guinea-pig and rabbit lungs. Scanning electron microscopy of the arterioles on both sides of the lodged microspheres indicated that the endothelial cells are spindle shaped. In contrast, the endothelial cells of equal diameter venules are polygonal. Furthermore, the nuclei of the arteriolar endothelial cells were significantly (P=0.019) narrower than those of endothelial cells in venules of equal diameter. Finally, it was observed that the differences between arteriole and venule endothelial cells persisted distally to the capillaries.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 121-123
ASTIGMATISM OF A THICK CYLINDRICAL OBJECT IN REFLECTIVE MODE CSLM
By CHANG-GUI WANG, H. RAIKKONEN & M. LUUKKALA Department of Physics, PO Box 9, 00014 University of Helsinki, Finland
Summary The axial response of a basic confocal microscope is determined when the sample is a thick cylindrical or tubular structure. The response from the back wall of the cylindrical sample is split into two separate signals due to basic aberration or astigmatism effects.
Journal of Microscopy, Vol. 174, Pt. 2, May 1994, pp. 125-127
Short Technical Note SEVERE SHRINKAGE OF MICROFIL DURING TISSUE CLEARING WITH THE SPALTEHOLZ TECHNIQUE
By J. MOLLER, K. ROBERTSEN & E. S. HANSEN Institute of Experimental Clinical Research, Department of Orthopaedics, University of Aarhus, Randersvej 1, DK 8200 Aarhus N, Denmark
Summary The effect of two commonly used tissue clearing methods on the morphology of vascular casts by Microfil, a silicone rubber injection compound, was investigated. Microfil undergoes extreme shrinkage when the casted tissue is cleared by the alcohol-methyl salicylate clearing technique. No shrinkage is observed when the alternative glycerine clearing method is used. The alcohol-methyl salicylate clearing technique should be avoided in studies employing Microfil.
PLEASE SEND YOUR QUERIES CONCERNING THE JOURNAL TO RMS-at-VAX.OX.AC.UK. WE ARE ALSO PLEASED TO ACCEPT LETTERS TO THE EDITORS BY EMAIL.
Does anybody know where I could find SJ Singer, the man who introduced EM immunocytochemistry by a paper in Nature (London) 1959. I would like to present him by his portrait at a meeting. All advices are welcome. Thanks, Sverker Enestr|m
Has anyone worked with a type of species in the bacteria field known as a marine cytophage type? I'm trying to fix the cells on agar and the cells keep floating up off the surface of the agar. I would like them to stay put. (maybe I need to put them in a training school for bacteria!). I've tried several methods suggested by members of the microscopy board but no success. Hopefully this problem can be taken care of. It's driving me nuts! Thanks, Phil
Message-Id: {9404251801.AA02278-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
Like a dumbbell I forgot to mention I need to do SEM on these little suckers. Fixing them and processing for TEM is no problem. That's easy. Trying to keep the little creatures attached to the agar surface and fixing them without them floating off is a #!$#%$$^&(^*& of a problem. I need to see the surface of the bacteria and how they are oriented in their growth on the agar without losing ANY of the cells. I should have become a mechanic or plumber 27 years ago, EM can be a real -at-$%^%& ! Thanx, Phil
Have you tried attaching them to a millipore filter by passing them from a syringe onto the filter (using millipore syringe fittings) and then fixing them onto the millipore surface by squirting fixative through the filter? This works for suspension culture cells and many small critters. The millipore filters can then be CPD and attached to a stub.
On Mon, 25 Apr 1994, rutledge phil wrote:
} Like a dumbbell I forgot to mention I need to do SEM on these little } suckers. Fixing them and processing for TEM is no problem. That's easy. } Trying to keep the little creatures attached to the agar surface and } fixing them without them floating off is a #!$#%$$^&(^*& of a problem. I } need to see the surface of the bacteria and how they are oriented in } their growth on the agar without losing ANY of the cells. } I should have become a mechanic or plumber 27 years ago, EM can be a real } -at-$%^%& ! } Thanx, } Phil }
Try this sandwich method - it works great for suspensions:
use the thinest layer of agar that you can remove with the colonies intact
- take a 13- 15 mm dameter nylon washer (you can use brass if you are not going to use OsO4) - I suppose you can use a larger diameter washer & filter so long as it will fit in your cpd unit - place an appropriate pore size filter on the washer - place a "spacer" on the filter (for cell suspensions I punch a hole in the blue spacer papres packed with the filters - you might want to try an o-ring) - cover with a second filter - top off the sandwich with another washer
clip the whole thing together with a paper clip (yours may be a triffle thick for that, so you may have to be inventive
we have used relatively short fix/wash/dehydrate times for suspensions with good results
the only catch is taht some of your cell may stick to the top filter (ours do) so you might need to make sure that "chamber" height exceeds the height of your sample and try to keep it right side up during processing
(an easy way to remove small sections of colonies from plates is with a cork borer or a glass pipette)
good luck & please excuse the typos
feel free to contact me if this is unclear
Marcelle A Gillott Univ Wisconsin-Milwaukee Department of Biological Sciences 414-229-4186
First: Thanks to all for their suggestions. Now: Let me go into this problem a little deeper. We have unknown bacteria of marine cytophage species that autofluoresce red and green. We want to look at the surface by SEM to see: 1: surface structures, if any 2: orientation of the bacteria as it grows (there is a definite orientation as seen after some of the cells have floated off) Need: a way to fix the cells without ANY cells floating off the surface Tried: 1: suspensions 2: growing on nucleopore filters 3: growing on coverslips Result: no autofluorescence, no definite orientation Fix: 1: direct addition of glutaraldehyde 2: cutting out pathways in the agar and fixing by letting the agar absorb the fix and fixing the cells from underneath the agar. 3: osmium vapor fixation then glut. Result: Some of the bacteria still float up when washing and dehydrating. Question: Is there a way to crosslink the cells by some fixation technique so they will remain in place on the agar surface?
John Mansfield North Campus Electron Microbeam Analysis Lab University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 08:59
Date:4/26/94 NC EMAL
Macro 'Read AFM File'; {reads header in and searches for the number of Samps/line, uses this value} {as the width and heigth for the image import (therefore supports 128,256 and} {512 squared images} var size, width, height, offset, slices, first, second, third, fourth: integer; icount: integer; temp, one, two, three, four: string; done: boolean; begin; done:= 'false'; width:=8192; height:=1; Offset:=0; slices:=1; SetImport('8-bit'); SetCustom(width, height, offset,slices); Import(''); icount:=1; repeat temp:=chr(getpixel(icount, 0)); if ((temp='S') and (one {} 'S')) then one:=temp; if ( (temp='a') and (one='S') and (two {} 'a')) then two:=temp; if( (temp='m') and (one='S') and (two='a')) then three:=temp; if( (temp='p') and (one='S') and (two='a') and (three='m')) then four:=temp; if (four='p') then done:='true'; icount:=icount+1; until done='true'; first:=(getpixel((icount+8), 0))-48; second:=(getpixel((icount+9), 0))-48; third:=(getpixel((icount+10), 0))-48; fourth:=(getpixel((icount+11), 0))-48; if (first {} 1) then size:=(first*100)+(second*10)+third; if (first=1) then size:=(first*1000)+(second*100)+(third*100)+fourth; Dispose; width:=size; height:=size; offset:=8192; SetImport('16-bit signed,swap bytes'); SetCustom(width, height, Offset); Import(''); end;
Phil, Can you overlay the agar/bacteria with another thin layer of agar then fix as usual. When osmium is added the bugs should turn black (if in colonies) making them visible after emebedding.
Royal Microscopical Society & Oxford Brookes University
26 - 31 March 1995
Location Oxford Brookes University - Oxford's new University, situated 1 mile from the centre of historic Oxford. Full board accommodation will be available at Morrell Hall, 5 minutes walk from the campus.
Cost Registration and full board accommodation will cost no more than œ440.0 pounds sterling. Receptions, the Conference Dinner and a copy of the special edition of the Journal of Microscopy are included in the cost. There are a very limited number of double rooms available.
Delegates To preserve the informal nature of the Botanical Microscopy Meeting, the number of delegates will be restricted to 150. Places at the conference will be allocated on a first come, first served basis with a preference given to those offering to present a poster. (Please note that places will only be guaranteed after payment has been received.)
Booking forms and abstract instructions will be available from June 1994.
Organizing Committee Nick Harris - Durham Chris Hawes - Oxford Nick Read - Edinburgh Peter Shaw - John Innes Institute
Format of Meeting Following the success of Botanical Microscopy 1991 in Durham, the same team are organizing the meeting to be held in Oxford. The scientific programme will be based around presentations from keynote speakers.
A call for abstracts will be made late 1994 and the organizing committee will select suitable papers from these and invite the authors to present them orally. However, posters on any aspect of plant cell biology involving microscopy may be presented.
Social A reception is planned for the opening evening of the meeting and an informal Conference Dinner will be held on the Thursday evening. A number of short tours around Oxford and its environs will be offered on the Wednesday afternoon of the Conference.
Publications Keynote and selected papers will be published as a special edition of the Journal of Microscopy which will be distributed to all delegates upon publication. All manuscripts will be subject to peer review.
Travel Air: Oxford Brookes University is a 60 minute bus ride from Heathrow airport and 130 minute ride from Gatwick airport.
Road: Coaches run from Victoria Bus Station in London every 20-30 minutes. Journey time is approximately 60 minutes.
Rail: There is a good train service from Paddington Station, London to Oxford.
Queries If you would like any further information, please contact Miss Karen Hale at the Royal Microscopical Society, 37/38 St Clements, Oxford, OX4 1AJ. Tel: +44-865-248768. Fax: +44-865-791237. Email: rms-at-vax.ox.ac.uk.
Programme to Include: þMicrotubule and cytoskeletal dynamics
þMicroscopy of living cells - ion imaging, cell-cell signalling
þPlant cell organization - cell walls, meiosis, low temperature techniques
þMolecular mechanisms of plant development - cell cycle, floral development
þPlant microbe-interactions
Keynote Speakers B Gunning - Canberra H Shibaoka - Osaka J Hush - Sydney S Giroy - Penn State K Oparka - Invergowrie K Roberts - Norwich M Parthasarathy - Cornell Z Cande - Berkeley J Doonan - Norwich R Howard - Wilmington
Please complete this coupon indicating your requirements, and return to the Royal Microscopical Society, 37/38 St Clements, Oxford, OX4 1AJ, UK.
I would be interested in attending the 5th International
Does anybody know where I could find SJ Singer, the man who introduced EM immunocytochemistry by a paper in Nature (London) 1959. I would like to present him by his portrait at a meeting. All advices are welcome. Thanks, Sverker Enestr|m
======================================================================= I send this mail again because of local configuration error: "Service unavailable" =======================================================================
Hi, We are considering the purchase of an Energy Dispersive X-ray Analysis (EDX) system for attachment to our SEM within the next year. Our applications are in two areas; paper making and corrosion of materials.
I would like to know if anyone is aware of mailing lists etc., accessable on the Internet, which are specifically interested in EDX. I would like to follow discussions on relative merrits of detector and window types , vacuum requirements for windowless detectors, pulse processor requirements, software options and in particular, read any horror stories associated with a given manufacturer.
If anyone has opinions on what is currently available from recent purchase deliberations or would like to recommend a review paper on current technology or a periodical which they go to for this sort of information it would be greatly appreciated.
My first priority is always dependable service, but having said that, I will be looking for a high quality detector capable of light element analysis, high pulse throughput, good quantitation with ease of use , beam control and image acquisition and replay capabilities.
Thanks for your help.
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
I am looking for some help regarding pore sizes in the secondary cell wall of wood. The wood has been partially degraded by fungi, and I want to see how the porosity of the lignin-cellulose substrate has changed. I plan on infiltrating wood with proteins of different molecular weights (my probes), then locating them with immunolabling. I have already decided on a 40000 M.W. (ovalbumin, and MN dependant peroxidase), and a 20000 M.W. (myoglobin) probe. I am still looking for an adequate probe for M.W. 5000, 10000, and 15000. By adequate probe I mean a protein that does not denature easily, and also I can purchase antibodies against. Any help would be appreciated.
Eugene Krueger, Dept of Plant Pathology, University of Minnesota.
-- Eugene Krueger Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: Spencer Stereoscope ------------------------------------------------------------------
Is anyone acquainted with a good repair service for AO Spencer stereomicroscopes? We have the "gray standard issue", step magnification, ubiquitous model, probably vintage 1959 or so. One of the prisms has come loose, and we've spent too much time already trying to get it EXACTLY back into position. Maybe there's a trick or tool we're missing. Thanks in advance for any help.
I am looking for some help regarding pore sizes in the secondary cell wall of wood. The wood has been partially degraded by fungi, and I want to see how the porosity of the lignin-cellulose substrate has changed. I plan on infiltrating wood with proteins of different molecular weights (my probes), then locating them with immunolabling. I have already decided on a 40000 M.W. (ovalbumin, and MN dependant peroxidase), and a 20000 M.W. (myoglobin) probe. I am still looking for an adequate probe for M.W. 5000, 10000, and 15000. By adequate probe I mean a protein that does not denature easily, and also I can purchase antibodies against. Any help would be appreciated.
Eugene Krueger, Dept of Plant Pathology, University of Minnesota.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
A question: To detect dim backscattered electron diffraction patterns, one generally uses a SIT (Silicon Intesified Tube) camera behind a phosphor screen. Hamamatsu's model of SIT has a quoted sensitivity limit of 10^-4 lux. Hamamatsu also sells (for a modest premium) an "intensified" CCD which is sensitive down to 10^-5 lux.
We know a SIT camera will work, but imagine the CCD system will work a little better.
Has anyone used an intensified CCD (Either Hamamatsu's or others')? Are there any drawbacks besides the price? Are there better SIT systems available that would rival the intensified CCD?
Thank you, Chris Krenn Graduate Student UC Berkeley Dept. of Materials Science
Are you working with tissue or purified amyloid? If tissue you need to permeablize the tissue with a detergent (such as Triton-X 100). It seems accesability is your problem. Try various concentrations of detergent, 0.1 to 1.0% for various times. Detergents are used after fixation and in all blocking, Ab's, and probe solutions. Hope this helps.
Wayne England has asked: *********************************************************************** I am having a problem getting Unicryl to polymerize properly and am left with a very brittle block or no polymerization at all. I have tried all of the manufacturer's methods (heat and UV covering all the variables) but am not satisfied with the results. Do you find this resin to be normally brittle or am I missing something? Also, does anyone know how this resin is at infiltrating plant material? It's properties sound too good to be true!! *********************************************************************** We have some experience using this polar resin, which has some ad- vantages over Lowicryl K4M but shares the general disadvantages inherent in hydrophilic resins. The labeling intensity is high but more varying than for Epon. I polymerize at -10C by direct UV in the low temperature chamber of Balzers FSU 010 freeze substitution unit for at least 2 days and using the low temperature UV polymerization insert, filled with 10 ml ethanol. The blocks become hard rubber-like and are more easily sectioned than Lowicryl. The ultrathin sections are more resistant to the electron beam than Lowicryl but not as good as Epon. The tecnicians are anxious about hypersensitivity reactions after exposure for some time. What are yours opinion? Sverker Enestr|m Dep of pathology Link|ping, Sweden
Brij 58 is another detergent that works well for immunocytochemistry. It is not as harsh as Triton and preserves a little more ultrastructure. Downside is that it often doesnt permeabilize all cells in a single preparation.
On Wed, 27 Apr 1994 EMLAB-at-opus.mco.edu wrote:
} Monica, } } Are you working with tissue or purified amyloid? If tissue you need to } permeablize the tissue with a detergent (such as Triton-X 100). It seems } accesability is your problem. Try various concentrations of detergent, } 0.1 to 1.0% for various times. Detergents are used after fixation and in all } blocking, Ab's, and probe solutions. Hope this helps. } } Ed Calomeni }
Message-Id: {9404271519.AA00742-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: microscopy-at-anlemc.msd.anl.gov Cc: dennis-at-odin.morph.med.umich.edu
One of my library patrons is studying corrosion of metal by bacteria. She needs techniques for preparing samples of bacteria on metal for SEM microscopy. She is also looking for methods of staining or gold-labelling to improve SEM sensitivity. The methods need to be applicable to a wide variety of bacteria species. Any information on best SEM conditions - voltage, angle, windows, etc. would also be helpful.
Thanks for your help.
Replies can be made directly to me at smiths-at-mlc.lib.mi.us or you can contact my patron, Cathy Stewart at 313/676-5292.
Susan Smith R&D Tech. Information Center National Steel Corp. Trenton, MI 48183
Message-Id: {MAILQUEUE-101.940427100951.630-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
We inherited a microscope slide awhile back which we would like to replace, but do not know of a source. The slide had a grid of letters on it (black background with white letters) and it was used for finding particular locations of structures on other slides; ie a certain cell on your slide was at position Xy on the reference slide. Ours is getting so beat up that it is going to fall apart any day. I am not sure if it was commercially available, or made by someone. Any help would be appreciated.
Mark Elliott UBC-Pulmonary Research Lab St Pauls Hospital Vancouver
We currently use Agfa Scientia film for (300kV) HREM. It is a little slower (maybe) than Kodak SO163, faster than 4489. It has a slightly finer grain than Kodak SO163, which helps. Two questions: 1) What are people using for faster film. The Agfa film is quite old (i.e. it has been around for at least ten years), and is there anything better at a reasonable price? 2) We have noticed some scratches at times which seem to be a quality control issue in the film. Is this common?
Scanning: the journal of scanning microscopy is one journal that you may want to check. It is published by FAMS Inc., Box 832, Mahwah, NJ 07430-0832 Phone 201/818-1010 FAX: 210/818-0086 Hope this is helpful.
Susan Smith National Steel Corp. Trenton, MI 48183
On Tue, 26 Apr 1994, Laurie Frederick wrote:
} Hi, } We are considering the purchase of an Energy Dispersive X-ray } Analysis (EDX) system for attachment to our SEM within the next year. } Our applications are in two areas; paper making and corrosion of } materials. } } I would like to know if anyone is aware of mailing lists etc., accessable } on the Internet, which are specifically interested in EDX. I would like to } follow discussions on relative merrits of detector and window } types , vacuum requirements for windowless detectors, pulse } processor requirements, software options and in particular, read any } horror stories associated with a given manufacturer. } } If anyone has opinions on what is currently available from recent } purchase deliberations or would like to recommend a review paper on } current technology or a periodical which they go to for this sort of } information it would be greatly appreciated. } } My first priority is always dependable service, but having said that, I } will be looking for a high quality detector capable of light element } analysis, high pulse throughput, good quantitation with ease of use , } beam control and image acquisition and replay capabilities. } } Thanks for your help. } } ______________________________________________________________________ } Laurie Frederick, A.SC.T. PAPRICAN } Corrosion Control Group 3800 Wesbrook Mall } The Pulp and Paper Research Vancouver, B.C. } Institute of Canada Canada V6S 2L9 } } Email: frederick_laurie-at-vanlab.paprican.ca } Tel: 604-222-3200 Fax: 604-222-3207 }
On 27 Apr 1994 MARK-at-prl.pulmonary.ubc.ca wrote of his need for a slide with a special grid on it.
My 1992 McCrone Accessories catalog has a similar slide called an "England Finder" (p/n 313). I would call them and see what they have for you. Their phone nos. are 708-887-7100 and 800-mac-8122.
1994 PACIFIC NORTHWEST EM SOCIETY SPRING MEETING on "Evolution in Microscopy: Interfacing with Computers" Fred Hutchinson Cancer Research Center - (South Lake Union) Pelton Auditorium, Building B 1100 Fairview Ave. N. Seattle, WA 98109 Friday, May 13th
12:55 p.m. Welcoming Remarks Charles Meshul, President, PNEMS VA Medical Center, Portland, OR
1:00 p.m. "The Merging Roles of EM, Microanalysis, & Computers for Characterization of Materials." Nestor Zaluzec, Materials Science Division, Argonne National Lab
1:45 p.m. "The Evolving Role of Hardware in Image Processing: What are the Requirements?" Leonard Pagliaro, Bioengineering, Univ. of Washington
2:15 p.m. Tour of New Fred Hutchinson Facility and Coffee Break
3:15 p.m. "Analysis and Handling of Cosmic Dust." Don Brownlee, Astronomy, Univ. of Washington
4:00 p.m. "Biological Applications of Microscopic Image Analysis." Grace Bartoo, Bioengineering, Univ. of Washington
4:30 p.m. "SEM Photography: Analog/Digital; B&W/Color." David Scharf, Los Angeles, CA
5:15 p.m. Adjourn
5:30 p.m. Social -at- Benjamin's on Lake Union Sponsored by: PNEMS & Corporate Members (EMS/Diatome US, Gatan Inc., JEOL USA Inc., Link Analytical/Oxford Instruments Inc., Palmborg Associates, Philips Electron Optics)
1994 PACIFIC NORTHWEST EM SOCIETY SPRING MEETING on "Evolution in Microscopy: Interfacing with Computers" Fred Hutchinson Cancer Research Center - (South Lake Union ) Rooms B1072 and B1074, Building B 1100 Fairview Ave. N. Seattle, WA 98109 Saturday, May 14th
11:00 a.m. Adobe Photoshop Software Demonstration David Scharf , Los Angeles, CA 90039
12:00 p.m. Lunch
1:00 p.m. PNEMS Business Meeting
1:30 p.m. Video Microscopy Demonstration Gary Crawford, Mideo Systems, Huntington Beach, CA
2:30 p.m. Particle Atlas Electronic Edition CD-ROM Demonstration Steve Shaffer, MicroDataWare, Hayward, CA
3:30 p.m. Real Time Imaging and Electron Diffraction Demonstration Dave Joswiak, Astronomy, Univ. of Washington
4:30 p.m. Adjourn
* This software exchange will consist of the MSA, MAS and EMC Public Domain Library having an excess of 100 megabytes of software (Mac and PC). If you are interested, bring formatted disks to make copies of the available software.
Contact : Mike Rock -at- (206) 685-7073 or Barbara Reine -at- (206) 543-1955 for Information.
PNEMS Spring Meeting Registration Form
This meeting, (May 13 & 14) covering the topic of computers interfacing with microscopes, promises to be exciting and educational. Our speakers representing a wide range of disciplines and interests, all share an expertise in using computers in their microscopic investigation. Please note the correction of the address for the meeting (the NEW Fred Hutchinson Cancer Research Center on South Lake Union, 1100 Fairview Ave. N.) Please fill out, and return, the following form to help us plan for your attending the meeting.
Registration in advance: Students...FREE Others...$ 15.00 Registration on site: Students $ 5.00 Others $ 20.00
Name:_____________________________________
Employer: Company __________________________________________
Chris Krenn asked about experiences with intensified CCD cameras. We have a Pulnix on our high-voltage electron microscope, albeit for a different application. We use it primarily for scanning grids and low dose focussing for beam-sensitive specimens. Under conditions where the illumination is barely visible on our high-brightness screen, but no details are visible, we can clearly see all we need with the video system. We can focus to the nearest setting on the objective fine control (.99 microns per step) in a few seconds, and can focus on the vernier control (1.6 microns full scale) in a few more seconds. This system is great for our applications. There is a trade-off be- tween sensitivity and resolution--especially for the inherently low-contrast images seen at high voltage. We opted for high sensitivity, and, within that constraint, maximized the resolution. We do NOT use the system for collecting ED data, since the intense cen- tral spot would damage the intensifier, even at low-dose conditions. Further- more, it is not at all clear that the CCD gives as good quantitation and sensi- tivity as LoDose x-ray film. The first point may not be a consideration for Chris's application, but the second might. Does anyone know a system with the sensitivity to quantitate small num- bers (1 or 2) of electrons, with the ability to produce a signal proportional to electron number for the more intense reflections (i.e. no saturation)? Since each electron hitting a film grain exposes that grain, it's hard to beat that quantum efficiency. Positional accuracy is less important for us than accurate quantitation, including background subtraction.
"The preparation of samples for [electron] microscopy was still a delicate task requiring skilled human hands; the preparation of a good sample was as demanding a craft as that ever practiced by an artisan -- and took almost as long to learn." Michael Crichton The Andromeda Strain
When this quote first surfaced during lunch a few weeks ago, microscopists were asking:
"Why is specimen preparation for microscopy, whether electron or light, not simple?
"Do I have to be an artisan? After all, I've got microwaves and high tech equipment - and hey, who's got the time?
"Is electron microscopy really only science fiction?
If these and similar questions have troubled you way past midnight, then this year's Spring Symposium may be for you. The application of microwaves is revolutionizing the preparation of biological specimens and, as a bonus, is drastically reducing the time involved. New techniques for minimizing damage in the preparation of materials such as ceramics, polymers, composites and biomaterials are here. Our speakers will be telling us all about these methods.
SPECIMEN PREPARATION
SHERATON INN, MIDWAY I-94 at HAMLINE AVENUE ST. PAUL, MN THURSDAY - MAY 26, 1994
SCHEDULE OF EVENTS
8:00 - 8:30 AM Coffee and Late Registration
8:30 - 8:35 AM Welcoming and Opening Remarks. Dr. Mark Cavaleri, Research Specialist, 3M Analytical & Properties Research Labs, 3M Company, St. Paul, MN.
8:35 - 9:25 AM Preparation of Advanced Materials (ceramics, composits, biomaterials) for the Microscope Dr. Don Zipperian, Manager, Long Range Planning and Development, Buehler, LTD., Lake Bluff, IL.
9:25 - 10:15 AM Specimen Preparation of Polymers for TEM Analysis and Special Applications Ms. Jacqueline Aguilera, Senior Physicist, 3M Company, Corporate Research Analytical, St. Paul,MN.
10:15 - 10:35 AM \ Coffee Break \
0:35 - 11:25 A.M. Quantitative Imaging in the Cereal Industry: Minimizing Specimen Preparation Dr. Gary Fulcher, Professor of Food Science, University of Minnesota, St. Paul, MN
11:25 AM - 1:00 PM 5 LUNCH 6 Box lunches will be provided to participants by MMS and catered by the Sheraton Inn.
1:00 - 1:10 PM Short Business Meeting Ratification of Bylaws, Election of Officers (read Bylaws below)
1:10 - 2:00 PM Rapid Microwave Fixation of Biological Specimens for Light & Electron Microscopy Dr. Gary Login, (Part One), Departments of Pathology at the Harvard School of Dental Medicine, Harvard Medical School, and the Beth Israel Hospital, and the Charles A. Dana Research Institute, Boston,MA.
2:00 - 2:50 PM Impervious Biological Specimens: Techniques and Tricks Ms. Virginia Lindley, Dept. of Agronomy, University of Arizona; Consultant for Industrial and Federal Agencies; President of Arizona EM Society.
2:50 - 3:10 P.M. \ Coffee Break \
3:10 - 4:00 P.M. A Toolkit for Calibrating and Standardizing Microwave Fixation and Staining Dr. Gary Login, (Part Two).
------------------------------------------------------------------------------- Please make your reservation in advance, POSTMARKED NO LATER THAN MONDAY, MAY 23, if you plan to attend the Symposium. Symposium Fee: $20.00 current regular MEMS/MSOM members 93/94, $30 non-member(confers regular membership), $10.00 student members 93/94, $15.00 non-member students(confers student membership). Fill out the form near the end of this newsletter and send it in as directed(prefered) or pay at the door. -------------------------------------------------------------------------------- - REGISTRATION FOR MEMS/MSOM SPRING SYMPOSIUM, MAY 26, 1994, MIDWAY SHERATON (if your registration includes a new membership, fill out and include MMS Membership Form, below). Please postmark your reservation no later that Monday, May 23.
Name__________________________Phone____________Affiliation______________________ __ # Individual Members -at- $20.00 each = $_______. # Student Members -at- $10.00 each = $________. Total Amount Enclosed = $________. Above cost for current 93/94 MEMS/MSOM members only. Non-members and renewals please include membership dues and form(see above).
Make out your check to MMS and mail it together with this form to: Dwight Erickson, MMS Treasuresr, 3M Center, Bldg. 251-1A-03, Saint Paul, MN 55144. Late reservations may pay at the door.
------------------------------------------------------------------------------- MMS(MEMS/MSOM) Membership Form: 1993-94 All microscopists are urged to support their Society at one of the membership levels offered below. The more dues-paying members we have, the more likely we are to attract sustaining corporate member- ships which form the financial backbone of our Society. Often, supervisors will support MMS member- ships out of their project budget because they recognize that it is a very inexpensive way to maintain and increase the skills of their microscopists. If you have been a member over the years and recognize the of MMS to the community of microscopists it serves, consider upgrading your membership this year to the patron or sustaining level. Thank you. Name_______________________________ Dr____ Mr____ Ms____ Phone ( )__________ Affiliation_________________________________________Position____________________ _ Address_____________________________________________________ ZIP__________ Describe your areas of interest; state manufacturer and model of instrumentation below: Bioscience___________________________________________________________________ Materials Science ______________________________________________________________ SEM____________________ TEM______________________ X-ray_____________________ Are you an MSA Member?_______ MAS Member?_______ Other Professional groups?___________ Basic $10___ Patron $25___ Sustaining $100___ Student $5___ Due by Dec 31, '93. Make checks payable to MMS and mail to our treasurer: Dwight Erickson, MMS Treasurer, 3M Center, Bldg. 251-1A-03, Saint Paul, MN 55144.
-- Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable To: Microscopy-at-anlemc.msd.anl.gov
Bill Tivol open the discusion about CCD cameras to the possible alternatives to phtographic film recording.
Did you consider the electron images plates (Fuji / Kodak / JEOL)? As far as I know (from the litterature), the DQE is about the same or better than for photographic films. The linearity is better, the dynamic range larger and they are suitable for electron diffraction. If somebody is interested I can look in my files to find more details and some references to papers (Ultramicroscopy ... a couple of years ago). Of course the complete system is a quite expensive investment(although compared to a microscope....) and seems to be available only for JEOL microscopes.
It would be nice to hear how they perform in practice from people who use them daily.
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Institut Interd=E9partemental de Microscopie Electronique Address: EPFL-I2M, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 (Central European Time) Fax: +41(21)693 44 01 ______________________ Eudora 2.0.2 __________________________________
I also had regular problems with scratches on Dupont Cronar film. I have seen no scratches on 4489.
On Wed, 27 Apr 1994, L. D. Marks wrote:
} We currently use Agfa Scientia film for (300kV) HREM. } It is a little slower (maybe) than Kodak SO163, faster than } 4489. It has a slightly finer grain than Kodak SO163, which } helps. } Two questions: } 1) What are people using for faster film. The Agfa film } is quite old (i.e. it has been around for at least ten years), } and is there anything better at a reasonable price? } 2) We have noticed some scratches at times which seem to } be a quality control issue in the film. Is this common? } } Thanks } } Laurie Marks } Northwestern University
} From: IN%"pataky-at-bcu.ubc.ca" } Subj: Cryostat help! } } Return-path: {BIOSCI-REQUEST-at-net.bio.net} } Received: from net.bio.net by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id } {01HBOOXBZ5DS8X4B47-at-gnv.ifas.ufl.edu} ; Wed, 27 Apr 1994 22:18:06 EST } Received: (from daemon-at-localhost) by net.bio.net (8.6.8.1/8.6.6) id TAA23358 } for cytonet-list; Wed, 27 Apr 1994 19:04:06 -0700 } Received: (from news-at-localhost) by net.bio.net (8.6.8.1/8.6.6) id TAA23346 for } cytonet-arpanet; Wed, 27 Apr 1994 19:04:05 -0700 } Date: Wed, 27 Apr 1994 23:50:46 +0000 (GMT) } From: pataky-at-bcu.ubc.ca (Dave Pataky) } Subject: Cryostat help! } To: cytonet-at-net.bio.net } Message-id: {pataky-270494154951-at-steevlab.generes.ca} } Content-transfer-encoding: 7BIT } Followup-To: bionet.cellbiol.cytonet } NNTP-Posting-Host: steevlab.generes.ca } } Anybody out there know why Tissue-Tek, presumably designed for embedding } samples for cryostat cutting, is so $##-at-***%* annoying to work with? The } problem: I'm cutting chick embryonic brain tissue, anywhere from 10-40 } microns, at -20 and no matter what I try the sections won't stop curling } up, rolling into tubes as soon as I lift the antiroll plate. I've tried } adjusting the blade angle, the "anti-roll" (that's a joke) plate, the } temperature, speed of cutting, new blade (disposable), nothing works. } Ideally I'd like to see nice flat sections that stay that way, preferably } "ribboning" off the blade so I can mount several at once. Know any tricks } or alternatives to Tissue Tek which may work better? Any ideas how to deal } with static electricity? (sometimes the sections stick to the "anti-roll" } plate). Feels like I'm battling the antichrist (and losing!), } } } -- } Dave Pataky } Dept of Zoology, UBC } pataky-at-bdc.ubc.ca } Brain can be particularly difficult. I suggest cryoprotecting in 20% sucrose with 3% PEG MW 400. Cut at -25. Don't expect to get a ribbon. We are usually satisfied with one section at a time. Rarely do we get two. I have a nice handout on cryosectioning that I picked up at a meeting. Give me your FAX # or mailing address and I will send a copy.
We were using SO163 but switched back to 4489. The SO163 seemed to have emulsion problems (much grosser defects than mere scratches, we had big chunks of emulsion fall out). We could never identify anything we were doing wrong and it seemed to be specific to certain Kodak lot numbers. We have not had problems since switching back to 4489 but sometimes need the faster film. Anyone else having problems with SO163 or should we try it again? We can't find a supplier of the Agfa film.
On Thu, 28 Apr 1994, Rodney L Kuehn wrote:
} } I also had regular problems with scratches on Dupont Cronar film. I } have seen no scratches on 4489. } } On Wed, 27 Apr 1994, L. D. Marks wrote: } } } We currently use Agfa Scientia film for (300kV) HREM. } } It is a little slower (maybe) than Kodak SO163, faster than } } 4489. It has a slightly finer grain than Kodak SO163, which } } helps. } } Two questions: } } 1) What are people using for faster film. The Agfa film } } is quite old (i.e. it has been around for at least ten years), } } and is there anything better at a reasonable price? } } 2) We have noticed some scratches at times which seem to } } be a quality control issue in the film. Is this common? } } } } Thanks } } } } Laurie Marks } } Northwestern University } } } }
Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable To: microscopy-at-anlemc.msd.anl.gov
Bill Tivol open the discusion about CCD cameras and the possible alternatives to phtographic film recording.
Did you consider the electron images plates (Fuji / Kodak / JEOL)? As far as I know (from the litterature), the DQE is about the same or higher than for photographic films. The linearity is better, the dynamic range larger and they are suitable for electron diffraction. If somebody is interested, I can look in my files to find the references of some (old) papers (Ultramicroscopy ... a couple of years ago). Of course the complete system is a quite expensive investment(although compared to a microscope....) and seems to be available only for JEOL microscopes.
It would be nice to hear how they perform in practice from people who use them daily. Yours
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Institut Interd=E9partemental de Microscopie Electronique Address: EPFL-I2M, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 (Central European Time) Fax: +41(21)693 44 01 ______________________ Eudora 2.0.2 __________________________________
We have been using Kodak SO163 without difficulty for years and we now use it in our JEOL JEM-4000EXII. We started using it because it is faster than 4489 and it can be pushed. The slightly larger grain does not seem to be a problem for our HREM users, but most do want increased film speed.
Russell Cook Electron Microscopy Center for Materials Research Argonne National Laboratory Argonne, IL 60439
Can someone offer suggestions for photographing Bacillus subtilis at the optical microscope level? These bacteria are small, motile and contain refractile bodies. In a fluid media there is a lot of Brownian motion and the refractile bodies create diffraction rings. We were thinking of using methyl cellulose. Can anyone suggest the proper viscosity to use? Does anyone know of a book or book chapter describing photographing bacteria?
I am just getting started trying to do low-dose HREM at high (1 MV) voltage. I have been told that SO163 developed in undiluted D-19 for 12 min is the best way to go, and I plan to try it as soon as the film arrives. Mean- while, I have been working at high mag (200kx-400kx) with Dupont LoDose. It is at least an order of magnitude more sensitive than SO163, but the grain size is also an order of magnitude larger (makes sense, equal sensitivity per grain and all that). Other drawbacks are the blue backing for LoDose and the tendency to fog and to show arrowhead-like marks from static electric discharges when very dry films are separated. Working in total darkness is also required with Lo- Dose. Murray King, at our lab, did a systematic study of the developing and fixing conditions for 4489 and LoDose to produce the best combination of sens- itivity and low fog. As a result we use 4 min in D-19 (4489) or GBX (LoDose) and 5 min fix. I'd be interested in anyone else's results with SO163. Although tedious, I believe such systematic studies are very valuable. If this particular wheel has not already been invented, I'll probably undertake the study in my copious free time (to quote from T. Lehrer).
Chris Krenn asks about using SIT cameras for detecting backscattered electron diffraction patterns.
As I understand this problem, you may not need the full sensitivity of the SIT (10^-4). A good CCD camera with exposure control may do the trick. A sensitive camera would have about .5 lux sensitivity, or less, with a nod given to the noise floor, at 1/60 sec exposure. Integrating on the chip for under a second would give around 10^-2 lux, with more available at longer times. This may be enough.
This camera would cost, maybe, $1400, or around 8% of your SIT.
My company makes a device called the OMNEX which can control these cameras, as well as do real time averaging, memory functions, measurements, digital contrast control, psuedocolor, zoom, frame storage, and lots of other things. We have a lot of people using it for applications where a little longer exposure time is all that's needed. (We also have people using it with SIT cameras for all of its other functions.)
Good luck. Send me a note if you would like any more info.
Bill Tivol posted a message regarding films for low-dose HREM. Perhaps I can suggest to use CCD camera (if one can afford it) to do a much better job. I have been doing low-dose HREM for several years now, and after taking the pains and frustrations with films, we started using slow-scan CCd camera 3 years ago. We are very pleased with the performence of the camera, and able to record low-dose HREM images with much confidence. I think CCD cameras offers the following advantages over conventional films: (1) Digital recording, i.e. on-line evaluation of image quality and fine tuning microscope operating conditions; (2) Very high sensitivity (low-dose). I am going to give a talk on low-dose HREm in the upcoming MSA meeting. If anyone is interested, please send me an email.
Ming Pan CSSS, Arizona State University panm-at-csss.la.asu.edu
Laurie asked for help on EDX selection, in this case windows:
Disclaimer: MOXTEK makes beryllium windows and ultra-thin windows for Si(Li) detectors, and supplies about half of the Si(Li) windows used world wide. I honestly tried to be objective in the following, but needed to warn you! I would be very interested in any comments, anecdotes, etc. from the microscope community on this subject.
I just wrote a chapter on thin windows for light element analysis for the book that Dave Williams and Dale Newbury are editing for the MAS. The book is entitled "X-ray spectrometry in electron beam instruments." It should be out this fall. A few items from my paper may be helpful:
If you are interested in light element analysis you should buy the spectrometer as a whole instrument for light element analysis. There is a lot more to light element sensitivity than the window (detector, FET preamp, processing electronics, software). It is possible to buy the best window, but still not get the best detection limits.
Windowless detectors have generally not lived up to their potential because of icing problems. A layer of ice can absorb as much as a thin window. If your microscope is very clean and scrupulously maintained vacuum-wise, however, this may be a good option. (This subject needs more text to treat fairly: manufacturers have done a good job to provide de-icing cycles, etc. to solve these problems.)
It is essential to maintain a steady regimen of standards testing to know the condition of the detector with all Si(Li) detectors. Two major things that can go wrong are detector icing and loss of vacuum (which will warm the detector, causing an increase of noise.)
Sometimes, to obtain better sensitivity at Be and B, a thin window is used that does not have an aluminum light blocking layer. Consider whether you need to block light from the instrument, room, or sample before selecting one of these. Even with aluminum coatings ultra-thin windows leak more light than beryllium windows do.
Since you are putting the spectrometer on an existing SEM it is a good idea to discuss your selection with both the electron microscope company and the spectrometer company. The biggest failure mechanism of thin windows is impact of particles on the window during venting. Some microscope models never have a problem, and some have a big problem. There are 'fixes' for the venting systems of problem microscopes.
The traditional reason for using something like dry nitrogen is degassing from the walls of a vacuum system, which is the major vacuum limitation (plus to some extent) of an unbaked system. If one uses dry nitrogen, then the initial chemisorbed layers tend to be quite nitrogen rich; nitrogen desorbs easily. If instead one uses air, then water and hydrocarbons (car exhaust fumes, people's breath etc) chemisorb on the walls and only very slowly leave. I must admit that with a modern microscope there is probably not that much of an effect since pumps have come a long way in pumping speed/$. However, if you are really concerned with UHV systems or are fighting contamination problems nitrogen (with very low hydrocarbon content) is probably going to help. I am not sure that the water level will be very relevant for most people.
Addendum: My mailer ate part of my last message. Leaks are also important, but one should worry more about backstreaming from roughing pumps in my experience.
Subject: Time:4:10 PM OFFICE MEMO Re: Backfill gases Date:4/29/94 This is a topic that I have discussed in some detail in a book on "Vacuum Methods in Electron Microscopy" that will be pub- lished early in May as the latest volume in Audrey Glauert's series PRACTICAL METHODS IN ELECTRON MICROSCOPY. Briefly, the purpose of backfilling with a dry gas is to reduce the amount of water vapor that is adsorbed onto the surfaces inside the vacuum system when the system is let up to atmospheric pressure. In even a moderately humid environment, or under conditions of prolonged exposure to the atmosphere, a surface can become covered with up to a hundred molecular layers of adsorbed water. Because water molecules are highly polar they adsorb to most surfaces quite tenaciously, and then desorb only very slowly when the system is pumped down again. This greatly prolongs the time required to pump down to an operating vacuum. Furthermore, unless a system can be baked out at temperatures above 200#161#C, the desorption of water also makes it very difficult to reach pressures much below the upper end of the 10-7 Torr range. You are correct, however, in concluding that there is little benefit to admitting a dry gas into the photographic chamber of an electron microscope, simply because the water evolved by the film will quickly coat the surfaces with water anyway. The same applies to bell jars, the specimen chambers of SEMs, electron guns, and other systems which are left standing open to the atmosphere for long periods of time. However, in situations where only a small hole will be opened in a vacuum system, such as when an aperture manipulator is being serviced, it is beneficial to use the dry gas, and to plug the opening loosely with aluminum foil and to maintain a slow flow of dry gas through the system while it is at atmospheric pressure. The primary concern in selecting the gas to use is that it be free of both water vapor and oil vapor. Gas pumped with an oil-sealed compressor will quickly lead to a very high rate of hydrocarbon contamination on the specimen. Other than that, nearly any dry gas will work, even dry air. Dry nitrogen is so commonly used because it is so readily available. As described in my book, it is even possible to capture the nitrogen that boils off from a Dewar flask in a plastic balloon and to use it for this purpose.
Comments: Converted from PROFS to RFC822 format by PUMP V2.2X
Thanx for the info about the image plate and CCD talks at MSA 94. I'll be at both. In both cases, the delay in readout, etc. is nothing compared to the time it takes to scan a negative with 10*10 micron pixels and to prepare the file to be small enough so that our computer doesn't choke on it. For HREM, digitization via CCD is really the way to go, and we intend to go that way if the next renewal of our grant allows us to purchase the equipment. For quan- titation of ED patterns, I don't know whether the CCD might not be overloaded at the center spot--our intensified CCD can be damaged by trying this. From the viewpoint of "making every electron count", can either method sense a single electron--actually, if each electron produces n photons, where n} } 1, this sensitivity is not as tough as it might first appear. There might be an interesting idea in trying a thicker phosphor or YAG for ED and a thinner (thus better resolution) one for HREM. Has anyone investigated this yet? In parti- cular, for our 1.2 MV electrons this makes a lot of sense to me.
Re the discussion of water-cooled xma detectors, does anyone make or use a de- tector cooled by a Peltier device? I used such a detector many years ago for proton detection. There, the detector was a silicon junction detector and was cooled in order to reduce noise; whereas, the LN cooling of Si(Li) detectors is essential to preserve the drift profile.
Alex King asked about the reasons for the use of a dry gas for airing the vac- uum in a microscope. Others have answered the why. The grade we use is the "HP nitrogen"; the UHP seems to be no better and much more $.
The detector in an EDX system is cooled to liquid nitrogen temperatures to lower the noise due to thermally generated carriers. A second reason the detector is cooled is to keep the lithium from drifting around, but modern systems can be warmed if the detector is biased.
The systems that seem to be water cooled are actually cooled with thermoelectric coolers to cryogenic temperatures. The water is used to carry away the heat from the TE coolers, which is considerable. The TE coolers cannot get the detector to 77K, which is why these systems are noisier than the LN cooled systems.
regards Mark W. Lund, PhD Director MOXTEK, Inc. Orem UT
On 27 Apr 1994 MARK-at-prl.pulmonary.ubc.ca wrote of his need for a slide with a special grid on it. I was unable to reach him with e-mail.
My 1992 McCrone Accessories catalog has a similar slide called an "England Finder" (p/n 313). I would call them and see what they have for you. Their phone nos. are 708-887-7100 and 800-mac-8122.
We use another method to provide dry nitrogen for venting, valves etc.. Rather than use bottled gas, we take the gas from our liquid nitrogen vessel. It is dried and as yet (touch wood) we have had no problems. This is after approximately 18 months operation.
The vessel is large, 2000 litre capacity but the gas is used for a TEM, SEM, quadrupole mass. spec. and a few other instruments. It is also used to vent ALL vacuum systems in the E.M. lab.
It sure beats having to change gas bottles!!
##################################################################### ********************** * Between the idea * 0------* And the reality * } ---|--- { * Between the motion * | * And the act * / \ * Falls the Shadow * _/ \_ * T.S. Eliot * ********************** Colin Veitch Tel + 61 (0)52 47 2611 CSIRO Division of Wool Technology Tel + 61 (0)52 47 2891 (dir.) P.O. Box 21 Fax + 61 (0)52 47 2657 BELMONT Vic 3216 Australia
To biological light microscopists, I want to make hydrophobic rings on glass microscope slides so that tissue-processing and immunocytochemistry can be carried out on tissue on the slide. To date, I have been smearing a thin ring of silicone (auto and caravan sealant!) and allowing it do dry. The difficulty with this is that the silicone has to be shaved off with a razor blade before the tissue is cover-slipped. I have heard of a product called a PAP pen which does the same thing, with the advantage that the hydrophobic layer is very thin. I found one such product made by "Agar" Essex, UK, distributed by "Alltech" (Australia) but the asking price seems outrageous, approx $Aus120 per pen (approx $US80). 1. Is there a similar product available from other companies (addresses, fax. nos appreciated)? 2. Is there a "home-brew" alternative, perhaps using liquid silicone?
Thanks for any advice, Dave Merritt Zoology University of New England Armidale NSW Australia dmerritt-at-metz.une.edu.au
I used to use a Sorval MT2-B. The company that serviced it for me was "RMC" in Arizona. Their phone number is 602-889-7900. They have service people all over the US. You may want to try them.
I've been charged with purchasing a high resolution coating system to use with both scanning probe and electron microscopes. My lab is primarily a scanning force microscope lab where we look at proteins and polysaccharides on polymers and cell membranes. We are interested in starting a substantial correlative microscopy effort using field emission SEM's on the same samples that we've previously probed with AFM or STM. The only coating systems currently available to us are mechanically pumped DC sputtering units. I have ~$25k to spend and have spent some time looking at the options. It looks like a turbo pumped sputtering system with a rotary tilt stage would best suit our needs. However, I'm in the dark concerning what parameters to assess in determining an optimal system. I know that grain size varies with the sputtered material. Does it also vary between different sputtering systems? Is a thermal evaporation source something to consider? Is it important to spend the money on an oiless backing pump for the turbo? What questions am I not asking that I should be asking?
To make hydrophobic rings on glass microscope slides an old homebrew method is to dilute a resin mountant such as DePex or Permount or Canada Balsam in the appropriate solvent and to use this as ringing agent. Try diluting the mountant 10 to 50 times with the recommended solvent (usually xylene or toluene) and apply with the sharpened end of an applicator stick. The advantage is that you do not have to remove the stuff before mounting under a coverslip. It is also a useful method to keep serial monitor sections in the correct order, each on its own separate little water droplet inside its own hydrophobic ring.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
We are using fluorescent dyes on bleached wood pulp fibres (ie cellulose) for confocal microscopy. We are having problems with "bleeding" or leaching of dye. Are there any fluorescent dyes which are reactive with cellulose or can be fixed similar to textile dyes (RIT, etc.) so that they will not leach out in aqeous environments? Thanks in advance.
James Drummond Pulp and Paper Research Institute of Canada Vancouver, B.C. Canada
We are using fluorescent dyes on bleached wood pulp fibres (ie cellulose) for confocal microscopy. We are having problems with "bleeding" or leaching of dye. Are there any fluorescent dyes that bind to cellulose or can be fixed similar to textile dyes (RIT, etc.) so that they will not leach out in aqueous environments? Thanks in advance.
James Drummond Pulp and Paper Research Institute of Canada Vancouver, B.C. Canada
} There is a potential alternative for bottled N2. There are driers for } compressed air that dry it to a lower dew point than lab grade N2. In } theory bleeding some of this into the tank instead of N2 from a } cylinder should work.
I haven't tried this, but you still would probably have the problem of all the hydrocarbons from a standard air compressor... How do these driers work? If they use some kind of cold trap, you might be okay. Liquid nitrogen will freeze both water and hydrocarbons out of an atmosphere.
Chris Krenn Graduate Student UC Berkeley Dept. of Materials Science
At the risk of swimming upstream (and against the apparent popular opinion), I have used PAP pens for slide mounted tissue processing, and if that is your method of choice, I think the PAP pen works just fine. I say if that is your choice only because i much prefer processing immunocytochemical material as free-floating sections rather than slide-mounted - better penetration, more uniform staining, and (extremely important) better rinsing (at least in my experience). The PAP pen, as best as I can smell, is a mixture of bees wax dissolved in xylene (or some similar concoction). It goes on easily if you have a dry area around your tissue, and it comes clean in a xylene rinse before mounting. It requires a little practice to get the right pressure so you don't get too much liquid out of the pen, but it is not brain surgery!
The PAP pen is available for about $30 US from RPI:
Research Products International 410 North Business Center Drive Mount Prospect, IL 60056
Ph: 1-800-323-9814
Let me know if you have any further questions. Cheers,
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio, TX 78284
Some of the problems can be due to using a disposable blade. We cut routinely serial cryosections of young chick brains cyroprotected in 30% sucrose and snap frozen in dry-ice or nitrogen chilled heptane and mounted on the chuck with OCT. Fixation is with just about every imaginable fix possible. Crank the temperature down, I like -25 to -35, use a real blade and keep it clean, especially the back of the blade. Sometimes I swing the anti-roll plate out of the way and use a #2 paintbrush (keep it inside the cryostat) to prevent curl - touch it to the base of the section as it gbegins to come off the blade and coordinate your arm so that you can move the brush in rythmn with the section as it moves with the cutting stroke. I've tried disposable blades for paraffin, to cut serial paraffin sections, without success. But the standard blades will allow me to cut ribbons as long as my arm can stretch holding the free end of the ribbon.
On Thu, 28 Apr 1994, Greg Erdos ICBR EM Core Lab Univers wrote:
} From: TOWER::GWERDOS "Greg Erdos ICBR EM Core Lab Univers" } To: IN%"pataky-at-bcu.ubc.ca" } CC: GWERDOS } Subj: Re: Cryostat help! } } } From: IN%"pataky-at-bcu.ubc.ca" } } Subj: Cryostat help! } } } } Return-path: {BIOSCI-REQUEST-at-net.bio.net} } } Received: from net.bio.net by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id } } {01HBOOXBZ5DS8X4B47-at-gnv.ifas.ufl.edu} ; Wed, 27 Apr 1994 22:18:06 EST } } Received: (from daemon-at-localhost) by net.bio.net (8.6.8.1/8.6.6) id TAA23358 } } for cytonet-list; Wed, 27 Apr 1994 19:04:06 -0700 } } Received: (from news-at-localhost) by net.bio.net (8.6.8.1/8.6.6) id TAA23346 for } } cytonet-arpanet; Wed, 27 Apr 1994 19:04:05 -0700 } } Date: Wed, 27 Apr 1994 23:50:46 +0000 (GMT) } } From: pataky-at-bcu.ubc.ca (Dave Pataky) } } Subject: Cryostat help! } } To: cytonet-at-net.bio.net } } Message-id: {pataky-270494154951-at-steevlab.generes.ca} } } Content-transfer-encoding: 7BIT } } Followup-To: bionet.cellbiol.cytonet } } NNTP-Posting-Host: steevlab.generes.ca } } } } Anybody out there know why Tissue-Tek, presumably designed for embedding } } samples for cryostat cutting, is so $##-at-***%* annoying to work with? The } } problem: I'm cutting chick embryonic brain tissue, anywhere from 10-40 } } microns, at -20 and no matter what I try the sections won't stop curling } } up, rolling into tubes as soon as I lift the antiroll plate. I've tried } } adjusting the blade angle, the "anti-roll" (that's a joke) plate, the } } temperature, speed of cutting, new blade (disposable), nothing works. } } Ideally I'd like to see nice flat sections that stay that way, preferably } } "ribboning" off the blade so I can mount several at once. Know any tricks } } or alternatives to Tissue Tek which may work better? Any ideas how to deal } } with static electricity? (sometimes the sections stick to the "anti-roll" } } plate). Feels like I'm battling the antichrist (and losing!), } } } } } } -- } } Dave Pataky } } Dept of Zoology, UBC } } pataky-at-bdc.ubc.ca } } } Brain can be particularly difficult. I suggest cryoprotecting in 20% sucrose } with 3% PEG MW 400. Cut at -25. Don't expect to get a ribbon. We are usually } satisfied with one section at a time. Rarely do we get two. I have a nice } handout on cryosectioning that I picked up at a meeting. Give me your FAX # } or mailing address and I will send a copy. } } ********************************************************** } * Greg Erdos ** * } * Director, ICBR EMCL ** Phone 904-392-1295 * } * 218 Carr Hall ** FAX 904-392-8598 * } * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * } * Gainesville, FL 32611 ** * } ********************************************************** } ********************************************************** } * Greg Erdos ** * } * Director, ICBR EMCL ** Phone 904-392-1295 * } * 218 Carr Hall ** FAX 904-392-8598 * } * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * } * Gainesville, FL 32611 ** * } ********************************************************** }
FOR THOSE OF YOU INVOLVED IN EDITING JOURNALS, WRITING BOOKS, ETC. IN THE FIELD, AND ANYONE ELSE WITH AN OPINION, WHAT DO YOU THINK ARE THE MOST ACCEPTABLE ACRONYMS TODAY FOR: HIGH RESOLUTION (TRANSMISSION) ELECTRON MICROSCOPY " " (SCANNING) " " LOW VOLTAGE, FIELD EMISSION, SEM HIGH PRESSURE OR ENVIRONMENTAL SEM
WOULD APPRECIATE SHORT RESPONSES WITH SUGGESTED ACRONYMS, TKS, LINDA SAWYER
We have a JEOL JSM-35C SEM in outstanding condition for sale. It was purchased in 1980 and was on service contract until April 21, 1994. It comes with a Tracor Northern (Noran) EDS detector and TN-2000 analyzer. Asking $8,000.00. Contact Dr. Stanley L. Flegler Center for Electron Optics Michigan State University Flegler-at-pilot.msu.edu
FYI, using FETCH, I reached your ftp volume at pub/ncsu/jruss, not pub/jruss as indicated in your note to the NIH Image list. Would this be a better place to exchange things rather than mailing?
Bill ========================== Bill Heeschen/Analyt. Sci.- Mat. Char. ----- 1897-F Building / The Dow Chemical Co. --- --- Midland, MI 48667 U.S.A. --- D O W --- phone: (517)636-4005 fax: (517)636-5453 --- --- Email: waheeschen-at-dow.com ----- ========================== R
Microscopy list: My apologies for that last note. It was intended for John Russ in response to one of his helpful explanations on the NIH Image mail list.
Again, my apologies.
Bill ========================== Bill Heeschen/Analyt. Sci.- Mat. Char. ----- 1897-F Building / The Dow Chemical Co. --- --- Midland, MI 48667 U.S.A. --- D O W --- phone: (517)636-4005 fax: (517)636-5453 --- --- Email: waheeschen-at-dow.com ----- ========================== R
If there are any groups in the preperation of pulp that bind feulgen, the material will give a brilliant red signal appropriate for most confocal instruments. It is very "fast" if it binds.
Bill Tivol asked: Does anyone know a system with the sensitivity to quantitate small num- bers (1 or 2) of electrons, with the ability to produce a signal proportional to electron number for the more intense reflections (i.e. no saturation)? Since each electron hitting a film grain exposes that grain, it's hard to beat that quantum efficiency. Positional accuracy is less important for us than accurate quantitation, including background subtraction.
I don't have any inherent bias towards Hammamatsu; it is merely the only vendor whose literature I've gotten so far, and whose name I was familiar with because of their image processing boxes. They have a series of CCD based photon counters which also have a wide dynamic range (10^6)
Chris Krenn Graduate Student UC Berkeley Dept. of Materials Science
Thanks for your reply. Word of your good results came through the grapevine, and was one of the reasons we are looking at CCD instead of SIT. Hopefully we will have a somewhat functional system by the end of the year.
Received: from MAILQUEUE by MICROBIO1 (Mercury 1.11); Tue, 3 May 94 11:36:44 EST Return-path: {Stanley_Hayes-at-rml.niaid.pc.niaid.nih.gov} Received: from emoryu1.cc.emory.edu by transporter.microbio.emory.edu (Mercury 1.11); Tue, 3 May 94 11:36:37 EST Received: from anlemc.msd.anl.gov by emoryu1.cc.emory.edu (5.65/Emory_cc.3.4.21) via SMTP id AA14758 ; Tue, 3 May 94 12:34:03 -0400 Return-Path: Stanley_Hayes-at-rml.niaid.pc.niaid.nih.gov
Dear Alfred,
We have a Pall air dryer, not to air the HVEM, but to pump dry air through our high-voltage tanks in the event that they have been opened. We find that using the dried air makes a big difference in residual water in the tanks after the air is pumped out and replaced by the SF6 insulating gas which is the normal fill gas for the tanks. These tanks are 2-3 meters in diameter and about 4 meters tall with a connecting piece about 1 meter**3. The air dryer has a pre-filter to remove oil, the dessicant, an oil vapor filter, and a particle afterfilter. The specified dewpoint reached is -60o F or below. It's also noisy enough that I'd hate to have to turn it on each time I wanted to air the microscope, but it could be mounted in a sound-absorbant cabinet.
Message-Id: {199405040807010548-at-qmgate.secrc.abb.se} X-Mailer: InterCon Dispatcher/SMTP for QuickMail X-Priority: 4
Looking for someone that has been working with oxidation, hydriding and wear properties of surface coated or surface modified zirconium base alloys (like Zircaloy 2 or 4). The environment is water or steam at ]300!C. I am interested in all topics related to the subject; coating or surface modification methods ( how to get a coating without defects going through the coating?), corrosion tests, investigation of tested samples (LOM, SEM, TEM, SAM, XRD, electrochemical impedance spectroscopy etc.).
Bengt Stridh ABB Corporate Research S-721 78 Vasteras Sweden E-mail: best-at-secrc.abb.se Fax: +46-21-13 41 00 Phone: +46-21-32 30 67
================================================ An Update Message from the Microscopy Listserver ================================================
G'day All Microscopy Listserver subscribers....
In sorting out a mailserver problem, today, I just noticed that about 2 weeks ago we received our 1000th subscription request. As in previous milestones, I hearby offer to by a beer (or any other appropriate concoction) for
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at the next microscopy meeting that we both attend. I'll definitely be at the MSA New Orleans meeting so Mikey whoever you are, make sure you look me up if your there. I hear tell there may be a pub/bar or two in town. Hmm.. at this rate (1000+ in 6 months of operation), I may be buying more than 1 or 2 beers in New Orleans, hope I don't exceed my limit being the quiet, unassuming person that I am. ;-)
We now have subscribers on every continent except Antarctica anyone know a frozen microscopist/microanalyst down there that's on Email??
As promised earlier in the year a new computer system has arrived, and I'm slowing getting around to shifting software to that machine. I'm having some trouble linking the MSA BBS into the new machine so things are taking longer than expected (as usual). The transition should be seemless at your end as there will be no changes in addresses or functionality just new hardware.
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FOR THOSE OF YOU INVOLVED IN EDITING JOURNALS, WRITING BOOKS, ETC. IN THE FIELD, AND ANYONE ELSE WITH AN OPINION, WHAT DO YOU THINK ARE THE MOST ACCEPTABLE ACRONYMS TODAY FOR: HIGH RESOLUTION (TRANSMISSION) ELECTRON MICROSCOPY " " (SCANNING) " " LOW VOLTAGE, FIELD EMISSION, SEM HIGH PRESSURE OR ENVIRONMENTAL SEM
WOULD APPRECIATE SHORT RESPONSES WITH SUGGESTED ACRONYMS, TKS, LINDA SAWY
Does anyone know of a low to moderately priced sharpener for carbon coater rods? The motorized ones I've seen in microscopy supply houses are going for about $1000 which seems a bit over-priced to me. I saw one once that was based on an ordinary manual type pencil sharpener, have not been able to find one. Any help on this subject would be greatly appreciated.
********************************************************************** * Glenn Poirier glenn_p-at-geosci.lan.mcgill.ca * * Electron Microprobe Lab Phone: (514) 398 6774 * * Earth and Planetary Sciences Fax: (514) 398 4680 * * McGill University THERE ARE THREE SIDES TO EVERY STORY; * * Montreal, Quebec YOUR SIDE MY SIDE AND THE TRUTH * **********************************************************************
The X-ray lab at the US Bureau of Mines has a carbon coater rod sharpener that is like a pencil sharpener. Where they aquired it i am not sure. You should contact Gary Van Landyut at PO Box 280, Rolla, Mo 65401 or call 314-364-3169. C
This may sound a little crazy but... Try a theatrical lighting company-one that specializes in high intensity spotlights. these apparently use carbon rods which are sharpened in a similar manner.
Mark Elliott UBC-Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, BC
I'm interested in any recommendations for cooled CCD cameras and associated MAC software for light-level immunofluorescent studies. We currently use a Hamamatsu SIT camera and NIH Image. The sensitivity is OK for our studies, but I'd like better resolution. Our cells are very small and we want to image several monoclonals against antigens in the same cell. What are the advantages of the various CCD brands? Has anyone used IPLabs software with a Power Mac? Will we see a big improvement in resolution over the SIT camera (an NTSC-based video camera) we now use? Thanks for any replies.
Does anybody have a proven method (recipe and conditions) for the electropolishing of Ta single crystal samples? Thanks. Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Date 5/6/94 Subject Carbon Rod Sharpeners From Chris Bowser To Magnavox Internet
Subject: Time:7:03 AM OFFICE MEMO Carbon Rod Sharpeners Date:5/6/94 smtp%"microscopy-at-anlemc.msd.anl.gov"
I have a carbon rod sharpener that I purchased about 10 yr ago from the Ernest F Fullam company. You might try them. Also the Ted Pella company has a small hand sharpener for about $40.00.
We are planning on using freeze-substitution as our standard protocol for specimen preparation for thin sections. I would like to get an idea of how many subscribers to this newsgroup use freeze-substitution and what general comments you may have. I have several questions, some of which cannot be answered by reading published methods. We will be freezing our specimens (bacteria) in liquid nitrogen cooled liquid propane. Should a special grade of propane be used for this? What safety precautions should be used with liquid propane? What types of substitution media are preferred? I have seen that both methanol and acetone are commonly used as solvents. Most substitution media contain glutaraldehyde, osmium tetroxide and uranyl acetate. Are there any tricks to making this mixture up? Once the substitution medium has been made up, should it be stored frozen in liquid nitrogen? Thanks in advance for any comments.
J. W. Austin Microbiology Research Division Food Directorate Health Protection Branch Health Canada Ottawa, Ont.
The Microscopy Listserver has been down due to hardware problems for the last 2-3 days. Things should be back to normal shortly. Please report any major problems to: Zaluzec-at-anlemc.msd.anl.gov
Sometimes ago I posted a questions about difference between digital and other type confocal. I got an address for a server group. I tried subcribing, but could not. Does anyone has that server address, and can someone give me information about confocals.
S. D. Walck's contribution to the backfill question had a lot of good things to say. I'd like to add two cents worth: Using teflon tubing, available, e.g. from Cole-Parmer works better than tygon in LN2.
i'm looking for a U.S. distributor, if there is one, for vertical slide staining dishes and racks. like standard dishes + racks, but the slides go in vertically and use less solution, and can hold more slides (25x75)than a coplin jar - 20 or 30 i think. they're evidently standard in Japan, and we have a quote from a Matsunami Trading Co. in Osaka for them at $10.70 for racks and $11.36 for dishes, which is reasonable, but Air Parcel Post at 2/3 of the list price isn't. so i was wondering if anyone might know of a U.S. source for these things. haven't come across any in the obvious places.
------------------------------------------------------------------ |Glenn Holm Internet:karuzis-at-wccf.mit.edu| |M.I.T Dept. of Brain + Cog. Sci. Bitnet:karuzis-at-mitwccf | |Cambridge, MA 02139 "Real Neuroscientists don't do gels!" | ------------------------------------------------------------------
Try Fisher Scientific - the staining sets are plastic, hold from 100-250 ml depending on how much of the slide you need immersed, and the racks hold 25 slides. The only problem I have found using these setups is that evaporation of solution is serious if you leave them sit. I keep my solutions in bottles and pour them back when I'm finished staining (the ones that aren't made up fresh of course). The sets are made by Tissue-Tek.
Cheers
David Morilak Dept Pharmacology UT Health Science Center San Antonio morilak-at-uthscsa
I was wondering if anyone could recommend a couple of good books on TEM and SEM of plant materials. I've always worked with animal and human tissue, and microorganisms so all of my books are related to these topics.
Thanks,
Phil Rutledge p.s. If you know the publisher and ISBN that would help also.
Message-Id: {MAILQUEUE-101.940511084750.751-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
Does anyone know of an easy way to fix a scratch on a 40X dry objective lens for an Olympus scope, other than returning it to Olympus??? Someone here scratched their's and are on limited budget and need it fast. Any suggestion greatly appreciated.
Thanks, Mark Elliott UBC-Pulmonary Research Lab, Vancouver
Message-Id: {MAILQUEUE-101.940511095225.756-at-prl.pulmonary.ubc.ca} To: MICROSCOPY-at-anlemc.msd.anl.gov
a FEW SUGGESTIONS; Introduction to Biological Electron Microscopy: Theory and Techniques, By Clinton J. Dawes Ladd Research Industries, Inc, Burlington Vermont, Library of Congress # 86-082930-not sure of ISBN #
Also Books by Robards, not sure of title-if find will let you know. Can also check for books by Hyatt, not sure which one but one has plant material in it.
Depends on what type of plant material you are dealing with-Fungi, marine algae or vascular plants-they are all different and their methods of preparation are different.
We have been using Denka LaB6 {100} 60 deg filaments in our JEOL 4000 with less than great results. Brightness is poor and after a week of use the center of the filament is small and dim and most of the illumination is from the lobes. Has anyone tried the LaB6 filaments from Kimble Physics or FEI in a JEOL 4000? Do they seem better or worse? Do they last?
Thanks Roseann Csencsits Electron Microscopy Center Argonne National Laboratory Argonne, IL
If anyone has a Noran 5500 no longer in service with good display boards and wants to make a deal. Please contact me directly by e-mail or voice.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
} We have been using Denka LaB6 {100} 60 deg filaments in our JEOL 4000 } with less than great results. Brightness is poor and after a week of } use the center of the filament is small and dim and most of the } illumination is from the lobes. Has anyone tried the LaB6 filaments } from Kimble Physics or FEI in a JEOL 4000? Do they seem better or } worse? Do they last? } } Thanks } Roseann Csencsits } Electron Microscopy Center } Argonne National Laboratory } Argonne, IL
We were trying the Kimball filaments when I first got here two years ago. After blowing a few, about which they were VERY understanding and generous, we decided to go back to tungsten, until everyone could sit down and figure out what was happening.
Peter Sewell, who makes the filaments, has been very helpful and accessible. At the time, he told me that they were doing some more testing on why they were getting runaway heating in JEOL scopes, but not Philips. It has to do with the control circuitry of the gun and what is being regulated. (I'm neither a physics nor an electronics whiz, so please don't ask for more specifics from me!)
The bottom line is that conditioning of the filament to be used in a JEOL, as well as conditioning of the gun, is essential if you're going to use their filaments. We have a JEOL 2000 EXII and have adopted their procedure at installation of tungsten filaments. We haven't gone back to trying the Kimball ones yet.
Filament conditioning: after installation, set gun bias to zero, turn up filament heating SLOWLY, when the vacuum starts to degrade stop, when it stabilizes continue turning up till you get to near your normal operating conditions. Only after heating the filament and getting beyond the outgassing should you turn up the bias to get emission. After conditioning this way, you shouldn't get any more outgassing. From what I can tell, you should be able to do this at any accelerating voltage. This conditioning has taken me one and a half hours. Be patient. I'm expecting very long filament life after this treatment.
I'd be interested in others experiences with this kind of procedure.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
} From: IN%"jgoodhouse-at-molecular.Princeton.EDU" "Goodhouse, Joseph" } Subj: Silver enhancement, ultra structure } } Return-path: {jgoodhouse-at-molecular.Princeton.EDU} } Received: from anlemc.msd.anl.gov by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id } {01HC7XIRCF4G8X5IJZ-at-gnv.ifas.ufl.edu} ; Wed, 11 May 1994 16:49:27 EST } Date: Wed, 11 May 1994 09:48:00 -0400 (EDT) } From: "Goodhouse, Joseph" {jgoodhouse-at-molecular.Princeton.EDU} } Subject: Silver enhancement, ultra structure } To: Micrscopy {Microscopy-at-anlemc.msd.anl.gov} } Message-id: {2DD0E2AB-at-molecular.princeton.edu} } X-Mailer: Microsoft Mail V3.0 } Content-transfer-encoding: 7BIT } Encoding: 10 TEXT } } } I am trying to do silver enhancent on 3nm gold probes in Drosophila embryos } for ultra structure morpholgy. The problem I am experiencing is that the } reaction is occurring too rapidly and is blowing the embryos apart. Can } some one direct me to a few good references on this technique for cells and } tissues, or provide me with a working protocol. Much appreciation } Joe Goodhouse } Dept. of Molec. Bio. } Princeton University } jgoodhouse-at-molecular.princeton.edu #################%%%%%%%%%%%%%%%%%%%%%%%%%%%##################%%%%%%%%%%%%%% If you are using one of the daylight kits like the one from BioCell you might try diluting the reagents with water to slow the reaction. Their rep. told me this at a meeting one time. Maybe lowering the temp would work too ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * ********************************************************** ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
} From: IN%"KARUZIS-at-wccf.mit.edu" "GLENN HOLM" } Subj: RE: U.S. supplier for vertical staining dishes? } } Return-path: {KARUZIS-at-wccf.mit.edu} } Received: from WCCF.MIT.EDU by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id } {01HC7V1A7RVK8X8BBU-at-gnv.ifas.ufl.edu} ; Wed, 11 May 1994 15:38:11 EST } Received: from wccf.mit.edu by wccf.mit.edu (PMDF V4.2-14 #2603) id } {01HC7UYC8CK48WXF5V-at-wccf.mit.edu} ; Wed, 11 May 1994 15:37:26 EST } Date: Wed, 11 May 1994 15:37:26 -0500 (EST) } From: GLENN HOLM {KARUZIS-at-wccf.mit.edu} } Subject: RE: U.S. supplier for vertical staining dishes? } To: GWERDOS-at-gnv.ifas.ufl.edu } Message-id: {01HC7UYC8CK68WXF5V-at-wccf.mit.edu} } Organization: Mass. Inst. Tech. - Whitaker College } X-VMS-To: IN%"GWERDOS-at-gnv.ifas.ufl.edu" } MIME-version: 1.0 } Content-transfer-encoding: 7BIT } } greg- } } thanks for the reply. i looked at the Fisher staining dish, but what we } want is a bit larger and holds the slides vertically in a rack, not in } grooves in the side of the dish like a coplin jar. will keep hunting. } ------------------------------------------------------------------ } |Glenn Holm Internet:karuzis-at-wccf.mit.edu| } |M.I.T Dept. of Brain + Cog. Sci. This VAX doesn't do NeXTmail | } |Cambridge, MA 02139 "Real Neuroscientists don't do gels!" | } ------------------------------------------------------------------
############%%%%%%%%%%%%%%%%##############%%%%%%%%%%%%%%########## I just remembered where I saw it. Shandon & Lipshaw Catalogue. Purveyors of goods for pathology, cytology and mortuary. Call 800-547-7429 and they will send a catalogue that smells like a morgue. Check out page 184 for a rack that holds 38 slides vertically on end.
Or if you are close to a hospital pathology lab or autopsy lab they may have the catalogue.
A few years ago, I used a very nice silver enhancement kit from Janssen called IntenseII. I used it only for light microscopy, but I know that people have used it successfully for EM as well. It was simple and quite forgiving with regard to development time. I also believe the reaction can be slowed by diluting the reagents. I don't remember who the US distributor for Janssen is now, but for some reason I'm thinking it might be Vector.
Cheers
David Morilak Dept Pharmacology UT Health Science Center San Antonio morilak-at-uthscsa.edu
Dr. Avalos, I lost your E-mail address. Please send it. Thank you. My apologies to the others on the list. Dr. Stanley L. Flegler flegler-at-pilot.msu.edu
I can't afford a new scanner but the thought of upgrading my Hitachi S450 with the Semicaps imaging system has excited me. My questions are...does anyone have the system on an analog scope? How does it grab the image off the scope? Can I use it with my TEM also? Is it as good as the other systems out there?
John Grazul Rutgers University Electron Microscope Facility
I saw a request recently from Michael Winton concerning TEM sample preparation of silicon on sapphire, but I saw no responses posted to the listserver.
Did anyone out there have a good (or reasonably good!) answer for him?
Thanks!
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
Now I remember. The comercial daylight kits for silver enhancement require the addition of gum arabic in order to slow the reaction. I can't immediately recall the deatails but maybe it will ring a bell in a younger brain ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
} Does anyone have proven sample preparation techniques for thin film } silicon on sapphire substrates?
Hi, Michael:
I tried to post this mail last weekend but the listserver was down. I hope this can be any help to you.
I made such a sample once. It came out beautifully. I assume that you are talking about cross-sectional TEM sample because it is easy to make plane-view sample. The recipe is as following:
1. Stack two pieces face to face together with a very thin layer of M-bond. Curing samples at 170 degree C for two hours. 2. Grind and polish one side. (with SiC paper first then finish with diamond paste). The thickness of the sample is 100 um now. 3. Dimple the other side to less than 25 um. 4. Ion mill the sample.
The experiences of grinding, polishing and using dimpler are necessary. Since sapphire is brittle and hard, you need patience. Good luck. -- Tan-Chen Lee Cornell University tanchen-at-msc.cornell.edu
Dear Clayton Smith: Thank you for your letter of May 4, 1994 and the catalog. Dr. Curzon will make a decision. My apologies to the others on the list. Sandy Burany
Subject: Time:1:49 PM OFFICE MEMO N2 from LN2 Date:5/12/94 In the recent discussions about using dry nitrogen to backfill vacuum systems, several of us have mentioned collecting the gas that boils off liquid nitrogen storage vessels and using it for this purpose. There is a very inexpensive way to do this that I devised some 20 years ago and used for many years with good success. What you do is to run a flexible, non-collapsible plastic tube (I prefer polyethylene tubing to Tygon, because Tygon seems to exude so much plasticizer - which might find its way into the vacuum system) from the liquid nitrogen container to the gas inlet of the vacuum system. At a convenient site, insert a tee joint into this tube. A large, flexible, inflatable, plastic container (a beach ball or childs toy animal) is attached to this tee joint by means of a length of soft, highly-flexible surgical rubber tubing. Before installing this tubing, however, take a sharp scalpel or razor blade and make a clean slit about 100 mm long in it. This slit will ordinarily close tightly enough so that the nitrogen from the storage vessel will flow into the plastic ball. When the ball becomes full, however, the slit will serve as a primitive pressure release valve by opening slightly to allow the gas to escape, thereby preventing the ball from rupturing. When the gas inlet is opened, the dry nitrogen in the ball will flow into the system under atmospheric pressure, and so there is no danger of over-pressurization. A small weight can be placed on the ball to sustain flow after the system reaches atmospheric pressure, if desired. A ball that is a half meter in diameter will store enough gas to fill most laboratory systems several times. This is a very inexpensive arrangement, but one that works quite well, and an inflated dinosauer that is hooked to your SEM will provide a topic of conversation for everyone who visits your lab. Again I mention that this technique, and many others, are described in my recent book on Vacuum Methods in Electron Microscopy, which can be ordered from Portland Press Ltd., c/o Ashgate Publishing Co, Old Post Road, Brookfield, VT 05036-9704, USA. (Fax: 802-276-3837); or Portland Press Ltd., Commerce Way, Colchester, CO2 8HP, UK (Fax: 0206-799331)
check Y_D Stierhof, et al J of electron microsc tech 17:336 for a nice comparison of different silver enhancement techniques
------- Forwarded Message Follows - - - - - - -
Now I remember. The comercial daylight kits for silver enhancement require the addition of gum arabic in order to slow the reaction. I can't immediately recall the deatails but maybe it will ring a bell in a younger brain ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Hello We're looking at living cultured cells in a perfusion bath maintained at 37 deg C. To minimise heat loss through the objective lens, we heat the lens during the experiments. I'm looking for a source of an appropriate immersion oil that does not change refractive index over the range 20-40 deg C to reduce any abberations in images of the cells. Any suggestions would be appreciated. Thanks Malea
************************************************************************ Malea Kneen Department of Physiology The University of Melbourne Grattan Street, Parkville, 3052 Victoria, AUSTRALIA
Re: Donald Grimes request for microscopy software details:
There are four PC packages published by the UK Institute of Materials,
titled
Scanning Electron Microscopy by John Humphreys Transmission Electron Microscopy by Peter Goodhew Electron Diffraction by Peter Goodhew Analysis in the EM by Peter Goodhew, John Humphreys and Graham Cliff
There is also a nice package on Stereographic Projection by John Humphreys, which is useful for those working with crystals.
They are all intended for introductory use (for people new to EM) and employ quite a lot of graphics. They are NOT research tools for competent microscopists. We use them with 2nd and 3rd year undergrads and people attending short courses on EM.
Each package costs about US$145. They are distributed by
Ashgate Publishing Co Old Post Road Brookfield VT 05036 USA Tel 802 276 3162 Fax 802 276 3837 (for the world except Europe)
and
Institute of Materials 1 Carlton House Terrace London SW1Y 5DB Tel 071 976 1338 Fax 071 839 2078 (for UK and Europe)
Work is in progress to upgrade these programs from DOS to Windows versions but there are no immediate plans for Mac versions (unless someone would like to volunteer to translate/re-emgineer them).
Peter Goodhew (series editor) University of Liverpool goodhew-at-liv.ac.uk (44) 51 794 4665 Fax (44) 51 794 4675
How are publication quality trace analysis results generated? I can get a stereogram with all the poles etc plotted on a computer, but how do you go about drawing the great circles (which are best done using a Wulff net} ? Some Bezier curves can be drawn to get the right curvature etc. but this would be pretty inaccurate, generally. Drawing by hand, unless done by a professional is not publication quality. Any suggestions? Thanks, Sharath dakshs-at-rpi.edu
Message-ID: {MAILQUEUE-101.940513102236.352-at-bmg.bhs.uab.edu} To: microscopy-at-anlemc.msd.anl.gov
Don, Signal Analytics Corporation makes several exellent digital imaging packages sold under the name IP Lab Spectrum for the Macintosh. Their forte is that the software is easy to install, configure, and use and the price is relatively (?) inexpensive, their base package starting around $1500. We have such a system up and running in our digital imaging facility at the University of Alabama at Birmingham and have had great success with it. Their address is:
Signal Analytics Corporation 440 Maple Avenue East Suite 201 Vienna, Virginia 22180
Phone 703-281-3277 Fax 703-281-2509 They are not on Internet yet but anticipate entering the network in the near future. I am currently working with the company to set up a user's group archive that will be accessable via Internet using Gopher. We hope to have this up an working in the next month or two. Any input from any interested parties would be greatly appreciated
Kevin McCarthy Kevin McCarthy Assistant Professor Department of Cell Biology University of Alabama at Birmingham Birmingham, Alabama 35294 Phone 205-934-9923/9924 Fax 205-934-7029
Message-Id: {MAILQUEUE-101.940513084352.998-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
To Malea Kneen could you please repeat the last part of your message about an appropriate immersion oil. For some reason I did not recieve the complete message so am not sure what type of oil you are looking for. Thanks Mark Elliott, UBC-Pulmonary Research Lab Vancouver, Canada
As many of the readers of this list have funding grants from DoE(Energy & Water Budget), NSF, and NASA (VA-HUD-IA Budget) I thought that you might want to know about this gem which is going through the US Congress.. It's not microscopy, however, it will likely impact our research funding. Apologizes in advance if you've already seen this information, or if you are one of our International Subscribers who isn't interested in the US research budget.
Nestor J. Zaluzec ANL EMCenter
---------- WHAT'S NEW by Robert L. Park Friday, 13 May 94 Washington, DC
1. BUDGET: CONGRESS TURNS OFF THE LIGHT AT THE END OF THE TUNNEL. Yesterday, the House Appropriations Committee divvied up the FY 95 budget among the 13 Subcommittees. Only Military Construction was up from last year. Energy and Water came out $81M below the President's request. VA-HUD-IA, which includes both NASA and NSF, came out $361M below the President's request; Subcommittee chair Louis Stokes (D-OH) said he had "grave concerns about whether this allocation can fund the space station." The specter of the SSC hangs over the space station debate; supporters of the space station like to point out that the money saved from the SSC last year did not go to science. Actually, no one in their right mind expects savings from the space station to go to science either-- the important thing is stop the money from going the other way.
Alexey Sidorenko phone: (7-095) 932-88-61 Moscow State University E-mail: avs-at-srdlan.npi.msu.su Institute of Nuclear Physics http://www.npi.msu.su/people/avs/avs.html
I just got my hands on the softcover book "The internet Companion-A beginner's guide to global Networking", and recommend it highly to anyone who is not a computer expert. Even though, I used the internet now for over two years, not until browsing through the text, did I finally understand some of the things I was doing without knowing why. If everyone in the userlist were to read the book, we would probably not get certain message-types that are explicitly discouraged in chapter three of the book.
***** ************ ************** ************ *Cesar D. Fermin, Ph.D |Fax (504) 587-7389 *Tulane Medical School |Answ. Mach.(504) 584-2618 *Pathology/SL79 |Secretary (504) 584-2436 *New Orleans, La 70112 | Lab (504) 584 2521 ***** ***************** ***********************
Message-Id: {MAILQUEUE-101.940516144653.198-at-prl.pulmonary.ubc.ca} To: microscopy-at-anlemc.msd.anl.gov
Does anyone know of a similar network to the microscopy one that deals with tissue culture problems???? We are having problems with guinea pig lymphocyte stimulation with phytohemaglutinin and need advice. Any help would be appreciated. Thanks Mark Elliott Pulmonary Research LAb Vancouver Canada
Have you checked out the "Big Dummies Guide to the Internet"? It is a Hypercard stack which covers most aspects of using the Internet. It is available via ftp. I think I got my copy from sumex-aim.stanford.edu.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On 16 May 1994, Fermin, Cesar wrote:
} } Hello: } } I just got my hands on the softcover book "The internet Companion-A beginner's } guide to global Networking", and recommend it highly to anyone who is not a } computer expert. Even though, I used the internet now for over two years, not } until browsing through the text, did I finally understand some of the things I } was doing without knowing why. If everyone in the userlist were to read the } book, we would probably not get certain message-types that are explicitly } discouraged in chapter three of the book. } } ***** ************ ************** ************ } *Cesar D. Fermin, Ph.D |Fax (504) 587-7389 } *Tulane Medical School |Answ. Mach.(504) 584-2618 } *Pathology/SL79 |Secretary (504) 584-2436 } *New Orleans, La 70112 | Lab (504) 584 2521 } ***** ***************** *********************** }
Mark Elliott queries about a similiar discussion group about tissue culture. In looking through a list of numerous listservers/discussion groups out their I can up with this: MEDCC-L-at-UAFSYSB Issues related to the study of media, culture, etc. Hope this helps.
I'm sorry for my annoying message to this mailing list. I wanted to subscribe and expected to get an automatic response with information.
Thanks to everyone who helped me.
Alexey
Alexey Sidorenko phone: (7-095) 932-88-61 Moscow State University E-mail: avs-at-srdlan.npi.msu.su Institute of Nuclear Physics http://www.npi.msu.su/people/avs/avs.html
you might try any of the bio. or sci. news groups. Reading "news" can be a real chore, and you have to wade through a lot of noise, but you do find the occasional gem. There are a handful of bionet news groups that function more or less like our mail lists, ie posting of questions and answers that all interested parties get to "eavesdrop" in on. I believe you can ftp a utility called TheNews from sumex-aim which holds your hand as you custom,ize your newsgroup subscriptions and set up your sessions. It also gives a listing of the zillion or so groups that are available. Good luck.
David Morilak UT Health Science Center Dept Pharmacology San Antonio morilak-at-uthscsa.edu
I have just tried to access sumex-aim.stanford.edu to obtain a copy of "Big Dummies Guide to the Internet", but the server is not available. Does anyone know of another ftp site?
_____________________________________________________________________ | James V. Jester | Dept Ophthalmology | | jester-at-crnjjsgi.swmed.utexas.edu | UT Southwestern Medical Center | | (214)648-7215 | 5323 Harry Hines Blvd | | | Dallas, TX 75235-9057 | |__________________________________|__________________________________|
Hi there, I am sorry about not sending the complete reference for the book. I hope that the following is of help.
RE:Internet Companion-Laquey
Source for Internet comp-Laquey The publisher is Addison-Wesley Publishing Co. ISBN 0-201-62224-6 Fourth Printing March 1993 ($11.00)
However, the text can be downloaded by anonymous FTP from FTP.STD.COM. If you have a Word Wide Web (WWW) browser like Mosaic you can get it from many sources, including our here at Tulane, by looking in the category internet information and is free, I like the book because is a nice ready reference. Good luck.
P.D. Other commonly recommended texts are also available from the same source.
Just received a demo disk for the Electron Flight Simulator program from Small World. It looks interesting. Does anyone have any experience with, or comments about this product? They're asking $449. Is it worth it?
.............................................................................. John Hudak hudakjm-at-mcmaster.ca I.M.R. - Electron Optics (905) 525-9140 Ext.24890 ABB Rm. 331 FAX: (905) 521-2773 McMaster University 1280 Main St. West Hamilton, Ont. L8S 4M1 Canada
Does anyone know of a commercial source for various thin ceramic (150 - 300 angstrom thick) films on salt flats (1" x 1" minimum area)? We need these for some "non-microscopy" experiments we are considering. The compositions required are SiO, SiO2, and/or SiN. Non-stoichiometric films are OK, we only need the average molecular weight to be correct.
Of course, we need these yesterday! Thanks to all in advance.
Regards, Bill Lamberti.
William A. Lamberti Office LA-196 Exxon Research and Engineering Company Route 22 East Annandale, NJ 08801 (908)730-2144 office (908)730-2262/2104 labs (908)730-3042/3051 fax Email: walambe-at-erenj.com
John I recieved the same package yesterday. The program does indeed look useful, but I have a serious problem with the price. $500 seems like a lot to pay for a nice interface, especially when non-Window versions are available free on the EMMPDL. Just my two cents. ********************************************************************** * Glenn Poirier glenn_p-at-geosci.lan.mcgill.ca * * Electron Microprobe Lab Phone: (514) 398 6774 * * Earth and Planetary Sciences Fax: (514) 398 4680 * * McGill University THERE ARE THREE SIDES TO EVERY STORY; * * Montreal, Quebec YOUR SIDE MY SIDE AND THE TRUTH * **********************************************************************
I am shopping for a new SEM and wanted to know what active users would recommend or not recommend. Ideally, I need an actual resolution of 75 A at 25K magnification. Our expected budget will be $200,000 and we would also like a microprobe. This microscope will be multi-departmental (biological sciences and chemistry). Thank you in advance, Page Owen
Page Owen Dept. of Botany Connecticut College 270 Mohegan Ave., Box 5555 New London, CT 06320 (203)439-2147
SPECIAL ISSUE - ELECTRON SPECTROSCOPIC IMAGING AND ANALYSIS TECHNIQUES. A SELECTION OF PAPERS FROM THE 4TH EUROPEAN WORKSHOP HELD AT THE INSTITUTE OF PHYSICS, UNIVERSITY OF MUNSTER, GERMANY
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 133 - 142.
KOHLER ILLUMINATION IN THE TEM: FUNDAMENTALS AND ADVANTAGES
G. BENNER & W. PROBST Electron Optics Division, Carl Zeiss, 73446 Oberkochen, Germany
Summary
Kohler illumination is the most favourable design for the illumination path of an electron microscope with a condenser objective lens. The new illumination system of the EM 910 and EM 912 OMEGA allows both wide area (Kohler) illumination for TEM operation and spot illumination for analytical investigations. Compared to conventional systems and objective lenses with a condenser mini lens, this system offers many advantages. In addition to the homogeneous, highly coherent and parallel illumination of every point in the specimen, it offers advantages for selected area diffraction and spot scan mode. Combined with the electron optical selection of a condenser aperture, this illumination system provides the flexibility necessary to achieve optimum illumination for the specimen.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 143 - 148.
INFLUENCE OF ZERO-LOSS FILTERING ON ELECTRON OPTICAL PHASE CONTRAST
P. HIRSCH & L. REIMER Physikalisches Institut, Universitat Munster, Wilhelm-Klemm- Strasse 10, 48149 Munster, Germany
Summary
Complex scattering amplitudes are used to calculate the phase contrast of colloidal gold particles.Comparison of measurements of the phase contrast intensity at the centre of the gold particle as a function of defocus for unfiltered and zero-loss filtered images demonstrates the increase in phase contrast achieved by zero-loss filtering even for a thick carbon substrate film. The granulation of amorphous germanium films is measured by the spatial root mean square values of image intensity in a defocus series.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 149 - 159.
IMAGE-EELS: SIMULTANEOUS RECORDING OF MULTIPLE ELECTRON ENERGY- LOSS SPECTRA FROM SERIES OF ELECTRON SPECTROSCOPIC IMAGES
K.-H. KORTJE Institute of Zoology, University Hohenheim, Garbenstrasse 30, D- 70593 Stuttgart, Germany
Summary
A new approach for element microanalysis with energy-filtering transmission electron microscopy (EFTEM) is presented which was accomplished with the CEM 902 electron microscope (Zeiss, Germany). This method is called Image-EELS, because it is a synthesis of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI). Series of energy-filtered images at increasing energy losses are recorded from one area with a TV camera. In a second step the intensity of selected regions in the image stack is measured with an image analysis system and plotted as a function of the energy loss. Thus many spectra from different objects can be calculated from one image series and compared with each other. The spatial resolution of EELS is considerably enhanced, the noise is decreased because many pixels from irregular objects are integrated, and the information from ESI can be analysed as a function of the energy loss.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 161 - 169.
OPTIMAL CONDITIONS FOR THE USE OF CORRESPONDENCE ANALYSIS FOR ELEMENT DETERMINATION IN EELS IMAGES
E. S. GELSEMA, * A. L. D. BECKERS* & W. C. DE BRUIJN + * Department of Medical Informatics, and + AEM-unit Pathological Institute, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands
Summary
Correspondence analysis is used for the determination of element localization and element concentration in electron energy-loss imaging. The introduction of a new method for the quantification of element concentration embedded in the protocols of correspondence analysis may be regarded as a useful replacement of the power-law estimation technique, especially in regions of the spectrum where the latter method cannot be used. Conditions for the optimum use of the method are established.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 171 - 182.
QUANTITATIVE ELECTRON SPECTROSCOPIC IMAGING IN BIO-MEDICINE: METHODS FOR IMAGE ACQUISITION, CORRECTION AND ANALYSIS.
A. L. D. BECKERS,* W. C. DE BRUIJN,+ E. S. GELSEMA,* M. I. CLETON-SOETEMAN# & H. G. VAN EIJK# *Department of Medical Informatics, +AEM-unit Pathological Institute, #Department of Chemical Pathology, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Summary
Many questions about the metabolism of specific elements in the human body might be answered if elemental concentrations could be measured in situ in cells. With electron energy-loss spectroscopic imaging (ESI), concentrations can potentially be determined with high spatial resolution. The theory of the quantification procedure has already been derived. Many practical instrument-related problems, however, have to be solved. In the current research an energy-filtering TEM is used and the image-acquisition chain is examined in detail. Quantification requires images to be recorded over a large dynamic range. To solve this problem, the use of optical attenuation filters has been introduced. The use of the combination of a scintillator screen and a TV-camera as a detection system has consequences for the processing of the data. Corrections for the camera photometric sensitivity and , to some extent, for shading are necessary. Further consequences of such a detection system for the correction of the element-aspecific spectral background and element detection are discussed. The derived methodology is tested in several ways and finally applied for the quantitative analysis of iron in liver parenchymal cells of a porphyria cutanea tarda patient.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 183 - 193
EFFECT OF SPECIMEN THICKNESS AND CARBON CONTENT ON THE CARBON PEAK REGION IN EEL SPECTRA
R. DOOR, K. D. HABERLE & R. MARTIN Sektion Elektonenmikroskopie, Universitat Ulm, D-89069 Ulm, Germany
Summary
Amorphous carbon films of different thicknesses and combinations of carbon films with CaF2 films of different thicknesses were used to create a standard reference library of EEL spectra. The relationship between the size and shape of the carbon peak region and the low-loss part of the spectrum was determined. The steepness of the background of the C-K edge, the size of the carbon peak and the plasmon shoulder on top of the carbon peak were investigated as a function of the thickness of the films and of the relative amount of carbon after normalization of the zero- loss peak. The minimum at the C-K edge and the maximum of the carbon peak were considered as examples for a modelling of the carbon peak region in different specimens. Methodological aspects which affect the accuracy of the measurement (characteristic lines of the detector, calculation of specimen thickness, method of t/lamda measurement, mass loss, energy resolution) were examined. The results indicate that modelling of the whole carbon peak region, considering its dependence on mass thickness and carbon concentration, should be possible from our standard reference spectra without use of the low-loss region. The use of modelled standard reference spectra for background extrapolation in biological specimens with unknown calcium content is proposed.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 195 - 206.
APPLICATION OF RECORDING AND PROCESSING OF ENERGY-FILTERED IMAGE SEQUENCES FOR THE ELEMENTAL MAPPING OF BIOLOGICAL SPECIMENS: IMAGING-SPECTRUM
JEAN-LOUIS LAVERGNE, * + COLETTE FOA, # PIERRE BONGRAND, # DOMINIQUE SEUX ++ & JEAN-MICHEL MARTIN* * Ecole Centrale de Lyon, Laboratoire de Tribologie et Dynamique des Systemes, URA CNRS 855, Departement de Technologie des Surfaces, F-69131 Ecully Cedex, France + KODAK-PATHE, Laboratoire d'Analyses, Z.I. nord, F-71102 Chalon sur Saone, France # INSERM U 387, Laboratoire d'Immunologie, Hopital St Marguerite, F-13277 Marseille Cedex 9, France ++ Laboratoire de Developpement et de Pathologie des Tissus Dentaires, UPR CNRS 412, Faculte d'Odontologie, F-69372 Lyon Cedex 8, France
Summary
Computerized energy-filtered transmission electron microscope (EFTEM) permits the recording and the processing of energy- filtered images, allowing a part of an electron energy-loss spectrum for each picture element to be obtained. This method, called 'Imaging-Spectrum', uses a Zeiss CEM902 coupled to several image analysis systems. The actual configuration records sequences of 48 images, 256 x 256 pixels, in steps of the energy loss, þE . Processing these sequences results in part of a core- loss EELS-spectrum for each pixel. This approach produces elemental maps with a short processing time. We have implements three kinds of background calculation for the image subtraction. The influence of the irradiation dose and of the energy selecting slit width on the quality of the spectra is investigated. The method is applied to the analysis of some biological specimens (pericellular coat behaviour during adhesion between macrophages and red blood cells and location of calcite microcrystals in dental pulp cells). The Imaging-Spectrum method appears to be suitable for the analysis of large areas.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 207 - 223.
EVALUATION OF LANTHANIDE TRACER METHODS IN THE STUDY OF MAMMALIAN PULMONARY PARENCHYMA AND CARDIAC MUSCLE BY ELECTRON ENERGY-LOSS SPECTROSCOPY
H. FEHRENBACH, A. SCHMIEDL, F. BRASCH & J. RICHTER Abt. Elektronenmikroskopie, Zentrum Anatomie, Kreuzbergring 36, D-37075 Gottingen, Germany.
Summary
Lanthanum (La) has widely been used as a tracer to study the integrity of plasma membranes. With conventional transmission electron microscopy (cTEM), the absence of electron scattering deposits from the cytoplasm has generally been assumed to reflect an intact cell membrane. However, the application of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) reveals that electron scattering deposits may be present which do not contain La. However, La could be detected in regions of pulmonary parenchyma and cardiac muscle that were devoid of electron scattering deposits. Therefore, to exclude misinterpretations based on CTEM the application of microanalytical techniques is strongly recommended for the study of the integrity of plasma membranes by means of La tracers. In addition, ESI and EELS are shown to distinguish between different tracers in simultaneous applications of La and terbium (Tb) which were used at the different faces of the pulmonary air-blood barrier. The analysis of the distribution of both tracers which form electron scattering deposits, indistinguishable by cTEM, may help us to understand the different functional significance of cellular alterations of both cellular borders of the barrier. As was shown for La, however, strictly controlled conditions are mandatory during the fixation procedure because an increase in the incubation time to more than 1 h in samples of pulmonary parenchyma may result in the occurrence of La deposits within the cytoplasm. In the absence of electron scattering deposits, the presence of La in glycogen granules and ribosome-containing areas of various types of alveolar septal cells even after 15 min incubation indicates that the absence of deposits does not necessarily correspond to the absence of the tracer.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 225 - 232.
DEMONSTRATION OF ALUMINIUM IN POLYPHOSPHATE OF LACCARIA AMETHYSTEA (BOLT. EX HOOKER) MURR. BY MEANS OF ELECTRON ENERGY- LOSS SPECTROSCOPY
I. KOTTKE * & F. MARTIN + * Universitat Tubingen, Botanishches Institut, Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, D-72076 Tubingen, Germany + INRA, Laboratoire de Microbiologie Forestiere, Centre de Recherches Forestieres, 54280 Champenoux, France
Summary
Toxic aluminium species in acidified soil damage the rootlets of trees. The symbiotic association with ectomycorrhizal fungi may, however, protect the rootlets by sequestration of aluminium in polyphosphates. Electron energy-loss spectroscopy was used to identify the relative amount of aluminium in the polyphosphate of the mycelium of the ectomycorrhizal fungus Laccaria amethystea. A three-window method at the L-2,3 edge yielded results which were in agreement with the spectra obtained at the aluminium K edge. The aluminium net distribution images were recorded at 85 eV, the backgrounds at 70 eV and 60 eV, setting the energy selecting slit to 5 eV and the beam current to 15 microA. The method can be used to study the sequestration of aluminium in mycelia of ectomycorrhizal fungi under different stress situations.
Journal of Microscopy, Vol. 174, Pt. 3, June 1994, pp. 233 - 238.
ESI IN SITU ANALYSES OF EXTRACHROMOSOMAL RNP GRANULES
S. ABOLHASSANI-DADRAS, * G. H. VAZQUEZ-NIN, *# O. M. ECHEVERRIA, *# V. BOUTINARD ROUELLE-ROSSIER* & S. FAKAN* * Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland # Laboratory of Electron Microscopy, Department of Biology, Faculty of Sciences, National Autonomous University of Mexico, Mexico City, D.F. 04510, Mexico
Summary
Observation of unstained ultrathin sections of salivary gland cells of Chironomus thummi and C. tentans, by means of electron spectroscopic imaging (ESI), has revealed that phosphorus is distributed in two types of granular structures in the nucleoplasm of these cells. In addition to a specific type of premessenger ribonucleoprotein (RNP) particle, known as the Balbiani ring (Br) granule, ESI revealed a new type of phosphorus-rich small granular component. Examination of unstained sections by energy-filtering transmission electron microscopy (EFTEM) offers the opportunity of obtaining the signal from the specimen alone, thus avoiding the possible contributions of heavy metals present in any staining product.
I need a reviewer for a short manuscript on the use of electron microscopy in the examination of minerals and soils. If any subscribers are qualified and willing to help in this matter please contact me directly.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
A recent posting listed the FTP site at STD.COM for obtaining the Internet Companion. It would be a great help if the posting individual could provide pointers to the directory where it is listed. The search engines always seem to be tied up when I try to log on, so this would be a help. Thanks.
Does anybody have any information regarding the thickness and roughness of nitrocellulose in amyl acetate (0.1%)? The procedure we used was to take 2 drops of the solution in a pasteur pipet and cast onto a very clean glass coverslip. This technique is used for a motility assay of actin filaments on myosin absorbed on the nitrocellulose layer. Any references or protocols would also be helpful.
Margaret (Peggy) Bisher
NEC Research Institute, Inc. 4 Independence Way Princeton, NJ 08540
Subject: Time:3:32 PM OFFICE MEMO LM Date:5/19/94 I am doing basic morphological studies on rat kidneys. I am looking at 1 micron epon sections that have been processed for standard electron microscopy. I have a few questions on what I am looking at, and I am looking for someone that I can talk to that might be able to answer some simple questions such as what kind of artifacts will you get when you perfuse, or is a perfused kidney even preferred over a biopsy, how much variation is there from rat to rat, etc., etc.. If anyone has experience working with rat kidney, I would really appreciate it if you could contact me. Thank-you, Jeanne
There is a new Macintosh software for electron diffraction and crystallographic calculations developed by Drs. J. M. Zuo and H.R. Zhu. Preliminary report can be found in last year MSA proceedings. This program plots crystal structure, stereoprojection electron diffraction pattern (SAD and CBED) and backscattered kikuchi pattern with a simulated microscope control panel. Intensity of kinematic approximation is used. The program also has calculator for complex numbers and vectors of real and reciprocal space, and functions for calculating electron and x-ray structure factors and Bragg angles etc. For more information contact
We have an Akashi/ISI EM-002A TEM (1988), one of only eight or nine made. I am keen to get in touch with any other owners of these instruments with regards to sorting out technical problems and obtaining spare parts. This would include owners of the EM-002B, which is similar in many ways. Yours faithfully,
Richard Easingwood EM Unit,School of Medicine University of Otago Dunedin New Zealand
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
I recently downloaded the Internet Companion successfully, as follows:
site: ftp.std.com dir: \ftp\OBS\The.Internet.Companion files: internet.companion (349 910 bytes) (Text version) internet.companion.Z (zipped version, Unix) COPYRIGHT Order.form Tracy.gif (I assume a .gif of the author, Tracey Laquey)
Note: if you are downloading to a DOS machine, you will have to save the files to a DOS-acceptable filename..(eg internet.txt) The ftp-site filename of {internet.companion} is OK in Unix, but not in DOS. [The same applies if you ever download uuencoded or zipped files. If the filename within the uuencoded file is more than 8 characters or contains unnacceptable characters, the file may not be uudecoded on a DOS machine. It is then necessary to change the filename within the compressed file (using any text editor) before uudecoding etc.]
Hello Ca imagers! We have been trying to image Ca transients in some cells (neutrophils and some mouse macrophages) following cross-linking of surface receptors but we have been running into some difficulties. Our hardware configuration is as following: We are using an intensified Hamamatsu CCD camera mounted on the side port of a Zeiss Axiovert. We are using the following filters: excitation 340nm 10nm FWHM or 350nm 10nm FWHM dichroic 375nm emission 405nm 45nm FWHM 490nm 45nm FWHM
The objective is a Zeiss 40X UltraFluar. We also use OD filters in the excitation path to minimize bleaching of the probe (usually 25 or 50% transmission). The dynamic range of the measurement, in terms of the ratio between the Ca-free and Ca-bound ratios, is only ~10, which may be insufficient to give good accuracy at Ca concentrations away from the Kd of the probe! This problem is compunded by the 8-bit accuracy of the imaging system (Image 1/FL, Universal Imaging Corp. using a Matrox video board). Does anybody have comments? Since most of the papers report values of [Ca]i and not the raw ratio values, I cannot judge whether we are doing something wrong! Is this a common observation? Comparable measurements with a spectrofluorometer on cells in suspension give us a dynamic range (as defined above) of ~40. Is anybody willing to share her/his experience? Any comments/suggestions will be greatly appreciated.
Thank you in advance!
************************************ *Massimo Sassaroli * *Dept. of Physiology and Biophysics* *Mount Sinai School of Medicine * *New York, NY 10029-6574 * *Tel: 212-241-9512 * *sassaroli-at-msvax.mssm.edu * ************************************
P.S. : Is there any other interest group dealing specifically with Ca measurement techniques? Thanks again
Hello, Last week the results of a poll on how people record their images was posted. After briefly scanning the replies, I deleted the file. Now I find myself wishing I hadn't. Could someone email the file to me? Is there an archival site where I could retrieve it from (seems to me I saw this listed here before). The reason I would like to recover this file is that we are contemplating interfacing a PC with our Hitachi S-4000 FESEM. Anyone who has any suggestions, please reply.
___________________________________________________________________ Randy Nessler rnessler-at-uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
We have an Akashi/ISI EM-002A TEM (1988), one of only eight or nine made. I am keen to get in touch with any other owners of these instruments with regards to sorting out technical problems and obtaining spare parts. This would include owners of the EM-002B, which is similar in many ways. Yours faithfully,
Richard Easingwood EM Unit,School of Medicine University of Otago Dunedin New Zealand
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
We have an Akashi/ISI EM-002A TEM (1988), one of only eight or nine made. I am keen to get in touch with any other owners of these instruments with regards to sorting out technical problems and obtaining spare parts. This would include owners of the EM-002B, which is similar in many ways. Yours faithfully,
Richard Easingwood EM Unit,School of Medicine University of Otago Dunedin New Zealand
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
Message-Id: {1994May23.141113.859926163-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy list)
I am trying to subscribe to the NIH-Image Listserver and was told that the address was listserv-at-soils.umn.edu and the message should be subscribe NIH-IMAGE {your name}
However when I did that I got a return receipt from listproc-at-soils.umn.edu that indicated that there was no NIH-IMAGE to subscribe to. Does anyone know the correct address for subscription. Thanks Judy Murphy San Joaquin Delta College Dept. of Microscopy Stockton, CA 209/474-5284 murphy-at-ms.sjdccd.cc.ca.us
The message, "No matches to nameserver query," is generated whenever the ph nameserver fails to locate either a ph alias or name field that matches the supplied name. The usual causes are typographical errors or the use of nicknames. Recommended action is to use the ph program to determine the correct ph alias for the individuals addressed. If ph is not available, try sending to the most explicit form of the name, e.g., if mike-fox fails, try michael-j-fox.
--------Unsent Message below:
Received: from anlemc.msd.anl.gov by nexus.uiowa.edu (1.38.193.4/931201) on Mon, 23 May 1994 23:41:13 -0500 id AA04787 with SMTP
Message-ID: {MAILQUEUE-101.940513102236.352-at-bmg.bhs.uab.edu} To: microscopy-at-anlemc.msd.anl.gov
Don, Signal Analytics Corporation makes several exellent digital imaging packages sold under the name IP Lab Spectrum for the Macintosh. Their forte is that the software is easy to install, configure, and use and the price is relatively (?) inexpensive, their base package starting around $1500. We have such a system up and running in our digital imaging facility at the University of Alabama at Birmingham and have had great success with it. Their address is:
Signal Analytics Corporation 440 Maple Avenue East Suite 201 Vienna, Virginia 22180
Phone 703-281-3277 Fax 703-281-2509 They are not on Internet yet but anticipate entering the network in the near future. I am currently working with the company to set up a user's group archive that will be accessable via Internet using Gopher. We hope to have this up an working in the next month or two. Any input from any interested parties would be greatly appreciated
Kevin McCarthy Kevin McCarthy Assistant Professor Department of Cell Biology University of Alabama at Birmingham Birmingham, Alabama 35294 Phone 205-934-9923/9924 Fax 205-934-7029
= = POSITION AVAILABLE = = Lab Manager for the Electron Microscopy Core Laboratory of the =Interdisciplinary Center for Biotechnology Research at the University =of Florida. = =DUTIES: Preparation and examination of biological specimens for = transmission and scanning electron microscopy in a facility that = serves the entire university community on a fee for service = basis. Collaborative research possible as part of the program. = Incumbent is expected to deal directly with the clients on = experimental design, execution of the project and preparation of = the data for publication, in consultation with the Scientific = Director of the laboratory. General lab management. = =QUALIFICATIONS: = experience in most aspects of biological electron microscopy. = Good communication skills, both oral and written. Ability to = deal on a one to one basis with investigators from a wide variety = of disciplines and backgrounds. = =STARTING DATE: Immediately = =APPLICATION DEADLINE: Open until position is filled. = =SALARY:$22,633 - 26,500. = =Inquiries by E-mail or full resumes to the address below = =********************************************************** =* Dr. Greg Erdos ** * =* Director, ICBR EMCL ** Phone 904-392-1295 * =* 218 Carr Hall ** FAX 904-392-8598 * =* University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * =* Gainesville, FL 32611 ** * =**********************************************************
To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: AO Spencer Stereoscope ------------------------------------------------------------------ I am interested in knowing if anyone has used a repair service for AO Spencer stereomicroscopes. We have a vintage 1959 model with a detatched prism, and find it nearly impossible to repair ourselves, using makeshift alignment procedures. There must be a better way. If you know of a good repair service, or a method for doing it that we may not have thought of, or have gone through a similar experience, I'd love to hear from you. If you've seen this message before, I apologize, but I got several undeliverable mail notices when I sent it out about a month ago and received no replies. Please reply to: DRStadde+aCENTRAL%ARMSTRONG-at-MCIMAIL.COM or phone: (717)-396-5109 THANKS!
Hi I received the J. of Microscopy, June issue summary, but I have no clue on how it was encoded so that I may be able to decode it and read it!! My mail is received by a VAX but it is automatically rerouted to a SGI workstation and, finally, to a Mac running Eudora v. 1.4. The header of the message does not include the encoding method. Thank you for your help!
Dear Nestor, After posting to sci.microscopy about the poll, I realized that it was mailed to me personally by the person who conducted the poll. I guess the reason that I received the results was that I had participated in the poll. As far as the below message, it is all I recieved. Was a file to be included? I have the address of another contributor to the poll, so I will see if they kept a copy of the file. ___________________________________________________________________ Randy Nessler rnessler-at-uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
On Mon, 23 May 1994 MICROARCHIVE-at-anlemc.msd.anl.gov wrote:
} Randy } } I'm not sure if I sent you what you wanted, but from your description } it was the only thing that looked right in the archives.... } } Nestor Zaluzec } ANL EMCentee } r
I was told that there is a new Polaroid product called 'Polapan 400' which is a medium-speed (ISO 400), coaterless, black-and-white professional film. The Polaroid company claims that this film is a new choice for T52 and T53 applications.
Does anyone have used this new film before? Can I get some comments from you? I use a SEM and take a lot of T53 films on rocks and minerals. Thanks.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
The polaroid polapan 400 film works fine as a replacement for type 52, we use it with the same settings as the type 52. I don't know how they compare as far as cost, we were given 8-10 sheets to demo. I'm sure they will provide you a sample if you ask.
Does anyone have any experience with or sources for the electropolishing of indium alloys, specifically InCd or InPb? I need to prepare TEM specimens of these materials, and keep all the steps taken during preparation below room temperature. Any suggestions will be greatly appreciated. ************************************************* F. Scott Miller smiller-at-umr.edu Electron Microscope Lab University of Missouri-Rolla voice: 314 341 4727 fax: 314 341 6934 *************************************************
A postdoctoral position is available in TEM characterization of high temperature superconducting (HTSC) materials in the Metals and Ceramics Division of Oak Ridge National Laboratory (ORNL). Job responsibilities would include characterization using advanced EM techniques (EDS, HREM, EELS, CBED, etc.) of a variety of oxide-based HTSC materials. Candidates *must* have a PhD in materials science or related field with a solid background *and* extensive experience in characterization of HTSC materials.
*** DO NOT REPLY ELECTRONICALLY *** *** ELECTRONIC RESPONSES WILL NOT BE CONSIDERED *** *** RESPOND ONLY BY MAIL***
To apply, send a resume, publication list, and names of at least three references to:
G. Sims Oak Ridge National Laboratory P. O. Box 2008 Building 4500S, MS- 6116 Oak Ridge, TN 37831-6116
Oak Ridge National Laboratory is an Equal Opportunity/Affirmative Action Employer.
A postdoctoral position is available in TEM characterization of high temperature superconducting (HTSC) materials in the Metals and Ceramics Division of Oak Ridge National Laboratory (ORNL). Job responsibilities would include characterization using advanced EM techniques (EDS, HREM, EELS, CBED, etc.) of a variety of oxide-based HTSC materials. Candidates *must* have a PhD in materials science or related field with a solid background *and* extensive experience in characterization of HTSC materials.
*** DO NOT REPLY ELECTRONICALLY *** *** ELECTRONIC RESPONSES WILL NOT BE CONSIDERED *** *** RESPOND ONLY BY MAIL***
To apply, send a resume, publication list, and names of at least three references to:
G. Sims Oak Ridge National Laboratory P. O. Box 2008 Building 4500S, MS- 6116 Oak Ridge, TN 37831-6116
Oak Ridge National Laboratory is an Equal Opportunity/Affirmative Action Employer.
I use Polapan400 exclusively, the contrast is as good as Type 52, IMHO, and not needing coating is too good to pass up.
You can get a discount if you order the "bulk pack" - 10 boxes/case (no outer packing)
It does not, however, have a negative with it. If you need a good 4X5 neg, shoot sheet film!
James Long (no relation ;-))
On 24 May 1994, Liang, Long wrote:
} } I was told that there is a new Polaroid product called 'Polapan 400' } which is a medium-speed (ISO 400), coaterless, black-and-white } professional film. The Polaroid company claims that this film is a new } choice for T52 and T53 applications. } } Does anyone have used this new film before? Can I get some comments from } you? I use a SEM and take a lot of T53 films on rocks and minerals. } Thanks. } } } Long Liang } Electron Microprobe and SEM Lab } ARCO Exploration and Production Technology } 2300 West Plano Parkway } Plano, TX 75075 } E-mail : LLIANG-at-is.Arco.COM } } } }
We were just discussing that same problem yesterday with those same scopes. The solution in this department has been to put them in a closet and buy new ones. I found there is a closet full..... and I thought we ought to be able to fix them. I couldn't get the prism straight myself... so if anyone knows of an economical way to fix them, let us know. Thanks ... Nina Allen
Your local microscope repair facility should be able to fix those easily. If not, McBain Instruments in Chatsworth, Calif. certainly can. Their phone # is (818)998-2702.
Following the protocol of Bendayan I complexed Dnas, phospholipase and prteinase_K to colloidal gold but totally failed with RNase A using ph 9.3 and 7.9 and 5.6. I tried to use Sigma Cat. # R 5503 ribonuclease A from bovine pancreas described as being essentially protease and salt free. How ever when I added 0.2ml of a 5mg/ml pure water solution to 10 mls of colloidal gold it immediately turned blue/purple and begain to settle out justas if I had added a high concentration of salt.
Where did I go wrong???? ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
I am unable to achieve uniform illumination and focus on my prints, particularly with 35 mm (T-max 100) film. Does anyone know if this is a characteristic of the Beseler CB-7 enlarger or is it something to expect with any enlarger? My 50 mm lens (a Schneider Companon-S) gives a sharp grain (as seen with grain magnifier) in the center but considerably more light and focus degradation at the periphery. The pattern is not a simple circle. The 100 mm lens (Rodenstock Apo-Rodagon) gives better uniformity of illumination and focus but the image is not crisp. The lens apertures are 1-2 stops from wide-open. All exterior lens surfaces are clean. I am using the standard enlarger head with recently cleaned condensers and heat shield. The Kodak polycontrast filters are, unfortunately, below the focusing lens. If the Beseler is inadequate for biological EM lab work using 4 x 5, 3.25 x 4, and 35 mm films, does anyone have recommendations for a better enlarger?
Message-Id: {MAILQUEUE-101.940526141013.256-at-bunyip.ph.rmit.edu.au} To: microscopy-at-anlemc.msd.anl.gov
SEM : viewing polyethylene and other "plastics"
Is it possible to gold coat polyethylene in a standard uncooled gold sputter coater without heating the surface so as to modify it ? We are looking specifically at surface detail and do not wish to have it modified at all. We also wish to look at other probably higher melting point plastics as well. John Millar jjmill-at-rmit.edu.au
Rodney L Kuehn reported problems with evenness of ilumination and focussing on an enlarger. I think that the main problem is that the aperture used in the enlarger lens is simply too large (too small an f number). This results in a small depth of focus. If a larger f number is selected, such as f16 or f22, then the depth of field will increase significantly, and the focussing problem should disappear. The unevenness of the illumination is probably also a problem of aperture size and should also be cured by increasing the f number. I hope this is of use.
Dr Douglas J Arrell Mechanical Performance and Joining Institute for Advanced Materials 1755 ZG Petten Netherlands
Greg Erdos asked about precipitation problems during RNase / colloidal Gold complexing. Our experience suggests that concentrations of 0.5mg of most proteins per 100ml of gold (Frens method) is adequate. If the Sigma RNase is not completely salt free there may still be enough salt in there to aggregate the colloid. Try 0.01ml of the 5mg/ml protein solution per 10ml gold at pH5 - it may work - and still give you adequate label(l)ing.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
We have used EMS for some time here without any great problems. The bu1 program does take some time to get used to, and building crystals using the space groups can be rather confusing. If the unit cell looks incomplete, try displaying a 2x2x2 block, as the structure may then become clearer.
Alternatively (the method I prefer) use the option which allows you to specify the lattice type (PFIC etc) and then the positions of the atoms in the motif. Thus by specifying F lattice and giving atoms at +- 1/8,1/8,1/8 the silicon crystal can be built very quickly. (I think, from memory, that the option needed is /b)
The bu1 program will provide a file containing all sorts of useful/useless information. In this is a count of the number of atoms in the unit cell. Whichever way you build the crystal, check this file to see that you have the right number.
George Burgess Materials Science & Metallurgy Cambridge CB2 3QZ
We have two versions of EMS, one running on a VAX and one on a PC. In both versions it works fine with silicon in x,y,z=1/8,1/8,1/8. If you look at the .prt file created by bu1, you can check if all atoms are there, and in the right place.
Anna Carlsson National Center for HREM Inorganic Chemistry 2 Chemical Center Lund University P.O. Box 124 S-221 00 Lund Sweden Phone: +46 46 108231 Fax: +46 46 104012 e-mail:Anna.Carlsson-at-oorg2.lth.se
} We were just discussing that same problem yesterday with those same } scopes. The solution in this department has been to put them in a closet } and buy new ones. I found there is a closet full..... and I thought we } ought to be able to fix them. I couldn't get the prism straight } myself... so if anyone knows of an economical way to fix them, let us know. } Thanks ... Nina Allen
If you want, you can contact Ron Roos at Leeds Instruments, 800-444-5333. He is supposed to be some kind of repair modification specialist. Leeds buys, sells, and trades all different kinds of older microscopes. Basic service is ~$60 an hour and parts. On site service is available. Other than liking the service this company provides, I have no connection with Leeds.
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Anatomy * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-2706 voice * | | * 806 743-2707 fax * | | * Email hecub-at-ttacs.ttu.edu * | | * * __| |__ * * |_______| *************************************************
The symptoms that Rodney Kuehn described for his enlarger sound to me exactly like a condenser out of alignment; they do not sound like a depth of focus problem. The Besslers that I am familar with have to be adjusted between 4 x 5 use and 35 mm use. If this is not done correctly, or if the bulb is not well centered, you get problems of focus and illumination that resemble those described by Kuehn. HTH, Tobias Baskin
If there is anyone out there still trying to nurse along a JEOL JEM100B TEM, we have just declared ours surplus. With the exception of a few parts that I have already committed, parts are free to a good home. If it's light, I'll ship it to you; if it's heavy, you need to pay the freight. Julian Smith III Biology Winthrop University Rock Hill, SC 29733 803-323-2111 (Vox) 803-323-2246 (Fax)
I was wondering if anyone has any information on film printers that can be hooked up to a computer (that is also connected to an SEM). Can anyone share their experiences (if any) with the use of these film printers with me and of course how much they cost. We had a refurbished Mirus film printer donated to us awhile back, but it didn't work right after awhile.
I hope that these are appropriate questions for this list. I sent almost the same questions to the NIH-Image list as well, but was not sure if that was the appropriate list either.
Thanks in advance for any information, Peling Melville
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
Lowicryl users, I have heard that HM20 resin is no longer available. Can anyone confirm this and/or offer any explanation as to why it is no longer sold.
Thank you.
Allan Mitchell
per Richard Easingwood, South Campus EM Unit University of Otago, Dunedin New Zealand.
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
I haven't kept up with this aspect of the field for the last few years, however, many moons ago when I was a younger lad, John Colby (of Magic IV fame) did some work on the shifts in light element K lines. Duncumb et al did work in the mid 60's, Holliday did work on carbide phases around the same time. These shifts are best measured by WDS (but as a uprobist you know this already). The chemical shifts are due to the fact that the emission lines are due to transitions of valence electrons to Kshell vacancies rather than deeper core levels (ie. L's or M's) to the K shell which we see in higher atomic number elements.
My suggestion is to look into some of the more recent books on Bulk X-ray analysis. Love and Scott, or the rewritten book by Reed (which should be on the market soon). You should also probably check out the last few years conference proceedings of the Microbeam Analysis Society.
Message-Id: {MAILQUEUE-101.940526162512.448-at-ahabs.wisc.edu} To: microscopy-at-anlemc.msd.anl.gov
Proteins below their isoelectric points are positively charged. Addition of the positively charged protein to the gold collapses the negative charge layer which keeps the gold sol in suspension. Raise the pH of the gold and the problem should disappear. Unfortunately, it is sometimes impossible to resuspend the gold in a buffer of much lower pH than that at which it will conjugate to the protein.
Scott Simmons University of Wisconsin
HELP!!\
Following the protocol of Bendayan I complexed Dnas, phospholipase and prteinase_K to colloidal gold but totally failed with RNase A using ph 9.3 and 7.9 and 5.6. I tried to use Sigma Cat. # R 5503 ribonuclease A from bovine pancreas described as being essentially protease and salt free. How ever when I added 0.2ml of a 5mg/ml pure water solution to 10 mls of colloidal gold it immediately turned blue/purple and begain to settle out justas if I had added a high concentration of salt.
Where did I go wrong???? ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
You said you had problems with focusing and uneven illumination with your enlarger. What type of enlarger and light source are you using? The aperture has very little to do with an image being out of focus. If you use a focusing aid to focus on the emulsion grains, make sure the focusing aid is in focus for your eyes. Check the focusing knob and make sure it stays put when you focus. Sometimes the focusing knob will have a tendancy to slip after a period of time if it is an older enlarger. You may be able to tighten the knob or have to have it replaced. Also, make sure the height adjustment knob is locked in place. Make sure the easel is nice and flat on the enlarger base. As far as uneven illumination, if you use a point light source make sure it is properly aligned in the enlarger head. Make sure your condenser lenses are clean. Make sure the lens you are using is the proper lens for the film. 35mm.......50mmlens 21/4.......90mmlens 4x5.......150mmlens Make sure the condenser lens matchs up with the film and lens you are using. Make sure the lens is in properly on the lens board and the board is in properly. Sometimes I have that problem with my enlarger. Someone else will change the lens and not get it in place properly. I use a Durst 45 enlarger and putting the lens in wrong is easy to do. F-stops has very little to do with uneven illumination or focus. F-stops are primarily for better depth of focus. If the image is out of focus nothing will help to bring into focus unless you use something like f:64 for a long enlarging time dependant on the density of the neg. If you a use point light source, you don't have to worry about f-stops anyway. Hope this helps! If you have any problems, you can reach me at: Phone: (410) 455-3582 Fax: (410) 455-3875 Email: prutle1-at-gl.umbc.edu
To: Rodney L Kuehn {kuehn002-at-maroon.tc.umn.edu}
Dear Rodney:
A common problem in our daily operation is unleveled upper condenser. This is particularly troublesome in the old Omega enlargers lacking clips for holding the upper (collecting) condenser in place. If the enlarger is moved, etc. the condenser will occasionally slide just enough to cause the problem you describe. This is less troublesome with enlargers that have motorized adjustments. I correct the problem by measuring the distance between the upper rim of the enlarger condenser housing and the glass edge of the condenser with a metric ruler. It takes some times to move it around until is all even. Then you must ensure that it will not move when returning it to the enlarger housing.
This happens here all the time. Agree, it is a pain.
***** ************ ************** ************ *Cesar D. Fermin, Ph.D |Fax (504) 587-7389 *Tulane Medical School |Answ. Mach.(504) 584-2618 *Pathology/SL79 |Secretary (504) 584-2436 *New Orleans, La 70112 | Lab (504) 584 2521 ***** ***************** ***********************
= =i am from the Sedat and Agard labs where more most of these observations have =been made, and the notion that one needs to wait until the 7th cleavage =division is entirely bogus. i believe it is everyone's experience that histone =incorporation into incorporation can be observed after a single round of =DNA replication. = =you may have some technical problems depending how deep your early clearvage =stage nuclei are in the embryo. i suspect that a conventional, wide-field =microscope would probably work ok (especially with a decent digital camera), =nut you could always use a confocal if necessary. certainly, being across the =Bay from Agard and Sedat's lab, there is no excuse for you not to call them, =arrange for a visit for some technical advice, and maybe even try some data =collection on their system with your embryos. = =i have no experience with the new molecular probe dyes. this would be another =good question for sedat (or perhaps a few other groups at ucsf like andrew =murray's or tim mithchison's). let me know what you find out... = ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
VCR Group uses the Ion Tech ion guns in its ion mills. You can contact them at VCR Group, Inc., 250 East Grand Avenue, Suite 31, South San Francisco, CA 94080, (415) 875-1000, FAX (415) 875-7111.
Russ Cook Electron Microscopy Center for Materials Research Argonne National Laboratory
There might be a chemical solution meeting your requirement, but I think it is hard to find. For my personal opinion, the best way to thin heterostructural cross-section TEM specimen is firstly mechanical grinding and polishing down to less than 10 um, then uses ion milling for final thinning.
We are considering purchasing either a Hitachi S4200 or an Amray 1845 FE-SEM. The SEM will be used in a research environment with multiple users and a wide variety of samples ranging from metallic alloys including hard magnets to ceramic powders. We will either purchase a Link ISIS or Noran Voyager EDS system. We would like comments from users on their likes versus dislikes of these systems, in particular the day to day performance in terms of SEI and BSE resolution, analytical integrity (i.e., beam stability, spatial resolution (beam scatter), and XRF behavior of the finial imaging system) and reliability of the vacuum system. Also, performance integrity (down time), willingness of service representative to trouble shoot and timeliness and performance of service personnel. Any other suggestions on what options or requests you would have done differently on the machine you have purchased would also be helpfull.
I am using an LKB cryoultramicrotome to demonstrate antigens in skeletal muscle and would appreciate any comments and suggestions from biologists using this technique to improve techniques.
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable To: Microscopy-at-ANLEMC.MSD.ANL.GOV
A couple of years ago, the company PLANO made for us fluorescent screens on glass disks to capture TEM pictures on a video camera. The glass (dia 12 cm, 1cm thick) was set as a window at the bottom of the observation chamber. The coating uniformity was good enough to the level of in situ experiment recording. It was produced without (or with very little binding agent), thus it was very sensitive to fingerprints and scratches, but relatively resistant to burning. We lightly coated the powder layer with aluminum in a standard vacuum coater. For some they used their own powder and our P22G one for the others.
Ask for Mr. Noli, PLANO W. Plannet GmbH, Marburger Strasse 90, D-35043 Marburg 7 Phone ..+64.21.41077 =46ax ..+64.21.51173
Best regards Philippe Buffat
} Hello microscopists, } I need your help on the following point: } I URGENTLY would like to make a fluorescent screen (50 micrometers thick of } Y2O2S-Eu powder on circular glasses -diameter 36 mm and 53 mm). I have the } fluo powder and the glasses. I don't have the know how. } Do you know the address-phone-Fax-email of any commercial company } (France-Europe-Rest of the world) who could make it? } Thank you in advance } Don't bother anyone with this. Please reply directly to me. } Yours, } Pierre } -------------- } Pierre Trebbia, LASSI/GRSM, Universite de Reims, F51062 Reims cedex, France } Email : pierre.trebbia-at-univ-reims.fr } Phone : (33) 26 05 33 62 } FAX : (33) 26 05 32 50
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Institut Interd=E9partemental de Microscopie Electronique Address: EPFL-I2M, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 (Central European Time) Fax: +41(21)693 44 01 ______________________ Eudora 2.0.2 __________________________________
User involved in cryosectioning muscle samples for TEM. Would appreciate any suggestions from users actively involved in this techniqe. Having trouble demostrating anti-dystrophin antibody (nova castra dys1) with immunogold methods after fixing tissue in 2% paraformaldehyde for one hour as suggested by suppliers. Any helpful hints would be appreciated.
Terry Robertson
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
Dear EM microscopists, We will be studying gecko oviducts with TEM and SEM. If anyone has any information regarding fixation of this lizard tissue we would like to hear from them.We are particularly interested in appropriate the buffer/fixative osmolarity to use.
Mark Gould
per R Easingwood
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
Although this may sound trivial, I refer to an experiment we had couple of years back. A researcher was studying bacteria and wanted to have immuno-gold labelling on it. She processed the samples according to the latest recipes but there was no labelling on the samples. After a month or so she showd the samples to me asking why. We checked the specimen processing step by step and finally I asked if she had mixed the immuno-gold bottle thoroughly. When the answer was no, we mixed it well and the problem was solved.
I am curious wether anyone else has been unsuccesfully trying to find gold.
With wishes for a good summer,
Jouko Maeki
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Maeki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
More on CPD. Currently I am buying a package which includes CPD 030 and magnetron sputter coater CSD 050 from BALZERS Australia , originally Balzers Lichtenstein, at a very reasonable price of Aus $19,700. CPD has got incorporated a refrigerator unit and apparently performs very well. Worth considering , perhaps? Cheers, Wis Jablonski EM Unit
M. J. Kramer Having used both types (manufacturer wise) I would recommend getting the Hitachi. I currently use Hitachi, Zeiss and JEOL scopes in my lab. Service from all three is outstanding. I used Amray many moons ago and even the service rep had problems getting replacememt parts that worked. If they corrected their problems with parts and service, I wouldn't know. The three I use have been around for a long time and Amray is still relatively new ( in my opinion) to the field. I think they have only been around for about 20 years. Hitachis' FE scope is a beautiful scope to work with. I tried it once at a demo and it seemed easy to use and the photos were very nice from low kv to higher kvs. The scope, as a whole felt "right".
I see that someone has once again forgotten one of the few rules on this listserver, namely that concerning the requesting or posting job search information.
Please remember, that requests by individuals for help in job location and/or placement is NOT a function of this listserver. Individuals are welcome to use the MSA/EMC BBS system for posting specific requests and/or resume's and to potentially help in job search/placement. There is also the MSA Statistical/Placement Office which can sometimes help.
Only general search information, such as announcing a call for candidates for EM related position by an organization (educational, government, or commerical) is within acceptable bounds of topics for posting onto this listserver. All such announcements should, of course, indicate some appropriate address or phone number for replies as they should not be directed to this listserver.
Although X-ray spectra can do this sort of work, isn't it true that for the most part the electron spectroscopies tend to do the job easier? I'm thinking here of mainly of ESCA/XPS (i.e. the photoelectron spectroscopies).
Once you have collected all the wisdom of the world on Screens please post a summary here for all to read.
The same should apply to everyone that posts a question and collects answers. Sometimes as Pierre pointed out not all replies go to the listserver and you would be "paying your dues" by providing a concise summary of all reponses for all subscribers to read and possibly file for future use.
Subject: Time:12:22 PM OFFICE MEMO Ion Tech Address Date:6/1/94 I have an address for Ion Tech Inc. at 2330 East Prospect, Ft. Collins, CO, 80525. Tel: 303-221-1807; Fax: 303-493-1439. My source gives offices in Germany and Japan, but none in the UK, so this may not be the organization you are interested in.
Is there fluorescent stain that can be applied as easily as DAPI or Hoescht stains that have excitations and emissions in the same wavelength as FITC or Rhodamine?
Thanks, David
=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= David L. Hirschberg bnhirsch-at-wicc.weizmann.ac.il Department of Neurobiology (972) (0)834-2127 (0)834-2412 work Weizmann Institute of Science (972) 847-4805 home Rehovot 76100 Israel (972) 834-4131 fax
} Although X-ray spectra can do this sort of work, isn't it true } that for the most part the electron spectroscopies tend to do } the job easier? I'm thinking here of mainly of ESCA/XPS (i.e. } the photoelectron spectroscopies). } } Nestor Zaluzec } ANL EMCenter } It depends on the spatial resolution you require. Here in Bristol we have a VG Escascope small area/imaging x-ray photoelectron spectrometer. The smallest area you can obtain XPS spectra from is 50um diameter, so that is the smallest area from which you can obtain chemical shift information (as long as you are prepared to wait a considerable time for the spectrum to appear out of the noise). In the imaging mode, {10um resolution can be obtained,where you image a chosen photoelectron energy, so the distribution of a particular chemical shift can be seen, at that sort of resolution. Newer instruments canimage down to 1 -2um. Nevertheless, *if* you can obtain the data you want on an sem or probe, then maybe, and I don't really have any experience of looking for chemical shift in wdx or edx spectra, you can more easily do so in small regionsof the sample, and perhaps with shorter acquisition times.
Keith Hallam Interface Analysis Centre University of Bristol England
Yesterday I sent a message about the Hitachi vs AMRAY problem. Unfortunately after sending it I read it again and it seemed as if I was giving a dim opinion of the scopes AMRAY produces. That was not my intention at all. I got the company mixed up with another company I had dealings with and with the other company I did have bad experiences with. It was another American company and I haven't heard anything about that company in a long time. It could be that company is no longer in business. I don't know. To AMRAY I humbly apologize. I know your sales people and have a deep respect for them. It was totaly unprofessional for me to make a mistake like this without reading what I sent out. Again, I humbly apologize.
There is a magnificent handbook of fluorescent probes/dyes published by Molecular Probes, Inc. of Eugene, OR.: FAX (503) 344-6504 or telephone (503) 465-8300. It serves as their catalog, and gives really extensive, comprehensive descriptions AND bibliogr aphies. They do have worldwide distributors, but I don't see any listed for Israel--let me know if I can help any further. Good luck. Grace Kennedy, Scripps Inst. of Oceanography/UCSD (neurobiology, too.)
Hello, humble Masters student here. My question is regarding the standardization of one samples x-ray spectrum to another. If my SEM was nice and stable I suppose I could just take a spectrum for a certain period of time. Or if I had some nice low noise amplifiers I could look at the sample current and integrate that. However, my SEM isn't very stable and I'm not sure I want to mess around with the sample current. Has anyone heard of any other methods or can point to some articles regarding this subject.
Thanks in advance. Dan Henne Simon Fraser University Vancouver, Canada. email: henne-at-sfu.ca
This is in response to the message regarding DAPI nucleic acid stain. The filter set needed for the DAPI fluorescent stain (bound to DNA) is the UV excitation filter set; EX max 359 nm and EM max 461.
I have a Hitachi SEM and TEM and service is good. The rep is very responsive to my scheduling problems and works hard to get the scope running quickly. There is no question about his competence. Neither scope has ever been down for more than a few days for lack of parts. Yet I have still not been totally satisfied with Hitachi service because they depend on the user to know when there is a problem. When Hitachi services a scope, they fix that problem and leave. They perform no other routine checks to catch problems early or to deal with problems that might go unrecognized by a microscopist, particularly if new to EMs. Further, they have no PM checklist to demonstrate to the microscopist that the scope really is running at peak performance. In short, the Minnesota rep is competent and very helpful but is not fully supported by the organization. AMRAY used to have a marginal reputation but their company seems to have undergone a complete transformation. I attended one of their user meetings and it was apparent that their discipline and attention to user needs was unmatched by other organizations I had examined. I spoke to several participants and they were extremely enthusiastic about AMRAY service and many of these people had prior experience with other big-name manufacturers. For example, after AMRAY services a scope, they do a routine check on all switches and other items that are reasonably easy and fast to check. AMRAY also uses an extensive check list on their PM which is presented to the microscopist along with test photos. I also had the impression that AMRAY reps are better trained before they are sent into the field solo. When my rep started here, he didn't know how to stigmate the TEM! Of the service meetings I attended (Hitachi, AMRAY, Cambridge, and JEOL), AMRAY was by far the most aggressive in extracting grievances from their users. The AMRAY people running the meeting worked their way down a rather long list, "Has anybody had trouble with response time? Has anyone had trouble getting parts." They spelled out their standards for response times so people knew what to expect. I was impressed. If this meeting (now about 2 years in the past) was typical of the organization, they must have good scopes and service. I must note that I have no personal experience on an AMRAY or with their service. My impressions result from this one AMRAY user meeting.
Are there any ultrastructural biologists in the email network that have used any techniques for demonstating acetyl cholinesterase receptors at the ultrastructural level ? Does anyone know of any source for peroxidase conjugated, gold conjugated alpha bungarotoxin or an antibody that has been raised to alpha bungarotoxin?. I would appreciate any information.
Terry Robertson
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
After 2 years of debating about this I decided to start seeking a position out of the U.S.A. The areas I narrowed it down to are Costa Rica or Australia. I was wondering if anybody knew of any universities or private companies in these areas that might have need of an electron microscopist with 27 years experience. Actually, somewhere in Europe I would consider also, preferably Sweden, Germany or Switzerland. Thanks,
Does anyone know of a book that lists emission wavelengths for the elements in the 1000 to 2000 angstrom range? We can find some info where the wavelengths are listed by element, but we would like a listing of WLs in order of WLs, in order to check for interferences.
TIA,
Sue Smith National Steel Corp. smiths-at-mlc.lib.mi.us
Try and get in touch with Prof. Brian Gunning, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra ACT 2601. He may able to suggest the names of persons who may know about openings for electron microscopists.
Can I please once again remind you that the listserver is not to be used by Job hunters looking for positions. I am sensitive to this issue as I have turned down many requests from both ANL personnel as well as outside users all qualified and many being personal friends, all who have ASKED to post this type of information and were turned down. Given that I just posted a notice reminding people of this only a week ago I am extremely disappointed in the recent postings. Once again, you are welcome to use the MSA/EMC BBS for these purposes, but NOT the Microscopy Listserver.
If you are not sure about a posting, I would be glad to have a quick look at it first before forwarding it to the listserver especially, if you are not sure whether or not it is appropriate. I have already done this many time for subscribers (most of which were by the way posted without modification). BUT, I donot want to convert this to a moderated, subscription only list as I really don't have the time. So please remember or refresh you memory on the "ground rules" you were sent when you first subscribed. I didn't ask for much, and your getting alot for almost nothing. I will also consider the suggestion that was sent to me by several subscribers of removing repeat "offenders" from the listserver, if someone gets abusive.
Dan Henne asked about standardizing beam current for taking x-ray spectra:
First of all I agree with what Bob Craig said in his nice n' concise summary of the do's and don't's of collecting "calibrated" x-ray spectra. I'll specifically address Dan's question of how to set up a calibrated, reproducible beam current with out using any kind of current meter. Of course, you won't know the *value* of the beam current, but at least you'll know it is the *same* value from sample to sample and day to day.
We do not have a specimen current meter nor a Faraday incident beam current meter, so what we do in my lab to standardize beam current is the following: Mount a small metal foil, like aluminum or copper, onto the edge of each stub that the samples are mounted on using carbon paint or double stick tape. Run a little collodal graphite around an edge or two to provide additional adhesive support and a conductive bridge to the foil. Then before taking spectra, move the electron beam over to the foil and look at the metal foil count rate. Then adjust the beam current using the condensor lens, or spot size, control to set an arbitrary rate, say 3,000 counts per second, near the high end of x-ray through-put for your system (especially for biological samples; may not need a high end setting for materials samples), but not more than, say, 33% deadtime. This value can be checked for stability periodically and should be checked for each new stub you put into the SEM. Thus you will be delivering the same incident beam current to your unknown samples. Of course, there is always some indeterminate error with any kind of instrumental setting, and the count rate fluctuates a bit from second to second due to detection and counting statistics in the electronics and the physics of beam current production. With our set-up, the fluctuation is no more than 100 counts per 3,000 and over a 100 second analysis time the fluctuations average out. Also, our SEM a Philips 500X, is quite stable. Provided the filament doesn't shift, the same 3,000 cps will be there hours later. We set the rate when we put a sample in, and check it about every 30 minutes to monitor any changes that may occur. If a change has occured, we must throw out some data and collect over again.
A few precautions: use only clean foils; actually image the foil surface at moderate magnification to make sure that you place the beam on a clean area without microscratches. Also, must set the stage tilt and detector-to-sample distance to some standard and reproducible setings, etc. as Bob Craig pointed out.
But if your SEM is as "unstable" as you make it sound, you will have to check the foil count rate immediately befor and after collecting a spectrum and keep only those spectra where the rate did not change during the analysis time. If it does change frequently, you may only be able to do limited relative quantitative analysis based on ratios.
One comment about the use of specimen current (if you're lucky enough to have a specimen current meter on your SEM) to set incident beam current. As Bob Craig said, you can't do it by monitoring the current on the actual specimen, UNLESS you pick the appropriate specimen; for example, keep a margin of the aluminum stub clean and free of specimen, paint, tape, scratches, etc, and measure the "specimen" or absorbed current there. This value is the total incident current minus the backscattered fraction, and this fraction should stay the same as long as you use the same metal target, kV, geometry, etc, etc, etc, etc. So you are sampling the same fraction of the total and by setting the same value of specimen current, you are then standardizing the incident curret. I would think that this would be another way, like my above mentioned indirect technique based on foil x-ray production, to consistently set a constant incident beam current.
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Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
We are in the market for a backscatter detector for our Cambridge 180 SEM. I ask your opinions on makes,models, etc. for such an item. We mainly are a life science (pathology) lab thus any info relating to this area is appreciated. Also any info, opinions, etc. on multi-channel-plate detectors vs backscatter detector? Are they worth the extra dollars? (for a life science lab)
I was not sure wheather you are looking at plant cells or animal cells. If it is plant cells, there several publications. But if you want a single source perhaps the one by Behnke would be most helpful:
Behnke, H-D. (1991). Nondispersive protein bodies in sieve elements. A survey and review of their origin, distribution and taxonomic significance. Internatl. Assoc. Wood Anat. (IAWA) Bulletin 12:143-175.
We have been dealing with AMRAY for 17 years now. There service has been outstanding here in the Bay Area: they respond quickly, home in on the problem, and do what needs to be done. They do routine maintenance. Our 1000A microscope is getting along now but it has always made very good pictures and remains an excellent low-resolution instrument: it runs and runs with little down time.
Jacob Bastacky Jacob Bastacky, MD Lawrence Berkeley Laboratory EMail: sjbastacky-at-lbl.gov University of California Phone: (510) 486-4606 Berkeley, California 94720 Fax: (510) 486-4750
Message-Id: {9406040024.AA03054-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: XEDS;SEM;Standardizing spectrum Orig-Author: {Daniel Henne {henne-at-sfu.ca} }:ddn:wpafb ----------------------------------------------------------- Hello, humble Masters student here. My question is regarding the standardization of one samples x-ray spectrum to another. If my SEM was nice and stable I suppose I could just take a spectrum for a certain period of time. Or if I had some nice low noise amplifiers I could look at the sample current and integrate that. However, my SEM isn't very stable and I'm not sure I want to mess around with the sample current. Has anyone heard of any other methods or can point to some articles regarding this subject.
Thanks in advance. Dan Henne Simon Fraser University Vancouver, Canada. email: henne-at-sfu.ca
Ion Tech Ltd was taken over a few years ago - I cannot remember by who. However, they are still in business but trading under the name of Atom Tech Ltd.
Atom Tech Ltd can be reached in the UK at:
Island Farm Avenue, West Molesey, Surry KT8 2UZ, England.
I have been unsuccessful in locating ANY information on the International Symposium on the Structure and Properties of Dislocations in Semiconductors to be held in 1995 (I believe in Italy). Would anyone happen to have anything on this conference or direct me to someone who does? Any assistance would very much be appreciated. Thank you.
H. R. Kolar Center for Solid State Science Arizona State University FAX: (602) 965-9004
Apologies to anybody who received this twice due to cross posting.
Every once in a while somebody asks for a survey of user fees for multi-user facilities involving LM or EM.
The discussion of fees has been very informative.
We were wondering, in addition to fees, how these facilities are administrated.
For instance: o What is the administrative hierarchy? o What is the salary and rank of the director of day-to-day operations? o What are the responsibilities of this person or people? o Who contributes the equipment to the facility and how are these resources allocated to users?
Any comments would be appreciated.
Thank you.
Michael Cammer cammer-at-aecom.yu.edu cammer1-at-telico.bioc.aecom.yu.edu
I have a microscope that came with a camera adapter that, among other things, has a film holder for 2 3/4 x 3 1/2 sheet film. Has anyone ever used film of this size? I would certainly appreciate any information that I could gather. I am not familiar with this size, or why it would be used.
At the HVEM we use 6.5 * 9 cm film (~= 2 3/4 x 3 1/2). This film is satisfactory for our purposes and the larger standard size will not fit in the camera. Either Kodak 4489--a very fine grain film which is our usual choice-- or SO163 can be obtained in this size. We also have some old x-ray film cut to this size which is used primarily for recording diffraction data. If you de- cide you don't want to get into this size film, could you please send me a de- scription of the holders you have? By some miracle, they may be compatable with the AEI holders we use. Thanx.
The EMMPDL has a contributed MC program called MacSim. Here is the abstract...
Nestor
Title :MONTE-CARLO SIMULATION (Z's MC v. 1.1) Keywords :.Electron-beam, Monte-Carlo simulation, energy loss, backscattered electrons, Rutherford cross section, Mott cross section. Computer :.Macintosh Operating System :6.0 or 7.0 Programming Language :Pascal (Think Pascal) Hardware Requirements :standard Author(s) :.Zbigniew J. Radzimski. Correspondence Address :.Analytical Instrumentation Facility North Carolina State University Raleigh, NC 27695-7916 Abstract: The current program is the further development of the program written many years ago by R. Myklebust and D.C. Joy and modified later by J. C. Russ (J. of Computer Assisted Microscopy, 1990, 2(2), p. 59). It calculates various parameters related to electron-beam interactions with solids related to absorbed and backscatetred electrons. It accepts multi-element and multi-component structures. It uses Rutherford or Mott cross section for scattering. Special thanks to Zbigniew Czyzewski and David Joy for providing Mott tables (see J. Appl. Phys., 1990, 68(7), p. 3066).
I know someone who is helping to set up an EM lab. They are interested in finding out where and how they might send someone to get training. I know that the various makers of EMs offer courses on how to use their instruments, but where can they get training on processing, sectioning, and other techniques used for boilogical TEM. (used for examining ultrastructure). Any information will be helpful.
There are several universities that offer courses in the techniques of biological electron microscopy including Cornell. Some years ago, I think the Microscopy Society of America (MSA) sponsored a survey of EM courses offered in the U.S. universities. If you get in touch with MSA they may be able to send you a copy of the survey.
Partha M.V. Parthasarathy Section of Plant Biology Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734 Fax: 607-255-5407
Our facility is designed to be totally self funded. Administration heirarchy is:
Faculty director (25% time) Sets goals, policy, etc. Consults with users and helps design projects Sees to overall day to day administration
Facilities engineer (full time) Maintains microscopes (we 2 SEMs, TEM, IVEM) Trains users Developes film Helps with specialized procedures
2 EM technicians (full time, each) Processing and embedding Specialized procedures Immunocytochemistry Enzyme cytochemistry Autoradiography Etc. Sectioning Printing Maintain EM records Billing
Contact Kodak in Rochester, NY. Ask for the Scientific Advisor/Advice Department.
Sue Smith for Sam Purdy -at- National Steel, Trenton, MI
On Fri, 3 Jun 1994 ARGIL-at-delphi.com wrote:
} I have a microscope that came with a camera adapter that, among } other things, has a film holder for 2 3/4 x 3 1/2 sheet film. Has } anyone ever used film of this size? I would certainly appreciate any } information that I could gather. I am not familiar with this size, or } why it would be used. } } Thanks, } } Arthur Gillman } Princeton, NJ } }
Message-Id: {MAILQUEUE-101.940607084003.448-at-bunyip.ph.rmit.edu.au} To: microscopy-at-anlemc.msd.anl.gov
Re : Fees for use of facilities in Em Units
Since we have only recently joined this group, we missed the comments on user fees which have apparently been circulating from time to time. Does anyone have a summary ? We are interested in not only fees for individuals or groups external to the university, but also we are obliged to cross charge for all teaching and research within the university to support the Unit. What scale of fees are commonly used for SEM, TEM,EPMA, spec prep from simple to complex ? Are there "discounts" for local users or are there different rates ? Are the rates different for expert users ? etc, etc.... THank you in advance for the comments.
John Millar EM Unit, RMIT, Melbourne Australia jjmill-at-rmit.edu.au
There are at least 2 very good BSE detectors assemblies namely Robinson BSE and Oxford Instruments P/L TETRA BSE. Robinson BSE is well known for its performance including biological subject but I was most impressed by per formance of TETRA BSE. It is auto-biased with four independent quadrants, which can give variety of BSE signal combination and working with a electron beam treshold of 0.1 nanoamp--I think-- it is a jewel. Have seen it working at ElectroScan Corp- Boston installed with E-SEM. Regards and good luck with the purchase , Wis Jablonski CSL Uni Tas ,Tasmania, Australia
Greetings, These are follow-up questions for Jay Jerome's posting regarding his unit: how many people does your lab serve? What percentage are from "in-house" (your department/division) vs. those from "outside"? Do you charge different fees based on the affiliation of the user and their level of expertise? How do you control the "quality" of the user? I have worked in the Cornell TEM Facility run by M.V. Parthasarathy, which seems somewhat similar to yours (with the exception of no in-house technical support for the instruments), where a user could pay a flat fee for a certain period, plus extra costs for special equipment, e.g., high-pressure cryofixation. A user could also pay on a per hour, plus materials basis. Do you have such a scheme as well? My department, which has a simple facility with an SEM and a TEM, charges virtually no fees to department users, except for the materials (films, stuff used in processing material, etc.) and we are evaluating the idea of initiating additional user fees. I realize that this thread has been carried for quite a while in this group, so thanks for the patience. Because this could be rather a contentious proposal for the department users, I'd like to hear from as many of you who have or had such user fee programs in place. Thanks very much!
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
In regard to general info on user fees - I think that there was a survey a few years ago through MSA (maybe the Education Committee or Tech Forum?)
depending on the size of your operation, and whether you are the only EM facility on campus, you can charge for everything (which can make people feel like you ar nickel&diming them to death) or roll the cost of coaters into the SEM beam time, etc; a two tiered scope charge for assisted vs unassisted usage is an incentive for folks to learn how to operate the beast - but be sure you retain the right to decide who is qualified for unassisted usage; a third level, where you are performing the work might be the same as the assisted usage charge or different, depending on your philosophy
***************!!!!!!!!!!!!**********
An important point to ponder in regard to the question of different rates for different users - be very careful ....... most of us are on federal funds and many of the instruments were purchased on federal funds - therefore there are some "rules" that have to be followed - these are the ones I am aware of - there are probably others (if anyone knows where these are concisely enumerated I would like that info)
- If the equip was purchased through government funding 0r is supported by govt funds and you are offering services to the "general public" (industry, hospitals etc), you cannot undercut the prices of any locally available commercial service- check the yellow pages for labs in your area (clinical & industria testing - few of these folks are in MSA)
- Anyone who's research is funded through the feds (don't know if this also applies to state grants) has to be charged the same price - there are ways to give the folks in your dept a break if the dept is providing financial support to the facility, but be sure it is clearly spelled out
- An additional charge = the % overhead your institution takes from fed research grants may be added to the fee for academics from other institutions who are grant funded
- there are also restrictions on using depreciation as a charge basis if the instrument was bought on govt funds and the users are govt funded
- i am sure there are more rules than this but these are the ones I have stumbled across
maybe one of the msa committees should undertake an investigation into this question as we all seem to stumbling around in the dark - to my knowledge no one has yet gotten in hot water on an audit, but as more and more of us are going (at least in part) to a charge basis, it might be a good idea to have some guidelines
1. Our laboratory servers approximately 80 major users (repeat more than 2X a year). 9 from our department, 41 from other departments within our institution, 24 from outside our institution (4 from industry), and 6 from foreign countries.
2. We charge less for departmental users because our department pays part of the service contract on the instruments.
3. We do not charge less based on user expertise. We provide initial training in any aspect of microscopy the user desires. After that we charge a fee for technical time if one of our staff is involved in the microscopy.
4. As outlined in a previous communique (Nestor- do we have a FAQ section?), we breakdown our fees for each individual technique done and cKcharge what it costs us to complete the task.Determining these fees is a major headache, but simplifies administration later and avoids hurt feelings since all are charged based only on what they use. We of course do not nit pick (i.e. charge for each grid, each cc of osmium, etc). Rather we have an average cost for negative stain, cost for cryosectioning, cost for cyroEM, etc.
5. Microscope usage is on a sign up reservation basis. Inexperienced users can only sign up during hours when resource staff are available for consultation. Once we (as a group) are convinced a user can handle themselves we make the microscopes available to them 24 hours a day. Rarely, but it has happened, we encounter someone who is either incapable or unwilling to use the laboratory in a responsible way. These people are denied all access.
6. Marcelle Gillot's summary of the problems of determining users fees is excellent and is almost identical to the constraints we use for determining fees. However, our legal department has a slightly different interpretation of allowable charges for outside, non-grant supported work. They advise that it is allowable to undercut the cost of locally available commercial services as long as you can document (and that may be a problem) that you do so while still recouping all costs of the work without relying on subsidies (goverment grants, institutional support for service contracts, salaries, building maintenance and rent etc.) to cut your cost. In other words, if you are willing to take less profit margin or pay your labor sweat shop wages you can charge less than your commercial competition. You just need to prove that you and your competition are playing on a level playing field.
I hope this is helpful. I would support a movement within MSA to survey and document the issues involved with fee for service operation. Business is clearly something most of us where not traine in and really don't want to spend our time on- however the world being what it is today.......... Regards Jay Jerome
Jim Romanow asks about techniques to produce "black backgrounds"
To possibly state the obvious, you could digitize the images (say using a standard flat bed image scanner ~$1500) apply a digital mask to the area you want to preserve and then "Paint" the background black. That technique is used by commerical artists all the time, many of which are going digital. I know of some "Electron Microscopy Artist's" who basically do the same thing using for example Adobe Photoshop on a MacIntosh.
If you want to go analog, then let me assume that you have tried all the usual tricks like black level shifting gamma processing and other various electronic signal manipulations and they don't get you the image contrast you want.
Have you tried exploiting the directional nature of Backscattered Electron Imaging? Here's what I'd try if I were really pressed to make the background signal as low as possible.
1.) Use a Low Z stub/base this minimizes the BSE signal 2.) Mount the sample on a fine wire stand. 3.) Tilt the stub/holder so that it is tilted away from the BSE detector 4.) Bend the support wire so that the sample faces the BSE detector and so that the sample is between the wire stand and the detector. 5.) Turn off the secondary electron collection field and put a negative bias on the collection grid.
Using this configuration, most secondary electrons from the "background" stub will be rejected by the BSE detector field (I'm of course thinking of a standard PMT detector with a negative retarding field used to reject low energy secondaries) and the BSE from the stub/holder will be for the most part going in the "wrong" direction hence will contribute neglegibly to the signal. You should get a very dark background signal here, however, all edges etc will be enhanced on your specimen and your image may not show the features that you are more comfortable seeing using conventional secondary imaging.
You could of course get fancy with more digital technology and some image processing alogrithms. For example, if you want to bring back the average signal level, record a normal secondary electron image and :-) digitally add/merge the BSE and SEI signals together on your computer, to restore some of the lost SEI information.
If the Coates and Welter does produce true NTSC TV rate signal you could take the signal directly into a TV rate frame grabber board on a computer and the image process to your hearts content. The problem will likely be that the "TV-rate" image that your SEM generates is probably not NTSC but just a fast rate which appears TV like. Slow scan digital imaging is of course do able, however, it's expensive. but you said that you didn't have a slow scan mode, so that option is not viable for you.
I'd probably go the image masking route using Photoshop. If you have patience then you can get excellent results, but so do those guys with the scissors and paste!
Lets talk EDS Detectors! Within a few months time, we will be adding an EDS detector with a thin window for light element detection capability to an existing TEM (Philips CM12, LaB6 electron source, no scanning coils (STEM), + or - 60 degree tilt stage, double tilt analytical grid holder). We are looking at a variety of detector characteristics and are considering: 1. resolution, 2. crystal area, 3. liquid nitrogen cooled vs. peltier cooled (dry), 4. silicon vs. germanium detector crystals, and 5. thin window characteristics. These aspects of detectors are somewhat linked together so its a matter of determining needs and priorities and convolving them with the available budget. We have 16 years of experience running EDS (7.0 micron beryllium window, also soon to be replaced with a new thin window detector) on an SEM, but the addition of EDS to a TEM, with light element detection, will be new territory for us. We are based in a college of agriculture and study a wide variety of biological samples, soils and minerals, and occasional metallic inclusions. We have been discussing these issues with vendors of EDS equipment to enable us to become familiar with the current product line. Now we want to put some of our reasoning and musings in the above areas to the network for feedback from our colleagues. We hope that some experienced hands in the EDS buisness will take a few minutes to respond with the kinds of helpful comments that only someone who has done real, practical work can give. We apologize for the length of this communication, but with the advent of Peltier cooled detectors and the recent introduction of germanium crystals, there is plenty more to consider when buying a new EDS system these days.
1. Resolution. After years of looking at flabby peaks on our 16 year old EDS system (resolution about 162 eV -at- Mn Ka), naturally our inclination is to go for all the resolution we can afford. This seems to imply using a LN cooled premium quality crystal with a 10mm area. Considering the sensitivity to low energy x-rays (0.1 to 1.0 KeV) possible with thin windows, it seems one would particularly want very good resolution to help sort out K,L, M shell overlaps in this low energy region. For light element (or low x-ray energy) detection, a seperate resolution at Fluorine (e.g., 68 eV -at-F) is usually given and we've seen values from 65 to 115 eV -at-F in currently available thin window detectors. Some say "not to worry" about resolution so much because todays peak deconvolution software will sort it out for you, but is it really that good in this energy range?
2. Crystal area. We are aware that the prevailing wisdom suggests that a 30mm (mm squared) crystal should be used on a TEM (with some loss of resolution?) for the larger solid angle of x-ray collection that they have; necessary for thin samples which generally yield lower count rates that a bulk sample in an SEM would give. We wonder if the LaB6 electron source in our TEM will provide enough beam current to get the count rate up (without frying the sample!) to a reasonable level, meaning maximum accumulation times of usable spectra in, say, 200 seconds? Also, do 10mm crystals come mounted in a smaller diameter "snout" which can be moved closer to the sample to increase collection solid angle?
3. LN cooled vs. Peltier cooled ("dry"). (There was a question on the net in late April about "water cooled" detectors but only one response was offered which explained that these detectors are Peltier cooled and water is simply used to carry away heat from the Peltier device). Tho generally a bit more expensive to aquire initially, the main argument for purchasing a Peltier cooled detector given by vendors (Noran, Kevex/Fisons) is the economy of operating them; no hassle with liquid nitrogen to fill twice a week, so saves time and money. Well, we're running a 10 liter dewar on our older EDS right now, and we use LN for cold traps and freezing procedures almost daily. So another 15-20 liters per week(estimate includes loss due to cooling down transfer dewar) isn't going to break the budget. In fact, I would think an EDS dewar is a better place to store LN than the industrial-grade 160 liter dewar that it comes in which vents off more gas than a pack of ardvarks on steroids. Vendor economy calculations apparently assume that you are using LN exclusively for your EDS dewar so that LN cost, delivery fees, tank rental, and LN technician salary, etc, are not spread out over other equipment that consumes LN, so its a maximum LN expense estimate. The down side seems to be higher initial cost and less resolution tho some figures we've seen for some 10mm Peltier cooled crystals, however, range between 138-149 eV (silicon crystals, Noran, Kevex). Now 138 is not bad, but such a detector lists for about $25,000, the 149 eV for $18,300. And if you go to a 30mm Peltier cooled crystal (Noran, generally recommended for TEM applications, see 2. above) the resolution drops to 155 eV -at- MN (112eV -at- F). Is anyone out there operating a Peltier cooled detector on TEM or SEM? They havn't been around as long as LN cooled ones, so we wonder about performance and reliability over time. They also require a seperate water cooling and circulating system to remove the heat being pumped out by the Peltier device. Any problems or commens about that? How's the resolution , etc.?
4. Silicon vs. germanium crystal. The new kid on the block is germanium. And to look at its resolution and countrate through-put values, one wonders if silicon's days are numbered. For SEM only, Link-Oxford offers a thin window 10mm germanium detector with a resolution of 115 eV -at-Mn at 1,000 counts per second (cps), 65 eV -at-F; it rises to only 133 eV -at-Mn at 10,000 cps, ($22,200). It has another slightly lower cost 10mm Ge crystal wth 120 eV -at-Mn, 68-70 eV -at-F ($20,800). For TEM, Noran offers a 30mm germanium crystal with 129 eV -at-Mn. These resolution values look pretty terrific to us!! Less important to us, but another advantage of Ge crystals, is that they absorb higher energy x-rays much better than Si crystals. Silicon absorption efficiency drops rapidly above about 20 KeV (the unabsorbed x-rays pass through the crystal without interacting); Germanium absorption efficiency doesn't begin to drop significantly until x-ray energies get above about 60 KeV. So good detection of high energy K-shell x-rays is available with Ge crystals. But how about at the very low x-ray energies of the light elements; does germanium do OK there? Does it have a dead layer like Silicon crystals do? Are there reasons why germanium detectors with thin windows are not the choice to make for serious light element analysis? Other than being a bit pricey, what are the drawbacks, if any, to Ge crystal detectors?. They havn't been on the market very long, so perhaps there are the usual problems of maintaining performance and reliability until the bugs are worked out. Other than that, it seems to have that "wave of the future" look to us.
5. Thin windows. We read Mark Lund's helpful, but brief, comments on thin windows in late April and look forward to his upcoming book chapter on the subject ( For the MAS, edited by D. Williams and D. Newbury). There are thins and ultra-thins, depending on how sensitive you need to be to the lightest elements, Boron and Beryllium. We only need to see down to Carbon here so a thin window is probably best for our needs. We are concerned about thin window failure rates, especially on the SEM. Do they need to be cleaned often to maintain good low energy x-ray transmission? What other concerns should we have about their care and operation? As Lund pointed out in his comments, if you are specializing in light element analysis, you really have to look at the over-all EDS system in addition to the window material. We do not intend to specialize in light element analysis, but we do want reasonably good detectability, qualitative and "semi-quantitative" of C, N, and O. But we also need to look a lot at the range Z=11 to Z=30, and occassionally above. Does one need specialized software for light element analysis and does it compromise analysis of heavier elements that may be in the same spectrum?
Enough already! Lets give EDS detectors a bit of network time. I'll compile responses into a single file and pump it back out in a few weeks. Thanks in advance to all who respond to this query.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Any input as to good grey-scale printers, as in TRUE grey scale. I'm interested in one that can be accessed from the Mac environment, not thermal paper, and makes nice transparencies. $$ is of minor concern.
I apologize if this repeats a thread.
Tim Foecke, Metallurgy Division, NIST tfoecke-at-nist.gov
___________________________________________________________________ Randy Nessler rnessler-at-uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
On Tue, 7 Jun 1994, Gib Ahlstrand wrote:
} Lets talk EDS Detectors! } Within a few months time, we will be adding an EDS detector with a thin } window for light element detection capability to an existing TEM (Philips CM12, } LaB6 electron source, no scanning coils (STEM), + or - 60 degree tilt stage, } double tilt analytical grid holder). We are looking at a variety of detector } characteristics and are considering:
My question is, without STEM, how are you planning to position your beam on the area of the specimen that you would like elemental information from? I am embarrassed to ask this question, as I obviously haven't researched this in publications. When I asked about collecting x-ray data while performing diffraction, I was told by a service engineer that it would flood the detector with BSE's. Also, there is the problem of diffraction flooding a rather large area of the specimen, and the selected area diffraction aperture allowing the pattern of information to be viewed, while blocking out extraenous information. Are you just interested in average area analysis? I hope I am not the only one curious about this. This is a little off of the topic, and I hope not to detract from the conversation. We are looking to upgrade a detector on a SEM, so I am very interested in following this discussion. Randy
The ANL EMCenter uses a Tek Phaser IISDX dye sublimination printer for all high quality gray scale (& color) work from computers. It is on an Ethernet backbone and directly usable from your Mac "chooser". The quality and price performance for both glossy prints & transparencies is excellent. Cost ~ $10K for the unit then the price/print for 8x10" $2 B&W, $3 Color add an extra $1 to each if you want a transparency.
I'll bring examples to the computer workshop at this year's meeting in New Orleans if anyone want's to have a closer look at real EM data versus manufacturer's demo prints.
Jim Romanow asked about how to get black backgrounds in SEM images:
I've accomplished that using a few simple specimen preparation techniques. The basic trick is to produce a very smooth featureless background, low Z composition prefered:
1. I've done the following for pollen grains or rust spores (dried down onto glass coverslip support surface from water or ethanol, they just stick naturally), should work with insects too, provided that any paint you use to mount the insect to the glass coverslip does not spread out beyond the edges of the insect so you don't see it in the background. Your pin mount technique should work here too: Mount a cleaned (EtOH rinse, air dried) 1 cm diameter round glass coverslip onto a standard aluminum stub using double stick tape and edge the coverslip with carbon paint to provide a conductive path to the aluminum and for extra adhesion. Mount your insect to the coverslip using a tiny dot of carbon paint, or using your pin mount method. Heavy metal coat on a rotary stage as usual and view in the SEM. In spite of the fact that the glass(silicon & oxygen) coverslip is dense and coated with gold-palladium, the pollen grains are brighter than the background and when printing the neg its easy to drop the featureless background into the black, perhaps with a bit of doging of the subject, if it has a simple profile, to burn in the background. The key here is that the background is featureless so that there are no edges of paint globs or other debris which will light up in secondary electron imaging.
2. Similar to the above, is to use a smooth surfaced double stick tape to mount your samples to avoid using paint (for very small samples).Or as a smooth background for your pin mount technique. Some double stick tape surfaces develop tiny cracks or hole patterns in them as a result of being in the vacuum or whatever and may not provide a feature-free background. Spreading carbon paint with a brush to get a low Z background may work if the paint is quite thin but sometimes it leaves too many "brushstrokes" visible and that detail may show up in the background.
3. Another method to get a fairly good low-Z background with a high-Z coating on your sample: Mount samples (directly or pin-mounted) on double stick tape or with a spot of carbon paint onto a stub that has been previously painted with carbon paint (and dried) to hide the aluminum, so you have a low-Z background. Set up your vacuum evaporator rotary stage to zero degrees tilt if you can: We have an old Ladd rotary tilt stage whose "whirling disk" can be re-mounted to zero degrees tilt, that is, horizontal. Set up the evaporator for an overhead carbon rod evaporation on one set of electrodes; set up another set of electrodes for a low angle metal (Au-Pd) coating of about 7 to 10 degrees elevation. After pump-down, set the stage rotating and do the two coatings, in either order. Very little metal coating will hit the stub surface because of the low angle of coating. A lot of metal will hit the target, tho it may be a bit thin on top of the target, hence the overhead carbon coating to get some extra conductivity on top surfaces. This method has worked very well for imaging seeds from 0.05 to 0.4 mm diameter in backscattered imaging (I wanted even "overhead" illumination that BSE gives) with black backgrounds. Should also work OK for secondary electron imaging (gives typical "side" illumination).
Good luck!!
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Stuart McKernan suggested that a set of images be organized for testing of printers. The results for which could be put on display at the Computer workshop at the MSA New Orleans meeting. I'm willing to organize this if there is enough interest. If you want to participate Email me seperately (Zaluzec-at-anlemc.msd.anl.gov)
I will collect a set of images from the MSA/EMC library and then setup a FTPable account where interested parties can download the files.
My first impression is that we should have the following in the collection for testing.
SEM-gray scale - Life Science (a bug &/or plant) SEM-gray scale - Physical Science (a computer chip &/or fracture surface) SEM-gray scale - BSE Channeling Pattern
TEM-gray scale - Life Science (cells & collected) TEM-gray scale - Physical Science (Dislocations & Grain Boundary) TEM-gray scale - HREM image of a crystal TEM-gray scale - Electron Diffraction SAED TEM-gray scale - Electron Diffraction CBED
I'm open to more suggestions for test images, make them off line and I'll collect the responses over the next 2 weeks or so. I'll then post the results to the mailing list for further comment before we actually do anything else. I'll attempt to pick micrographs which push the limit of the printers (max contrast & resolution) rather than just a pretty picture. I'll also make sure text is present on each micrograph so that users can also judge the capabilities of the printer to reproduce high quality text as well as an image.
} I think it would be worthwhile to set up an informal meeting related to } this list server. Of course there will be folks coming to the computer } lab, but do you think there would be a place for a presentation of the } server and handouts of how to subscribe/unsubscribe, access, etc? This } might be a chance for exchange of this kind of information, or a way to get } some of the interested parties together. Just a thought.
There will be a tutorial session at the MSA meeting entitled
" A HitchHiker's Guide to Microscopy & Microanalysis Using Telecommunications,Email and the Internet"
by "some guy who wears a hat"
At it I'll have a bunch of handouts about the listserver, the EMMPDL, MSA BBS, a run down of other aspects of Internet (EMail, Telnet, newsgroups, FTP, Mosaic, ......) and anything else I can fit in given time & space & my fortitute. We also plan on-line demos of each if the phone lines can handle the links.
In addition, John Mansfield will be also doing a Tutorial on Image Processing using NIH Image as an example.
These tutorials are set for Thursday Afternoon August 4th, from 1--} Closing. (i.e. ~ 3-4 hours), depending on the audience.
Jay Jerome & John Posthill get the credit for twisting John's and my arms into doing these tutorials. I guess they figure there might be as many as a dozen people that might be interested, anyway, I know of at least 2 people that will be there. So Listserver subscribers, you're welcome to stop by, it's as good a time as any.
The workshop has a reasonably large room again this year, and will be set up for informal as well as formal presentations throughout the week. Of course, I'm more than willing to participate in an even more informal get together at any local pub, so John C. just set a time & place! ;-)
} } The workshop has a reasonably large room again this year, and will be set up } for informal as well as formal presentations throughout the week. Of course, I'm } more than willing to participate in an even more informal get together } at any local pub, so John C. just set a time & place! ;-) } } Nestor Zaluzec } ANL EMCenter } FAQ- Who's buying? ;-} Jay Jerome
As various contributors have pointed out, a black background depends on having the highest possible difference in Z between foreground and a {featureless} background.
With biological samples it is useful to expose the specimen to Osmium (fix or postfix in osmium tetroxide for wet specimens, expose dry specimens to osmium tetroxide vapour in a sealed container for a few hours) and then to mount on a stub coated with gold size.
Japan Gold Size (the stuff that artists use, obtainable from any art supply shop, we use Winsor & Newton brand but others would also be ok) is a sticky liquid, containing mostly linoleic acid polymers. A very thin layer of this on a stub becomes very tacky within 5 or 10 mins at 50 deg C, depending on the characteristics of the batch, and can then be used in the same way as double-sided tape. The thin layer on the stub is made by wiping a slight smear of the liquid over the face of the stub. This soon polymerizes into a glassy, featureless, nonsticky, non-outgassing, low Z layer.
The polymerization can be hastened by a further period at 50 deg C. If your specimens are heat sensitive this further bakeout is not necessary, the process will go to completion at room temp within a few hours. Sputtercoat the sample with a minimum of gold and the high Z osmium in the sample on the smooth low Z gold size will give a nice dark background. Even a very thin layer of gold size on the stub is thick enough to keep the beam away from the relatively high Z stub.
The gold size idea is not our own, it was published long ago in a paper that we cannot now find, but it works really well for a wide variety of specimens. In addition it is cheap, natural, biodegradeable, nontoxic and apparently also non-allergenic! Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Free to a good home. Philips EM 300 Electron Microscope now surplus to requirements due to rationalization of Faculty EM Services. Very Good Condition. Philips maintain the instrument. Further details: Mr J N Brown, Department of Physiology, University of Edinburgh, Teviot Place, Edinburgh, UK. Telephone (031) 650 3273, fax (031) 650 6527.
Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable To: Microscopy-at-anlemc.msd.anl.gov
=46ollowing the question from R. Nessler: My question is, without STEM, how are you planning to position } your beam on the area of the specimen that you would like elemental } information from? I am embarrassed to ask this question, as I obviously } haven't researched this in publications. When I asked about collecting } x-ray data while performing diffraction, I was told by a service engineer } that it would flood the detector with BSE's. Also, there is the problem } of diffraction flooding a rather large area of the specimen, and the } selected area diffraction aperture allowing the pattern of information to } be viewed, while blocking out extraenous information. } Are you just interested in average area analysis? I hope I am not } the only one curious about this. This is a little off of the topic, and I } hope not to detract from the conversation. We are looking to upgrade a } detector on a SEM, so I am very interested in following this discussion. } Randy
In the CM12, you can work under normal TEM observation and reduce step by step the size of the illumination spot down to about 20 nm while still retaining enough beam current in the probe to perform EDS microanalysis in a reasonable time. For that purpose you will use the spot size knob (physically the 1st condenser lens). This way is available on most modern TEMs too. DON'T FORGET TO REMOVE THE OBJECTIVE APERTURE, which otherwise will produce a hudge amount of X-rays and dazzle the EDS detector (it may take some minutes to fully recover its normal dead time!). It can be helpful to switch to diffraction observation if you want to analyze a tiny precipitate that give a different diffraction pattern than the surroundings. Be careful, a probe shift may happen due to the weak coupling of magnetic fields of the diffraction lens and the objective lens. So you will have to slowly shift (blind) the probe until you find again the caracteristic diffraction pattern of your phase. Then start microanalysis. If you see the pattern changing during the analysis, you should stop acqisition, shift the probe again and then continue the analysis. I desagree entirely with your service engineer. Switching to diffraction do not change anything to the magnetic field around the sample (ie, the objective lens) within the limit of the weak coupling mentioned above. So there are no more BSEs reaching the detector in one mode than in the other. Thus there is no reason not to use diffraction instead of image if you feel easier to decide wether or not you are firing on the right phase. He was probably thinking to the observation in low magnification mode (LM mode for Philips) where the excitation of objective lens is switched off or very strongly reduced. Then BSEs do no longer spiral in the magnetic field and then are allowed to escape toward the EDS detector.
I hope this will help you. Do not hesitate to contact me again if I wasn't clear. But I am leaving Friday evening for the next two weeks. Best regards
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Institut Interd=E9partemental de Microscopie Electronique Address: EPFL-I2M, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 (Central European Time) Fax: +41(21)693 44 01 ______________________ Eudora 2.0.2 __________________________________
} ___________________________________________________________________ } Randy Nessler } rnessler-at-uiowa.edu } The University of Iowa Central Electron Microscopy Research Facility } } } On Tue, 7 Jun 1994, Gib Ahlstrand wrote: } } } Lets talk EDS Detectors! } } Within a few months time, we will be adding an EDS detector with a thin } } window for light element detection capability to an existing TEM (Philips } } CM12, } } LaB6 electron source, no scanning coils (STEM), + or - 60 degree tilt stage, } } double tilt analytical grid holder). We are looking at a variety of detector } } characteristics and are considering:
Firstly, thanks for the useful info on EDS detectors. One question: Has anyone had success with annealing detector crystals to combat incomplete charge collection? } } My question is, without STEM, how are you planning to position } your beam on the area of the specimen that you would like elemental } information from? I am embarrassed to ask this question, as I obviously } haven't researched this in publications. When I asked about collecting } x-ray data while performing diffraction, I was told by a service engineer } that it would flood the detector with BSE's.
Switching to diffraction mode focuses the intermediate/diffraction/P1 (nomenclature depends on manufacturer) lens onto the back focal plane of the objective. It does (should) not influence the illumination seen actually at the specimen. Maybe your service engineer is thinking of fried (jellied?) EELS detectors ? :-) (which company is he from, btw?). I've lost the post now (hereafter referred to as the "lost-poster"), but someone mentioned weak coupling between INT and OBJ lenses - surely this problem, re spot shifting, is not a problem with correct rotation centring. Life starts getting very tricky, however, when your specimen is charging/magnetic/unstable - I won't go into details here though, since that's another issue. For precipitates/inclusions/nano-crystalline materials, viewing the diffraction pattern is indeed a good way to check the spot position, as already pointed out. Quantitative microanalysis of these features is a headache, though, but that's also another thread.
} Also, there is the problem } of diffraction flooding a rather large area of the specimen,
Use a smaller spotsize (C1 and C2 controls). CBED often is performed with tiny (~1nm or less FWHM) spot-sizes. LaB6 or FEG will help here. In a darkened room, you should be able to see sufficient information in the image formed with the smallest C1 and slightly defocussed C2 to orient the specimen. Chances are that if you cant see anything, your count rate will be too low anyway.
and the } selected area diffraction aperture allowing the pattern of information to } be viewed, while blocking out extraenous information.
Not sure what you are getting at here. The SAA, being far down the column (position varies - any post-specimen image plane will do), is well removed from anything to do with EDS, although possibly it may contribute X-ray counts if sufficiently high. It's a simple matter to find out if it is contributing and whether you need to make sure all users remove it prior to analysis. As the lost-poster said, it is *very* important to remove the Obj Aperture (OA) or you may start encountering incomplete charge collection (which manifests itself as an asymmetric, low-energy tail to each peak) soon afterwards. :-(
} Are you just interested in average area analysis? I hope I am not } the only one curious about this. This is a little off of the topic, and I } hope not to detract from the conversation. We are looking to upgrade a } detector on a SEM, so I am very interested in following this discussion. } Randy
The comments above refer to TEM/STEM. Life is quite different for SEM. We too are considering SEM detector upgrade/purchases so are also v interested in this thread (re detector crystals).
I'm working on analyzing dislocation arrays in ferroelectric PbTiO3 thin films. I'm searching for any software, preferably for Macintosh or DEC Alpha UNIX, that will simulate dislocation contrast under various diffraction conditions. I'm aware of ONEDISANL and TWODISANL (available through the EMMPDL archive), but would like to try other options if they are available. Suggestions?
Thanks in advance.
Stephen Streiffer Max-Planck-Institut f. Metallforschung Stuttgart, Germany
In the TEM literature there is a technique called pseudo-replica formation where formvar or collodion is poured in a thin layer over a surface and alloed to dry. It is then floated off, havin trapped particulates and large molecules which can then be observed with TEM. Has anyone done or does any one know of a similar technique for SEM. I need to enumerate particles attached to moist surfaces that cannot be processed by conventional means for SEM (living skin). I wonder if pressing scoth tape to the surface would pick up all the particles down to the several micron range???
We're seriously considering starting a user-fee type of arrangement for our electron microscopy facility. I was wondering if and how much other similar facilities charge for use of microscopes and equipment as well as consulting, etc..... Our facility is staffed by a faculty advisor and a half-time facilities manager.
I would even be willing to run a survey and compile the results if that is what it takes to get this information.
Thanks,
Todd Voiles Central Facility for Electron Microscopy Center for Materials Research and Analysis University of Nebraska, Lincoln, City Campus
} Hi, } } } We're seriously considering starting a user-fee type of } arrangement for our electron microscopy facility. I was wondering if } and how much other similar facilities charge for use of microscopes } and equipment as well as consulting, etc..... Our facility is staffed } by a faculty advisor and a half-time facilities manager. } } I would even be willing to run a survey and compile the results } if that is what it takes to get this information. } } Thanks, } } Todd Voiles } Central Facility for Electron Microscopy } Center for Materials Research and Analysis } University of Nebraska, Lincoln, City Campus } } tvoiles-at-unlinfo.unl.edu } Todd: Here at UMBC we charge for EM services but since we are a state institution we can't make a profit. All of our charges cover just the costs. We are probably on the low end as far as charging for services go. If it will help, send your fax number and I will fax a copy of our charges to you. Phil Rutledge Phone: (410) 455-3582 FAX: (410) 455-3875
A minor point, but potentially an important one for the future of Microscopy Listserver.
The Microscopy Listserver, and the MSA BB system are not the same entity. Although I basically run both (and also the EMMPDL, & Coordinate the MSA Computer workshop), the funds for each do not come out of the same pot.
To most of you things are transparent and (relatively?) seemless. I've tried to keep it that way intentionally as I've tried to make sure that everyone has access with the maximum amount of versatility (of course not everything works like it's supposed to but we're getting there slowly). The reason for this note is that I've seen some references lately where these admittedly subtle distinctions are accidently blurring, for example, in references to where things are located or how to get access to some information. This may not seem like a big deal to most of you, however, ultimately, I do need to document things to present and future funding sources (i.e. there is no free lunch) so when and if you talk about these different systems please try acknowledge them as different beasts.
Just for the record:
The MSA Computer Workshop is funded totally by MSA, except during joint society meetings, when our sister societies chip in a proportion of the costs. It is a week long interactive workshop held each year at the annual meeting of MSA.
The MSA BBS is jointly funded by MSA and ANL EMC. It is a regular BBS system runing on a PC with both Internet and Telephone links. MSA supports an 800 number (1-800-MAS-EMSA) as well as having purchased the PC and some of the software. The ANLEMC pays local costs (power, phone lines, network lines) and spends time & effort to keep it updated and running.
The EMMPDL is mainly funded by the ANLEMC, MSA contributes toward some expenses when local/sister societies want copies, or when the library is distributed at meetings (for example in the MSA computer workshop at New Orleans, or local society meetings).
The MICROSCOPY LISTSERVER (i.e. this EMail distributed discussion group) is run by by the ANLEMC but is not "officially" supported yet by any funding agency, basically I started it on the side as an experiment (call it a voluntary service to the microscopy community). Right now the ANL EMCenter ultimately pays the network costs through it's general overhead costs (so ANL and DoE should get some credit).If it is referenced, especially if you want to acknowledge it please simply use the phrase "The ANLEMC Microscopy Listserver". Try not to confuse it with the MSA BBS, while I'll know what you mean, if I need to raise support funds in the future (and I will need to) then it will need to be acknowledged appropriately.
Those of you who are reading the Microscopy Listserver via Newsgroups are getting a mail feed from ANLEMC, via a mailserver at the University of Michigan arranged by John Mansfield. Also,if you are exclusively on a newsgroup system you should know that the newsgroup replies do not presently filter back to the Microscopy Listserver subscription list. This means that your replies and/or questions do not reach most of the users of this system. This is on the list of things to fix, but it's one of those things that takes time and $$... If you want to reach the maximum coverage, you should post to:
We've just received an Alden 9315ctp continuous tone printer, but we were faced with a surprinsigly poor information for operating the printer, specially having in mind its price. The only executables they send are a tprnt.exe, presumably for TIFF files (no indication for the version), and a couple of conversion executables like t2r.exe and x9pc.exe. Unfortunaly in our hands these programs seems to have quite an random behaviour, either printing the image correctly, either producing truncated, layed, or tottaly corrupted images. So far we've tested a number of programs to generate the TIf's and Raster's (PSP, XView, PStyler, etc.) and so far we could not find a pattern for this behaviour. Being in a small european country, the local rep's can't be of much help (actually it's the first one they sold). Therefore I would to ask help to anyone who could provide me with any info on the operation of such printer, including other drivers for direct printing from the software (Windows for instance), info on net sharing drove by UNIX machines, and any possible internet contact with the guys from Alden. Thank you very much for any help.
________________________________________________________________ Jose A. Feijo Dept. Biologia Vegetal, Fac. Ciencias Lisboa Ed. C2, Campo Grande, P-1700 LISBOA, PORTUGAL
I am working on CaM localization and quantification in plant cells, and for that I have been using polyclonal antibodies raised in rabbit against bovine CaM. These antibodies were sold by SIGMA, but now they only sell MAb's, which does not detect the plant CaM.
So is there anybody out there with a stock of polyclonal anti CaM. It does not matter if the antibodies are raised against plant or animal CaM, but i would prefere if they were raised in rabbit.
Gred Erdos was inquiring about making replicas for SEM of living skin
One method you could try is to use dental molding compound - (the stuff they use to make impressions for orthodontics etc) - you mix it up, press it onto the surface of interest and it hardens up very quickly and can be removed without damaging the original surface -
We have done this a a class exercise - using the replica as a mold to make a "positive" with Spurrs resin - we generally did 2 applications, the first being used to remove the small surface debris which you want to look at so I suspect it should work for you
The stuff we used was called "Cuttersil" and was available through Columbus Dental PO Box 620 St Louis Mo 63188
} } *Received through multiple hands across the sea and along the internet } } } } INTERNET VIRUS ALERT! Date: Wednesday, May 25, 1994 } } } } A Virus has been discovered on Internet that is disguised as CD-ROM shareware. } } } } Unknown hackers have illegally put the Chinon name on a destructive shareware } } file and released it on the Internet. This catastrophic virus is named } } "CD-IT." --DO NOT DOWNLOAD. IT WILL CORRUPT YOUR HARD DRIVE. The program, } } allegedly a shareware PC utility that will convert an ordinary CD-ROM } } drive into a CD-Recordable (CD-R) device, which is technically impossible, } } instead destroys critical system files on a user's hard drive. The program } } also immediately crashes the CPU, forces the user to reboot, and stays in } } memory. } } } } Widest dissemination is requested. } } } } } } } } (Mr) Lindsey Thomas Martin (lmartin-at-sfu.ca) } Dialogue: Canadian Philosophical Review } Vancouver Studies in Cognitive Science } International History Review } Simon Fraser University, Burnaby BC Canada. }
---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
A visiting scholar at the Friday Harbor Labs is looking for parts for a JEM transmission microscope (model #100B). If anyone knows of a source please reply to Scott Schwinge at schwinge-at-fhl.washington.edu
Just so you know, part of the problems which caused a large number of Undeliverable mail messages has been cured. A server at the Univ. of Michigan had gone batty and was fixed by John Mansfield. We still have another server somewhere in the system which cannot deliver some mail to "previously" valid addresses. I'm investigating the problem.
___________________________________________________________________ Randy Nessler rnessler-at-uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
On Wed, 15 Jun 1994, MICHAEL DELANNOY wrote:
} I am looking for software that will drive a confocal microscope but doesn't } save image files in the .pic form but something more useful for other } Mac imaging programs such as Photoshop. Alternatively (and more likely) a } program that converts .pic to .gif or PICT without loss (at least significant } loss) of image quality. The clumsyness of manipulating images for figures in } comos is not aceptable } } } } Shawn Burgess } c/o Mike Delannoy
Talk to Biorad about the program .pic to .tiff. We have it on our MRC-600. Lately, people have been copying files to floppies and taking them to Adobe Photoshop. They are able to manipulate the .pic images knowing that the header is 76 bytes, there are 768 columns, 512 rows, and bytes/pixel = 1. Granted, it is a little cumbersome to do this. It appears that you have the confocal and Mac already, and is proably most economical to research the means to interface/import images. Randy
...... am looking for software that will drive a confocal microscope but doesn't save image files in the .pic form but something more useful for other Mac imaging programs ....
You should seriously look into getting a copy of NIH-Image. The current version level is 1.55 and 1.56 is in beta. You may download a copy from zippy.nimh.nih.gov using anonymous FTP. Alternatively if you attend the Microscopy Society of America Meeting in New Orleans in August you may get a copy at the Computer workshop & software exchange.
Message-Id: {MAILQUEUE-101.940615150052.256-at-vanlab.paprican.ca} To: Microscopy-at-anlemc.msd.anl.gov
Are there any microscopists out there who've worked on morphology differences between wood species? We're trying to find data on northern and interior British Columbia wood species with respect to fibre dimensions. If anyone has any insight out there I'd be grateful to hear from you, perhaps directly since this is a topic of extremely narrow interest and needn't tie up the listserver (unless someone else wants the info too; let me know). This is also kind of a shot in the dark to draw some of you forestry related microscopists out of the woodwork (pun unintentional). Thanks.
James Drummond Pulp and Paper Research Institute of Canada Vancouver, B.C.
First let me say that I AM NOT LOOKING FOR A JOB! But I would like to see a discussion of the future of technical jobs in EM. I always look at the classified ads in the EMSA (now MSA) bulletin. I have always found it interesting to see where tech jobs were and if my qualifications were keeping me competitive with the market. The last few MAS bulletin didnot have many, if any, EM job.
I have had scientist out side EM tell me that EM had had it peak, as all tecnologies do, and now has less and less uses. I believed that although pure ultrastructual studies of biological tissue has been pretty well covered, new advances would renew the need for trained people.
Is it true that EM is past its prime? EMSA did take out the "E"
Are people advertising job some where else?
Is money so tight that EM has become to expensive to be widely used?
Are service facilities taking the place of numerous smaller labs?
Are old EM tech. so full of Glut and OsO4 that they are not dying off?
Someone please give me some comfort. I still have 28 more years until retirement.
We will be purchasing a pc operated SEM with the intent of being able to remotely control the instrument. Initially, the remote site will be in our building and then a second phase is to be able to remotely control the instrument from a sister campus. Is anyone doing remote control of a SEM? If so, could you address the following questions?
Have you been able to accomplish remote control other than serial?
What functions can you perform?
What software interfaces have you used to access the SEM from elsewhere? (carbon copy, pc anywhere, etc).
What are the speed considerations?
Have you attempted to send both video and the data signals in real time?
How have you accomplished it?
If you have not accomplished this task, do you have any ideas or suggestions about how it could be done?
In times of very tight money, I don't think any of us can provide much comfort. However, I think Larry's points are good ones. I do think more of us are employing light microscopy (or going back to our original roots in LM). I think this is good because often LM/EM combined studies can be much more powerful than single technology. EM provides the resolution, but LM provides greater sample area and dynamic information. Our scheme to survive (contact me again in 5 years to see if it works), is to attract non-traditional EM users (biochemists, physiologists, etc) by helping design their experiments and making use of EM as easy as possible. All that is required of them is the ability to focus and capture the images. This has worked well for us over the years, the biochemistry department rather than the anatomy or pathology department constitutes our biggest users. It takes more of my time but it keeps us solvent. So, yes ithink there is still a large need for us dinosaurs (molecular genetics still won't answer all questions and a picture is still worth a thousand words even with inflammation) but you have to be more of a slalesman and gear your services to the community needs. My humble opinion.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Wed, 15 Jun 1994, Larry Hawkey wrote:
} } First let me say that I AM NOT LOOKING FOR A JOB! } But I would like to see a discussion of the future of technical } jobs in EM. I always look at the classified ads in the EMSA (now MSA) } bulletin. I have always found it interesting to see where tech } jobs were and if my qualifications were keeping me competitive with the } market. The last few MAS bulletin didnot have many, if any, EM } job. } } I have had scientist out side EM tell me that EM had had it peak, as } all tecnologies do, and now has less and less uses. I believed } that although pure ultrastructual studies of biological tissue has } been pretty well covered, new advances would renew the need for } trained people. } } Is it true that EM is past its prime? EMSA did take out the "E" } } Are people advertising job some where else? } } Is money so tight that EM has become to expensive to be widely used? } } Are service facilities taking the place of numerous smaller labs? } } Are old EM tech. so full of Glut and OsO4 that they are not dying off? } } Someone please give me some comfort. I still have 28 more years until } retirement. } } Larry Hawkey } Hawkey-at-neuro.duke.edu }
EM certainly has a future if our experience is any indication. We run a central service lab and we have never been without work to do for more than half a day. Seems just as we catch up (which rarely happens) the phone starts to ring.
We may not be seeing positions advertised for tech's due to the time lag involved in the publication of professional society newsletters. When I have a vacancy in the lab it usually occurs with little more than a few weeks notice and I need to fill that spot quickly and cannot wait for the next MSA Bulletin to come out. So I always turn to Jungle Drums for faster communication. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
} } First let me say that I AM NOT LOOKING FOR A JOB! } But I would like to see a discussion of the future of technical } jobs in EM. I always look at the classified ads in the EMSA (now MSA) } bulletin. I have always found it interesting to see where tech } jobs were and if my qualifications were keeping me competitive with the } market. The last few MAS bulletin didnot have many, if any, EM } job. } } I have had scientist out side EM tell me that EM had had it peak, as } all tecnologies do, and now has less and less uses. I believed } that although pure ultrastructual studies of biological tissue has } been pretty well covered, new advances would renew the need for } trained people. } } Is it true that EM is past its prime? EMSA did take out the "E" } } Are people advertising job some where else? } } Is money so tight that EM has become to expensive to be widely used? } } Are service facilities taking the place of numerous smaller labs? } } Are old EM tech. so full of Glut and OsO4 that they are not dying off? } } Someone please give me some comfort. I still have 28 more years until } retirement. } } Larry Hawkey } Hawkey-at-neuro.duke.edu } Larry,
My own bias is that EM use for biology will increase in the future. We have all seen a very striking renaissance of morphology at the LM level over the last few years. For example, everyone in molecular biology now wants to do immunocytochemistry, in situ hybridization and to localize reporter gene expression. And molecular biologists are finding out that they can utilize these methods intelligently only if they have some understanding of microscopes, tissue organization, cells, ultrastructure and basic morphological methods and strategies. New technologies are greatly expanding the capabilities of morphological methods. For example, the confocal LM can show where fluorescent ICC activity is in cells with much greater clarity than previous fluorescent microscopes could. All this is a far cry from a few years ago, when graduate students thought that if they learned how to clone genes their futures would be assured.
I really believe that these trends will continue, and will extend more and more to the EM level. It is often important to localize antigens within cells, which can be done by immunocytochemistry with gold particle labels. When lacZ (yielding beta galactosidase) is used as a reporter gene, it is often very difficult to identify the blue cells seen with the Xgal reaction in a complex tissue. In that case, localizing the Xgal staining reaction at the EM level (where it can be seen as dark blobs in the cells) allows one to utilize ultrastructural critera for the cell identification. There are many other examples.
My own convictions along these lines are clear from the fact that for the last two years I have offered a graduate course, "Morphology for Molecular Biologists" here at the University of Michigan. The course is held once a week (a three hour session), and includes lectures and demonstrations covering LM, TEM, SEM, a 2-hr "minicourse" in histology, ultrastructure, and then immunocytochemistry, autoradiography, in situ hybridization, localization of reporter gene expression, and finally confocal LM. Grad students from various departments and schools at the U-M have seemed highly motivated in the course.
So I am betting that the electron microscope does indeed still have a future.
A. Kent Christensen Department of Anatomy and Cell Biology University of Michigan Medical School akc-at-umich.edu
Microscopists: I operate a core facility here at UMBC and we are continually getting new contracts for EM services. I even get requests from universities that have EM facilities on campus. EM did take a nose dive but I think EM is on the way back up. I know this may cause an argument, but even molecular biologists are starting to see that instead of looking at gels all the time for information, additional and important information can be found by doing EM. Yes, I do have a bias against molecular biologists. When you have been constantly told by molecular biologists that there is no future for EM, you sort of get biased. I guess they forget that molecular cell biology is just another TECHNIQUE as is EM. Have a cold one for EM! Phil
The future of EM also concerns me. Molecular biology is in it's prime and the investigators seem to be following the trail of grant money that follows the latest technological fad. Who can blame them? However, I am still optimistic about the future of all microscopy. Similar to other labs, we have expanded our LM capabilities and entered into confocal microscopy. Further, we are also responsible for flow cytometry. Contracts with industry have been very important to the lab's fiscal health. Diversification enables us to select the most relevant technology for the client's needs. Correlation of molecular work to the morphology will bring the investigators back--in a different way. Immuno, fluorescence, digital imaging/analysis etc., are all going to be a part of it. Look at the environmental SEM, or SFM, or AFM. Actually, there is a lot to be excited about. What do the material microscopist's think? Eh, Nestor?
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick (Chuck) * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Anatomy * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-2706 voice * | | * 806 743-2707 fax * | | * Email hecub-at-ttacs.ttu.edu * | | * * __| |__ * * |_______| *************************************************
We would like to record some information with our images, perhaps in a header. It is getting to cumbersome to keep matched doc files with their images. We would like to be able to move and store one file per image and have that file also contain all of the documentation, instrument parameters, etc. We would like that information to be in ASCII so that it can be read easily.
I'm looking into using a TIFF tag such as imagedescription, to designate a long string or narrative that would describe the image. That string might look something like this: ((operator Dave Bright)(Instrument JEOL 8500)(Magnification 1000x 5=B5 field width)(date 16 June 1994)(Image_mode transmission bright field)(comment Sample #3 lot 445x2. 100nm gold coated after fine polishing. etc. etc.))
I presume we would have to write a small application that would append/edit/extract this part of the TIFF file. Other tiff file readers such as NIH Image would ignore it - the actual text would probably be put after the image, although this should not make any difference.
Anyway, for you image documenters out there: How do you do it? Are there already utilities around for keeping information along with the images, other than database type utilities?
Thanks :8o) Dave
------------------------------------------------------------- David S. Bright bright-at-enh.nist.gov Microanalysis Research Group Bldg. 222 (Chem.) A113 National Institute of Standards & Technology (NIST, formerly NBS) Gaithersburg, MD 20899-0001 / USA 301-975-3911 (voice), 301-216-1134 (fax) "A false balance is an abomination to the Lord but accurate weights are his delight.", Proverbs 11:1
} } This is hardly microscopy but rather a long shot. I have a colleague here } looking for a copy of the TRIM (transport of ions in solids) program, which we } believe to be public domain or otherwise available, for computing } ion distributions inside a solid after ion implantation. Can anyone help } direct us in the right direction? We would prefer a Mac or PC version, } but we also have other workstations so could probably run almost any } version. } } Thanks. } } Tony Garratt-Reed } } }
The author is James F. Ziegler IBM-Research, 28-0 Yorktown, NY 10598
Greetings, A client wants to embed ants in plastic for both light and TEM and has had problems with the exoskeleton embedding adequately in paraffin. We have no experience with insects. Are there procedures for softening the chitin? Any help would be appreciated.
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick (Chuck) * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Anatomy * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-2706 voice * | | * 806 743-2707 fax * | | * Email hecub-at-ttacs.ttu.edu * | | * * __| |__ * * |_______| *************************************************
John Benci asked about suppliers of used microscopes. Since used equipment is my favorite kind I have collected a list of companies that include electron microscopes in thier inventory. Of course this list is not complete, and I would love to hear about any others:
Cusaco Phone 908-502-9246.
Scientific Equipment and instrument Company 408-428-0464
OOPS! Cusaco's phone is 908-502-0246!! ignore the one above!
The Source 1-800-722-7719
Techlink 408-922-0888
International Equipment Trading Company 708-913-0777
} Greetings, } A client wants to embed ants in plastic for both light and TEM and } have no experience with insects. Are there procedures for softening the } chitin? Any help would be appreciated.
You might look at a recently published method in which the cuticular or waxy surface is primed by treatment with gamma-glycidoxypropyl trimethoxysilane prior to resin embedment in LR White. the paper was in Microscopy Research and Technique 21:355-360 (1992). The silane is available from Polysciences,Inc. (800)-523-2575.
} John Benci } Materials Science & Engineering } Wayne State University
} e-mail address: jbenci-at-eng.wayne.edu
} ------------------ RFC822 Header Follows ------------------ } Received: by mse.engin.umich.edu with SMTP;16 Jun 1994 12:29:09 U } Date: Thu, 16 Jun 94 12:15:18 EDT } From: jbenci-at-hub.eng.wayne.edu (John Benci) } Message-Id: {9406161615.AA29719-at-ss0.NIS.ss0} } To: microscopy-at-anlemc.msd.anl.gov } Subject: Used SEM's
A gentleman by the name of Clark Houghton, located in Ohio, would be the best and closest source I know of. Like me, he offers maintenance services on a wide range of electron microscopes. Unlike me, he also deals in used, refurbished instruments and also has leasing options. There are others offering used instruments, but if I were looking for a used instrument, he's probably the only one I would talk to.
Clark Houghton Phone (513) 927-5373 FAX (513) 927-5557
BTW, although I don't generally deal in used equipment, I often help my customers find new homes for their old stuff. None that I know of currently have equipment available, but I expect at least a couple of machines on the market in the next 6 months to a year. If your needs are not immediate, send me email with a more specific listing of your requirements.
Just to clarify - I have no financial relationship to Mr. Houghton, and only a passing friendship. He is well known and respected in the field.
Allen R. Sampson
Advanced Research Systems 317 North 4th. Street St. Charles, IL 60174
Could you please tell me if you are a state or private institution. Who bought the microscopes, who pays the tech salaries and who buys the supplies at priori. Finally who pays for the service contrast of each instrument. Do your charges to customers reflect these categories? How much let us say you would charge for embedding a piece of heart cuttting at three levels and making 30 pictures (5X7 inches) 10 at low mag (1-3) 10 at 5-20 and ten at 20-100? Thanks.
***** ************ ************** ************ *Cesar D. Fermin, Ph.D |Fax (504) 587-7389 *Tulane Medical School |Answ. Mach.(504) 584-2618 *Pathology/SL79 |Secretary (504) 584-2436 *New Orleans, La 70112 | Lab (504) 584 2521 ***** ***************** *********************** _______________________________________________________________________________ Cc: Microscopy Group
In answer to Dwight Beebe's additional questions:
1. Our laboratory servers approximately 80 major users (repeat more than 2X a year). 9 from our department, 41 from other departments within our institution, 24 from outside our institution (4 from industry), and 6 from foreign countries.
2. We charge less for departmental users because our department pays part of the service contract on the instruments.
3. We do not charge less based on user expertise. We provide initial training in any aspect of microscopy the user desires. After that we charge a fee for technical time if one of our staff is involved in the microscopy.
4. As outlined in a previous communique (Nestor- do we have a FAQ section?), we breakdown our fees for each individual technique done and cKcharge what it costs us to complete the task.Determining these fees is a major headache, but simplifies administration later and avoids hurt feelings since all are charged based only on what they use. We of course do not nit pick (i.e. charge for each grid, each cc of osmium, etc). Rather we have an average cost for negative stain, cost for cryosectioning, cost for cyroEM, etc.
5. Microscope usage is on a sign up reservation basis. Inexperienced users can only sign up during hours when resource staff are available for consultation. Once we (as a group) are convinced a user can handle themselves we make the microscopes available to them 24 hours a day. Rarely, but it has happened, we encounter someone who is either incapable or unwilling to use the laboratory in a responsible way. These people are denied all access.
6. Marcelle Gillot's summary of the problems of determining users fees is excellent and is almost identical to the constraints we use for determining fees. However, our legal department has a slightly different interpretation of allowable charges for outside, non-grant supported work. They advise that it is allowable to undercut the cost of locally available commercial services as long as you can document (and that may be a problem) that you do so while still recouping all costs of the work without relying on subsidies (goverment grants, institutional support for service contracts, salaries, building maintenance and rent etc.) to cut your cost. In other words, if you are willing to take less profit margin or pay your labor sweat shop wages you can charge less than your commercial competition. You just need to prove that you and your competition are playing on a level playing field.
I hope this is helpful. I would support a movement within MSA to survey and document the issues involved with fee for service operation. Business is clearly something most of us where not traine in and really don't want to spend our time on- however the world being what it is today.......... Regards Jay Jerome
Does anyone have a procedure for producing skeletal carbon TEM grids with holes sufficiently small to capture particles ranging in size from 200 nm to 30 nm?
Thanks.
-- Diandra L. Leslie-Pelecky Center for Materials Research and Analysis PH: (402) 472-9178 University of Nebraska FAX: (402) 472-2879 Lincoln, NE 68588-0113 diandra-at-unlinfo.unl.edu
I wish to confer with someone knowledgeable about the cryogenic aspects of EDS detectors. e.g., loss of insulating capacity of the Dewar, effects of warmup - quantitative, i.e., resolution with time and temperature- activation energy of the kinetics, effects of operating at dry ice -acetone temperatures.
A really good reference will suffice.
Please reply direct. I will prepare digest of any information that I receive for the list.
Message-Id: {9406201136.AA17097-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Software / Font needed Orig-Author: {mwinton-at-nosc.mil (Michael J. Winton)}:ddn:wpafb ----------------------------------------------------------- I apologize in advance for a post which is somewhat related to microscopy but not very closely. Please skip this message if that bothers you.
I am looking for a font for the Macintosh which has overlined numbers (Miller indices) built in. I have been searching and searching, and am tired of "drawing" the indices and pasting them into my documents. I'm sure that someone out here must have such a font. A public domain font editor would also be useful-- then I could create such a font and make it available to others. I have yet to find a font editor in an FTP site--font utilities are usually useful for little more than cataloging. I thought that there would be enough scientists in this newsgroup who have to deal with this regularly that one of you might know of something.
Please let me know if you can help.
Michael Winton (mwinton-at-nosc.mil / -at-zazen.lbl.gov)
Does anyone have a procedure for producing skeletal carbon TEM grids with holes sufficiently small to capture particles ranging in size from 200 nm to 30 nm?
Thanks.
-- Diandra L. Leslie-Pelecky Center for Materials Research and Analysis PH: (402) 472-9178 University of Nebraska FAX: (402) 472-2879 Lincoln, NE 68588-0113 diandra-at-unlinfo.unl.edu
Dear Light Microscopists, Does anyone know of a specific LM stain for fibrocartilage? A student is doing a study on a part of the skull and joints and would like some help. Any suggestions?
Thank you Piotr Swierczynski
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
Dear EM people, Does anyone know of any company (other Fisons UK) manufacturing gold or gold/palladium ring targets for Biorad sputter-coaters? We have just been informed (after waiting two weeks and having being informed that it was on its way) that we have a 3-4 week wait for a new one from Fisons UK, a slight problem as due to an "administrative oversight" there is no spare. Any suggestions for an alternative source in future would be gratefully received. Yours faithfully RE
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
In Word for Macintoshes, Miller indices are easy to make using the formula editor.
For those using Macs and don't know how to use the formula editor I can send a small Word 5.1 document with some examples of Miller and Weber indices. (BTW Word for Macs is much easier to use than WordPerfect!!).
Cheers, Timon
------------------------------------------------ Timon Fliervoet, Timonf-at-earth.ruu.nl, Faculty of Earth Sciences, Department of Geology, Utrecht University, P.O. Box 80.021 3508 TA Utrecht, the Netherlands. tel: (0)30 - 535054, fax: (0)30 - 537725
SPI Supplies carries the targets you are looking for and they should fit the Bio-Rad instrument. They are located in West Chester, Pennsylvania, USA. Tel # 1-800-242-4774 and FAX # 1-215-436-5755. Good luck!
In message Tue, 21 Jun 1994 17:07:32 +1200, richard.easingwood-at-stonebow.otago.ac.nz (Richard Easingwood) writes:
} Dear EM people, } Does anyone know of any company (other Fisons UK) manufacturing gold or } gold/palladium ring targets for Biorad sputter-coaters? We have just been } informed (after waiting two weeks and having being informed that it was on } its way) that we have a 3-4 week wait for a new one from Fisons UK, a } slight problem as due to an "administrative oversight" there is no spare. } Any suggestions for an alternative source in future would be gratefully } received. } Yours faithfully } RE } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 }
Message-ID: {MAILQUEUE-101.940621115228.256-at-eye1.eye.ufl.edu} To: microscopy-at-anlemc.msd.anl.gov
Several of our people are working with whole mounts of retinas removed from cow eyes, and are looking for a stain that will differentiate between dividing cells and inactive ones. We could use any suggestions for LM or fluorescent stains that do not require embedding and sectioning. Thanks! Evelyn Clausnitzer U of Florida Ophthalmolgy
Piotr Swierczynski was asking for specific stain for fibrocartilage. Don't think there is one-no-one here knows of one. Suggest you stain for collagen-thick bands in amongst cartilage would differentiate it. Possible stains would be Wright's, Masson-Trichrome or H and E/alcian blue double stain. Could also try an alizarin red S and Toluidine Blue O stain (Dawson 1926, Stain Techn. 1:123-4; Williams 1941, Stain Techn. 16:23-5). Let us know what you find out-which one works better.
Mark Elliott, UBC-Pulmonary Research Lab, St.Paul's Hospital Vancouver BC
Evelyn Clausnitzer was asking about stains for whole mounts for mitotic activity-try DAPI, Hoechst or acridine orange. They all work on tissue cultured cells which have not been embedded. Should be able to tell mitotic figures easily. Check with Molecular Probes to see if there are others, but these are fairly easy to use and look great.
Mark Elliott UBC-Pulmonary Research Lab St. Paul's Hospital Vancouver BC Canada
Have you tried just a basic H&E stain? It works well on fibrocartilage from a human intervertebral disk. Is the brain damaged? If so, have you tried: "Foot's Ammoniated Silver Carbonate Method"? From Foot (1924). It will show: vascular reticulum, tumor cells , and connective tissue around a brain tumor. Here is the procedure if you want to try it:
Fix: formalin (1:10) recommended; Cajal's FAB and Bouin's fluids also satisfactory.
Embed: in paraffin
Solutions: A. Ammonical silver carbonate: To 10 ml of 10.2% AgNO3 add conc. NH4OH, drop by drop until precipitate is almost redissolved; then add 10 ml of 3.1% Na2CO3, concentrated formalin, 1 ml; water, 100 ml. B. Reducing agent: 1% Na2CO3, 3 ml; conc. formalin, 1 ml; water, 100 ml. C. Intensifying solution: oxalic acid, 2 gm; conc. formalin, 1 ml; water, 100 ml.
Staining schedule: 1. remove paraffin from mounted sections and treat for 24 hr at room temp. in a mixture of pyridine and glycerol (2:1 by volume). 2. rinse in 95% etoh, then in water. 3. impregnate for 2.5 hr at 40 C in silver sol. A. 4. wash in distilled water. 5. reduce 5 min in sol. B. 6. wash in tap water. 7. tone 5 min in 0.2% gold chloride. 8. wash in tap water. 9. intensify by treating 5 min in sol. C. 10. rinse in tap water. 11. fix in 5-10% Na2S2O3.5H2O. 12. wash in tap water. 13. dehydrate. 14. cover in balsam or synthetic resin.
Note: The stain is not recommended for normal brain. It has considerable value in demonstrating connective tissue reaction around tumors if reticulin fibers are laid down by this connective tissue. If FAB fix is used, the reticulin staining is said to be suppresed.
ANNOUNCEMENT: Tribology and Coatings Technology Listserver/Mailreflector
Dear Colleagues:
With this EMail message we announce the startup of a Tribology Listserver/Mailreflector System. The purpose of this system is to allow the scientific community a centralized Internet address to which questions/comments/answers in the various fields of Tribology and Coatings Technology can be rapidly distributed to a list of interested individuals.
For the purposes of this discussion forum, Tribology should be considered to include all aspects of the study of coatings which involve friction, wear, lubrication, corrosion, strength, stability, design, and fabrication of materials and/or processes which involve the interaction of a surface (or surfaces) with its environs.
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To register with the system
1.) Send an Email message to "LISTSERVER-at-ANLEMC.MSD.ANL.GOV" within the body of the Email message include the following line:
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where "EMailaddress" is the electronic mail address of your host computer, and "UserName" is the username by which you are registered on that system.
PLEASE NOTE: The word TRIBOLOGY is important in your subscription request as this LISTSERVER software processes Email for several different mailing lists.
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3.) Anyone may post messages to this list, however, only subscribers will receive copies. One may post messages/comments to this list using any conventional Email system by sending a message to:
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This Tribology Listserver suplements the ANL Microscopy ListServer and MSA (Microscopy Society of America) electronic bulletin board system, and the EMMPDL (Microscopy and Microanalysis Public Domain Library) as a means of electronic communications which are also accessible over Internet as well as conventional telecommunications (i.e. modem) lines. Details of these are available upon request from EMMPDL-at-ANLEMC.MSD.ANL.GOV.
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Also, as this is an experimental and FREE, service to the commmunity please understand that some system problems will occur. They will be dealt with as (my) time permits but it is being done on a purely voluntary (& masochistic) basis.
========================================================================== Nestor J. Zaluzec Materials Science Division , Bldg - 212 Argonne National Laboratory, Argonne, Ill. 60439 USA
Message-Id: {9406221358.AA27889-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb Subj: Re: Software / Font needed Orig-Author: {Brian Gregory Demczyk {temcom-at-engin.umich.edu} }:ddn:wpafb ----------------------------------------------------------- No one in their right mind would use Word Perfect instead of Microsoft Word! Of course one is able to overstrike in the latter!
Group - Per a recent request, the winners of the Polaraid International Instant Photomicrography Competition are: Grand Prize: Dr. Gerald T. Baker, Mississippi State Univ. B&/W Light Micrography: Floyd E. Alberts, Ford Motor Company Microscopy Techniques: John Georgiou, University of Toronto Material Sciences: Vito Giannini, Italcementi SpA (Italy) Student: Jim Wetzel: Clemson Univ. Color Light Micrography: Wutian Wu, Eastern Virginia Medical School Should any wish a copy of the full 3-page announcement, send me your fax number and I will be pleased to supply. I expect that we will do an article on the full winners list in a future issue of Microscopy Today. Regards, Don Grimes, Microscopy Today
Does anyone have a procedure for producing skeletal carbon TEM grids with holes sufficiently small to capture particles ranging in size from 200 nm to 30 nm?
Thanks.
-- Diandra L. Leslie-Pelecky Center for Materials Research and Analysis PH: (402) 472-9178 University of Nebraska FAX: (402) 472-2879 Lincoln, NE 68588-0113 diandra-at-unlinfo.unl.edu
I need to purchase an inverted epi microscope to conduct membrane kinetic studies. Because of the experimental setup I can't use immersion optics. I have about 22K to spend and I already have a good quality video camera. Are there any thoughts on which product lines are the best and recomentations for objectives?
} I need to purchase an inverted epi microscope to conduct membrane kinetic } studies. Because of the experimental setup I can't use immersion optics. I have } about 22K to spend and I already have a good quality video camera. Are there } any thoughts on which product lines are the best and recomentations for } objectives? } } Thanks } } Carlo Montemagno } } Try a Nikon, if you want a rotating stage a Nikon will not do. Clarissa
Subject: Time:2:46 PM OFFICE MEMO Re:Overbars in MS Word Date:6/22/94 It is possible to print nearly any selected character centered over nearly any other character by using, in combination, the 'Overstrike' and 'Superscript' typesetting commands provided in Microsoft Word. These commands are described in the section on producing formulas (which appears on pages 98 to 105 in the instruction manual for MSW V4.0). As an example, working with New York font, the following command string will produce a capital 'X' with a bar neatly centered above it: .\O.\AC(X,.\S.\UP11(_)), while the command string: (.\O.\AC(1,.\S.\UP11(_))0.\O.\AC(1,.\S.\UP11(_))) produces the set of Miller "bar one-zero-bar one". In these strings, the special character (.\) telling MSW to enter the typesetting mode is produced by holding down the command and option keys while typing a backslash (\). Command strings must be prepared by activating the "Show-#166#" mode , and the final printed format can be displayed by the "Hide-#166#" mode (obtained in the Edit menu or toggled by pressing the command and 'Y' keys simultaneously). Now, you may complain that it is inconvenient to type long command strings such as these as frequently as might be needed in many manuscripts. However, the process can be simplified by first typing the manuscript using a dummy variable (e.g. VX for vector X, 1B for overbar one, etc.). When completed in this manner, prepare the command string for a particular symbol and save it on the clipboard (command-c). Then use the Find utility (command-F) to find the first occurrence of the corresponding dummy variable, click in the ULH square to release the Find window, replace the dummy variable with the command string (command-V), find the next occurrence of the dummy variable (option-command-A), replace it, and so proceed through the entire manuscript. Try it, you'll find it works rather well. You will also find the full set of typesetting commands to be very useful for inserting formulas into manuscripts - they require a little practice, but turn out to be quite convenient and versatile, once you get familiar with the system.
Message-Id: {9406221911.AA29215-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb Subj: overstriking... Orig-Author: {"Daniel L. Callahan" {dlc-at-owlnet.rice.edu} }:ddn:wpafb -----------------------------------------------------------
If you find out how to overstrike in Word (Mac), please post to the microscopy listserver so all will know! I too have hunted this down unsuccessfully.
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
Scott, To overstrike in Word for the Mac is staggeringly simple. To put e.g., a slash through a zero similar to one of the Scandinavian characters, you should i) hold down COMMAND and OPTION together and type a backslash ii) type an o (for overstrike strangely enough) iii)) type a LH bracket iv) type the two characters, separated by a comma v) close the brackets. (Of course the Scandinavian letters are available anyway so you would not bother doing this particular example) Congratulations, you have just overstruck! If you want to put a bar above an index, just make the second character a superscript hyphen.
Happy overstriking Rick Hall Mat.Sci., U. of Delaware
Okay folks, I'm more than a little suprised that Nestor hasn't chimed in here and I might be out of place, but this is _NOT_ the place to either discuss the pros and cons of a particular piece of word processing software, particularly when it has no direct bearing on microscopy, nor is this forum the place to flame someone for the signal to noise ratio of their posts. I have been the unfortunate participant (vicarious) in other flame wars on different forums and they serve _NO_ purpose. If you find the comments of someone offensive and wish to respond, please do so in private. Please, please, don't let this highly useful and informative discussion group dissolve into acrimonious shoot-outs over message content or perceived attitude. Thanks, I'll now fade back to my usual semi-transparent level of participation.
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
NB the command key is the key marked with an apple sign NB2 hold down the option, command and backslash (\) key simultaneously (this will activate the formula editor). NB3 it might be a bit longer then option-command-\O(1,_) but can be put in a macro
Cheers Timon
------------------------------------------------ Timon Fliervoet, Timonf-at-earth.ruu.nl, Faculty of Earth Sciences, Department of Geology, Utrecht University, P.O. Box 80.021 3508 TA Utrecht, the Netherlands. tel: (0)30 - 535054, fax: (0)30 - 537725
I just want to thank both Nestor & Dwight for bringing out the fact that this IS a microscopy list. I agree wholeheartedly that this is NOT the place for discussions (shall we say) on things such as word vs. wordperfect. Let's get back to microscopy please.
Just venting.
Thanks, Peling Melville
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
Reply_ Overstrike FONT Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu A company in Ann Arbor, Allotype Typographics sells two fonts that are great for Miller indices. They are called Haber and Thompson, they are based on Helvetica and Times respectively. I have been using these fonts for about 5 years now and have been very happy with them. Allotype can be reached at 313-480-3666. I am just a satisfied customer. Jfm.
We are trying to use a procedure that was established over 20 years ago. One of our problems is that a chemical was used to treat the glass slides in order to prevent the wood pulp fibres from sticking to them during drying. The chemical used was dichlorodimethylsilane which is considered highly toxic. We would like to hear any suggestions for an alternative way to prevent the fibres from sticking.
Greetings, I would like to say that the discussion on overstriking has been very helpful. While I agree that those parts of the discussion that "flamed" were not needed, the main discussion had a lot of content, and I don't see why this disscusion should not be allowed to continue on this group.
I bet word processing is the ONLY technique that EVERYone on this list uses. Word processing certainly is an indespensible technique for the professional microscopist. I guess it is reasonable to have some limits: certainly flames are unwanted, and so too discussions of an ethically dubious nature might be shunned, such as how microscopists can avoid income taxes, and finally discussions that are wholly irrelevant, such as on making the best cheese fondue could be canned. My view is that word processing is relevant for microscopists to talk about. I might add, as a light microsopist in a biology department, there are many subjects covered on the list that I ignore. Three cheers for clear subject lines.
To be fair to the individual who started the discussion thread on "Miller Indices" (not word processors!), the originator of the message, first sent the text to me and I was asked if it was appropriate for this forum. Since it was a serious question, and one which was related to microscopy and he was clearly in need of an answer (& and a listing of the various options for solving his problem) I concurred and told him to post the question to this forum. It's now run it's course and unless there is something else to add specifically on Miller Indices that has not been covered let's move on.
I will continue to be a behind the scene's filter for anything which the subscribers to this list may wish an opinion on prior to posting and a traffic cop when things appear to be getting shall we say "out of hand". Feel free to abuse my screen all you like. You will neither be the first and I'm sure you will not be the last, and as you are all aware the electronic trash can is only a button away.
I would have to agree with Tobias Baskin's comments on acceptable content of this bulletin board. As long as the material is something used by the readers on a professional level, why not include it as fair game? In an open system, such as this, it's up to the contributors to remain professional, and keep in mind what may be of general interest to the rest of the readers.
I find this forum interesting to read, and it's provided a wealth of information that I've squirreled away for future reference. I'd really like to see it kept open...
Thanks
Margaret E. Hogan, PhD Electron Microscopy Service The Jackson Laboratory 600 Main Street Bar Harbor, Maine 04609 (207) 288-3371 #1450 (207) 288-5079 FAX meh-at-aretha.jax.org
Message-ID: {MAILQUEUE-101.940623184732.288-at-UNDERBANK.shef.ac.uk} To: microscopy-at-anlemc.msd.anl.gov
Hi, I would have kept quiet but for some users suggesting that it is a good idea to exchange trivia about word processing. I joined a microscopy board, not a software panel - I get a lot of useless mail and I don't need a pile of messages about word processing. May I politely suggest that people who want to talk about anything except microscopy on this valuable service should form their own club.
As long as everyone is getting in the act, I will also jump in. I am a Mac user and I subscribe to a variety of newsgroups, many of which specialize in discussions of topics such as WP programs. I suggest that other Mac users seek out such discussion groups and PC users seek like discussions. Let's talk scatterin' events here...
Larry Sutter Michigan Technological University Department of Metallurgical & Materials Eng. 1400 Townsend Dr. Houghton, MI 49931
Does anyone have/know of a Photometrics Star 1 thermoelectrically-cooled CCD camera in good condition which is available for purchase? They are apparently no longer commercially available (that's why I am bothering this list about it.)
Bill ========================== Bill Heeschen/Analyt. Sci.- Mat. Char. ----- 1897-F Building / The Dow Chemical Co. --- --- Midland, MI 48667 U.S.A. --- D O W --- phone: (517)636-4005 fax: (517)636-5453 --- --- Email: waheeschen-at-dow.com ----- ========================== R
Subject: Time: 4:42 PM OFFICE MEMO Is EM art? Date: 6/23/94 Are we scientists are artists? If the answer is the first then why are there so many micrograph competitions?
} Subject: Time: 4:42 PM } OFFICE MEMO Is EM art? Date: 6/23/94 } Are we scientists are artists? If the answer is the first then why are there } so many micrograph competitions?
} To answer this. EM is an art! You either have it or you don't!
} OFFICE MEMO Is EM art? Date: 6/23/94 } Are we scientists are artists? If the answer is the first then why are there } so many micrograph competitions? This question has no simple answer. For starters, the question of who (or what) an artist is may need to be answered. Answering the question, "What is a scientist?" is not easy, but at least science is a collection of fairly well pre/proscribed paradigms. Art, on the other hand, is a free-for-all. It is now generally accepted that photographs may be art; I say generally accepted because there are some very conservative/reactionary/retrograde critics who refuse photography art status, but if we relegate these people to the status of bonkers, then we'd all agree (as any reasonable person would) that photographs can be art. So what makes a micrograph, a specific class of photograph (although this definition must be broader to include video, computer representations, etc.) a work of art? Let's look at a more general question, what makes (an object) art? Or a question that attempts to seek boundaries: does art have to be made by an artist? If art can be defined by an audience's reception of an object or event and not by a performer (e.g. artist-- regardless of intent), then a micrograph can be a work of art without there being an artist, in this case a scientist. The danger this position raises, however, is that it may not priviledge only the relics of human endeavor; for instance, a rock or sunrise could be misconstrued as art. Here is the center of the issue: the objects to which the questions really refer (micrographs) are compelling aesthetic objects. Just because something looks good or touches that ineffable something in us and is made by a person or people does not mean it is art.
None of these musings lead directly to the question of why there are micrograph competitions. Entering a competition implies desire for exhibition or need for public recognition or motivation to WIN or the expectation of another line on the cv, but these apects of showing work, although the same or similar to concerns in the arts, really have nothing to do with art specifically.
The only way, logically, to answer the general question whether scientists are artists and to, pragmatically, avoid exploding the term "artist" so that it is meaningless, is to answer NO. More simply: There are scientists who are artists. Not all scientists are artists. The last two statements have nothing to do with whether micrographs, in general, are art. Some micrographs are art and some are not.
On Thu, 23 Jun 1994, rutledge phil wrote: } On 23 Jun 1994, Paul Webster wrote: } } OFFICE MEMO Is EM art? Date: 6/23/94 } } Are we scientists are artists? If the answer is the first then why are there } } so many micrograph competitions? } } To answer this. } EM is an art! You either have it or you don't! EM is a craft.
} Subject: Time: 4:42 PM } OFFICE MEMO Is EM art? Date: 6/23/94 } Are we scientists are artists? If the answer is the first then why are there } so many micrograph competitions?
From the for what its worth dept., since ya asked...:
I don't think that being a scientist should exclude one from appreciating art. Hopefully, its in the study of the symmetry and beauty of nature (be it biological or material, with a light or electron 'scope) that scientists can learn and "see" more of the world around them. Having such competitions, to myself anyway, allows one to view others' work; it gives a forum for the display of different photomicros that otherwise I might not ever see for myself. It also allows others to see the best of different techniques/methods.
Besides, its a way to win neato prizes :) :) But I suppose that ya have to enter 'em first, 'eh?
My {$0.02, -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /\v/\ | | Metallographic Lab. | Missouri Speleological Survey \-v-/ | | Rolla Research Center | Bat Conservation International /\v/\ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /-v-\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
From HOLONET What's Appropriate? Please stick to microscopes! Reply to Tobias Baskin's message:
I was away from my office for one week and came back to over 150 E-mail messages from the Confocal newsgroup and from this newsgroup. It's going to take a long time to read and save the useful messages, and delete the redundant replies, and delete the all too large number of off-topic messages. Come to think of it, this reply is probably off-topic, so I would aprreciate your re-reading the Moderator's announcements of the purpose of this newsgroup instead of replying to this message.
} I would like to say that the discussion on overstriking has been very helpful...
I deleted all of those with extreme prejudice without reading any.
} I bet word processing is the ONLY technique that EVERYone on this list uses....
So what. I'm on here to learn about MICROSCOPES. I'll go as far as anti-vibration tables for acceptable hardware. Questions and comments on software not directly related to to your microscope should be asked elsewhere.
} there are many subjects covered on the list that I ignore...
Yes, but the junk mail still shows up in my mailbox.
} Three cheers for clear subject lines.
Agreed. I will delete without reading all future messages that have "None" or garbled subject lines (my apologies to any customers who don't get a reply from me because of this policy).
Message-Id: {MAILQUEUE-101.940624083209.352-at-bunyip.ph.rmit.edu.au} To: microscopy-at-anlemc.msd.anl.gov
I agree entirely John Millar RMIT Australia ------- Forwarded Message Follows ------- To: microscopy-at-anlemc.msd.anl.gov
Hi, I would have kept quiet but for some users suggesting that it is a good idea to exchange trivia about word processing. I joined a microscopy board, not a software panel - I get a lot of useless mail and I don't need a pile of messages about word processing. May I politely suggest that people who want to talk about anything except microscopy on this valuable service should form their own club.
Paul Fons asked about HREM image simulation software. In this context I would like to float an idea for responses. In the past, Hitachi has been selling our software at prices which (in my opinion) are ludicrously high. We are in the process of rewriting it for a RISC/UNIX workstation, exploiting X-windows and UNIX wide system calls. I am very tempted to make it freeware, either via ftpanon or otherwise when this is finished; in other words it should be compilable on any modern UNIX system (and PC/Mac systems as they move to be compatible with workstations).
As you know, it is a fallacy to imply that the two are mutually exclusive. EM is used as a tool in a great many areas of science. The yearning to use such a tool with some elegance (rather than in a manner that is dull or sloppy) is not unique to EM. I know biochemists, molecular biologists, and others, whose work has elegance. They are not satisfied with a mere dumping of required information (in any form) into a paper. They want the information to be well organized, tight, clear, a pleasure to read. One might say they do science with some art. I hope we can have more science involving EM that has this kind of elegance. If there is an occasional EM art show on the side, it is certainly not a problem.
Regards, Kent (A. Kent Christensen, Dept. of Anatomy and Cell Biology, Univ of Michigan, {akc-at-umich.edu} )
-----------------------------------
On 23 Jun 1994, Paul Webster wrote:
} Subject: Time: 4:42 PM } OFFICE MEMO Is EM art? Date: 6/23/94 } Are we scientists or artists? If the answer is the first then why are there } so many micrograph competitions? } } }
Reply_ RE} HREM simulation software Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu Absolutely excellent idea!!!!! There are too many people who write software in the microscopy field and think they are going to get rich on it. They try to sell it for exorbitant amounts of money and it is frequently buggy as all hell.They are usually unable to support it adequately since they are typically only selling it as a part time job. Software marketing and support is difficult to do properly! I think making it available free would be an excellent service to the user community.
-------------------------------------- Paul Fons asked about HREM image simulation software. In this context I would like to float an idea for responses. In the past, Hitachi has been selling our software at prices which (in my opinion) are ludicrously high. We are in the process of rewriting it for a RISC/UNIX workstation, exploiting X-windows and UNIX wide system calls. I am very tempted to make it freeware, either via ftpanon or otherwise when this is finished; in other words it should be compilable on any modern UNIX system (and PC/Mac systems as they move to be compatible with workstations).
Comments ?
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Why not? How can anyone be against this? If you can cover your costs somehow, and you want to do it, consider me a cheerleader!
On Fri, 24 Jun 1994, L. D. Marks wrote:
} Paul Fons asked about HREM image simulation software. In this } context I would like to float an idea for responses. In the past, } Hitachi has been selling our software at prices which (in my opinion) } are ludicrously high. We are in the process of rewriting it for a } RISC/UNIX workstation, exploiting X-windows and UNIX wide system calls. } I am very tempted to make it freeware, either via ftpanon or otherwise } when this is finished; in other words it should be compilable on } any modern UNIX system (and PC/Mac systems as they move to be } compatible with workstations). } } Comments ?
A synthetic Ni-olivine (Ni2SiO4) from the Smithsonian (USNM #717) may be available. We have some of this material that was used in a thermodynamic study by R. Robie et al (Amer. Mineralogist v.69, p.1096, 1984). You may be able to obtain some through Gene Jarosewich at the Smithsonian.
Jim McGee
James J. McGee email: jmcgee-at-lunatic.er.usgs.gov U.S. Geological Survey Phone: (703) 648-6782 959 National Center Fax: (703) 648-6789 Reston, VA 22092
Dear John, I assume the unknowns are silicate minerals. If not, you should make an attempt to have the same average Z-value for unknowns and standards--Chuck Fiori made a point of this in referrence to biological specimens. I'd try to mix a nickel compound (any one which is handy and doesn't have other peaks in the energy region of interest will do) with a molten silicate glass or with quartz if you can heat it enough to get thorough mixing. I'd make a range of different nickel fractions from ~1% to whatever you expect at maximum. The average Z and thorough mixing are the keys. Good luck.
As long as we are all voting, I agree that the discussion of word processors has run its course. However I did learn some useful things applicable not just to miller indexes but to microscopy writing in general. Since we are a varied background of users (and many of us could care less about subscribing to a bulletin board on word processors or operating systems, etc.) . IMHO the occasional inclusion of helpful but peripheral information and questions should be encouraged. Like others, my mailbox is too cluttered and I throw out a lot without reading. But I would like to keep things open. Occasionally a gem comes along.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
RFC-822-Headers: Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII On Tue, 21 Jun 1994, Carlo Montemagno wrote:
} I need to purchase an inverted epi microscope to conduct membrane kinetic } studies. Because of the experimental setup I can't use immersion optics. I have } about 22K to spend and I already have a good quality video camera. Are there } any thoughts on which product lines are the best and recomentations for } objectives? } } Thanks } } Carlo Montemagno
On June 22nd Clarissa wrote in response to Carlo Montemagno's question:
} Try a Nikon, if you want a rotating stage a Nikon will not do.
For those who want a rotating stage for the Nikon inverted microscope, as of a few weeks ago, a rotating, centrable, gliding stage is available for the Nikon Diaphot 200/300. It attaches by the same 3 screws as the standard stages, it is not graduated, but works well for DIC. Cost: $1600.00 It is manufactured by Micro Video Instruments of Avon, Ma. USA For more information: 508 580 0080 or FAX 508 580 8623
I also heartily agree that open software would be a big advance. Don't limit it to HREM. The errors in commercial packages are virtually uncorrectable and we wind up with the situation bulldust in (the code) = bulldust out. Sadly only known after a lot of pain and wasted time. eg the K-line simulation problem tracked down by Alwyn Eades and his collaborators.
In another "hot" topic which could set off a mine is better than yours email war.....
JEC of GIT asked about imaging programs (and possible platform competition). Well let me try to be impartial and start the discussion ;-) ------------------------------------------------------------
There are many commerical programs available for handling image data, all of these do good to excellent jobs, (on both Mac's and PC's -- Oops just showed a preference didn't I--) unfortunately you will have to purchase them.
But to answer your question . No one has bothered to port NIH image to the PC platform. The lastest version 1.56beta9 runs on virtually all Mac's but not the PC. The source code is available on Zippy.nimh.nih.gov if you are interested in a challenge.
On the PC side the only freeware programs I am familiar with are NCSA Image for the PC, and UCFImage for the PC. There are also a couple of programs which I would call Viewers, the just let you put images up on the screen and not much else. All of these are freeware and will be available at the computer workshops at the PARIS ICEM13 meeting (July 17-22), as well as the at the New Orleans MSA/MAS meeting (August 1-5).
The NCSA suite of programs were not designed as an image processing programs but then again neither was NIH-Image, rather they might be better described as a data set visualization programs. I've occassionally used the NCSA code but not frequently, since I've found that I can more readily customize NIH Image to Microscopy related tasks.
UCFImage is a older image processsing type program out of the University of Central Florida, the version (5.0) I have lacks the polish of a true GUI program (it runs under DOS) and hence to most users who have been wedded to WINDOWS or Mac's (there I've now balanced my comments) might be disappointed. I found it clumbersome to use, however, if you are running on an older DOS machine then you may wish to check it out.
If anyone is still interested I can dig up the readme files and post segments tomorrow.
I'd be interesting in hearing about any other public domain programs which can be added to the software library database. So if you know of such please post the information here. as well as include the information on how to get copies. I'll then add the information to the EMMPDL libraries and try to insure that copies are available next month at both ICEM and MSA.
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I also heartily agree that open software would be a big advance. Don't limit it to HREM. The errors in commercial packages are virtually uncorrectable and we wind up with the situation bulldust in (the code) = bulldust out. Sadly only known after a lot of pain and wasted time. eg the K-line simulation problem tracked down by Alwyn Eades and his collaborators.
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I also heartily agree that open software would be a big advance. Don't limit it to HREM. The errors in commercial packages are virtually uncorrectable and we wind up with the situation bulldust in (the code) = bulldust out. Sadly only known after a lot of pain and wasted time. eg the K-line simulation problem tracked down by Alwyn Eades and his collaborators.
Anyone have a K-1905-1 board out of a Kevex micro-7000 EDX analyser spare that they are able to give away / sell? Ours is faulty and hindering one of our M.Sc. students.
Thankyou,
Keith Hallam
p.s. If I send a message out on the microscopy discussion group does it automatically get echoed to the usenet group too? If so, apologies for repeating myself!
We had a major network crash here at ANL over the weekend. FRIED fiber optics and lots of headaches all around. I apologize if messages got lost over the weekend. I think things are back to "semi-normal".
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I also heartily agree that open software would be a big advance. Don't limit it to HREM. The errors in commercial packages are virtually uncorrectable and we wind up with the situation bulldust in (the code) = bulldust out. Sadly only known after a lot of pain and wasted time. eg the K-line simulation problem tracked down by Alwyn Eades and his collaborators.
The image analysis facility down the hall is interested in the names of some software packages that a digitized or scanned electron diff pattern can be measured and indexed with. Are there any public domain/shareware programs available? Since the demand is small, they aren't willing to spend a great deal. Thanks in advance. Randy
___________________________________________________________________ Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
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Why can't EM be both science and art - we use this tool as scientist and in the process create some wonderfully beautiful images that have a definite aesthetic value - in my opinion the fact that we can appreciate the beauty and have a desire to share it in no way detracts from our seriousness as scientists - besides it may grab the attention and interest of a non-scientist and get him or her interested in learning more about the subject of the image or the process by which it was created
JULY 1994 ISSUE OF THE JOURNAL OF MICROSCOPY - VOLUME 175 PART 1
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 1-9
Differential imaging in confocal microscopy
T. WILSON, R. JUSKAITIS & J. B. TAN, Department of Engineering, University of Oxford, Parks Road, Oxford, OX1 3QZ, U.K.
SUMMARY A coherent detection system using a two-mode optical fibre is described which permits confocal, differential amplitude contrast and differential phase contrast images to be obtained simultaneously from a scanning optical microscope. the direction of differentiation may be chosen arbitrarily. The differential imaging modes possess an optical sectioning property.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 10-20
A novel method of Z-contrast imaging in STEM applied to double labelling
W. TICHELAAR, C. FERGUSON, J.-C. OLIVO, K. R. LEONARD & M. HAIDER, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany
SUMMARY A novel method of Z-contrast imaging in the scanning transmission electron microscope (STEM) is presented. The technique relies on the element dependence of the angular distribution of the scattered electrons, and is realized with a detector consisting of a set of concentric rings. It is possible to discriminate 9-nm colloidal gold and silver specifically distributed on thin sections. In addition to this practical work, numerical evaluations are used to assess the method. With two smaller markers, this approach will be useful in discriminating closely-spaced antigenic sites when steric hindrance occurs with double-labelling using probes of different sizes.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 21-33
A new method to correlate acoustic spectroscopic microscopy (30MHz) and light microscopy
A. F. W. VAN DER STEEN, J. M. THIJSSEN, J. A. W. M. VAN DER LAAK, G. P. J. EBBEN & P. C. M. DE WILDE, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands
SUMMARY A powerful new method is used to correlate between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into slices, between 250 and 10 micrometres thick. The thick sections were investigated acoustically, the thin sections by light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2.5 x 2.5 mm). Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30MHz, the slope of the attenuation spectrum (between 10 and 50MHz), backscattering at 30MHz, the slope of the backscattering spectrum (between 10 and 50MHz), and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 micrometres. The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen-rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 x 250 micrometres. These histological features were correlated with acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 34-43
High pressure freezing of cell suspensions in cellulose capillary tubes
H. HOHENBERG, K. MANNWEILER & M. MULLER*, *Laboratory for Electron Microscopy I, Institute of Cell Biology, Universitatsstrasse 2, CH-8092 Zurich, Switzerland. and Heinrich-Pette-Institute for Experimental Virology and Immunology, at the University of Hamburg, D-20251 Hamburg, Germany
SUMMARY A procedure for efficient cryoimmobilization of large volumes of cell suspensions or micro- organisms by high-pressure freezing is described. This procedure uses transparent, porous cellulose capillary tubes with an inner diameter of 200 micrometres, into which the suspensions are drawn by capillary action. The tubes are processed by high-pressure freezing and freeze substitution as if they were tissue samples. Centrifugation of suspensions at low temperatures is no longer necessary and cryopreparation is greatly facilitated. A very high yield of adequately frozen specimens is obtained due to the constant, defined sample geometry. This approach can also be used to process suspensions by conventional chemical fixation, eliminating the need to embed pellets in low-melting-point agarose, for example, prior to chemical fixation. The preparation procedure is demonstrated with suspensions of nematodes, paramecia and bacteria.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 44-53
Nucleotide and protein distribution in BrdU-labelled polytene chromosomes revealed by ion probe mass spectrometry
RICCARDO LEVI-SETTI, JAN M CHABALA & SARAH SMOLIK*, Enrico Fermi Institute, The University of Chicago, 5640 South Ellis Avenue, Chicago IL 60637, U.S.A., and *Vollum Institute for Advanced Medical Research, Oregon Health Services University, Portland, OR 97201, U.S.A.
SUMMARY Detailed chemical maps of BrdU-labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine-analogue BrdU within the chromosomal DNA is localized by detecting the Br- secondary ion signal, while both nucleic acid and protein content, are mapped through the abundantly emitted CN- signal. This novel approach supersedes, and helps to explain the origin of, the banding patterns that are observed by conventional staining techniques. the high spatial resolution and chemical and isotropic sensitivity of the technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 54-59
Application of backscattered electron imaging to the study of the contact zone between lichen and rock. Assessment preparative procedures
J. WIERZCHOS & C. ASCASO, Centro de Ciencias Medioambientales, Serrano 115, bis Madrid 28006, Spain
SUMMARY In the study of the lichen-rock interface, light microscopy, scanning electron microscopy (SEM) in secondary emission mode and transmission electron microscopy are the most commonly-used techniques. As these methods have some limitations, there is a need to explore other techniques for observation of the lichen-substrate interface. One of the most promising methods is the application of SEM in the back-scattered electron (BSE) emission mode. The thallus of Aspicilia intermutans (Nyl.) Arn. growing on granitic rock was examined by SEM in BSE mode. The detailed preparation of transverse sections of the lichen-rock contact zone is presented. The BSE scanning images of the lichen-rock interface obtained present new insights into the ultrastructural features of the biological components, providing more information about the biogeophysical and biogeochemical weathering of rock.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 60-69
Semi-automated measurement of true chord length distributions and moments by video microscopy and image analysis
EBEN H. OLDMIXON, JAMES P. BUTLER* & FREDERIC G. HOPPIN, Division of Pulmonary Medicine, Memorial Hospital of Rhode Island, 111 Brewster Street, Pawtucket, RI 02860, U.S.A. and *Harvard School of Public Health, Boston MA 02115-9957, U.S.A.
SUMMARY The distribution of lengths of airspace chords in pulmonary parenchyma characterizes many architectural features of the alveoli and alveolar ducts. Laborious to obtain manually, the distributions and density functions may be acquired semi-automatically by video microscopy, digitization and image processing. The accuracy of the estimation is influenced by the microscopical methods and also by the techniques used (i) to convert the digitized grey-scale picture to a two-valued image, (ii) to collect the chord lengths and (iii) to compensate for finite field widths. The last problem arises because some of the chords are completely visible within a field, while others are only partially seen, since one of the two air-space boundaries lies outside the field of view. The error systematically biases the observed distribution. This paper presents solutions to hardware, software and analytic problems encountered while developing the capability to measure airspace chord length density functions semi- automatically. Formulas for estimating the true chord length density function from samples of observed chord lengths are presented. Also given are formulas for the estimation of the first and second moments of the true chord length distribution from the means of observed chord lengths. These techniques of image preparation and analysis should be suitable for characterizing particle, grain or cell size distributions, especially where many profiles fall partially outside the field of view.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 70-83
A simple algorithm to measure the volume-weighted and number-weighted mean volume of particles
GUY J. LAROYE & K. GRANT, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada
SUMMARY An algorithm is presented which offers an alternative approach for measuring volume- and number-weighted mean volume and standard deviation of particles. Using a computer- assisted manual method the following intermediate steps are performed automatically: generation of linear probes emanating from the sampling point of the object and intersecting the profile periphery, measurement of their lengths, and measurement of the area of the transect required for estimating the standard deviation of the volume-weighted mean volume. By first tracing manually the outline of the periphery of the object with a cursor, on a magnetic tablet or on an image acquired into the computer with a video camera, the location of all pixels of the periphery is registered and the area of the transect is measured concurrently. The computer is informed of the coordinates of the selection point in the uniform random (UR) sampling grid by clicking the cursor. All ensuing random linear probes between the sampling point and the object profile periphery emanating from this selection point, radiating at angular intervals of 29-30 degrees to the periphery. In the case of vertical sections, similar lines are generated at intervals where the sine of the angle changes by a value of 0.33. The volume-weighted mean volume of the object is estimated from the average of all products. As the periphery is traced, the algorithm can automatically determine the area of the cross-section of the object, from which the standard deviation of the volume-weighted mean volume can be calculated. Some elements of the algorithm are also used for the measurement of the number-weighted mean volume. The latter procedure is facilitated using an acoustic vertical depth monitor attached to the microscope. The impact of truncation ('lost caps') on the precision of measurements is discussed. The algorithm is of particular use in light microscopy for measuring cell nuclei by direct visual inspection of the microscopic field using a side-arm mirror assembly interfaced with a magnetic tablet.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 84-89
Microspectroscopic measurement of optical properties of rat liver in the visible region
AKITOSHI SEIYAMA, SHENG-SONG CHEN, HIROAKI KOSAKA & TAKESHI SHIGA, Department of Physiology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
SUMMARY Microspectroscopy is used to investigate optical properties of haemoglobin-free perfused rat liver. Visible spectra of 20 micrometre diameter spot size were measured in transmission and/or reflection modes as a function of the thickness ( {1200 micrometres) of the liver edge. Optical density (OD) in transmission mode increased with increasing liver thickness, whereas in reflection mode OD decreased but became almost constant above a certain thickness (c.600 micrometres) of the liver. The Kubelka-Munk (KM) two-flux model, with a minor modification, was applied successfully to the analysis of the changes in OD as a function of the thickness. This approach estimates the KM absorption coefficient, KM scattering coefficient and effective penetration depth of the liver. The optical properties were similar to reported values, obtained with different methods.
Journal of Microscopy, Vol. 175, Pt. 1, July 1994, pp. 90
Letter to the Editors: A generalized terminology for multidimensional microscopy
MARK A BROWNE, C VYVYAN HOWARD, GLEN JOLLEYS & DUNCAN STACEY, Department of Fetal & Infant Pathology, University of Liverpool, PO Box 147, Liverpool, L69 3BX,
Gillian Wilson Executive Editor The Royal Microscopical Society Journal of Microscopy & 37/38 St Clements Proceedings of the RMS Oxford OX4 1AJ UNITED KINGDOM rms-at-uk.ac.ox.vax OR rms-at-vax.ox.ac.uk Telephone +44 (0)865 248768 Fax +44 (0)865 791237
Comments: Converted from PROFS to RFC822 format by PUMP V2.2X
} ALSO...we have an OMU-3 which we LIKE to repair, but I was told some time } ago that parts for it are also no longer available. Is anyone aware of } a parts inventory and/or service for this instrument. }
Try calling TEK-NET, Inc. I haven't used them, but spoke with someone there recently, so I'm pretty sure they're still in business. Their letter of introduction, sent out in '90, described the organization as a group of service engineers who used to work at Cambridge Instruments' East Region Service Center, until that center was closed. The list of equipment that they service is impressive, and Reichert is among them.
I've been tempted to post this information before, just to let folks know they exist, so here it is:
TEK-NET, Inc. 1985 Swarthmore Ave. Lakewood, New Jersey 08701 (908) 905-5530 (908) 905-0152 FAX
I have no affiliation with them, just wanted to get the word out.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
This fall we are starting a new graduate level course on all aspects of light microscopy ie. fluorscence(sp),polorized,bright/dark field etc. and need recommendations for a good textbook. This course will include epquiment and methods. Thanks in advance for any help.
Can someone please suggest stereology methods (assessing number of nuclei) at the light microscope level involving electronic imaging and appropriate software? We have concerns over randomization of fields...grid overlay?..x-y stage coordinates? Any good books on the subject?
Yes it does matter what you pay for the diamond knife. There are various qualities of diamonds which can be used for knives. Also the company's garantee(sp) will matter. Go with the higher price knives and little trouble will happen.
Weibel's book on stereology is still my favorite. It allows an intuitive understanding of the subject of stereology for those who only need this information or who are just beginning the subject, then presents the mathematics later for those who need or want it. As long as the subject is up for discussion, I will state a bias, related to automated (electronic) systems. The subject is peripherally related to the question under consideration. It also applies to both stereology and statistics where new automated or semi-automated systems are proliferating. 1. It is often easier and more efficient to carry out some of the analyses and tests using hand calculators, overlay sheets, and other low-tech means than using the fancy programs.
2. Both statistics and stereology require a lot on the user to know which is the best method, most useful, or most efficient means of carrying out data collections or analysis.
It follows from these then that a program alone is only a tool. The investigator must first invest in acquiring the knowledge of how to use the tool. i.e. Always buy the book and learn the techniques before buying the program.
For many ,this would appear to be very obvious. I state this "obvious" fact because of the number of papers I have reviewed lately where statistical methods were used incorrectly. In most cases the methods stated that such-and-such a program was used for the statistics. This suggests to me that far too many people are using these programs to generate numbers they do not understand because they failed to invest the time in understanding the discipline. Thats MHO, anyway.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Fri, 1 Jul 1994, BOBROWW wrote:
} Can someone please suggest stereology methods (assessing number of nuclei) at } the light microscope level involving electronic imaging and appropriate } software? We have concerns over randomization of fields...grid overlay?..x-y } stage coordinates? Any good books on the subject? } } Walt Bobrowski } bobroww-at-aa.wl.com } } }
In my experience over the years, diamond knives seem to differ mainly in 2 factors: price and quality control. Without mentioning any specific manufacturers, the most expensive ones are made by a unique process which does give them some desireable features...ie many resharpenings, better edge quality, greater durability, etc. All the others are made by essentially the same process and differ as to type of mounting, boat, etc. Most companies will give you several weeks following purchase to try it out and determine if the quality meets your standards. I myself have bought the cheapest (from a single manufacturer) for many years, and have only had to return a single knife. I also tend to buy the largest size available (4 mm or larger) as these tend to last longer, have better quality control, and seem to give you the most for your money.
consider a package called "Stereology" by Kinetic Imaging in Liverpool UK. It's a very useful windows based package, developed by a team led by V. Howard. It's very userfriendly and kind cheap. Give me a mail if you want specifics.
} Can someone please suggest stereology methods (assessing number of nuclei) at } the light microscope level involving electronic imaging and appropriate } software? We have concerns over randomization of fields...grid overlay?..x-y } stage coordinates? Any good books on the subject? } } Walt Bobrowski } bobroww-at-aa.wl.com } } }
While we are on the subject of diamond knives, does any one have a trick to get the knife edge to be a little more hydrophilic? my knife edge appears very clean but it is getting harder to keep the edge wet with out greatly increasing the water level in the boat. any thoughts on this would be appreciated.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Stereology Walt Bobrowski asks - Can someone please suggest stereology methods (assessing number of nuclei) at the light microscope level involving electronic imaging and appropriate software? We have concerns over randomization of fields...grid overlay?..x-y stage coordinates? Any good books on the subject?
I agree with Jay Jerome in recommending the book by Weibel (1979, Academic Press); Practical Methods for Biological Morphometry. vol 1 Sterological Methods. A more recent update can be found in Gundersen 1986 J. Microsc. 143:3-45. Look out also for other reviews by Cruz-Orive and by Gundersen. When using machines it is not easy to avoid bais. For example many counting frames will only score objects that are totally in the frame. This is OK for small particles but large objects may be under sampled. There are many alternative modern techniques which are simple to apply and give efficient and unbiased results. These techniques do not require the purchase of expensive machines or complicated software. The best way to find out about these techniques is to take a short course, one of which we are offering at Yale this August. Paul Webster, Yale School of Medicine.
Re} Diamond knife wetting The trick from the past is to run some siliva over the edge with an eyelash! A more modern way is to rinse out the boat, and the knife surface, with water containing a small amount of detergent. Try very low concentrations of Photoflo (Kodak).
Reply to: RE} Stereology Walt Bobrowski asks - Can someone please suggest stereology methods (assessing number of nuclei) at the light microscope level involving electronic imaging and appropriate software? We have concerns over randomization of fields...grid overlay?..x-y stage coordinates? Any good books on the subject?
I agree with Jay Jerome in recommending the book by Weibel (1979, Academic Press); Practical Methods for Biological Morphometry. vol 1 Sterological Methods. A more recent update can be found in Gundersen 1986 J. Microsc. 143:3-45. Look out also for other reviews by Cruz-Orive and by Gundersen. When using machines it is not easy to avoid bais. For example many counting frames will only score objects that are totally in the frame. This is OK for small particles but large objects may be under sampled. There are many alternative modern techniques which are simple to apply and give efficient and unbiased results. These techniques do not require the purchase of expensive machines or complicated software. The best way to find out about these techniques is to take a short course, one of which we are offering at Yale this August. You may also like to contact the International Society for Stereology in Freiburg, Germany for details of other courses. The secretary is Heimo Kurtz, tel 011 49 761 203 5092 and fax 5054. Good Luck Paul Webster, Yale School of Medicine.
For the very basics, Weibel Er. Stereologic principles for morphometry in electron microscopic cytology. International Review of Cytology, 1969, 26:235-302 has a nice presentation. Of course this predates the dissector and its offspring, so there is a lot more one can do than you could back in '69. Taking a course on stereology is an efficient way to learn, especially the pitfalls. The jump from 2-D sampling to 3-D numbers is not always intuitively obvious to the beginner. The number of times I have seen counts of the number of objects found in micrographs directly transposed to number of objects in cell is becomming legion. For those that don't realize it- You can't do that.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
Subject: Time:3:15 PM OFFICE MEMO RE:LM book Date:7/1/94
If I were looking for a book on light microscopy, I'd check with the McCrone Associates in Chicago (Ph. 708-887-7100; Fx: 708-887-7764). They are experts in all kinds of LM methods, and for years have offered courses on all phases of the subject. I believe they have written books for these courses, and perhaps you could adopt one of them for your course.
I use my saliva to wet the edge of the knife. Wet your fingers with saliva, run fingers over your eyelash "pushstick", then wet the knife edge. Works everytime. Best of luck.
XEI Scientific (Ph: 415-369-1133; Fx: 415-363-1659) market a complete nitrogen-purge system (SEM-Clean) designed to remove oil contamination from specimen chambers of SEMs. They can unquestionably give you details of how the system is optimally designed and operated, and would undoubtedly be willing to design a system specifically to meet your needs. Carl Henderson, manager of the Microprobe/EM lab in our Geology Department here has an XEI system on an SEM and has been very pleased with it. I don't know his email address, but his FAX is 313-763-4690. If you get in touch with him I am sure he will be able to give you considerable information on how this kind of a system performs and operates. Danielson Associates (Ph: 708-960-0086; Fx: 708-960-0546) market a device (Phototron) that produces ultraviolet light with a wavelength distribution optimal for desorbing molecules from surfaces inside vacuum systems. This might be of use to you, either alone or in conjunction with a nitrogen purge system, as discussed on page 201 of my recently published book on Vacuum Methods in Electron Microscopy (available from Ashgate Pub. Co., Fx: 802-276-3837)
Message-ID: {MAILQUEUE-101.940701154911.256-at-eye1.eye.ufl.edu} To: microscopy-at-anlemc.msd.anl.gov
We have the same problems with wetting of diamond knives, and have looked at an old one using SEM.. the amount of plastic adhering to the edge was unbelievable. I believe that this layer contributes both to the "dulling" effect after long use, i.e. that the diamond crystalline structure is not so much dull, just coated, and to the hydrophobic quality. We are all using what are really only partially polymerized epoxies, and the unpolymerized component ends up on our knives. Our best success has been to carefully cure the epoxies, and to soak tghe knives overnight in weak detergent, rinse, and run a styrofoam stick wedge across the edge as recommeded by one of the companies. This doesn't have to be done very often. Also in using photoflo as the detergent, make sure it is very dilute, we soaked off all of the finish coating on the outside of one knife boat! Evelyn Clausnitzer U of Florida Ophthalmolgy
Message-Id: {9407012222.AA07599-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: N2 Vacuum Cleaning Orig-Author: {Alan Wilson {wilsona-at-blackjack.cis.dsto.gov.au} }:ddn:wpafb ----------------------------------------------------------- Does anyone have experience with cleaning (FE)SEM vacuum chambers using a N2 gas bleed at around 100-150Torr. 1. How much gas is used in this process (1 G size bottle = 6.4m3 of gas)? 2. Presumably the cleaning depends on the quantity of gas passed through the system. Thus a lower speed rotary pump would take longer than a higher speed rotary pump? 3. Would a higher speed pump be better due to the greater gas flow speeds giving a greater "scouring" action?
Alan Wilson *:-{)} wilsona-at-blackjack.cis.dsto.gov.au Airframes and Engines Division DSTO-Aeronautical and Maritime Reseach Laboratory 506 Lorimer St Fishermens' Bend 3207 Victoria AUSTRALIA
-- John F. Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, 2455 Hayward St., Ann Arbor, MI 48109-2143 Phone: (313)-936-3352 FAX: (313)-936-3352 jfmjfm-at-umich.edu
We soak dirty/difficult to wet diamonds overnight in diluted triton X-100, .1% or less. Then run alcohol-0washed styrofoam cleaning stick alon g the edge.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
I am currently doing TEM on freshwater fish. The fish I am looking at is the "brown bullhead". Does anybody have a suggestion as to the fixative that would be best for this type of fish? The osmolarity and pH is important to the fixation of the fish. The fish can tolerate salinities from 0-8ppt. Should a different fix with different osmolarities and pH be used for fish taken from higher salinities or can the same fix be used for the fish taken in both 0 and 8ppt salinity? Thanks, Phil
I forgot to mention that I am also doing an exposure study at different salinities (5,10,15,25,35ppt) using both a Euryhaline and Anadromous species of fish in the larval and mature life stage. Is it necessary to adjust the pH and osmolarity for the differnt salinities given these criteria? i.e. habitat and life stage. This is for TEM studies also. Thanks again, Phil
Phil, Regarding the issue of osmolarity and TEM fixation: I have no experience with fish but I do a lot of fixation of platyhelminths (flatworms) from a variety of environments (including fish parasitic species). I use boiled, filtered water from the environment of the animal to make up the buffers for fixations and washing, and usually a pH of around 7.4 for marine and 7.2 for freshwater species. Good luck
Aquatic organisms tend to be a problem, and often considerable experimentation is necessary. In general though, here's what you have to consider with freshwater beasts: Invertebrates tend to be osmoconformers...they are about isotonic to their environment. The blood of some freshwater mussels I've worked with measures about 30 mOsm, which roughly conforms to their pondwater. Most buffers are ineffectual at this concentration, so my best results seemed to be from fixing in a glutaraldehyde-pondwater mixture. Freshwater vertebrates are hypertonic to their environment. I would suspect that under different salinities, their kidneys will keep their blood osmolarity rather constant. You should formulate your fixative based on blood osmolarity (as determined by freezing point depression), keeping in mind that tissues look better by TEM when fixed in a slightly hypertonic fixative. Keep in mind when formulating your fixative, the EOP (effective osmotic pressure) of your fixative. Formaldehyde is so membrane-permeable that you don't have to consider the osmotic pressure contributed by it. Glutaraldehyde is not so permeable and only contributes about half of its calculated osmotic pressure. I hope this will be of some help and at least get you started in the right direction. W.L. Steffens University of Georgia
Phil, Regarding pH and osmolarity for TEM of fish tissues: I used to work with lamprey in both salt and fresh water and we used the same buffer for both with no problems-Millonig's phosphate pH7.2-7.4 with 7% sucrose I believe. We made it up using distilled water from the lab.
Hi I'm the one that ask if there is a future in EM. Most of the responses I received thought that there is. (Although, these were from people who have a reason to hope there is.) I am, however, branching out. I have also been put incharge of the department's confocal microscope. I will take a class with Biorad in Sept. I heard on this news group that there was a confocal news group. Could someone send me the address. I have been unable to find it in my list of news groups. Also, is there anyone else who reads this group that will be taking the Biorad class in Sept. in New York? I would be interested in hearing from you.
I'm looking for additional ideas for a specimen which would be useful for training SEM novices. The idea is to have something which could be distributed to students with training materials (instructions and illustrations) keyed to this specimen. Qualities of importance include: 1. robust - capable of taking careless handling without major change in properties. 2. can be easily cleaned or rejuvenated with minimal equipment. 3. inexpensive. 4. reasonably consistent copies can be produced or obtained in quantity. 5. interesting structure from low magnifications to 10,000X or so to encourage exploration. 6. suitable structures to practice skills in focusing and stigmating. 7. produces interesting micrographs. 8. ideally -- interesting contrast in both SE and BSE modes.
I stress the word "novice". Assume that this specimen could be handed to a high-school student and would both survive and stimulate interest.
Also, I would appreciate any suggestions for a basic EDS training specimen to be used in the same way. Here one would like to have a segregation of elements to encourage "what's this ?" kind of questions and produce interesting dot-mapped images -- the goal here is strictly qualitative -- suitability for quant semi-quant analysis would be a bonus. Of course, it would be ideal if this could be the same specimen as the first!
I am familiar with some specimens which have been used for this purpose, but hope someone might have some imaginative suggestions.
I would like to make contact with anyone using MPM 201 micicrophotometer from Zeiss with MSP 21 processor. We do single cell Calcium measurements but have a number of specific problems concerning the procesors signals to the fast shutter. Specifically I would like to be able to vary the time between measurements in dual wavelength mode. Thanks in advance Gary Jamieson Haematology Austin Hospital Heidelberg VIC 3084 AUSTRALIA Internet gapja-at-austin.unimelb.edu.au Phone 613 496 5518 Fax 613 459 1674
Here's what I've used for the past 20 years: find an old windup mechanical watch, preferably a ladies model. On disassembly, you will have numerous very small screws, gears, bearings, etc. Many are excellent for learning mounting techniques, focussing, stigmation, demonstrating depth-of-field, secondary vs backscatter imaging, etc. And of course they are durable and recyclable.
Received: From MCONET(MAILER) by ANLNRH(ANJE6.10) for MICROSCO-at-ANLEMC; Thu, 7 Jul 94 08:28 Received: from opus.mco.edu by cutter.mco.edu (PMDF V4.2-14 #5375) id {01HEF4OFW0F4000NG8-at-cutter.mco.edu} ; Thu, 7 Jul 1994 09:26:38 EDT Received: from opus.mco.edu by opus.mco.edu (PMDF V4.2-14 #5375) id {01HEF4HT56U8001NKT-at-opus.mco.edu} ; Thu, 7 Jul 1994 09:21:19 EDT
Fred,
My recommendation for SEM training material would be, hair. It is always handy and in large quanties. If sample is taken at the end of a day, all sorts of things can be found on it, such as pollen, which is good material for high mag. and low mag work. I do not do much EDS work but any rusty bolt should work fine for this. Good Luck.
I need microscopy-oriented symbols to use when composing flyers, brochures for MSEM and for EM public relations work at Chicago State University. I use Harvard Graphics for Windows, but the symbol library is oriented more toward business use rather than science. Can anyone help out with this? I can use anything from atoms to a drawing of a JEOL TEMSCAN. A simple light microscope would be nice. Thanks. Joyce Craig Biology Department Chicago State University Chicago, IL 60628 Internet bafpjec-at-uxa.ecn.bgu.edu Phone 312 995 3800 FAX 312 995 3759bafpjec-at-uxa.ecn.bgu.edu
Mosquitoes and fruit flies make very useful specimens. While they are not robust, they have wonderful detail up to magnifications of 20,000 and provide excellent opportunites to demonstrate the effects of foreshortening, charging, background interference, and composition.
Rod Kuehn University of Minnesota
On 6 Jul 1994, Fred Schamber wrote:
} I'm looking for additional ideas for a specimen which would be useful for } training SEM novices. The idea is to have something which could be distributed } to students with training materials (instructions and illustrations) keyed to } this specimen. Qualities of importance include:
Well, may not be too imaginative, but may be samples worth considering: (ie: they are common and long-lasting, and fairly easy to prepare)
- a carbon steel with nice pearlitic and cementite structures etched w/ either Picral and/or Nital acids (~4%); (ex/ 1080, 52100);
- a cast iron, etched as above, revealing graphite nodules surrounded by ferrite in a matrix of pearlite (ex/ grade 80-55-06 ductile iron, or a class 30 gray iron that has graphite flakes in matrix of pearlite);
- may also desire to collect a few -rusty- bolts, nuts, etc. and prepare cross-sections of 'em to reveal and compare the corrosion products vs the remaining steel/iron (may be interesting to you/your "students" to have different amounts of corrosion to study on different items of the same grade of steel/iron);
- various aluminum alloys can be very interesting (ex/ samples of brazed joint areas, alloys of the 3xx series which have quite a few various precipates, and if can find 'em, a very-slow cooled Al alloy revealing many, large dendrites);
- other metals that could be very interesting and informative: tin alloys (Sn-Pb, Sn-Zn-Cu), lead alloys (Pb-Sn-Sb-Cu), and copper (brass);
- and lastly, some geological samples may be worth while, although harder to an extent to keep in good condition, and may be harder for you to acquire and prepare. Examples: ore samples, metamorphic & igneous rocks of various grades, and possibly sedimentary rocks with various mineral stainings.
Enjoy, -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /\v/\ | | Metallographic Lab. | Missouri Speleological Survey /\v/\ | | Rolla Research Center | Bat Conservation International /\v/\ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /\v/\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
John- try Diatome they are a proven product with great support. the size and thickness of your sections will determine the knife size, angle, and grade of diamond -Mike
On Thu, 30 Jun 1994, HARDY, JOHN wrote:
} } With such a wide range of prices - through various suppliers - for a } specific size of diamond knife (for thin sectioning) does the saying } "you get what you pay for" apply here, or is a diamond knife a diamond } knife? Thanks for any experienced comments. } } jhardy-at-smtplink.coh.org }
Tom- wetting the edge is easy if you add some sort of surfactant, saliva, dilute photo-flow, triton x-100, tween-80, etc. saliva on the tip of your (dog eye-lash) brush tool works best, just pull the water up with the spit laden brush tip. -Mike
On Fri, 1 Jul 1994, Tom Phillips wrote:
} While we are on the subject of diamond knives, does any one have a trick to } get the knife edge to be a little more hydrophilic? my knife edge appears } very clean but it is getting harder to keep the edge wet with out greatly } increasing the water level in the boat. any thoughts on this would be } appreciated. } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax) } } }
The most effective and flexible method of producing black backgrounds seems to be by blocking collection of background electrons. This can be accomplished on a regular stub by tilting the sample 90 degrees so the bottom of the chamber becomes the background. The chamber floor is coated with carbon paint to reduce secondary electron generation. On the side of the chamber floor, just to the collector side of the viewing area, tape a carbon-coated barrier. Ours is made of cardboard but in humid weather it absorbs water and requires extensive pump-down periods. Its dimensions are roughly 2 cm high x 3 cm long. For other samples, such as hair, we use a custom-designed stub made from a hollowed brass cylinder. The bottom is solid. The top is square with a square opening of about 1 cm. The cylinder is about 20 mm deep and is coated with carbon paint. The sample, of course, lies across the opening. No experimentation has been done with other sizes or shapes so it is likely that more compact designs would work as well. Both methods produce uniform jet-black backgrounds.
Also, higher kv expands the contrast range at the extremes. Light objects become white, dark objects turn black. Therefore, increase kilovoltage as much as the sample allows. Sorry for the tardy reply.
Rod Kuehn University of Minnesota
On Tue, 7 Jun 1994, Jim Romanow wrote:
} } I have been doing low magnification SEM (50 to 150x) on insects } using a 16 year old Coates and Welter HPS-50B FESEM (TV rate only). } Some of my clients desire a "black background" on the photographic } images (analog 4200 line photo monitor/ Polaroid Type 52) and I } find this is not possible in most cases due to imaging of the mounting } device and stage area around the sample. I have tried mounting on } pins which will eliminate holder interference but I still see gray } shapes in the background from several centimeters away. } } Is a black background the norm with some SEM designs, a } darkroom technique that can be applied to modify photographs that } originally looked like mine or achievable if I change my SEM } techniques? } } Jim Romanow } The University of Connecticut } Physiology and Neurobiology Dept. } Storrs, CT } bsgphy3-at-uconnvm.uconn.edu
You can try using a butterfly or moth wing for the SEM training. That's what we show students when they come in to see a demonstration of our SEM. As long as the student doesn't squash the wing with their finger, the wing should be a pretty good specimen to look at. You definitely can go from low to very high magnifications to see alot of different things and it can be used to practice focusing and stigmating. There are plenty of "holes" and microsculpture. I'm not sure if you can see interesting contrast in both SE and BSE modes. I've never tried it. Another thing is that most if not all people should be familiar with "the powder" that comes off the wing when a person touches it. Once they know what they are viewing, they can in a way, identify with it.
One thing that comes to mind that can be used for EDS work is flakes of paint. I know that paint is composed of various elements and the students can look at different colors or different shades of the same color. They should be able to find out for example, what makes one shade of red different from another shade of red. The paint samples would need to be carbon coated.
Just afew ideas.
Good luck! Peling Melville
On July 6, 1994 Fred Schamber writes:
} I'm looking for additional ideas for a specimen which would be useful for } training SEM novices. The idea is to have something which could be distributed } to students with training materials (instructions and illustrations) keyed to } this specimen. Qualities of importance include: } 1. robust - capable of taking careless handling without major change in } properties. } 2. can be easily cleaned or rejuvenated with minimal equipment. } 3. inexpensive. } 4. reasonably consistent copies can be produced or obtained in quantity. } 5. interesting structure from low magnifications to 10,000X or so to encourage } exploration. } 6. suitable structures to practice skills in focusing and stigmating. } 7. produces interesting micrographs. } 8. ideally -- interesting contrast in both SE and BSE modes. } } I stress the word "novice". Assume that this specimen could be handed to a } high-school student and would both survive and stimulate interest. } } Also, I would appreciate any suggestions for a basic EDS training specimen to } be } used in the same way. Here one would like to have a segregation of elements to } encourage "what's this ?" kind of questions and produce interesting dot-mapped } images -- the goal here is strictly qualitative -- suitability for quant } semi-quant analysis would be a bonus. Of course, it would be ideal if this } could be the same specimen as the first! } } I am familiar with some specimens which have been used for this purpose, but } hope someone might have some imaginative suggestions. } } Fred Schamber } RJ Lee Group, Instruments Division
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
As you suggested, a simple light microscope will indeed be very suitable, because more people are able to relate to a light microscope than an electron microscope. You have an excellent one already in Chicago - the pin designed by McCrone Research Institute for the Illinois State Microscopy Society. Call MRI at 312-842-7100 to see if they can send you a copy of the design or a pin itself.
Shu-Chun Su Hercules Inc. Research Center Wilmington, DE 19808 Phone: 302-995-3498 Fax: 302-995-4135
On Thu, 7 Jul 1994, Joyce Craig wrote:
} I need microscopy-oriented symbols to use when composing flyers, } brochures for MSEM and for EM public relations work at Chicago State } University. I use Harvard Graphics for Windows, but the symbol library } is oriented more toward business use rather than science. Can anyone } help out with this? I can use anything from atoms to a drawing of a JEOL } TEMSCAN. A simple light microscope would be nice. Thanks. } Joyce Craig } Biology Department } Chicago State University } Chicago, IL 60628 } Internet bafpjec-at-uxa.ecn.bgu.edu } Phone 312 995 3800 } FAX 312 995 3759bafpjec-at-uxa.ecn.bgu.edu }
I recieved a call this morning from Clint Mills. He works in the EM lab at a VA hospital in Alabama. It seems that his water chiller has been a source of chronic problems over the past two years. He was calling to inquire what brand/make of chiller we use. Since we are on a house chilled water system, I wasn't much help. If anyone would like to call him with comments and suggestions, I am sure he would appreciate it. His phone number is (205)-933-8101 ext. 6719. He has a Hitachi H-600, which came with a variety of specimen exchange rods. He would also like to sell the bulk specimen holder and the rotational holder. ________________________________________________________________
Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
Message-Id: {9407081619.AA06260-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: SEM Training Specimen Orig-Author: {Fred Schamber {73751.3677-at-compuserve.com} }:ddn:wpafb ----------------------------------------------------------- I'm looking for additional ideas for a specimen which would be useful for training SEM novices. The idea is to have something which could be distributed to students with training materials (instructions and illustrations) keyed to this specimen. Qualities of importance include: 1. robust - capable of taking careless handling without major change in properties. 2. can be easily cleaned or rejuvenated with minimal equipment. 3. inexpensive. 4. reasonably consistent copies can be produced or obtained in quantity. 5. interesting structure from low magnifications to 10,000X or so to encourage exploration. 6. suitable structures to practice skills in focusing and stigmating. 7. produces interesting micrographs. 8. ideally -- interesting contrast in both SE and BSE modes.
I stress the word "novice". Assume that this specimen could be handed to a high-school student and would both survive and stimulate interest.
Also, I would appreciate any suggestions for a basic EDS training specimen to be used in the same way. Here one would like to have a segregation of elements to encourage "what's this ?" kind of questions and produce interesting dot-mapped images -- the goal here is strictly qualitative -- suitability for quant semi-quant analysis would be a bonus. Of course, it would be ideal if this could be the same specimen as the first!
I am familiar with some specimens which have been used for this purpose, but hope someone might have some imaginative suggestions.
I am looking for a US contact that is affiliated with (BASF A. G., Ludwigshafen, Germany) or a source for tetcyclasis.
Any help will be most appreciated.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
On Fri, 8 Jul 1994, Francisco Javier H Blazquez wrote:
We are studying the absorption of macromolecules (proteins) by the intestinal cells of a freshwater fish (Prochilodus scrofa). We are using ferritin as an eletron microscopic and optical microscope marker and it can be observede in the enterocytes of the fish. When we tried to isolate the intestine and added ferritin to Eagle's medium where a isolated segment was incubated for 5 hours we fail to observe ferritin absorption by the enterocytes. The literature reports some insucess like this one. The time employed permits observations when this ferritin is given by oral route. Why isolated intestine don't phagocytes macromolecules? I think that problably some hormones are needed. May someone helps us?
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
JOHN CHANDLER ASKED ABOUT THE UNCURED EPOXY ON THE EDGE OF A DIAMOND KNIFE-- I AM TALKING ABOUT CURING THE EPOXY AS WELL AS POSSIBLE BEFORE SECTIONING. THIS ALL REFERS BACK TO SOME READING I DID A COUPLE OF YEARS AGO IN SOME BOOKS ABOUT THE CHEMISTRY OF PLASTICS (I'LL HAVE TO LOOK UP THE REF.) THE EPOXY PLASTICS THAT WE USE ARE ALSO USED IN INDUSTRIAL APPLICATIONS, AND THE POLYMERIZATION THAT THEY USE IS ON THE ORDER OF 200 DEGREES OR MORE FOR DAYS! WE WOULD NEVER BE ABLE TO SECTION STUFF LIKE THAT; WHAT WE USE IS BARELY STARTING TO CURE. IT SEEMS THAT ALL THE UNPOLYMERIZED MONOMERS IN OUR PLASTICS ARE WHAT COLLECT ON THE KNIFE EDGE, AND SLOWLY HARDEN. Evelyn Clausnitzer U of Florida Ophthalmolgy
My lab is beginning the hunt for the perfect FEG SEM. We're a service lab in a sizeable chemical company so we see all manner of samples - but the bulk of our work is with polymeric samples. Good low kV operation is an obvious must but the choosen machine had better perform well doing EDS and some high kv, high resolution work (catalytic materials). Ease of operation and reliability are also musts - like many of you, we really depend on SEM info and finicky instrumentation and downtime is not something we want to spend all that money on. So what's the experience out there? Any horror stories? Any very happy users (especially those working with polymers)?
Re: Discussion concerning the remains of uncured epoxy on the back of a diamond knife: Industrial epoxies are indeed cured at temperatures very much higher than those we use, but this is due to the absence of a catalyst in the industrial formulations. The ca. 1% catalyst that we use makes it possible to cure our plastics at 50 - 60 C within a reasonable time - BUT - it is quite true that our formulations are not fully cured after 12h at typically 60C.
When the increase in hardness of a variety of EM embedding epoxies is measured during curing, all of them need approximately 40-48h at 60C for a full cure. This result is true for the generally-used catalysts DMP30, BDMA and DMAE (or S1) at normal concentrations of 0.5 - 1%, used in conjunction with a variety of modified and unmodified epoxy formulations. The choice of anhydride hardener also does not influence the length of the curing times to any great extent.
With the above in mind we have, in this lab, been polymerizing our epoxy embeddings for 48h at 60C. The embedded material does not seem to be influenced by the increased time, but we get: more reproducible sectioning qualities, cleaner knives, increased beam stability and possibly less contamination in the EM. The blocks are slightly harder than with the short cure times, but still section well with a diamond knife. (All this courtesy of Chris van der Merwe in this lab.)
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Microscopy Forum {microscopy-at-anlemc.msd.anl.gov}
To microscopist everywhere:
My lab is beginning the hunt for the perfect FEG SEM. We're a service lab in a sizeable chemical company so we see all manner of samples - but the bulk of our work is with polymeric samples. Good low kV operation is an obvious must but the choosen machine had better perform well doing EDS and some high kv, high resolution work (catalytic materials). Ease of operation and reliability are also musts - like many of you, we really depend on SEM info and finicky instrumentation and downtime is not something we want to spend all that money on. So what's the experience out there? Any horror stories? Any very happy users (especially those working with polymers)?
I would like to get hold of (pay for) a late model freeze fracture device in good working order with all the gizmos and widgets etc.
If you, or anyone you know is looking to sell an instrument (Balzers, Cressington etc) which is, in the "single owner, well serviced, less that 20,000 miles group, preferably with A/C and electric windows" please could you drop me a line
Thanks
Simon C. Watkins Director SBIC University of Pittsburgh Pittsburgh PA 412-648-3051
I publish a monthly newsletter on analytical instrumentation, and I encourage subscribers to make use of Internet and other information sources on line. Could you send me information on your forum, so I can pass it on to my readers?
Our monthly survey for July 1994 covered SEMs and x-ray detection systems.
Jo Rita Jordan Analytical Consumer jjordan-at-world.std.com
In response to Dave Calvert's questions about FEG SEM instrumentation, I can't say much about the bad or the ugly, but we have had a very good experience here at NRL with a Hitachi S-800 that was purchased several years ago. It has proven to be very reliable over its years of service, and its operation is actually much simpler than most of the more conventional SEM's that we have scattered around. It has been used by a large number of researchers, ranging from high school students to senior PhD's, and most users are able to get good results the first day they are on the machine, assuming they need fairly standard operating conditions. The setup we have includes a thin-window EDS system and a Robinson backscattered electron detector.
Altough our machine is an older model, I think that two of the features that have contributed to its reliability are worth considering when evaluating a newer instrument. First, it has a fairly robust vacuum system with an easy-to-use specimen airlock and good isolation and differential pumping of the specimen chamber and the rest of the column. This is essential for polymer specimens and for cases that we occasionally see, when an over-anxious operator neglects to let the silver paint dry long enough. Second, the S-800 is arranged so that its more advanced features, such as gamma control and special conditions for low-voltage operation, do not get in the way of its basic operation.
I hope these thoughts are useful.
James Sprague Surface Modification Branch Code 6670 Naval Research Laboratory Washington, DC 20375
We have been using aminopropyltriethoxysilane coated slides for LM immunohistochemistry and in situ hybridization. We were using xylene as clearing agents at first but because of the health problems, etc with xylene we decided to try the substitutes, mainly Hemo-De and Histoclear. We seem to be having a problem with these however, and it has gotten worse in the last few months. We seem to be getting alot of water in our clearing agent, even after well dehydrating. Could this be an artifact of the silane coating?? Why not the problem with xylene?? Has anybody come across the same problem?? Thanks for any suggestions.
Mark Elliott, PhD UBC-pulmonary Research Lab St. Paul's Hospital Vancouver, BC Canada
The following recipes are reasonable guesses from Bernie Kestel at Argonne National Laboratory. They are guesses since he has not thinned cast aluminum. The results are from polishing done with a South Bay 550-B single vertical jet polisher. It supplies the higher voltage needed by some electrolytes, in-situ process viewing, and allows for rapid foil rinsing. Use only ethyl alcohol to rinse ploished aluminum (also copper) to avoid methanol's corrosive attack of the finished surfaces.
Suggestions:
1. 150 ml nitric acid, 350 ml methanol, 40 ml butyl cellosolve (2-butoxy ethanol). Try -40C to -45C (a colder solution leaves a textured surface), 80 volts, 50-75 mA, medium flow rate.
2. 5.3 g lithium chloride, 11.16 g magnesium perchlorate, 500 ml methanol, 100 ml butyl cellosolve. To produce this electrolyte safely, first mix the liquid components together, then slowly add the dry granular salts to the stirred liquid mixture. Do not mix the dry components together! Continue stirring until the solids dissolve completely. Use at -50C, 100-150 volts, 12 mA, and a slow flow rate. Different concentrations of the salts may also work in special cases, i.e. more dilute or more concentrated solutions may help.
3. The following change was made to recipe #2 to thin Al-6wt%Ge, and it retained precipitates which were electron-transparent. Add 200 ml (or so) of acetic acid to the cooled solution. Use at -25C, 100 volts, and 15 mA with a slow to medium flow rate.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
In message Mon, 11 Jul 1994 00:16:38 -0400, Dave Calvert {73363.1014-at-compuserve.com} writes:
} To microscopist everywhere: } } My lab is beginning the hunt for the perfect FEG SEM. We're a } service lab in a sizeable chemical company so we see all manner of } samples - but the bulk of our work is with polymeric samples. Good low } kV operation is an obvious must but the choosen machine had better } perform well doing EDS and some high kv, high resolution work (catalytic } materials). Ease of operation and reliability are also musts - like many } of you, we really depend on SEM info and finicky instrumentation and } downtime is not something we want to spend all that money on. } So what's the experience out there? Any horror stories? Any very } happy users (especially those working with polymers)? } } Thanks in advance for the information. } } } Dave Calvert - Eastman Chemical Company } ================ We have been very happy with the low kV operation of our HITACHI S4500 FESEM (turbo pump). We have successfully imaged many uncoated material including polymers, diatoms (silica shells) and frozen material at kVs ranging from 0.5 to 2.0. We have had the instrument for about 15 months and it has been very reliable and has not had any down time. Since ours is a multiuser facility as well as a teaching facility, the instrument has tolerated some mishandling. We have not yet had a great demand for X-ray analyses so I cannot as yet comment much about it. M.V. Parthasarathy Section of Plant Biology Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734 Fax: 607-255-5407
Message-Id: {9407111755.AA00390-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM specimen prep. of powder Fe ? Orig-Author: {dakshs-at-rpi.edu}:ddn:wpafb -----------------------------------------------------------
Greetings, I need to study the microstructure of Fe powders (ranging from 5 - 50 microns in size) under the TEM. What is the specimen prep. procedure for such powder materials? Also, is there a quick and dirty method for a first look? Any help would be appreciated. TIA, yours, Sharath dakshs-at-rpi.edu
If you can use a microtome such as the Reichert Ultracut E, very thin samples can be made using a diamond knife. I've used Araldite 502 to embed Ti-Al powder and microtomed the blocks at 0.2-0.5 mm/sec to produce 60nm-thick sections.
If you are interested in the details, call or drop me an e-mail note.
Dear Microscopists, We have been told to stop using molecular sieves to "dry" our solvents, ie ethanol, acetone and methanol. The reason is because molecular sieves have been implicated in damaging diamond knives if fine "grit" from the sieves ends up in the final resin block. When the block is cut the grit damages the knife edge.
The recommmendation was to use copper sulphate however I am not convinced that this won't present a similar problem. I am interested to hear from others how they keep their absolute solvents absolute.
For Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
I'm looking for a company that carries 0 or 00 coverslips. I've looked in all the usual sources (Fisher, VWR, Polysciences) and nobody has them. If anyone knows an address of a company that does please let me know.
Dear microscopists, Wanted your views/opinions on black and white automatic photographic printers. The features we need are: variable speed, variable temperture, automatic replenishing, an efficient washer and a dryer or dryer attachment.
for Deborah MacLead
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
} ...We have been told to stop using molecular sieves to "dry" our solvents, ie } ethanol, acetone and methanol. The reason is because molecular sieves have } been implicated in damaging diamond knives if fine "grit" from the sieves } ends up in the final resin block. When the block is cut the grit damages } the knife edge. }
I have for years been using four angstrom molecular sieves, activated by heating to 205-315 degC for two hours, to dry solvents and keep them dry for freeze substitution. I have had not problems with premature dulling of diamond knives, but I have always (a) let the "fines" settle for a day or two minimum before using the solvent, and (b) removed any remaining suspended matter by centrifugation or filtration before use. I and others have found that reagent grade acetone or tetrahydrofuran from a freshly opened bottle generally does not need to be further dried for use in freeze substitution.
We regularly use molecular sieves but we extract the solvent ONLY with a pipette to avoid stirring up dust. We have recognized no problems from this practice. However, I am also not convinced that the solvent needs to be this dry. Has anyone experimented with eliminating dried acetone/ethanol?
Rod Kuehn University of Minnesota
On Tue, 12 Jul 1994, Richard Easingwood wrote:
} Dear Microscopists, } We have been told to stop using molecular sieves to "dry" our solvents, ie } ethanol, acetone and methanol. The reason is because molecular sieves have } been implicated in damaging diamond knives if fine "grit" from the sieves } ends up in the final resin block. When the block is cut the grit damages } the knife edge. } } The recommmendation was to use copper sulphate however I am not convinced } that this won't present a similar problem. I am interested to hear from } others how they keep their absolute solvents absolute. } } For Allan Mitchell } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 } }
Message-Id: {MAILQUEUE-101.940712073223.320-at-vanlab.paprican.ca} To: microscopy-at-anlemc.msd.anl.gov
There was an article in "The Microscope" Vol 42 No.1, 1994 on the McArthur microscope and some other portable microscopes. According to the author, the McArthur microscope is available from Prior Scientific Inc. (80 Washington St., Bldg. 0-54, Norwell, MA 02061, U.S.A. (617) 878-8442. I hope this helps.
James Drummond Pulp and Paper Research Institute of Canada Vancouver, B.C.
We have been using the Ilford 2150 RC for the past several months. It fulfills all the reqyuirements except variable speed. Frankly we don't know how we ever managed with out it. The results are equivalent to tray development and you have a finished, dry print in 59 seconds. Standardizing our negatives exposure and development has been important so taht any given batch of negatives can usually be done with out readjusting for exposure. This makes it a real time saver when you can run them through without thinking about it ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
=From: TOWER::GWERDOS "Greg Erdos ICBR EM Core Lab Univers" =To: IN%"richard.easingwood-at-stonebow.otago.ac.nz" =CC: GWERDOS =Subj: Re: Solvent drying = =} From: IN%"richard.easingwood-at-stonebow.otago.ac.nz" =} Subj: Solvent drying =} =} Return-path: {richard.easingwood-at-stonebow.otago.ac.nz} =} Received: from anlemc.msd.anl.gov by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id =} {01HELE2ULG5S8WX95Z-at-gnv.ifas.ufl.edu} ; Mon, 11 Jul 1994 21:00:20 EST =} Date: Tue, 12 Jul 1994 11:42:29 +1200 =} From: richard.easingwood-at-stonebow.otago.ac.nz (Richard Easingwood) =} Subject: Solvent drying =} To: microscopy-at-anlemc.msd.anl.gov =} Message-id: {199407112342.AA19751-at-arwen.otago.ac.nz} =} MIME-version: 1.0 =} Content-type: text/plain; charset="us-ascii" =} Content-transfer-encoding: 7BIT =} X-Sender: st004718-at-brandywine.otago.ac.nz =} =} Dear Microscopists, =} We have been told to stop using molecular sieves to "dry" our solvents, ie =} ethanol, acetone and methanol. The reason is because molecular sieves have =} been implicated in damaging diamond knives if fine "grit" from the sieves =} ends up in the final resin block. When the block is cut the grit damages =} the knife edge. =} =} The recommmendation was to use copper sulphate however I am not convinced =} that this won't present a similar problem. I am interested to hear from =} others how they keep their absolute solvents absolute. =} =} For Allan Mitchell =} =} Richard Easingwood =} Department of Anatomy and Structural Biology, =} P.O. Box 913 =} University of Otago, =} Dunedin, New Zealand. =} Fax:64-3-479 7254 =} Telephone:64-3-479 7301 =} =} =############################################################# = I put the molecular sieve inside a dialysis bag, tightly tied at =both ends. It is made from 1 inch tubing and looks like a sausage at the =bottom of the vessel. Lets water in but won't let the grit out fd=********************************************************** =* Greg Erdos ** * =* Director, ICBR EMCL ** Phone 904-392-1295 * =* 218 Carr Hall ** FAX 904-392-8598 * =* University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * =* Gainesville, FL 32611 ** * =********************************************************** ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Thomas Scientific(Arthur THomas Co.) lists cover slips of 0 thickness of at= least two different brands. They have various 800 numbers, depending where= you are: Far West region is (800) 345-2102. Their standard for that= thickness, by the way, is 0.085-0.13mm. Good luck.
Does anyone out there know exactly how the WPE of a resin is determined?? = Please, no answers telling me what it is--I want to know what is involved,= physically, with the determination of its value..... Incidentally, my= apologies to Nestor-I accidentally sent this to him, instead of the= bulletin board.....Thanks, to anyone who can help. Grace Kennedy/UCSD
To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM sample prep for Fe powder Orig-Author: {COOK-at-anlemc.msd.anl.gov}:ddn:wpafb ----------------------------------------------------------- If you can use a microtome such as the Reichert Ultracut E, very thin samples can be made using a diamond knife. I've used Araldite 502 to embed Ti-Al powder and microtomed the blocks at 0.2-0.5 mm/sec to produce 60nm-thick sections.
If you are interested in the details, call or drop me an e-mail not
I stopped using molecular sieves about 10 years ago for all applications, and haven't seen any noticeable change. For critical point drying however, I do always use a freshly opened bottle of absolute ethanol. A chemist advised me some years ago that ethanol is far too hygroscopic for molecular sieves to keep it absolute. This requires a special distillation step in the manufacturing process. They will keep acetone dry, however I too question just how dry it needs to be for resin embedding.
} Wanted your views/opinions on black and white automatic photographic } printers. The features we need are: variable speed, variable temperture, } automatic replenishing, an efficient washer and a dryer or dryer } attachment.
In December, 1993, I asked a similar question about B&W print processors. I got 23 excellent responses to the query which I edited into a compact format. I will forward it to anyone who is interested. Just contact me by e-mail.
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Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
We use molecular seives for drying purposes. I also do have some problems with knife marks on sections. I returned a knife once and the manufacture said there were no chips out of the edge by it was faulty. Due to molecular seives?? Could be, I have never heard of this. When we change the seive material we do rinse it very well with solvents. Is there breakdown of the seive material during use? Will have to look into another dehydrant.
Malis and Steele have listed a reference for microtomy of Fe: L. Reimer, Z. Metallkunde, 50 (1959) 37-41. There are many references to the microtomy of materials in T. F. Malis and D. Steele, "Ultramicrotomy for Materials Science", in "Specimen Preparation for Transmission Electron Microscopy of Materials II", ed. Ron Anderson, Materials Research Society Synposium Proceedings, vol. 199, Materials Research Society, 1990.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
I can report only one negative incident with using molecular sieve. A client sucked up acetone from within the pellets during dehydration for critical point drying prior to SEM viewing. The plant leaf surfaces were litered with particulates. Subsequent comparative EDS analysis showed that the particles were sieve.
Since then, whenever I set up new sieve, or prior to heat-drying sieve, I always rinse it 3-4 times with the solvent to wash out as much dust as possible. Use relatively large bottles to put sieve and solvent into and replenish with pipet (dribbling solvent down inside wall of bottle) when 2/3 has been used, leaving 1/3 solvent as a buffer against turbulent mixing with the solvent near the sieve pellets. Handle the bottles gently to prevent stirring up the sieve. And NEVER take solvent from right above or within the sieve. Let settle overnight after adding new solvent to the bottle.
Now, I'm glad this subject came up as I've been wondering if there is any quick , cheap and reliable method to measure water in these solvents in the range .01 to 2.0% by volume, just to see if the sieve is really doing what we think it is. It seems that gravimetric methods based on specific gravity or weighing precise amounts on an analytical balance would not be easy to do or accurate. Is there some absorption method available?
How often do you heat-dry the sieve, how many bottle volumes do you run through before drying the sieve?
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Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Message-Id: {9407121551.AA05059-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM specimen prep. of powder Fe ? Orig-Author: {dakshs-at-rpi.edu}:ddn:wpafb -----------------------------------------------------------
Greetings, I need to study the microstructure of Fe powders (ranging from 5 - 50 microns in size) under the TEM. What is the specimen prep. procedure for such powder materials? Also, is there a quick and dirty method for a first look? Any help would be appreciated. TIA, yours, Sharath dakshs-at-rpi.edu
---------------------- Replied Message Body ---------------------- To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM specimen prep. of powder Fe ? Orig-Author: {dakshs-at-rpi.edu}:ddn:wpafb -----------------------------------------------------------
Greetings, I need to study the microstructure of Fe powders (ranging from 5 - 50 microns in size) under the TEM. What is the specimen prep. procedure for such powder materials? Also, is there a quick and dirty method for a first look? Any help would be appreciated. TIA, yours, Sharath dakshs-at-rpi.edu
Try Thomas Scientific (800) 345-2100, they have 0 cover slips in a wide range of sizes.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Mon, 11 Jul 1994, Mike Folsom wrote:
} } Folks - } } I'm looking for a company that carries 0 or 00 coverslips. I've } looked in all the usual sources (Fisher, VWR, Polysciences) and } nobody has them. If anyone knows an address of a company that } does please let me know. } } My thanks in advance - } } Mike Folsom } } } _______________________________________________________________________________ } M.W.Folsom/Biology/UNM/Albuquerque,NM~87131/505.277.4277/mwfolsom-at-hydra.unm.edu } }
Why do you dry solvents? Is this to be able tyo keep large quantities in opened containers? For EM and SEM I've always gotten by purchasing dehydrants in pint containers and relegating the last inch of an old opened bottle to less than anhydrous uses.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
} Does anyone out there know exactly how the WPE of a resin is determined?? Please, no answers telling me what it is--I want to know what is involved, physically, with the determination of its value..... Incidentally, my apologies to Nestor-I accidental ly sent this to him, instead of the bulletin board.....Thanks, to anyone who can help. Grace Kennedy/UCSD } } } Grace: If you can give me a fax number I will fax a copy of how to determine the WPE. I will also include a table for mixing. The table will be for epon 812. The system may work for other resins. You can get the WPE straight from the manufacturer if you can find a telephone number for the company. We used to do this when we used Epon 812. I started using Araldite 502 15 years ago and I like it better than the epon or the equivalents of epon. I can get beautiful serial sections and the staining of LM sections is very good. It's a very simple resin to make up and you can freeze it in batches like you can do with epon. Here's the receipe just incase you want to give it a try.
Araldite 502..............12.5g DDSA....bring to..........22.5g DMP-30.....................0.3ml (I use tuberculin syringe) Mix well. I use applicator sticks to mix.
I mix this up in the morning and by that afternoon when I get ready to embed, the air bubbles are out of the resin. Hope this helps. Phil 8-{)
Like Greg Erdos, we too have an Ilford 2150 print processor. Ours is 4 years old.
Yes it can give you dry prints in about 60sec. but if you want the prints to last for years you will probibly need to refix and rewash them. Some of ours began to turn brown after about a year.
Yes the prints are consistent. When you get the exposure right for one publication or grant photo you can print the next 8 as fast as you can change paper and expose it.
We have had a number of things that have had to be replaced in the last 4 years.
One heating element
one gear for one of the roler
the water selinoid switch
twice I replaced the nut on the heat sensor (this made a big mess both times with developer all over the counter) (I was most displeased.)
The contacts for the drier heaters kept corroding until I replaced them with the coated ones that now come with the instrument. I could not talk Ilford into giving me a set, so I had to buy them.
i also know that I will have to replace the drier rolers soon.
This processor is not used a great deal. We average about 300 to 400 prints a month.
It is nice to be able to print one print if you want and not waste chemical. We can print 16" X 20" prints, if we want. (That is fun.) We can print 8" X 10" EM prints as fast as we can change negs.
It costs us about 40 cents a print for 8 X 10s in chemicals. This is not to bad for multi uses darkroom.
This is all I know. I hope it is helpful to some one.
Larry Hawkey Neurobiology Duke Hawkey-at-neuro.duke.edu
I've done critical point drying for years using abolute alcohol from the bottle, not freshly opened but discarded if 3/4 empty or been sitting too long ie month or more. I have not observed any difference. Note that some cylindars of CO2 are contaiminated with water... this hows up by freezing in the output line of CO2 escaping to atmoshere, usually as start & stop flow, sort of snorting and popping. Test with pure CO2 (ie no specimen in chamber) and if its still snorts, repl;ace the CO2 (about 1 cylindar in 10 or more has had this.
I find that pollen grains are very good specimens with which to train students on SEM. You can fix them, but if you have a mature plant with a stamen, just shake it onto a stub with adhesive of some sort. Set it to dry in a box with drierite for 24 hours....and then sputtercoat and observe. Diatom shells are really good too. Nina Allen
We have had a Kodak Dektomatic print processor for the past five years. This unit has all the variables requested in the original posting.
Over the years this processor has been nearly unstoppable except for routine cleanings and one broken bearing. At various times during the year a variety of users will produce as many as 1000 prints in a month.
The recommended processing speed produces a completely dry and archive level print in 93 seconds. No further finishing has ever been required. melsen-at-MICROBIO.emory.edu
I am tempted to try benzyl dimethyl amine (BDMA) in place of DMP-30 but don't have the original reference. How much does one use? Anyone have the original citation? I usually use: 20 g EmBed812 + 10 g DDSA + 10 g NMA + 0.6 g DMP-30.
Thanks.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
We have used black and white print processers for the last 12 years. Our original Durst Variospeed finally died last year and we now use a Durst Printo model. This is a modular system for the advanced amateur and performs very well. It can be set up with all the functions you want apart from continuously variable speed (you can set up 4 fixed speeds for differing chemistries). We found with the variospeed that we hardly ever altered the speed .
We use a very simple and inexpensive version without an attached drier and this is excellent for all our printing needs.
We've been having an intermitten problem over the last 2 years that we have been unable to solve and hoped someone out their might have had the same problem and found a solution.[I am forever the optimist]. The problem is with 4489 film. Occasionally we process a batch which comes out with abnormally shaped densities in the emulsion. It is not on the surface of the film, but rather in the emulsion and is not related to exposure or silver grain distribution. These areas print light because of the extra density. It is intermittent enough that it is hard to track down. It seems to be related to certain batches of film, since changing lot numbers gets rid of the problem. We investigated every aspect of our handling and processing and can't figure determine anything we are doing that could damage the emulsion. We've even tried brutalizing the film in various ways to cause the problem. The problem comes in spurts and usually limits itself to our most important images- the one where the sample can't be reproduced. Its bloody maddening and has us stumped. Any suggestions for things to try would be helpful.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I am not much of a fan of molecular sieves. A long time ago when I was doing some freeze-substitution work, I put some radioactive H20 into various organic solvents with molecular sieves. what I found was that the sieves and water reached some consistent equilibrium dependent on the solvent. For instance, ethanol is commercially available with 0.008% water but I found molecular sieves were ineffective in removing trace amounts of added radiolabeled water. This is why 100% ethanol is so much more expensive than 95%; a simple solution like sieves isn't good enough. removing the last 5% is not as simple as using molecular sieves. acetone is commercially available with {0.5% water and sieves removed 76% of the water. THF comes at 0.005% water and sieves removed 95% of the water. for routine TEM, we simply use fairly recently opened pint bottles and have no problem. for freeze-substitution, we always use a freshly opened pint bottle.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Do any EM users know of a substitute petri-dish for the old Lux Permanox tissue culture dish for processing of cell cultures directly for resin embedding. I have tried several possible substitutes that the manufacturers assured me were the equivalent but as soon as propylene oxide was added to them they quickly melted into a plastic mess.
Terry Robertson
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
} We've been having an intermitten problem over the last 2 years that we } have been unable to solve and hoped someone out their might have had the } same problem and found a solution.[I am forever the optimist]. The } problem is with 4489 film. Occasionally we process a batch which comes } out with abnormally shaped densities in the emulsion. It is not on the } surface of the film, but rather in the emulsion and is not related to } exposure or silver grain distribution. These areas print light because of } the extra density. It is intermittent enough that it is hard to track } down. It seems to be related to certain batches of film, since changing } lot numbers gets rid of the problem. We investigated every aspect of our } handling and processing and can't figure determine anything we are doing } that could damage the emulsion. We've even tried brutalizing the film in } various ways to cause the problem. The problem comes in spurts and } usually limits itself to our most important images- the one where the } sample can't be reproduced. Its bloody maddening and has us stumped. Any } suggestions for things to try would be helpful. } } Jay Jerome } ************************************************************** } * aka: W. Gray Jerome * } * Pathology * } * Bowman Gray School of Medicine of Wake Forest University * } * 910-716-4972 * } * jjerome-at-isnet.is.wfu.edu * } ************************************************************** } } } }
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
My suggestion is to not use proplene oxide. Use ethanol for final dehydration and infiltration mixture. Works well. Might take a little longer but does not eat up petri dishes. Are you using Epon (or subitutes)? Spurr's Resins will eat up normal perti dishes. Good Luck.
Please send us details of your full name and address (The Royal Microscopical Society, 37/38 St Clements Oxford, OX4 1AJ (Tel: (01865) 248768/Fax: (01865) 791237)), indicating your requirements, if you would like further information on any of the RMS' events listed below:
1994 1 - MICRO 94 Conference and Exhibition (London) 12-15 September 2 - Immunocytochemistry Course (Oxford) 5-9 September 3 - Flow Cytometry Course (Cambridge) 19-23 September 4 - Microscopy and Catalysis (London) 27 October 5 - Ultrastructural Immunocytochemistry Course (Sutton) 14-18 November
1995 A - Electron Microscopy Course (Manchester) 9-13 January B - Microscopy in Geology (London) 8 March C - Annual Immunocytochemistry Meeting (London) 23 March D - Botanical Microscopy (Oxford) 27-31 March E - Microscopy of Magnetic Materials (Oxford) 3-5 April F - Annual Light Microscopy Meeting (London) 11 April G - Microscopy of Biomaterials (Oxford) 19 April H - Scanning Probe Microscopy Meeting (Nottingham) 24-25 April I - CYTO 95 Conference Cell Signalling (Southampton) 3-6 July J - Summer School in Light Microscopy (Leeds) 17-21 July K - Immunocytochemistry Course (Oxford) 4-8 September L - Immunophenotyping Meeting (London) September M - Cryotechniques Course (Glasgow) 11-15 September N - Flow Cytometry Course (Cambridge) 18-22 September O - Computers in Microscopy Course (Cambridge) 18-21 September P - Ultrastructural Immunocytochemistry Course (Sutton) 13-17 November
To N.L. Desmond: My attempt to send you the print processor summary keeps getting returned, "unrecognized host" message attached. Please contact me and send your e-mail address so I may try again. Thanks.
'pologies to all other netters for this intrusion.
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Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
A recent article by EA Stanley in Microscope 41: 15-27 (1994) lists a source for the McArthur (portable) microscope as Prior Scientific Inc., 80 Washington Street, Bldg. 0-54, Norwell, MA 02061, Telephone (617) 878-8442.
Dear Jay, 4489 is the usual film in use here; we've shot over 10**5, and I'd think we've seen everything, but I don't recognize the problem from your de- scription. By "abnormally shaped densities in the emulsion" do you refer to grains, clusters of grains, or larger-scale phenomena? Does the problem occur for several sheets of film from one pack (& pk/100 or pk/250?), or is it less predictable. Have you looked at a film in bright safelight to see if there are any visible surface characteristics? If so, and if the film is not uniformly OD 6 after this, do the lighter areas referred to correlate with anything noted on the surface? What voltage are your electrons, or, rather is V { { 100 kV? Our HVEM (V = 10**6 V) may penetrate the abnormalities leaving little to dis- tinguish them by.
Jay Jerome posted the message concerning problems with Kodak 4489 films. From my experience this type of problem causes a lot of pain and frustration, especially when it occurs to your most important electron images. Unfortunately you do not notice this until you developed the films.
Perhaps the best way to solve the problem is to stop using film to record images. If you can afford a commercial CCD camera, your pain and frustration are gone forever ! With such a device, what-you-see-is-what-you-get. You never miss any action ! Plus you got all the image processing capabilities either on-line, or off-line. We have had very pleseant experience with a commercial CCD camera. Our sample is radiation sensitive material.
Just my own opinion.
Ming Pan Center for Solid State Science Arizona State University
To all who responded to the announcement on the microscopy bulletin board:
Thank you for your interest! Analytical Consumer is available by subscription only; we do not accept advertisements. We sell all back issues, including the SEM one in July 1994. That issue is $40 in the USA (and territories) and Canada, and $45 elsewhere. Subscriptions are $260 in North America and $310 elsewhere (US funds).
We survey a different technology for analytical chemistry each month, asking labs what equipment (manufacturers) they own, why they bought it, and their opinion of the instrument and its service. The result is a customer satisfaction analysis for that equipment. In the four years of publication, we have covered a wide variety of instruments, supplies, and software, from gas chromatography to robotics or FTIR spectrophotometers to LIMS.
All those who gave me a mailing address will receive information, including a list of back issues, about Analytical Consumer in the mail. If you need something faster, please call me at (508) 369-9079 or e mail to jjordan-at-world.std.com.
Thank you for your interest, Jo Rita Jordan, PhD Analytical Consumer (508) 369-9079 jjordan-at-world.std.com CompuServe 76150,2171
In reply to my 7/6 posting requesting ideas for SEM training specimens there have been a number of responses -- all useful and appreciated. I intend to contact a number of individuals for more specifics. I would also invite any with an ongoing interest in this subject to contact me directly for further sharing of ideas.
In reponse to my posting, John Chandler of Colo State replied:
} } OK, spill the beans! Which specimens have you thought about? I'd be interested in knowing. This is a really good question. { {
Herewith my reply to John and others who might be interested -- things I had thought about:
(1) Prickly gold grids -- Pro: easy to get from supply houses, coarse and fine structures, good depth of field and focus/stigmation specimen. Con - somewhat expensive to buy, can't be cleaned, no useful BSE or EDS, doesn't have any everyday associations to interest students, BORING!
(2) Semiconductor circuit dies -- Pro: consistent and inexpensive, good macro structure, interesting and recognizable features, robust, easy to mount and clean, strong BSE and X-ray/mapping contrast. Con - little fine structure, limited contrast, rectilinear patterns are not very good for focus and stigmation practice, gets boring after a while.
(3) Burnt-out bulb filaments -- Pro: can make them oneself, can get really spectacular 3Dstructures with macro and high mag detail, very sharp edges and high contrast -- great pictures! Con -- haven't figured out how to make them consistently, a pain to mount, easy to destroy, can't be cleaned, not useful for BSED or EDS.
(4) Fracture surfaces -- Pro: not hard to obtain, interesting detail, very durable. Con - no major/obvious problems here -- if one could find the right material (displaying elemental segregation) and develop a cheap procedure for preparing and mounting, this may be a good bet.
(5) Insects -- Pro: great to look at and cheap to obtain. Con - have to be mounted carefully. Easily destroyed. Not useful for EDS.
(6) Aluminum dendrite structures -- Pro: durable, cleanable, lots of fine structure, and produce great pictures. Con: have to spend a lot of time finding the structures. Boring EDS and BSE.
For me a big issue is to be able to stamp out a whole lot of nearly identical specimens. I intend to use the specimen for printed or video-taped training materials which a student could take from ground-zero (turning on the SEM) through more advanced topics, and so it is important to be able to predict what the student will see. Of the above, the semiconductor sample and the fracture surface are probably the best candidates I knew of for my purposes. (However, I intend to look at hard at some of the new suggestions received.)
I spent 10 years trying to get TEM blocks that would thin section perfectly every time, and I succeeded by using Quetol. That formula is published in my book "Current Trends In Morphological Techniques, Volume I", 1981, CRC Press, Boca Raton, page 243. It is as follows: Quetol 651 - 30 gm, NSA - 50 gm, NMA 8 gm, and DMP-30 - 2 gm. I tested nearly 100 epoxy formulas to finally arrive at this one, which has high contrast, and sections easily. However, the problem I never solved, although I spent several years and dozens of tests, was the presence of precipitates in the tissues, apparently as a result of the reaction of phosphate and cacodylate buffers with uranyl acetate en bloc treatments. The technique I finally settled on was to get to the 100% alcohol stage for the treatment of the blocks of tissue with uranyl acetate. However, the results with this are less satisfactory in terms of unbroken membranes than when the tissue is stained en bloc before dehydration. My question is: has anyone run similar tests, and found a method of en bloc staining with uranyl acetate before dehydration without getting the pepper-like precipitates in the tissues (they are not caused by staining of the thin sections, but are present in the tissue, as they can be seen even on unstained thin sections)? Organic buffers such as HEPES and PIPES improve the situation, as they do not react with uranyl acetate, but the contrast is much lower. This is a significant problem, and I have seen the precipitates in published micrographs of various authors on numerous occasions. We have all probably had them occur from time to time, and it seems worse with dense tissues such as brain, and not so bad with tissues that are less dense.
John E. Johnson, Jr. Editor, Microscopy Research and Technique
Hi, This sounds too coincidental, but just before checking my mail I was reading the chapter by H.H. Mollenhauer in "Artifacts in Biological Electron Microscopy", edited by R.F.E. Crang and K.L. Klomparens. Mollenhauer's chapter _Dehydration and Embedding Artifacts in TEM_, pg. 43-64, is quite interesting, particularly his discussion of two types of "pepper", which he defines as section contamination of a dense particulate matter having a general appearance of black pepper. Of the two types, embedding pepper seems to be the one that might be contributing to your problem. Mollenhauer states that this type of pepper is influenced by the resin formulation, occurs when sections are stained with lead citrate, is much worse with Spurr resin than Epon or Araldite (unfortunately he doesn't discuss Quetol), and is "exacerbated" when en bloc UA is used before dehydration and embedding. He suggests that pepper may be formed in sections when stains become trapped within the section or react with one of the resin components. To remedy the problem, he recommends pretreatment (I assume before the lead citrate step) with either 1% EDTA or O.5% HCl. Both pretreatments will apparently eliminate the embedding pepper. The other type he calls fixation pepper. This type may be removed by pretreatment with 1-2% periodic acid prior to staining with UA or PbCit. BTW, are you using your resin mixture with animal tissues, plants or both? I work with plant tissue, primarily leaves, and have been using Spurr resin and Mollenhauer's Epon/Araldite formulation. How well does Quetol work with plant material? Hope this info helps. Dwight
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
There has lately been some discussion on the relative merits of various epoxies for embedding samples. It is of course impossible to formulate one resin that will satisfy all applications, but if a resin is formulated to enhance the desirable properties of epoxies and hardeners and to minimize the undesirable characteristics, a useful improvement in qualities can be obtained. Attributes that are important in formulation include: choice of epoxide (influences wettability, crosslinking, beam stability, contrast) anhydride to epoxide ratios (influences elongation after the yield point, which is a measure of sectioning quality) anhydride characteristics (influences hardness) choice of plasticiser (influences hardness and beam stability)
Use of micro-hardness testing to check the relationship between formulation and hardness, and tensile testing to relate Young's modulus, plastic deformation and toughness to sectioning qualities makes it possible to formulate towards a goal.
We have now been using the result of such a formulation (with Quetol 651) for a number of years on all types of material, for EM and for 0.5 - 1 micron sections for LM. It adheres exceptionally well to most sample surfaces, including such difficult samples as plant leaf cuticles, it sections well, has very little own structure, contrasts well and is stable in the beam. Specimens in this also stain well for LM with buffered Toluidin Blue. We like it and use it for more than 90 percent of our biological samples. It is hard - use a diamond knife for best results. It is also not the worst epoxy to use if you would like to do structure and immunolabeling on (in?) the same section. It contains: Quetol 651 19.4g MNA 22.3g DDSA 8.3g Araldite RD2 1.0g S1 (DMAE) 0.5g
We do not use U-acetate during the dehydration, so do not know if this resin formulation also shows pepper-like precipitates. We have occasionally seen pepper deposits after fixing in Glut. in 0.1M (or higher) phosphate buffer, but none if the phosphate conc. is lower. A reference to specimen pepper: Hendriks & Eestermans (1982), Electron dense granules and the role of buffers: artefacts from fixation with glutaraldehyde and osmium tetroxide. J. Microscopy, 126: 161-168.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Are you sure that the PO is necessary (or even useful)? We routinely use acetone for Embed 812 embedding and seldom have problems. On the other hand, PO adds extra steps, extra cost, and is reportedly carcinogenic.
Rod Kuehn University of Minnesota
On Fri, 8 Jul 1994, Terry Robertson wrote:
} Do any EM users know of a substitute petri-dish for the old Lux Permanox } tissue culture dish for processing of cell cultures directly for resin } embedding. I have tried several possible substitutes that the manufacturers } assured me were the equivalent but as soon as propylene oxide was added to } them they quickly melted into a plastic mess. } } Terry Robertson } } } } Dr Terry A. Robertson Telephone: 61-9-3462935 } Department of Pathology Fax: 61-9-3462891 } University of Western Australia } Nedlands WA 6009 } Australia Email: } troberts-at-eosin.path.uwa.edu.au }
John: As to your problem with "pepper", are you washing enough after your fix? If you don't wash the glut out of the specimen enough, especially doing enbloc staining, the osmium will bind to the glut left in the sample and form a pepper like precipitate. If someone needs enbloc staining, I sometimes wash my tissue overnight. For normal processing with post staining I never have a problem with precipitate in the sections. The best thing to do, in my opinion, is to WASH,WASH,WASH, after fix. Or post stain with uranyl acetate. For my post stains I use:
uranyl acetate..........5g 50% ETOH..............100ml stain for 4 minutes (6-700A sections) for 2-400A sections I stain 6 minutes
Hope this helps. If you have any questions you can reach me at: voice: 410-455-3582 fax: 410-455-3875 email: prutle1-at-gl.umbc.edu
John H. Johnson recently asked about "pepper" in ultrathin sections:
..."the problem I never solved, although I spent several years and dozens of tests, was the presence of precipitates in the tissues, apparently as a result of the reaction of phosphate and cacodylate buffers with uranyl acetate en bloc treatments."
In December, 1993, I asked a similar question of the Microscopy network and compiled 11 responses detailing experiences and solutions to this problem. If you would like a copy of this summary, contact me be e-mail and I will send it to you.
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Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Soem time ago, I asked if anyone else was using Quetol 651, and almost no= one replied. Why not?? I see several responses to the "pepper" problem= containing recipes for its use. I'd like to know the source and lot= numbers of this Quetol that you're currently using, plus the same info for= the NSA, if you use that: I've had a persistent, unresponsive problem for= the past year with Quetol system, and would like to solve it once and for= all. Thanks. Grace Kennedy, UCSD
Richard, For sea urchin gametes, we got very good results using a Karnovsky-type fix (glut-formaldehyde) at about 2%-2% in filtered seawater. Seawater itself is a good buffer, though something in it seems to react w/ UA if you use it en bloc. Since this mixture is about isotonic with the urchin cells, we ended up adding a bit of sucrose to make it slightly hypertonic so the mitochondria stayed nice. I don't recall right offhand how much sucrose we did add, but it was only a few grams per liter. Hope this helps.
Re: Suggestion to wash the tissue blocks for extended periods.
Yes, I have tried the washing and washing and washing procedure in a vain attempt to eliminate the pepper. I have tried leaving the blocks in buffer for up to a week before moving on to the en bloc staining with uranyl acetate (the specimens have been glutaraldehyde fixed, washed in buffer for up to 2 days, then osmicated, then washed in buffer for up to 7 days). No matter how long the blocks are washed in buffer after osmication and before en bloc staining, the precipitates are there in the thin sections, even with no lead or uranyl staining of the sections. It does not occur quite so badly on tissues which contain cells not so tightly packed together or in poorly fixed tissues (which would have more broken membranes), but I fix brain tissue very carefully, and it is typically very tightly packed together, which may result in retention of phosphate or cacodylate regardless of washing. The whole thing is more of an irritation than anything else, because it does not really interfere with scientific interpretation, but is rather unaesthetic. As I mentioned, the problem is eleminated by using organic buffers or staining en bloc at the 100% alcohol stage, before propylene oxide and infiltration with epoxy. However, organic buffers react very quickly with osmium tetroxide, and the resulting contrast is much lower than with phosphate buffer or cacodylate. Also, waiting until the alcohol stage results in lower quality of preservation than if en bloc stained before dehydration. Believe me when I say that I have tried EVERYTHING, with no luck in finding a reliable method of en bloc staining with uranyl acetate at the post osmium stage, before dehydration, which is the best place to do it, as uranium helps preserve membranes against the damage caused by dehydration. At present, I am tracing down a possible new technique with borohydride reduction suggested to me by a very brilliant biochemist. I will post the results when I have them.
Re: Referral to H. Mollenhauer's chapter in the 1988 Crang and Klomparens textbook.
Hilton also published on this topic in Microscopy Research and Technique, 1993, volume 26, number 6, pp. 496-512. The situation as I see it is to prevent the precipitates from forming in the first place rather than removing them after they have occurred. Still, the EDTA/HCl method can eliminate their visibility, and may be one of the best remedies so far. Hilton Mollenhauer spent decades dealing with such problems, and I would trust his findings and solutions implicitly.
Re: Quetol 651 problems.
If your difficulty with this epoxy is represented by holes in the thin sections (incomplete infiltration), I went around in circles over that one too. The problem is solved by leaving the tissue blocks in the 50/50 epoxy/propylene oxide overnight rather than just for a short time.
John E. Johnson, Jr. Editor, Microscopy Research and Technique
Message-Id: {9407141741.AA15674-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: OPTICAL MICROSCOPY - MICROSTRUCTURES IN STEELS . Orig-Author: {Griffiths Michael MJ {griffmj-at-msmailipx.bhpese.oz.au} }:ddn:wpafb -----------------------------------------------------------
Do any Materials Scientists / Metallurgists out there know of a reliable,reproducible etching technique to delineate bainite / tempered martensite structures in plain low carbon steel , [0.1% C] , which will yield nice coloured light microscope photomicrographs ?
We have an honors student here who despite trying a plethora of etchants : LePera's Reagent , Klemms Reagent , BASP , Cadmiun Sulphide Reagent , Beraha colour etchant , has had no luck.
Thank you
MIichael Griffiths B.H.P. Steel Newcastle Works Australia.
If this topic is a bit too esoteric for the general Bulletin Board maybe any responses can be E Mailed directly to me .
My Internet address is : griffmj-at-msmailipx.bhpese.oz.au
Greetings! While we are discussing resins, I'd like to get some feedback on the suggestion to replace proplyene oxide with acetonitrile (as mentioned to me by one of the resource people at Polysciences). She said that I could simply introduce acetonitrile in place of propylene oxide in the same ratios with the resin. Anyone tried this? I often use acetone as a dehydrant and so go right into resin from 100%, but I've also used EtOH and done the same thing without the transitional solvent (epoxy). Again, I work only with plant material, primarily leaves, so your mileage may vary.
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
John Johnson says: "...As I mentioned, the problem is eleminated by using organic buffers or staining en bloc at the 100% alcohol stage, before propylene oxide and infiltration with epoxy. However, organic buffers react very quickly with osmium tetroxide, and the resulting contrast is much lower than with phosphate buffer or cacodylate. ..."
does everybody use a buffer for their osmium step? I use HEPES without much problem but remember someone saying they did it in pure water since the osmium permeabilized the membranes and osmolarity no longer counted. In regards to John Johnson's comment, how about a short fixation in osmium/HEPES to ensure stability and loss of osmo-sensitivity then a second fix in osmium/water? is this crazy?
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
It is a coincidence that someone should ask about the use of acetonitrile as a substitute solvent in EM specimens. We just received an article on this subject, and if it is accepted, I will post the author's name and address so you can request a preprint.
John E. Johnson, Jr. Editor, Microscopy Research and Technique
On Thu, 14 Jul 1994, Griffiths Michael MJ wrote: } } Do any Materials Scientists / Metallurgists out there know of a } reliable,reproducible etching technique to delineate bainite / tempered } martensite structures in plain low carbon steel , [0.1% C] , which will } yield nice coloured light microscope photomicrographs ? } } We have an honors student here who despite trying a plethora of etchants : } LePera's Reagent , Klemms Reagent , BASP , Cadmiun Sulphide Reagent , Beraha } colour etchant , has had no luck.
Might try: [hint: I highly recommend #2 ;)]
1. Beaujard & Tordeux's: 21-28% aqueous NaHSO3; immerse 10-25 sec.
2. Villela's: 5 ml hydrochloric acid + 1 gr. picric acid + 100 ml Ethanol; immerse -few- seconds to minutes; -GREAT- STUFF!!! Works very good for CVD iron also; use polarized light, of course...; be -very- careful not to disturb the final "film" on the polished surface.
3. You say that Beraha's has been tried... which version(s)? There is: a) 1 gram Na2.MoO4 + 100 ml H2.O + 0.1 gram HH4.HF2; immerse 20-30 sec.; b) 3 gram K2.S2.O5 + 10 gram Na2.S2.O3 + 100 ml H2.O; immerse 1-15 min. c) many more...
4. and, tho' not too colourful, Picral [4 gr. picric acid + 100 ml H2.O, and sometimes + few drops (~4) 17% zephiran chloride (wetting agent)] -does- work... (may also need a few drops of HCl to enhance the action)
Good luck, -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /\v/\ | | Metallographic Lab. | Missouri Speleological Survey /\v/\ | | Rolla Research Center | Bat Conservation International /\v/\ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /\v/\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
Netters, We recently tried acetonitrile as a substitute for ethanol in a dehydration series. The application is immunocytochemistry at the light microscope level and the resin we embed in is a mix of butyl and methyl methacrylate. The tissue is a plant root. In our trial, the acetonitrile was inferior to the ethanol, leading to greater distortion of the tissue (cell separtation and wrinkling), and higher background. We did not "trouble shoot" this, but on the basis of this trial, we were not encouraged to pursue the matter further.
Bonjour! In reference to the posting by Tobias Baskin, my interest is not in _substituting_ acetonitrile for a dehydrant like ethanol, but in using it in place of propylene oxide. My guess would be that like ethanol and the lower percentages of acetone, considerable tissue shrinkage would occur if the acetonitrile is used as a straight dehydrant, as Tobias experienced. Yet another resin question: How do most people store the resin components? I have seen them kept in a large glass desiccator, at room temperature on the shelf, but are there particular tricks that will yield a longer shelf life? Do the different components have different lifetimes? I work with the Spurr formula and the Epon/Araldite mixture of Mollenhauer. I'm also considering trying the Quetol 651 formulation suggested to me by Jan Coetzee (quetol, araldite RD-2 [1,4-butanediol diglycidyl ether], MNA, DDSA, DMAE). Thanks
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
A trick for Mollenhauer's Epon/Araldite, that I got out of an old Harvard Anatomy Department set of protocols, is to put your customary proportions of Araldite, Epon (Poly/Bed 812 or other), and DDSA into a glass bottle, cap it loosely, put it in a 60 degree oven for about an hour, then tighten the cap and mix thoroughly by vigorous agitation (they mix very effectively, because the heat has made them less viscous). Then put the bottle in the refrigerator, where the stock will be stable for six months or more.
When you need catalyzed resin, take the stock out of the refrigerator and let it come to room temperature. Then take out the volume you want, put it in a suitable tricorn plastic disposable beaker, and return the stock to the refrigerator. Add DMP-30 (with a Pipetman) proportionately to 2% (in other words, if you took 10 ml out of the stock bottle, add 0.2 ml DMP-30 to it). You can mix with a metal weighing spatula. You then have the desired volume of catalyzed resin.
I have used this and it works very well. The thorough mixing of the stock components is a considerable advantage (they often don't get mixed properly because they are so viscous). I like being able to prepare only the amount of catalyzed resin that I actually need, often only 10 ml. It is very convenient. The instructions that come with the Polysciences Epon/Araldite kit (cat. #02595) confirm that "In the absence of DMP-30 the mixtures are stable for six months at 4 degrees C, and for several days at room temperature".
} Bonjour! } In reference to the posting by Tobias Baskin, my interest is not } in _substituting_ acetonitrile for a dehydrant like ethanol, but in using } it in place of propylene oxide. My guess would be that like ethanol and } the lower percentages of acetone, considerable tissue shrinkage would } occur if the acetonitrile is used as a straight dehydrant, as Tobias } experienced. } Yet another resin question: How do most people store the resin } components? I have seen them kept in a large glass desiccator, at room } temperature on the shelf, but are there particular tricks that will yield } a longer shelf life? Do the different components have different lifetimes? } I work with the Spurr formula and the Epon/Araldite mixture of } Mollenhauer. I'm also considering trying the Quetol 651 formulation } suggested to me by Jan Coetzee (quetol, araldite RD-2 [1,4-butanediol } diglycidyl ether], MNA, DDSA, DMAE). } Thanks } } Dwight U. Beebe } Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca } Universite de Montreal Voice:514-872-4563 } 4101, rue Sherbrooke est FAX:514-872-9406 } Montreal, PQ H1X 2B2 Canada } } } }
Received: From ANLVM(MAILER) by ANLNRH(ANJE6.10) for MICROSCO-at-ANLEMC; Sun, 17 Jul 94 21:29 Received: from ANLVM by ANLVM (Mailer R2.07B) with BSMTP id 5997; Sun, 17 Jul 94 21:28:09 CDT Received: from earwax.pd.uwa.edu.au by ANLVM.CTD.ANL.GOV (IBM VM SMTP R1.2.2ANL-MX) with TCP; Sun, 17 Jul 94 21:28:00 CDT Received: from [130.95.124.120] (awsj [130.95.124.120]) by earwax.pd.uwa.edu.au (8.1C/8.1) with SMTP id KAA08688; Mon, 18 Jul 1994 10:26:44 +0800
We currently considering setting up a facility to analyse high resolution TEM micrographs using Fraunhofer diffraction. The set up we are considering goes something like this:
He-Ne laser-} collimator-} diaphragm-} the film-} lens-} mirror-} camera or -} lens-} CCD-} image processing
Are there any suggestions, pitfalls etc, that we should avoid or consider. Are there any commercial apparatus available or is it a pick and mix from an optics catalogue?
All suggestions eagerly awaited.
Keith Moulding
Hong Kong University of Science and Technology Materials Characterisation and Preparation Centre.
We store all resin components (for Spurr's, Embed 812, Araldite and Quetol) except DMP-30 and DMAE at room temp. We store the catalysts at 4 degrees and discard them after a year or two. I've seen no hint of a limiting shelf life for the other components.
The complete Quetol resin is made up a gallon-at-a-time, aliquotted into 12 ml plastic vials and stored at -40C. The gallon lasts for about 6 months and is used long before it starts to set.
Rod Kuehn University of Minnesota
On Fri, 15 Jul 1994, Dwight Beebe wrote:
} Bonjour! } In reference to the posting by Tobias Baskin, my interest is not } in _substituting_ acetonitrile for a dehydrant like ethanol, but in using } it in place of propylene oxide. My guess would be that like ethanol and } the lower percentages of acetone, considerable tissue shrinkage would } occur if the acetonitrile is used as a straight dehydrant, as Tobias } experienced. } Yet another resin question: How do most people store the resin } components? I have seen them kept in a large glass desiccator, at room } temperature on the shelf, but are there particular tricks that will yield } a longer shelf life? Do the different components have different lifetimes? } I work with the Spurr formula and the Epon/Araldite mixture of } Mollenhauer. I'm also considering trying the Quetol 651 formulation } suggested to me by Jan Coetzee (quetol, araldite RD-2 [1,4-butanediol } diglycidyl ether], MNA, DDSA, DMAE). } Thanks } } Dwight U. Beebe } Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca } Universite de Montreal Voice:514-872-4563 } 4101, rue Sherbrooke est FAX:514-872-9406 } Montreal, PQ H1X 2B2 Canada } } }
Someone in our department is doing in situ hybridization at the EM level and is having some problems with contamination on the sections.
She is processing thin sections of Lowicryl-embedded material by floating on the hybridization solution. This is done at 42 deg C in a sealed container for 22 hours. After hybridization, grids are washed 5X10 min in PBS at room temperature to remove hybridization solution. This is followed by a histochemical stain.
In the EM, there is an amorphous "sludge" over the section, along with some electron-dense precipitate. Processing for the histochemistry alone, including potassium permanganate, UA and lead citrate stains, eliminates the sludge/ppt problem, so it looks like the in situ step is the culprit.
Does anyone have suggestions for eliminating this contamination? I can get more details, if that would help.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
I've had the best results with picral or nital. Sometimes a few drops of zephyrin chloride to the picral helps.
Let me know how this pans out or if you have tried this already. You can reach me via:
Sue Smith smiths-at-mlc.lib.mi.us
On Thu, 14 Jul 1994, Griffiths Michael MJ wrote:
} } Do any Materials Scientists / Metallurgists out there know of a } reliable,reproducible etching technique to delineate bainite / tempered } martensite structures in plain low carbon steel , [0.1% C] , which will } yield nice coloured light microscope photomicrographs ? } } We have an honors student here who despite trying a plethora of etchants : } LePera's Reagent , Klemms Reagent , BASP , Cadmiun Sulphide Reagent , Beraha } colour etchant , has had no luck. } } Thank you } } } MIichael Griffiths } B.H.P. Steel } Newcastle Works } Australia. } } If this topic is a bit too esoteric for the general Bulletin Board maybe } any responses } can be E Mailed directly to me . } } My Internet address is : griffmj-at-msmailipx.bhpese.oz.au } } } } } } }
In response to the several comments regarding embedding media, I would like to mention some experiences regarding embedding and the ease of sectioning as well as the quality of the sections themselves. I used EPON and Araldite for years, and then, in 1976, switched completely to Quetol 651. However, even though the sections cut easily and the contrast was high, there were often small holes in the sections. I thought there might be water in the media (I believe Quetol 651 is water soluble), or perhaps in the absolute alcohol, propylene oxide, acetone, epoxy plasticizers (DDSA, NMA) or polymerizer (DMP-30). So, I had a local chemical analysis company in Baltimore (this was when I was with NIH, and Johns Hopkins University) test the various reagents for the presence of water, including some bottles that I left open overnight. The results indicated that there was no significant amount of water in any of the reagents, including the ones that I left open overnight (and Baltimore is a humid city). So, I experimented with leaving the samples in various stages of dehydration and infiltration for different lengths of time. It turned out that the 50/50 stage (acetone/embedding media) was the critical step. By leaving the tissue in the 50/50 stage overnight, or longer, the holes were no longer present, and the technician said that the blocks cut better than anything he had ever seen. With EPON, the holes did not occur as often, but the blocks cut less evenly when standard infiltration times were used. I believe that we have been leaving our specimens (depending on the density of the tissue) in the dehydration and infiltration stages for much too short a period of time, and that the holes are caused by incomplete dehydration and infiltration due to this improper procedure. With Quetol, because of its water solubility, I theorize that water left over from incomplete dehydration, rather than any water in the absolute alcohol or media, cannot be displaced. EPON, on the other hand, is not water soluble, and probably displaces residual water. However, extending the dehydration and infiltration times will improve the sectioning quality of any embedding media in my experience. On the other hand, regarding my recent correspondence with all of you on the TEM embedding pepper problem, no amount of rinsing or extended dehydration has eliminated the pepper when the specimens are stained en bloc before dehydration. Because we all use different types of specimens, I would be interested in your comments regarding the above theory, as well as experiences with embedding pepper using different types of specimens (I use brain which is very tightly packed tissue; it does not occur in my lab when I use monolayer cell cultures).
John E. Johnson, Jr. Editor, Microscopy Research and Technique
Your proposed setup--L-} C-} D-} F-} L'-} M-} Cam--looks OK; it's what we put together from components. I don't know of a commercially available kit, but I'm sure anyone who sells one will let us know. If you go the CCD-} Im. proc route, there is no need to do optical diffraction; you can get the same information from FFT of the image. One great convenience is to have a right-angle viewer on the camera--this prevents painful contortions when checking that the pattern is in the right position to show up on the film. Good luck.
I have seen a reference for using acetonitrile as a dehydrating agent as well as substituting it for the PO in epoxy resins - I only tried it once and was not very impressed with the results
*** CAUTION *** Acetonitrile combined with water releases hydrogen cyanide gas !!!
while it is touted as being considerably less toxic than PO users should be aware of the above reaction if it is being used as a dehydrant
I have an Agfa B&W print processor and I have a most annoying problem with it and would appreciate some help from anyone who has had & solved this problem.....
If the processor sits unused for more thatn a few days, I get this icky black mold(?) that grows in the baths - I can keep it somewhat under control by adding a small amount of bleach to the wash water, but I worry about what this is doing to the prints and their longetivity - it seems to originate in the activator- but I can't be certain - also it stains the trays to the extent that they cannot be completely cleaned
Message-Id: {1994Jul19.090536.319743799-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy listserver)
Microscopy Training Trends-Your Input Solicited
Hi fellow microscopists, * We are having a special symposium at MSA in New Orleans (Aug 1-6, 1994) on training trends in microscopy. * I solicit your input on the subject that I will collate and make available at the meeting and summarize and make available to the microscopy list when complete. * Specific examples are encouraged. * Feel free to answer any of the questions or just make some general comments, although specific examples will be extremely helpful. * Send your answers to me directly at my e-mail address: murphy-at-ms.sjdccd.cc.ca.us. * If you have "reply" on your mail option, just click it, write your message, and send it. It should then be sent directly to my e mail address without bottling up the network. * Thank you in advance for your help. * Judy Murphy, PhD, Dept. of Microscopy, San Joaquin Delta College, 5151 Pacific Ave., Stockton, CA 95207, Phone 209/474-5284; FAX 209/474-5649
I am looking for information on the following: 1. Formal courses taught at your institution in electron microscopy (lecture, lab or lecture and lab) and # of students taking the courses (present vs past). Indicate if other types of microscopy courses. 2. Microscopy courses that have decreased in units or been completely eliminated because of lack of enrollment. 3. Informal training in microscopy (indicate type) and # of students taking the training (present vs past). 4. Service microscopy done at your institution A. for graduate students (#) B. for researchers (#) C. other 5. Funding for microscopy labs A. Has this decreased or increased for your lab? ballpark percentage or remained unchanged? B. Specifically, how has this affected your operation? 6. What are the general trends you are aware of in training for microscopists with respect to numbers, material covered, amount of lab, etc. 7. What implications do you think this will have in the job market, at the workplace, etc.? 8. Any other comments?
For those that send information, thank you sincerely.
We have tried unsuccessfully to collect dust mites for an SEM project. Does anyone have suggestions on how to collect and prepare such a sample? So far what we have done is attach double stick tape to stubs and either blot areas (floor corners, bed linens, various anatomical locations) or sweep and area and then press collection onto double stick tape. We then give a brief gold-palladium coat.
I'm having some problems with the Microscopy Listserver system. Please expect some "Old Mail" and "Duplicate" messages traveling across the net for the next day or so. We've had several crashes in the last few days and I've discovered a large queue of messages which may not have been delivered, some of which are several weeks old. I apologize in advance for the hassel of seeing a message twice but rather than check everyone against the archive I've decided the easiest thing to do is just to resubmit the lot. I'll do it gradually over the next day or so.
Once again excuse the test message. I'm still trying to correct the nameserver problems and this is the only way I can test all 1300+ subscribers/sites....
Once again. This is a Listserver test by your friendly neighborhood SysOp. I'm still trying to get the nameserver test to complete. Sorry for the traffic. Just delete this.
We're looking for a high speed, high resolution flatbed scanner that can be used to scan both prints and negatives. The scanner should have a Mac driver (It'll be a super plus if the scanner can be interfaced to a unix system). The price range is below $5000. Any suggestions, comments, or stories are very much appreciated. Thank you in advance.
Best regards,
Xiao Zhang GE Corporate R&D (518) 387-6709 internet: ZHANG-at-CRD.GE.COM
Zhang, I recently bought a Nikon "Coolscan" slide scanner (~$2,000). It scans 2x2 slides, 35mm negatives, in color or monochrome. Resolution is better than 2K dpi. It will interface to anything with a SCSI port and comes with software for MS-Windows and Mac. It serves my needs because just about everything I have ends up as slides or negatives. Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
Although I haven't tried it myself, it seems that the best place to look for dust mites would be in a vacuum cleaner bag (after several vacuum runs across a normal carpet).
We have just completed our latest survey called "BUYING A NEW MICROSCOPE/THE AGONY AND THE ECSTASY". We've had so many calls about the work that it might be helpful to send along this information. The article includes a quantitative system for a selection that is appropriate for any microscope. The survey encompasses 59 scanning electron microscopes. In every case, users were contacted directly. We assessed for Customer Satisfaction, Applications, Microanalytical Accessories, and Service History.
This survey, Volume 6, Numbers 6 and 7 is available to new and existing subscribers of our newsletter. If you are interested in subscriber information, we have an 800 number. 800-440-0311.
This study is a joint effort of MICROSCOPE TECHNOLOGY & NEWS and ANALYTICAL CONSUMER. Additionally, we are working on a similar study for Transmission Electron Microscopes. If you have a TEM and wouldlike to be considered for the survey, please call us.
Our next issue, published in early August, will feature a review of the MSA/MAS meeting in New Orleans. If you can't attend, call us, tell us what you would like to have reviewed, and we will try to accommodate you.
Regards to all, Ellie Solit, Formerly Scientific Marketing Manager for Polaroid
Many years ago (10) we purchased an entire optical diffractometer from Polaron. We still use it occasionally, although lately we have been doing FFT's of images captured by Gatan's slow scan camera. The last addresses I had for Polaron are:
Polaron Equipment Ltd. 53-63 Greenhill Crescent Watford Business Park Watford Hertfordshire WD1 8QS U. K.
Polaron Instruments, Inc. 2293 Amber Drive Line Lexington Industrial Park Hatfield, Pennsylvania 19440 U. S. A. Telephone: 215-822-2665
P.S. Sorry about that previous, garbled message.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
Does anyone know any technicques we can use prepare Dioscorea (yam) or other angiosperm material? We are interested in using EM to see polymorphism in chromosomes. We have been using root tips. Thanks. Joyce.
Please excuse this plea for help.I need to access WorldWideWeb on internet (for images) from my home Mac thru a remote workstation account running XWindows and Mosaic as the browser program .Is there a Mac program that will emulate XWindows for this purpose? Is there another way around this? My internet connection is only thru my workstation account. Are there other browser programs for Mac/WWW access via XWindows? Thanks for any help you can provide.
Marc C. Brande, M.S. Live Brain Cell Functioning in 3D Culture San Diego 3D Imaging Group 3840 Camino Lindo San Diego, CA 92122 Email: BRANDE-at-SDSC.EDU Voice: (619) 587-4830 SD3D Email Discussion List: All aspects of 3D Imaging To subscribe/unsubscribe,send request to: sd3d-request-at-sdsc.edu To post a message, send message to: sd3d-at-mailserver.sdsc.edu
} } Please excuse this plea for help.I need to access WorldWideWeb on internet } (for images) from my home Mac thru a remote workstation account running } XWindows and Mosaic as the browser program .Is there a Mac program that will } emulate XWindows for this purpose? Is there another way around this? My } internet connection is only thru my workstation account. Are there other } browser programs for Mac/WWW access via XWindows? } Thanks for any help you can provide. } } NCSA provides WWW Browsers for X-Win, MS-Win, and MAC's. So you don't need to setup an X-Server only to browse the WWW (though X might be a nice addition even to MAC's ;). For more information check the NCSA Mosaic Home Page at :
} Sorry for repeating the same subject, but I just couldn't let anything } dangling for too long. To wrap up the discussion on the necessity of using } dry nitrogen when venting the TEM camera chamber, I measured the time it } took for the camera chamber of our JEM-2000FX TEM to pump down to ~0.5 mPa } (at which point the air-lock opens automatically and the green light for } the filament comes on). The results are as follows: } } using dry N2: 6'2" } using room air: 6'15" } } I did not repeat this experiment to determine whether the difference is } within fluctuation, but given the 13 s difference, I do not think it is } worthwhile to do so.
Gary, what was the humidity level in your lab at the time? With relative humidities of 65 - 85% in my lab, I find a remarkable difference in pumpdown time with honest-to-gosh dry nitrogen! If the camera has been open for more than about 60 sec on a humid day (like today, with a hurricane lurking out there) I give the camera a few second burst of nitrogen just prior to pumpdown, as well. It makes a huge difference! So I guess the bottom line is - whatever works. But it pays to check out your own individual situation.
Aloha, Tina Weatherby Carvalho Biological EM Facility University of Hawaii
Applications are invited for the above position in the Department of Histochemistry, Royal Postgraduate Medical School.
The successful candidate will be expected to oversee and expand the existing molecular biology unit within the Department. The unit is mainly engaged in investigating the roles played by specific regulatory factors (hormones, neurotransmitters, enzymes, receptors, free radicals) in the pathogenesis of human diseases, namely vascular remodelling, asthma, osteoporosis and gut dysfunctions. Applicants should have experience in probe preparation, in-situ hybridization and PCR techniques.
Ambitious and well motivated, the successful candidate will be encouraged to develop their own research interests within the scope of the Department's work and take full advantage of academic career opportunities within this prestigious establishment
The salary level range for a this Senior Research Officer 1A would be between GPB 19,326 and GPB 20,953 plus GPB 2,134 London Allowance according to age and experience.
Application forms and further details are available from the Personnel Department, RPMS, Du Cane Road, London W12 0NN, tel 081 740 3204, reference AJAY1.
OUTLINE JOB DESCRIPTION
The Department of Histochemistry is an extremely active research based Department which uses an integrated, thematic approach to its investigation of regulatory factors (hormones, neurotranmsitters, enzymes etc) and their contribution to normal bodily function and disease processes. Research is centred on the study of growth and remodelling, blood flow, inflammation and neuromuscular interaction. The mainstream technology is based on microscopy. The successful candidate will have the opportunity of taking over the existing molecular biology unit and expanding it. He/she will take responsibility for the day to day management of the facility and will be able to develop their own research interests within the scope of the Department's work. As a post-doctoral researcher, they will be expected to contribute fully to the academic as well as practical aspects of the Department, participating in inter- and intra-departmental seminars and helping to maintain the Department's position as an internationally renowned research centre through the production of scientific publications.
Hands on experience of probe preparation, in-situ hybridization and PCR methods will be required along with experience of supervising the work of others.
This post will suit an ambitious individual who is sufficiently motivated to take full advantage of this opportunity to develop a career within this prestigious academic institution.
Here in the Department of Anatomy and Cell Biology in the University of Michigan Medical School, I offered an EM course ("Biological Electron Microscopy") for four years (1988-91). It was a lecture/laboratory course, a rather intensive "hands on" type of course for graduate students. It was limited to 8 students. It seemed to go well, but was very labor intensive, and very expensive (each student received the equivalent of about $500 worth of supplies and recharge time on instruments in our department central central research facility (called the "Cell Biology Laboratories"). It was discontinued because of the expense and the large amount of labor.
For the last two years, I have offered a broader course for graduate students, entitled "Morphology for Molecular Biologists". It is a lecture and demonstration course (students have some "hands on" experience in the course, but less than the previous course). As the name indicates, there is an emphasis on immunocytochemistry, in situ hybridization, localization of reporter gene expression, and confocal microscopy, but there is also a rather extensive introduction to light and electron microscopy (both TEM and SEM), including both instrumentation and specimen preparation. Since many students in molecular biology these days do not have much experience in interpreting light and electron micrographs, the course includes a 2-hour "mini-course" in histology, and a 2-hour introduction to EM of organelles. The course is limited to 16 students, and they seem quite enthusiastic and well motivated, because of the obvious importance of morphological insight for some approaches in contemporary molecular biology.
Our department has a central research facility, the "Cell Biology Laboratories", containing equipment including TEM (Philips CM10), SEM (ISI=Topcon DS-130), confocal facility (BioRad MR600, Meridian, extensive Unix-based image processing and analysis), Balzer freeze fracture, Leica-Reichert ultramicrotomes, photographic darkroom, etc. The CBL was set up in 1979, and is primarily a hardware facility, offering the use of equipment to anyone in the University (on a recharge basis). A CBL Manager keeps the equipment in excellent shape, and gives training and help to users. There is some service (again on a recharge basis), although this has not been a major emphasis. As is often the case for such facilities, it is difficult to generate enough recharge money to operate completely in the black, covering very high annual service contracts (for example, about $12K for the Philips TEM), Manager's salary and other expenses. However, it has generally done quite well.
My own bias is that EM in the future may see some of the same renaissance that LM has seen in molecular biology over the last few years. I think that the questions and localizations will be more and more intracellular, where the resolution of EM will be needed to provide answers.
Kent
A. Kent Christensen Department of Anatomy and Cell Biology University of Michigan Medical School Ann Arbor, MI akc-at-umich.edu
Our laboratory will be introducing a new technology during the Poster Session at the MSA meeting for relatively rapid 3-D reconstruction from a series of tilted images. Simpler than a complete computed tomography reconstruction, the software can be run on a Macintosh or DOS computer and is highly adaptable to extracting quantitative information. If you want to see it in action, We will have several examples for demonstration. The poster is PQ# 435 THREE-DIMENSIONAL RECONSTRUCTION OF ATHEROSCLEROTIC FOAM CELLS USING TOMOSYNTHESIS.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I need some advice from someone who has had success preparing TEM samples of metallic film / ceramic substrate systems. I have been trying to make samples perpendicular to the interface of the film/substrate and have been unable to keep the metal layer from debonding from the substrate during either cross sectioning or mechanical thinning. Any thoughts?
Eric Stach Grad Student Department of Materials Science and Engineering University of Washington email: stach-at-u.washington.edu
Help! On attempting to section blocks from a recent fixation, I encountered a frustrating series of problems with compression, wrinkling, and related faults. After checking my sectioning methods and not finding any noticable mistakes, I went back to my notebook and realized that I had added twice the accelerator (DMP-30) to my Epon/Araldite mixture. A look at some of the available texts yielded info about the necessity for correct catalyst amounts, but specific info about what to do wasn't there. Is this excess catalyst the cause of the sectioning problems (the blocks seem too soft)? Or is something else the culprit? I had thought that too much accelerator would have caused the blocks to become very brittle, but the opposite seems to be the case. The mixture was: 7.5 gm EMBed 812 12.5 gm Araldite 502 27.5 gm DDSA 1.4 gm DMP-30 (should be 0.7gm) Polymerization was at 70 C for 43 hr.
Thanks in advance,
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
I am about to start a new project doing TEM of inactivated (I hope!) HIV. I'll be doing straight morphology and immuno-gold labelling of viral surface glycoproteins. I haven't hit the library yet and was wondering if anybody out there had a good fix for straight TEM and/or IMC of HIV. Citations are also welcomed.
Thanks in advance.
John Aghajanian Worcester Foundation for Experimental Biology
jjerome: Try a small, low powere vacuum-cleaner like a dust-buster, or a whisk broom. Gather a buch of dust and run through a Berlese funnel. Also: have you tried plucking eyebrows and eyelashes for Demodex? Should be able to get several from any healthy person. Soft opisthosomas, so they are a good drying-test. Phil Oshel poshel-at-luc.edu
For the last few days I have been receiving multiple "error messages" and "returned mail" from the Post Office at ecf.toronto.edu which have the text of postings to the microscopy list from other individuals. i had previously received these postings. Why do we get all these error messages? Am i the only one?
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
} For the last few days I have been receiving multiple "error messages" and } "returned mail" from the Post Office at ecf.toronto.edu which have the text } of postings to the microscopy list from other individuals. i had } previously received these postings. Why do we get all these error } messages? Am i the only one? } } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax)
Nope! Me, too.
Mike Wilson
------------------------------------------------------------------------ | Mike Wilson | | | Otolaryngology Dept. | "Don't take life so serious, son -- | | Univ. of Texas Health Sci. Ctr.| it ain't nohow permanent!" | | San Antonio, TX 78284-7777 | -- Pogo (Walt Kelly) | | Phone (210)567-6507; FAX -3617 | | ------------------------------------------------------------------------
I hope that this doesn't sound corny but, I just wanted to say that I am glad that this list exists. It is nice to be able to have a place to write to if you are having problems with a fixation process or if you have questions on a particular type of microscope, etc.
Thanks to everyone for being helpful in answering various questions and of course, thanks Nestor for taking up the task of maintaining and monitoring this list.
This is to everyone attending the MSA meetings in New Orleans, see you there!!
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
John- We've done immunogold labeling of HIV gp120 and found that it was sensitive to all aldehydes and ethanol fixatives. We do now do immunostaining on unfixed tissue and then fix. For immunochemistry inside of cells we permeabilize with BRIJ 57 (Triton seems to extract everything). I hope this is helpful. I am anxious to hear the other answers you get since they may solve some of our problems too.
Best-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Fri, 22 Jul 1994 JOHNA-at-SCI.WFEB.EDU wrote:
} Greetings folks, } } I am about to start a new project doing TEM of inactivated (I hope!) HIV. } I'll be doing straight morphology and immuno-gold labelling of viral } surface glycoproteins. I haven't hit the library yet and was wondering if } anybody out there had a good fix for straight TEM and/or IMC of HIV. } Citations are also welcomed. } } Thanks in advance. } } John Aghajanian } Worcester Foundation for Experimental Biology } } JOHNA-at-sci.wfeb.edu } }
I use to work with HIV alot in my old job. I would fix viral suspensions with 1-2% glut. in 0.1M PIPES buffer. The problem with this is that the fixation would make the particles clump together, thus for your case hindering labeling sites. If you are using cell culture, fix with a 1% of less of glut. I would use 1% ammonium molybdate as the negative of choice. Some references to check out are by S. Hearn and by G. Herrera. There are many references out there on viral immunocytochemistry.
} } I am tempted to try benzyl dimethyl amine (BDMA) in place of DMP-30 but } don't have the original reference. How much does one use? Anyone have the original citation? I usually use: } 20 g EmBed812 + } 10 g DDSA + } 10 g NMA + } 0.6 g DMP-30. } } Thanks. } } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax)
I recently tried using BDMA as a catylst using the EMS formulation EMBed 812 16.2 mls DDSA 10 mls NMA 8.9 mls BDMA 1-1.4 mls (EM Sciences recommends using 2X the volume of DMP-30 when substituting BDMA for DMP-30)
The blocks shared similar characteristics of cutting etc as epon batches made with DMP-30. One significant difference was in the shelf life. I store mixed batches of epon plus accelerator in aliquots at -20 C. The batches with BDMA were significantly more viscous after 1 month at -20. Furthermore, leaving 100% BDMA-epon for 24 hours on the rotor produced a taffylike material that made final embedding more difficult. By comparison, the DMP-30 batches retained characteristics similar to freshly mixed batches.
Regarding the original citation, give Electron Microscopy Sciences a call at 800-523-5874.
steve ---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
} For the last few days I have been receiving multiple "error messages" and } "returned mail" from the Post Office at ecf.toronto.edu which have the } text of postings to the microscopy list from other individuals. i had } previously received these postings. Why do we get all these error } messages? Am i the only one? } } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax) }
Resent-Date: Fri, 22 Jul 1994 13:58:31 -0500 (CDT) Resent-From: microscopy-at-ANLEMC.MSD.ANL.GOV Resent-To: MICROSCOPYLIST-at-ANLEMC.MSD.ANL.GOV To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Metal Film / Ceramic Substrate TEM Prep Orig-Author: {microscopy-at-ANLEMC.MSD.ANL.GOV}:ddn:wpafb -----------------------------------------------------------
I need some advice from someone who has had success preparing TEM samples of metallic film / ceramic substrate systems. I have been trying to make samples perpendicular to the interface of the film/substrate and have been unable to keep the metal layer from debonding from the substrate during either cross sectioning or mechanical thinning. Any thoughts?
Eric Stach Grad Student Department of Materials Science and Engineering University of Washington email: stach-at-u.washington.edu
I have received a number of requests for information regarding permeabilization mentioned in my reply about HIV immunostaining and so I am passing on brief comments to the users group.
The method we use is described in a paper by one of our post docs: Landers et al, 1993. Am J Pathol. 142:1668.
it is a modification of a method first described by Schliwa. Schliwa et al, 1981. PNAS, USA 78:4329.
We use Brij 58 in a concentration range of 1-3 percent to vary the harshness of permeabilization. Addition of PEG 20000 can also soften the harshness. We use PHEM buffer for the solutions. Concentration of PEG is 0-5%. Not all cells will be effectively permeabilized and a few cells will be completely trashed. However, the cells which are permeabilized show very good structural preservaion. The timing of permeabilization should be brief but will be determined by the cells you use. I.E we hit or miss each time out. Why do you think they call it magic? ;-)
I hope this is helpful-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
In message {199407122042.QAA15951-at-ahab.rutgers.edu} Alan Pooley writes: }
...... Note that some } cylindars of CO2 are contaiminated with water... this shows up by freezing in the output line of CO2 escaping to atmoshere, usually as start & stop flow, } sort of snorting and popping. Test with pure CO2 (ie no specimen in chamber) } and if its still snorts, replace the CO2 (about 1 cylindar in 10 or more } has had this.
Note: above discussion in context of using ethanol as transition fluid .
Alan,(sorry for late response)
I may be wrong, but I always thought that the snorting and popping of ices out the exhaust tube was either CO2 freezing up (dry ice) in the needle valve and exhaust line as it expands, cools, and is released to atmospheric pressure (the ol' Joule-Thompson effect), or an ethanol slush forming in the cooled CO2. Usually, after 4-5 successive rinse cycles to flush ethanol out of samples, the ices dissapear, so I've assumed it is ethanol freezing up. Question is, does the ethanol really cool enough during expansion out the valve to cool to its freezing point, which is about -117.3 C, (from CRC Handbook)?. In fact, my Ladd CPD unit has heaters surrounding the valves to prevent such ice-up (but I seldom use them because they tend to heat up the champer during the CO2 flushing procedures). So my theory is that due to the high cooling rate of the liquid CO2 due to its conversion to gas as it expands through the exhaust or drain valve from a pressure of 500 to 1000 lbs/in2 in the CPD chamber to atmospheric pressure some CO2 cools enough to form dry ice, or cools the mixed in ethanol enough to freeze out.
To test for water in the CO2, do a blank run with no sample in the chamber. Introduce liquid CO2 into the chamber at a typical working temperature of 5-10 degrees C. Vent it out the chamber's drain valve and trap or collect the "ices" formed in the exhaust. If it is dry ice, it should sublimate quickly leaving no liquid behind. If it is water ice, it should melt into liquid water which will not readily evaporate because it is cold after just melting (artefact possible during humid summer months: the cooled CO2 vapor will cool the lab bench, filter paper, or whatever you collect the exhaust ices and vapors on, and moisture can condense out of the air, misinterpreted as water in the CO2. Thus each new tank of CO2 can be tested to see if it contains any significant water. When I do this test, I usually get no ices out nor accumulating; just a cool spot on the hood wall, with sometimes a bare trace of moisture which I usually attribute to condensation out of the air on the cool spot.(or...water in my CO2 tank???)
Then do a test with some ethanol in the cooled chamber (3 squirts from a pipet, again no actual sample) , fill with liquid CO2, vent out and see what you get out. When I do this test, I get little icy patches, which when they melt, sure smell like ethanol.
My Ladd CPD unit uses CO2 expansion into the chamber (with vents wide open to create quick flow-through to set up a large pressure drop into the chamber) for cooling and I usually see liquid CO2 bouncing around in the chamber when I cool it in this manner, but not ices.
Keep in touch.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
JOURNAL OF MICROSCOPY - VOLUME 175 PART 2, AUGUST 1994
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 93-99
Quantitative analysis of the microstructure of the human cornea and sclera using 2-D Fourier methods
Shahram Vaezy & John I Clark, Department of Biological Structure SM-20, University of Washington, Seattle, WA 98195, USA,
SUMMARY
A two-dimensional (2-D) Fourier analysis was used to characterize the microstructure of the human cornea and sclera. The average centre-to- centre spacing of collagen fibrils was found to be 59nm for the cornea and 285nm for the sclera. These results agreed with those obtained by direct measurement using the electron micrographs, and those reported in the literature. The spatial order in the microstructure of the cornea was much greater when compared with that of the sclera. The results of the 2-D Fourier analysis were consistent with the theory of transparency of the eye. The 2-D Fourier analysis will be useful in quantitative characterization and analysis of the complex microstructure of biological cells and tissue in normal development and abnormal pathogenesis.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 100-107
A high sensitivity CCD system for parallel electron energy loss spectroscopy (CCD for EELS)
Zizhou Tang, Ruoya Ho, Zengli Xu, Zhifeng Shao & Andrew P Somlyo, Molecular Physiology & Biological Physics, University of Virginia, Box 449 Jordan Hall, Charlottesville, Virginia 22908, USA
SUMMARY
A cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy-loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal-to-noise ration and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorous L edge generated from a phosphoprotein, phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required at elemental mapping for a given sensitivity.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 108-120
Kinetics of intracellular Ca2+ concentration changes and cell contraction of electrically stimulated cardiomyocytes as analysed by automated digital-imaging microscopy
Hubert Schneider, Marc Fallert & Ernst Dieter Wachsmuth, K 125.507, CIBA-GEIGY Ltd, CH-4002, Basel, Switzerland,
SUMMARY
Enzymatically disaggregated, electrically stimulated cardiomyocytes from adult rats were examined by television-mediated vital microscopy for intracellular Ca2+concentration and contractile activity. Using an inverted microscope in the epifluorescence mode, the Ca2+ signal was imaged with a low-light-level CCD camera and traced by means of the intracellular concentration of the fluorescent complex of Ca2+ with its indicator Fluo-3. Using the transmitted-light mode, cardiomyocytes that were not loaded were imaged with a conventional CCD camera with automatic gain control and traced by length measurements. Optical images of at least 40 cardiomyocytes per batch of cells from one heart were recorded in up to 20 microscopic fields of observation on videotape within 20min. They were consecutively analysed by a personal computer installed with an image analysis card at a time resolution of 20ms, employing a discrete convolution operation, filtering and threshold setting for fluorescence measurements, and contour description and vectorial analysis for length measurements. Frames of fluorescent images were corrected for the halo effect caused by the increase in the Ca2+- dependent fluorescence signal after electrical stimulation. The cell contraction had to be measured in the transmission mode without Fluo-3 due to the inhibition caused by the intracellular Fluo-3. the following coefficients of variation (V) were determined: Vfluorescence { 0.033 and Vtransmission { 0.003 for the precision of measurement, and Vfluorescence { 0.05 and Vtransmission { 0.04 for the reproducibility. The system was validated with isoprenaline and ouabain as agents to modify the Ca2+-signal and the contraction. The response of the cardiomyocytes of various rats to electrical stimulation, with respect to amplitude and its time point, has a V { 0.08 for both the Ca2+ signal and the contraction.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 121-134
Reduced carrier single-sideband microscopy: a powerful method for the observation of transparent microscopical objects
F Bretschneider & P F M Teunis, Comparative Physiology Neuroethology Gp, University of Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands,
SUMMARY
The theoretical and practical properties of different forms of contrast formation in the microscope based on anaxial illumination are investigated: so-called single-sideband (SSB) techniques. The use of anaxial illumination in transmitted light microscopy is by itself a form of phase contrast (asymmetric illumination contrast, or AIC), but needs enhancement via a video circuit coupled to the microscope. The addition of a partially absorbing mask, known as a carrier attenuation filter (CAF), in a proper, conjugate plane in the microscope, improves contrast substantially. The imaging properties of this reduced-carrier, single-sideband imaging method (RC-SSB) were tested using the transparent parts of a compact disc (CD); the tracks may be treated as small objects with a controllable phase shift. the results were compared both theoretically and experimentally with Zernike's phase contrast and with Nomarski differential interference contrast. The SSB technique has been shown to reveal transparent, submicrometre parts of living unstained tissue, such as the microvilli on sensory receptor cells of the transparent catfish, Kryptopterus. The high resolving power, together with the variable spatial-frequency contrast enhancement, makes it a powerful technique for the imaging of in vivo subcellular details.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 135-142
Particle-surface interaction in thin vitrified films for cryo-electron microscopy
Marek Cyrklaff, Norbert Roos, Heinz Gross & Jacques Dubochet, European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012 Heidelberg, Germany,
SUMMARY
The concentration of particles in thin vitrified films of suspensions is described as a function of various parameters such as the type of particles observed, the time the sample is left on the grid and the effect of different washing procedures. The thin films are prepared for cryo- electron microscopy by the classical single-side blotting method or by blotting both sides of the grid simultaneously. The single-side blotting method results in particles preferentially absorbing to the non-blotted surface. This has the advantage that the concentration of particles in the thin vitrified film is higher than in the original suspension. The energy involved in adhesion of particles to the surface appears to be generally small. In most cases, it does not cause significant deformation of the particles or of the surface of the film. However, there are cases, for example with lipid vesicles, where the particles are broken as a result of absorption. Since particles remain absorbed to the air-liquid interface, it is possible to wash or dialyse the solution directly on the grid with negligible loss of particles. This represents a very rapid and handy method for micro-dialysis. A thin film is then formed by blotting the specimen and vitrified by rapid cooling.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 143-153
Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections
Alice Warley, Katherine P B Cracknell, Helen B Cammish, Charles H C Twort, Jeremy P T Ward & Stuart J Hirst, Sherrington School of Physiology, St Thomas' Hospital Medical School, Lambeth Palace Road, London, SE1 7EH, U.K.
SUMMARY
Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X-ray microanalysis studies are presented. The cells are grown on Pioloform-covered gold grids supported on Thermanox coverslips. This provides a growth-compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze- drying. The effects of different washing media (0.3M sucrose, 0.15M ammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled-water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 154-161
Fluorescence bleach rate imaging
G J Brakenhoff, K Visscher & E J Gijsbers, Electron Microscopy & Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands,
SUMMARY
Bleach rate imaging on a (cooled) CCD can be easily achieved using a confocal microscope with bilateral scanning and detection coupled to a workstation; it is as easy as acquiring regular fluorescence images. Several analysis and display methods for bleach rate imaging are presented such as the bleach map (and its inverse) and a matrix-based decomposition method for multi-labelled specimens based on the bleach rate differences between the dyes used. With these tools, bleach rate based imaging can become a viable alternative to multiple labelling techniques for component identification in fluorescent specimens.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 162-165
Fluorescence saturation in confocal microscopy
K Visscher, G J Brakenhoff & T D Visser, Electron Microscopy & Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands,
SUMMARY
The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approaches that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 166-170
Calcium alginate encapsulation of small specimens for transmission electron microscopy
A M Page, J R Lagnado, T W Ford & G Place, Department of Biology, Royal Holloway, University of London, Egham, Surrey, TW20 0EX
SUMMARY
A technique of encapsulating small objects in calcium alginate for further processing for transmission electron microscopy is described. Five methods are outlined which enable a variety of specimens, including single cells (in suspension and on agar plates), small organisms and monolayers of tissue culture cells to be processed. A method for immunolabelling alginate-entrapped material is also outlined.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 171-174
Sequential treatment by phosphotungstic acid and uranyl acetate enhances the adherence of lipid membranes and membrane proteins to hydrophobic EM grids
Alexander N Barnakov, Biochemistry Biophysics Department, Washington State University, Pullman WA 99164, U.S.A.
SUMMARY
A simple procedure for screening by electron microscopic observations of conditions for the reconstitution of membrane proteins into lipid bilayers is described. This procedure consists of a 5-10s treatment of electron microscopic grids, to which the sample has already been applied, with 1% phosphotungstic acid before proceeding with final staining in uranyl acetate. The method substantially enhances the adherence of lipid membranes and membrane protein particles to hydrophobic collodion/carbon grids
ANLEMC is dying..... I've managed to get it back up and running, however, expect that sometime in the next few days that this mail server will have a new home.
Please keep an eye on your mail for the announcement of a new host address. I will have to permanently move the microscopy mailing list so that the problems of the last few days do not repeat themselves.
Sorry for the headaches many of you have had in the last week, it wasn't fun for me either.
Nestor -------------
Nestor J. Zaluzec Argonne National Lab. Materials Science Division
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from anlemc.msd.anl.gov by jarrah.itd.adelaide.edu.au with SMTP (5.61+IDA+MU+NF/UA-5.28) id AA03609; Thu, 28 Jul 1994 00:16:15 +0930
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Nestor, I am sure you hear about anyone who has problems however I just wanted to send a note to thank you for putting in the time and energy to provide this service to the microscopy field. It is an invaluable network for all of us. Judy M _______________________________________________________________________________
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
A few weeks ago I volunteered to prepare a set of test images for round robin testing of grayscale printers for images. The images are now ready and accessible via ANONYMOUS FTP. But before you all rush off to grab them let me spell out a few details.
1.) Only grab the images now if you intend to print them and bring copies of your output to the computer workshop at the MSA meeting in New Orleans. I will leave the images posted on the site for those of you who wish to download them later, but let those who are definitely intending to participate at MSA have first crack.
2.) Download the README file in addition to the images! It contains important information as well as a form for you to fill out and bring to the workshop.
3.) The images are B&W TIFF files. There are 2 copies one at 100 dpi (~1 Mbyte) and one at 300 dpi (~10Mbytes). These are not small images and you should realize that they will take time to download and requires approximately the disk space I have indicated to store. Both images should be downloaded and printed. They are identical, however, they WILL stress the limit of your printers.
4.) Each of the image files is stored both in BINARY and BINHEX format choose your pleasure.
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Our lab has been making cross sections for TEM analysis utilizing the Anderson tripod polisher technique. The good news is that this technique seems to work, the bad news is I know of only one supplier for the abrassive diamond films (South Bay Technology). Not that there is anything wrong with South Bay, in fact they are always quite helpful when called on. But as the only supplier of these films that I know of, I am limited by their in stock inventory. Does anyone know of other suppliers for the diamond abrasive films?
----Mail status follows---- Have been unable to send your mail to {gillen-at-[129.6.98.22]} , will keep trying for a total of three days. At that time your mail will be returned.
----Transcript of message follows----
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Message-Id: {9407271840.AA02306-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: MICROSCOPY-at-ANLEMC.MSD.ANL.GOV Cc: dennis-at-odin.morph.med.umich.edu
} ...Does anyone know of other suppliers for the diamond abrasive films? } ... } John Phelps } NIST, Boulder
3M used to sell adhesive backed paper with alumina and diamond coatings of various grit sizes. I believe that the product was called 'Imperial Lapping Paper.' Check with your local rep.
} ...Does anyone know of other suppliers for the diamond abrasive films? } ... } John Phelps } NIST, Boulder
} 3M used to sell adhesive backed paper with alumina and diamond coatings } of various grit sizes. I believe that the product was called 'Imperial } Lapping Paper.' Check with your local rep.
If you do buy 3M, I've been told to make sure to get the Type A 3M lapping discs. The quality of other types are not as high (diamonds pull out of disc too easy) and who wants to ruin their samples.
A grad student in my lab needs to do some counting using a light microscope. A grid would be most helpful, but at present we don't have the bucks to shell out for a new occular. Are there other alternatives out there, such as a transparent film with a grid or some such that one can place inside an occular? Thanks for your help.
Gail Celio Botany and Plant Pathology Michigan State University
I hear that 3M company make diamond lapping films (down to 3 micron), large pieces, bulk packs, at low prices. Sorry, I don't have a name or #.
On Wed, 27 Jul 1994 PHELPS-at-ENH.NIST.GOV wrote:
} Hello, } } Our lab has been making cross sections for TEM analysis utilizing the } Anderson tripod polisher technique. The good news is that this technique } seems to work, the bad news is I know of only one supplier for the abrassive } diamond films (South Bay Technology). Not that there is anything wrong with } South Bay, in fact they are always quite helpful when called on. But as the } only supplier of these films that I know of, I am limited by their in stock } inventory. Does anyone know of other suppliers for the diamond abrasive films? } } thanks in advance, } John } } John Phelps } NIST, Boulder }
Try to trim block down as small as possible. Then soak in propylene-oxide (pop) to remove soft plastic, then re-infiltrate stepwise, 1:1 pop:resin, 1:2 pop:resin, 1:3, 100% resin 2changes, embed in a hard resin mix polymerize hot. This may work, but no promises, I've had mixed results. Maybe microwave polymerizing the block you have might work?
On Wed, 27 Jul 1994 dennis%odin-at-odin.morph.med.umich.edu wrote:
} I have embedded some samples of domestic cat spermatozoa in an incorrectly } mixed batch of Poly/Bed 812. The plastic came out excessively soft with no } possibility to be hardened further. } } I have tried one of the re-embedding techniques recommeded in Hayat's 1989 } book on EM technique using 100% EtOH saturated with KOH, but it destroyed the } fragile sample. } } Any recommendations on how these samples can be re-embedded safely? They are } difficult to obtain, and I only have a few with which to work. Any } suggestions are appreciated! } } Thanks, } } Dennis Shubitowski } dennis-at-odin.morph.med.umich.edu }
Gail Celio asks about using a homemade grid occular. I have made some experimental grids with a photocopier and transparency film. Cut to the right size this could easily be used for your purposes. Two notes of caution: you have to have the right kind of eyepiece. Some eyepieces have a lens in front of the image plane, and you can't get access to it. If you take the eyepiece off the microscope and look through it while shoving a cotton swab in the other end you should be able to get the end of the swab in focus before hitting the first lens if you can't you are out of luck. Second, the reticle has to be exactly in focus, meaning that the image from the objective is at focus in the plane of the reticle. This also makes dirt, etc on the reticle in focus.
If you are lucky enough to have an eyepiece that was specifically designed for holding a reticle there will be a nice ledge to push the reticle up against. If not you need to make a spacer which will take some adjustment, which which is what graduate students do well. The reticle can be held against the spacer or ledge with a spring clip. regards, Mark W. Lund MOXTEK, Inc Orem UT
Greetings! Ed Basgall responded to my resin storage question and asked me to forward his note to the group as he's been having problems getting notes posted. In a second note, he wrote
"Joe Mascorro at Tulane Univ, New Orleans, LA (EMSA august 1992) did a comparison of viscosities of variuos resin components. and found that mixtures using BDMA instead of DMP-30 had less than half the viscosity even after 60 minutes post mix. EMSA proceedings 1992, p 746."
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
---------- Forwarded message ----------
Please unsubscribe me from the Microscopy list Thank you Daniel Dagan
I forgot to tell you that I have managed to save all your subscriptions. You will *NOT* have to resubscribe. Just change your local nicknames/aliases....
Can anyone tell me what the status of the electron microscope company Camscan is? It would probably be best to respond to me directly, rather than post to the list.
regards Mark W. Lund MOXTEK, Inc. Orem UT lundm-at-xray.byu.edu
We are thinking of upgrading an existing TEM to allow acquistion of digital images that could then be ported directly to our image processing and analysis software. Does anyone out there have experience with digital cameras for TEM? I am interested in what models people are using and the quality of the image. Are the digitized images suitable only for morphometry or can they be used for publication prints. Thanks
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Gail- If your occulars are either non-focusing, or you are anxious about taking them apart, let me suggest a simple fix. Photograph a grid (like that of a bright-line hemocytometer) using 35mm color film. Use several maginifications to make grid images with different spacings. I like to give the image a color cast, usually light blue to correct for the yellowing of many illuminators at low power. The developed transparancy can be placed in the light path at any point which also has an image of the field-limiting diaphragm. I'd put it right on top of the exit of the diagphram of a Zeiss, for instance. Now focus your condensor so that the image of the grid is focused onto the object plane.You should be able to sector the field of view without much confusion. Hope this helps- let me know if there are any problems or questions Hal Krider Biotechnology Center Image Analysis Facility The University of Connecticut 203-486-4860
Does anyone know off the top of their head the electric field required for room temperature field emission from a standard tungsten gun and what type of force this exerts on the filament?
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: No Need to Resubscribe Orig-Author: {"Nestor J. Zaluzec-Argonne Nat. Lab." {ZALUZEC-at-AAEM.AMC.ANL.GOV} }:ddn:wpafb -----------------------------------------------------------
Sorry Folks...
I forgot to tell you that I have managed to save all your subscriptions. You will *NOT* have to resubscribe. Just change your local nicknames/aliases....
a bit more on resin storage - I follow basically the same procedure as Ed Basgall (mix up a batch, place aliquots in syringes for storage) one way to minimize leaks is to plug the "needle-end" with a toothpich or shaved applicator stick, leaving just enough sticking out to be able to grab it later, then replace the original plastic tip cap and parafilm - I almost never get leakage, but just to be on the safe side I store the syringes in Ziplock freezer bags
----Mail status follows---- Have been unable to send your mail to {gillen-at-[129.6.98.22]} for one day, will keep trying for another two days. At that time your mail will be returned.
----Transcript of message follows----
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Does anyone have information on the durability of images printed on the Fargo Primera printer, and is it similar in quality to the $7K Alden Electronics 9315CTP? I have had a demo print from the Alden printer near my window on the wall for a few weeks, and it is REALLY brown. Any way to fix thermal paper?
christoffersen-at-snmail.jsc.nasa.gov wrote: } Can someone suggest some entry points (books, papers, EMSA proceedings etc.) } that can update me on the state of the art in cathodoluminescence spectroscopy } applications in SEM/TEM? A book or article on current CL theory would also be } most helpful. Thanks.
Try:
1. AUTHOR: Yacobi, B. G. TITLE: Cathodoluminescence microscopy of inorganic solids / B.G. Yacobi and D.B. Holt. PUBLICATION: New York : Plenum Press, c1990. DESCRIPTION: ix, 292 p. : ill. ; 24 cm.
or
2. AUTHOR: Marshall, Donald J. TITLE: Cathodoluminescence of geological materials : an introduction / D. J. Marshall, with a chapter contributed by Anthony N. Mariano. PUBLICATION: Boston : Allen & Unwin, 1988. DESCRIPTION: xiv, 146 p., {12} p. of plates : ill. ; 29 cm.
Hope this helps Roy
Ed Vicenzi tel (609) 258-1464 office Princeton University tel (609) 258-1406 lab Princeton Materials Inst. fax (609) 258-6878 70 Prospect Ave. Princeton, N.J. 08540-5211 email: vicenzi-at-phoenix.princeton.edu
A student was staining some arthropod antennae with methylene blue for a project. One day she couldn't find the stain, and so arbitrarily picked another off the shelf (which contained a bunch of really antique bottles). It was azocarmine and, fortunately, worked unbelievably well in staining the structures we were looking for (aesthetascs) a nice pink! Unfortunately, we don't know why. I can't find anything about azocarmine itself. It sounds like carmine will stain glycogen and mucous, but that doesn't explain why it did this type of chemoreceptor so well, and did not stain the other types at all. Does anyone have any clues? I'm an electron microscopist, so anyting that happens in color is beyond me!
Aloha, Tina Weatherby Carvalho Biological EM Facility University of Hawaii
Franklin RJM, Barnett SC, 1991. The electron microscopic appearance of the beta-galactosidase reaction product. Acta Neuropathol 81:686-687.
Engelhardt JF, Allen ED, Wilson JM, 1991. Reconstitution of tracheal grafts with a genetically modified epithelium. Proc Natl Acad Sci USA 88:11192-11196.
Bob Cardell, of the Department of Anatomy and Cell Biology at Univ of Cincinnati Med School has also published on this, but I can't find the reference.
Regards. A. Kent Christensen, Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor {akc-at-umich.edu}
--------------------------------
On Thu, 28 Jul 1994, Nestor J. Zaluzec-Argonne Nat. Lab. wrote:
} From: SMTP%"delannoy-at-welchlink.welch.jhu.edu" 25-JUL-1994 13:13:25.58 } To: MICROSCOPY } CC: } Subj: b-gal react. prod. EM } } Date: Mon, 25 Jul 94 14:13:53 EDT } From: delannoy-at-welchlink.welch.jhu.edu (MICHAEL DELANNOY ) } Message-Id: {9407251813.AA01300-at-welchlink.welch.jhu.edu.bubba} } To: microscopy-at-anlemc.msd.anl.gov } Subject: b-gal react. prod. EM } Cc: delannoy-at-welchlink.welch.jhu.edu } Mime-Version: 1.0 } Content-Type: text/plain; charset=us-ascii } Content-Length: 161 } } Does anyone have any experience detecting the beta galactosidase } reaction product at the EM level? } } Please respond to mwilson-at-welchlink.welch.jhu.edu. Thanks! }
1) It appears that the problems with the LISTSERVER have not been entirely sorted out, as I continue to recieve quite a lot of it at the moment. I don't know if this is a throw-back from the prevoius problems or something new.
2) Does anyone have any experience of stage control on optical microscopes linked to image analysis systems. We intend to purchase or develope a system to analyse cracks in material surfaces over an approximately 10x10mm area on a light microscope. This requires very accurate (preferably sub micron) stage positioning to link sets of video shots together into one large file. We had considered using ether an off-the-shelf system or building one based in NIH-Image. Any commentsor recommendations would be of help.
Thanks.
Dr Doug Arrell Mechanical Performance and Joining Institute for Advanced Materials 1755 ZG Petten Netherlands
As Diamond Lapping film seems to be the hot topic of the day, I thought it might be appropriate for me to give you all an update on the lapping film products from South Bay Technology.
It is important to note that the SBT films are not 3M films and that they are made through a different process. SBT films are considered to be a "closed coat" product whereas the 3M films are considered an "open coat" product. The differences between the products do not make one better or worse than the other - just different. If you have used a South Bay film and switch to a 3M, then you will notice a difference in the performance. Each type of film has a place in tripod polishing and in many other applications. On some materials a closed coat type film may work better than an open type film and vice versa.
To meet the varied needs of our customers, South Bay Technology plans to introduce a new line of "open coat" films that are designed as a direct replacement to 3M films (and at a much more reasonable price). We will continue to offer the "closed coat" film as we have many customers who are very pleased with this product and are reluctant to change. The new "open coat" product is not due out for several months. I normally would not make such a premature announcement, but the volume of diamond film postings here forced my hand. We currently have several beta sites lined up for testing of this film, but we would welcome requests from other people who would be interested in testing out the film as well.
I have seen several postings on this list server referring to the price and availability of these films. These films are expensive and when you need them - you need them. I'm the same as many of you - "if I needed the film tomorrow, I'd order it tomorrow". There are ways to minimize your cost and ensure that the film is available when you need it. At SBT we offer quantity discounts for as few as 25 pieces of film (assorted sizes). You can save money by prchasing a reasonable supply of film at one time rather than buying 2 or 3 pieces every 2 weeks. If it is not possible or desirable for you to stock the film yourelf, you can enter into a blanket or open order agreement.
A blanket order is an order for a specified group of items with a scheduled delivery. For example, you may want a total of 15 discs per month in 3 different sizes. While 15 discs doesn't qualify for a quantity discount, 15 discs x 12 months would qualify for a sizable discount if a blanket order was arranged. A blanket order offers the highest discounts as it helps us to plan our inventory more precisely. Also, with a blanket order, we maintain "reserved stock" just for you. This means that we always have the product available to ship - even if you decide to that you need it 3 weeks earlier than planned.
An open order is an order placed for a specific dollar amount, but without specific products or deliveries. You simply guarantee that you will spend x number of dollars within a specified period of time.
You may also want to consider purchasing all of your sample preparation supplies together. You can combine supplies from your met lab etc. to increase the size of your blanket or open order and therby increase your discount. As we do offer every type of sample prep supply, you can include much more than just your diamond films.
I realize that this has turned into a bit of an advertisement, but I felt that I had to say something to clarify the Diamond Film Debate. The concerns people seemed to have about diamond lapping films are those of price, performance and delivery. I hope I have provided some insight on these topics and made some valid sugestions that will enable you to save money and frustration. We are a small, family owned business that has been around for over 30 years. We've lasted that long by working very hard to offer our customers superior products and service at very reasonable prices. We take great pride in what we do and always strive to meet our goal of absolute customer satisfaction. We are always interested in hearing input from our customers (and those of you we haven't gotten to yet!). If any of you plan on being in New Orleans for the MSA/MAS Meeting, please stop by our booth (No. 534) and say hello. I'll be there myself and will look forward to meeting you. I encourage anyone who has any questions or comments to contact me directly as follows:
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
Mr-Received: by mta PETVAX.MUAS; Relayed; Fri, 29 Jul 1994 07:25:47 -0400 Mr-Received: by mta PETVAX; Relayed; Fri, 29 Jul 1994 07:25:47 -0400 Mr-Received: by mta SRVR01; Relayed; Fri, 29 Jul 1994 07:25:23 -0400 Disclose-Recipients: prohibited
Does anyone have experience embedding cell cultures plated out on your typical plastic multi-well plate (Falcon, Corning) using LR White as an embedding media? A 24 Hr Heat cure at 50C causes the plate to dissolve. We need to avoid catalytic cure for immunocytochemistry.
Walt Bobrowski Parke-Davis Research Ann Arbor, MI 48105
To all microscopists on the listserver: since there have been listserver problems over the last few weeks, I thought it would be useful to re-post this message, in case the original did not get through.
The following is an announcement for the INTERNATIONAL WORKSHOP ON ELECTRON HOLOGRAPHY. A circular for this meeting has recently been mailed to a selected mailing list, but the listserver may reach a number of people who may be interested and who were not included on our original list. I welcome all responses by e-mail (privately, of course). The workshop has an outstanding list of speakers, and, because of large private sponsorshop, will be very inexpensive for participants (in other words, a cheap ticket). Don't hesitate to reserve your spot; the attendance will be limited because of space limitations.
INTERNATIONAL WORKSHOP ON ELECTRON HOLOGRAPHY - Theory, Applications and Future Prospects -
August 29 -31, 1994 Holiday Inn World's Fair Knoxville, Tennessee, USA
Organized by
Exploratory Research for Advanced Technology (ERATO), Research Development Corporation of Japan (JRDC) Oak Ridge National Laboratory (ORNL) University of Bologna University of Tennessee
Purpose: This workshop offers an international forum for experts from all over the world to discuss recent developments in the techniques, theory and application of electron holography in materials and biological science research.
Organizing and Program Committee
A. Tonomura ......................................ERATO, JRDC and Hitachi Advanced Research Laboratory (HARL) L.F. Allard .....................................................Oak Ridge National Laboratory T.A. Nolan ......................................................Oak Ridge National Laboratory G. Pozzi ............................................................................University of Bologna D. C. Joy ......................................................................University of Tennessee Y. A. Ono ..........................................................................ERATO, JRDC and HARL
REGISTRATION: Free of charge (includes extended abstracts and hard-cover proceedings, to be published by Elsevier) ACTIVITY FEE: $100 (partial support for Sunday reception, 3 continental breakfasts, 2 buffet lunches, workshop banquet, box lunch and transportation to ORNL on Wednesday.) PROGRAM: The workshop will be comprised of technical sessions held over two and one-half days, with a tour of microscopy facilities and demonstrations of holography at ORNL scheduled for Wednesday afternoon. The workshop banquet, to be held at the Knoxville Museum of Art, will be highlighted by an after-dinner talk by T. Mulvey on Gabor and the early history of holography. The museum will be available entire evening for the workshop attendees. OFFICIAL LANGUAGE: English
Invited Speakers and Topics:
G. Ade (PTB, Braunschweig) .........................Digital recording and processing L. Allard (ORNL) .................................................................Holography of fullerenes J. Bonevich (NCEM,LBL) ............Observation of vortices in superconductors J. Chen (ERATO, JRDC) ........................................Real-time electron holography A. Datye (Univ. New Mexico)............................................Holography of catalysts V. Dravid (Northwestern Univ.) .......................Holography of electroceramics H. Fink (IBM Zurich) .......................................Electron point source microscopy B. Frost (ORNL)................................................Holography of electrostatic fields R. Herring (ERATO, JRDC) ...............................Diffracted beam interferometry T. Hirayama (ERATO, JRDC) ...........................Holography of magnetic domains K. Ishizuka (ERATO, JRDC) ........................Tilted single side-band holography D. Joy (Univ. Tennessee) ..................................New fields and future prospects G. Lai (ERATO, JRDC) .....................................Holographic computed tomography M. Lehmann (Univ. Tubingen) .................Holographic reconstruction methods H. Lichte (Univ. Tubingen) ........................High resolution electron holography M. Mankos (ASU) ..................................................................................STEM holography G. Matteucci (Univ. Bologna) ..............................Holography of magnetic fields M. McCartney (ASU) ....................................................Holography of p/n junctions T. Mulvey (Univ. Aston) ............................Early history of electron holography M. Op de Beek (Univ. Antwerp)..........................................Through-focus methods Q. Ru (ERATO, JRDC) .............................................Phase-shifting interferometry J. Spence (ASU) ......................................Projection microscopy and holography J. Steeds (Univ. Bristol) ..............................................Coherent beam diffraction T. Tanji (ERATO, JRDC) ...........................Atomic surface potential holography A. Tonomura (ERATO and HARL) ..................................ERATO and HARL research D. van Dyck (Univ. Antwerp) ....................Reconstructed image interpretation E. Volkl (ORNL) .....................................................Digital processing of holograms
Posters(tentative) K. Aoyama (ERATO, JRDC)................Holography of Thin biological filaments A. Carim (Penn State Univ.)...................................Holography of fine particles D. Joy (Univ. Tennessee) ........................Holography of ferroelectric domains T. Matsumoto (ERATO, JRDC).................Frozen-hydrated DNA super-helices Q. Ru (ERATO, JRDC) ..........................................Incoherent electron holography E. Volkl (ORNL)................................................................Extended Fourier analysis
Contributed Poster Papers
We have room for a few additional contributed papers which will be accepted as posters. Contributed papers will also be eligible for publication in the hardcover proceedings. Please e-mail your interest in submitting a poster to allardlfjr-at-ornl.gov and we will fax a circular with instructions to you. Any other queries about the workshop are also welcome.
Hi, I'm sectioning Spurr embedded (hard formulation) leaf tissue on an Ultracut using Diatech diamond knives (two different ones) and I've been encountering what appears to be a static problem. Every other section crumples and wrinkles badly. As the block approaches the knife the water in the boat can actually be seen to bulge or lift upward toward the descending block face. When the block touches the edge of the knife (water bulge?) the water attaction is instantly released, but the section appears almost to be cut almost under the surface. The section has water on top of it. The block face is quite small, { 0.5mm in both height and width; the edges are clean and smooth, the piece is oriented with its long axis perpendicular to the knife edge. I have tried adjusting: the speed of the cutting stroke, the clearance angle, the level of water in the boat, and have used two different knives. The instrument was recently serviced by a competent person and I have no reason to believe that the microtome is malfunctioning. I want to emphasize that this occurs regularly, every other section. I also tried cutting sections of different thickness, but the problem remained. I have tried touching a damp filter paper wedge to the block (not the face) during the return stroke and that seems to have a positive effect, but it is very awkward to do.
Any experience with a phenomenon like this? Any likely solution?
Thanks in advance!
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
this message repeats an announcement mailed during the recent period of problems with the host computer
OPENING FOR ELECTRON OPTICS FACILITY ENGINEER The Department of Metallurgical and Materials Engineering at Michigan Technological University has an opening for an engineer who will be responsible for operation, maintenance, repair, and supervision of microanalytical instruments in its Electron Optics Facility. This facility includes several scanning electron microscopes, transmission electron microscopes, and an electron microprobe. Successful candidates will have training and experience in operation, maintenance and repair of similar instruments in a research setting. Duties may include collaboration with faculty and graduate students in preparation of research proposals, development of new microanalytical methods and publication of research. Interested applicants should send a resume , including names and addresses of three professional references, to the following address:
Chair, Electron Optics Facility Engineer Search Committee Department of Metallurgical and Materials Engineering Michigan Technological University 1400 Townsend Drive Houghton, MI 49931-1295
Salary range for this position is $34,261-$54,818/yr, starting salary will depend on experience and qualifications. The search committee will begin reviewing applications on August 1, 1994. Applications will be accepted until the position is filled.
Michigan Technological University is an equal opportunity employer/educational institution and welcomes applications from all qualified applicants.
It sounds like you should spit in your boat! Really, the surface tension of the water can be reduced by a tiny bit of saliva. I keep toothpicks in alcohol, and before using them put them in my mouth. That little bit of saliva when you use the stick to push the water to the edge of the knife is enough to make a difference. The only problem this might cause is epithelial cells which will be visible in your thick sections. Also be careful about lipstick, but from your name I would as same that probably shouldn't be a problem. If you don't like that idea, you can use Photoflo, diluted by about 1000 I think-anyway enough so you don't get soapsuds in your boat. Also-don't fill your boat too full. Just enough so you can push the water up to meet the section. Good luck. Joyce.
----Mail status follows---- Have been unable to send your mail to {gillen-at-[129.6.98.22]} for two days, will keep trying for another 24 hours. At that time your mail will be returned.
----Transcript of message follows----
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
} A student was staining some arthropod antennae with methylene blue for a } project. One day she couldn't find the stain, and so arbitrarily picked } another off the shelf (which contained a bunch of really antique } bottles). It was azocarmine and, fortunately, worked unbelievably well in } staining the structures we were looking for (aesthetascs) a nice pink! } Unfortunately, we don't know why. I can't find anything about } azocarmine itself. It sounds like carmine will stain glycogen and } mucous, but that doesn't explain why it did this type of chemoreceptor so } well, and did not stain the other types at all. Does anyone have any } clues? I'm an electron microscopist, so anyting that happens in color is } beyond me! } } Aloha, } Tina Weatherby Carvalho } Biological EM Facility } University of Hawaii
Thank God for ignorant students. There are two types of azocaramine (B & G). Gomori 1939 Anat. Rec. 74:439 & 1941 Am. J. Path. 17:395 used azocarmaine G and orange G to stain alpha, beta, and D cells in Islets of Langerhans. Mollier 1938 Z. Wissen. Mikr. 55:472 (in German) used azocaramine G in a quadruple stain for elastic tissue, muscle, collagen and epithelium. Lille 1965 Histopathologic Technic and Practical Histochemistry (McGraw Hill 3 ed) reports a use of azocaramine B, orange G and aniline blue to stain muscle, glia fibrils, collagen erythroctyes and nuclei. good luck.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Could anyone tell me how to mount grains of sodalite for analysis in an AFM? Or point me to some work in this field?
Also, does anyone know the, or a source of, the atomic structure of H-ZSM5, in coordinates ( For an EMS file.) I have some references with angles and lengths etc but not enough to be able to calculate the coordinates.
Subject: Time:8:38 AM OFFICE MEMO Re} Round -robin Images Date:7/29/94
I was not clear in my earlier message. I know where the Images are posted, but need to know where and when the images will be compared at the meeting in New Orleans. Thanks for your help.
Mike Mike_Schwartz-at-qm.yale.edu Fax 203-785-5263
Michael Schwartz, Ph.D. Section of Neurobiology Yale University School of Medicine 333 Cedar St. New Haven, CT 06510
} Does anyone know off the top of their head the electric field required } for room temperature field emission from a standard tungsten gun and what } type of force this exerts on the filament? } } Daniel L. Callahan } Department of Mechanical Engg. and Materials Science } Rice University } dlc-at-owlnet.rice.edu
Assuming the following 1) a fixed angular intensity of 0.1 mA/sr 2) radius of tip = 0.1microns 3) T = 300K 4) W {310} work fct 4.5eV
The field on the tip is ~5 V/nm
A good reference is: "Point Cathodes for Use in Virtual Electron Optics," Tuggle D.W. Journal of Microscopy, Vol 140, December 1985.
For anyone who is interested, FEI has an extensive list of technical articles relating to thermal and field emission, which is available upon request.
Best regards, Damon L. Heer
FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830
----Reason for mail failure follows---- Sending mail to {gillen-at-[129.6.98.22]} : Could not be delivered for three days.
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Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
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----- Unsent message follows ----- Received: from cuchulain by diana.met.bham.ac.uk; Wed, 27 Jul 1994 16:30:22 +0100 Received: from bham.ac.uk by met.bham.ac.uk; Wed, 27 Jul 1994 15:30:26 +0100 Received: from anlemc.msd.anl.gov by bham.ac.uk with SMTP (PP) id {26371-0-at-bham.ac.uk} ; Wed, 27 Jul 1994 15:30:12 +0100
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Dear Microscopists, I am helping a student set up a cryofixation (plunge or slam on a KF80) and cryosubstitution project on blades of rye grass. She hopes to immunolabel for CI proteins in the blade. This protein is a Poty virus-induced protein. Our problem is that we can't find much published material suggesting the best substitution medium, best fixative and recommended Lowicryl to use. There doesn't seem to be much botanical cryofixation/crysubstitution literature out there. Can anyone offer some suggestions?
Allan Mitchell Technical Officer South Campus EM Unit Dept of Anantomy and Structural Biology Otago Medical School NZ
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
} Dear Microscopists, } I am helping a student set up a cryofixation (plunge or slam on a KF80) and } cryosubstitution project on blades of rye grass. She hopes to immunolabel } for CI proteins in the blade....There doesn't seem to be much botanical } cryofixation/crysubstitution literature out there. } Can anyone offer some suggestions? } ... As a starting point, try:
Zhang G.F. and L.A. Staehelin. 1992. Functional compartmentation of the golgi-apparatus of plant-cells - immunocytochemical analysis of high-pressure frozen-substituted and freeze-substituted sycamore maple suspension-culture cells. Plant Physiology 99:1070-1083.
Hippe-Sanwald, S. 1993. Impact of freeze substitution on biological electron-microscopy. Microscopy Research and Technique 24:400-422.
Galway M.E. 1993. Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozen freeze-substituted protoplasts of white spruce(picea-glauca) Journal of Cell Science 106:847-858.
} Dear Microscopists, } I am helping a student set up a cryofixation (plunge or slam on a KF80) } and cryosubstitution project on blades of rye grass. She hopes to } immunolabel for CI proteins in the blade. This protein is a Poty } virus-induced protein. Our problem is that we can't find much published } material suggesting the best substitution medium, best fixative and } recommended Lowicryl to use. There doesn't seem to be much botanical } cryofixation/crysubstitution literature out there. } Can anyone offer some suggestions? } } Allan Mitchell } Technical Officer } South Campus EM Unit } Dept of Anantomy and Structural Biology } Otago Medical School } NZ } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 } ====== Here are a few references:
M.K. Kandasamy, M.V. Parthasarathy and M.E. Nasrallah, 1991. High Pressure Freezing and Freeze-substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae of Brassica. Protoplasma 162: 187-191
Biao Ding, J.S. Haudenshield, R.J. Hull, S. Wolf, R.N. Beachy anf W.J. Lucas, 1992. Secondary Plasmodesmata are Specific Sites of Localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. The Plant Cell 4: 915-928.
Some references for Lowicryl embedding of cryofixed plant material:
Document 1 TI IMMUNOCYTOCHEMICAL LOCALIZATION OF NUCLEAR ANTIGENS IN THE UNICELLULAR GREEN ALGA CHLAMYDOMONAS-REINHARDTII PROCESSED BY CRYOFIXATION AND FREEZE-SUBSTITUTION AU TREMBLAY-S-D. LAFONTAINE-J-G. SO PROTOPLASMA 165 (1-3). 1991. 189-202.
Document 2 TI FLAVONAL RING B-SPECIFIC O GLUCOSYLTRANSFERASES PURIFICATION PRODUCTION OF POLYCLONAL ANTIBODIES AND IMMUNOLOCALIZATION AU LATCHINIAN-SADEK-L. IBRAHIM-R-K. SO ARCH BIOCHEM BIOPHYS 289 (2). 1991. 230-236. Document 3 TI IMMUNOCYTOCHEMICAL DEMONSTRATION OF THE PEROXISOMAL ATPASE OF YEASTS AU DUMAS-E. LHERMINIER-J. GIANINAZZI-S. WHITE-R-F. ANTONIW-J-F. SO J GEN VIROL 69 (10). 1988. 2687-2694. (This article also has localization in plant cells) M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu
Two who have done good work on cryosubstitution with plants are Rick Howard at DuPont (howardrj-at-esvax.dnet.dupont.com) or Howard Berg at Memphis State U. (Bergrh-at-msuvx1.memphis.edu) ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
You asked about azocarmine. I have not found a specific reference to azocarmine, however, the azo dyes are very common. Examples of azo dyes are congo red, CI Fast Red, CI resorcine dark brown, and CI naphthol blue black B. The general reaction is:
ArN2+X "+" HR to ArN=NR "+" HX
I do not know for sure, but carmine is an old art color for red. I would bet you have an old jar of something similar to Congo Red. If you want to read more about the way azo dyes work, there is a very good section in Kirk Othmer's Encyclopeadia of Chemical Technology, 3rd ed. Vol 3, Vol 3, "Azo Dyes".
Melanie Thom, Sr. Chemist AlliedSignal Aerospace South Bend, IN 219-231-2856
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G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
The program which best meets your criteria for area and perimeter measurement and cost is NIH Image. You will be pleasantly surprised at its capability and reliability. According to the manual, it is available from:
1) From a friend. The Image program, including source code and documentation, is public domain and may be freely copied, distributed and modified. However, if you modify Image, please update the about box before distributing your version of the program.
2) Via anonymous FTP from zippy.nimh.nih.gov[128.231.98.32]. Enter "anonymous" as the user name and your e-mail address as the password. The /pub/nih-image directory contains the latest version of Image (nih- image154_fpu.hqx or nih-image154_nonfpu.hqx), documentation in Word format (nih- image154_docs.hqx), and complete Think Pascal source code (nih- image154_source.hqx). The directory /pub/image/images contains sample TIFF and PICT images. The directory /pub/image/image_spinoffs contains versions of Image extended to do FFTs (ImageFFT), fractal analysis (ImageFractal), and to support quantitative evaluation of cerebral blood flow, glucose metabolism, and protein synthesis (Image/MG). There is a README file (0README.txt) with information on the file formats used.
3) Library 9 (Graphics Tools) of the MACAPP forum on CompuServe. Source code is in Library 6 of the MACDEV forum.
4) Twilight Clone BBS in Silver Spring, MD. The Clone has 16 lines on sequential rollover, starting with 301-946-8677. To guarantee a V.32 connection, call 946-5034. Image is currently available at no charge from the Twilight Clone.
5) Subscribe to the NIH Image mailing list by sending a message containing the line "subscribe nih-image {your name} " to listserv-at-soils.umn.edu. Next obtain a list of the available NIH Image archive files by sending an "index nih- image" command to listserv-at-soils.umn.edu. These files can then be retrieved by means of a "get nih-image filename" command. The files are Binhexed and broken into chunks less than 32K in size. The NIH mailing is maintained by the Soil Science Department at the University of Minnesota.
6) NTIS (National Technical Information Service), 5285 Port Royal Road, Springfield, VA 22161, phone 703-487-4650, order number PB93-504868 ($100 check, VISA, or Mastercard). Both the zippy.nimh.nih.gov FTP site and the Twilight Clone BBS are likely to have newer versions of Image than NTIS.
Azocarmine, despite what the name suggests is not an azo dye but a member of the quinone-imine group (azin subgroup). Azocarmine G is a sodium salt of a disulfonic acid of phenylrosinduline and has a C.I. number of 50085. There is a related dye, azocarmine B which is a trisulfonate (C.I. 50090).
I have been able to find little on the use of the dye as a stain apart from it being used as an alternative to acid fuchsin in Mallory's connective tissue stain and a couple of general purpose stains.
Most of my information comes from a volume entitled "Biological Stains" by H.J. Conn 7th edition 1961 which is a good guide to a wide range of dyes used for biological staining and was prepared in association with the Biological Stain Commission. I assume there are later editions out there somewhere but this is the latest in our library.
Hope this is of some help
Ian Hallett HortResearch Mt Albert Research Centre Auckland New Zealand
Dear microscopists, In June I posted a note asking if anyone knew of an alternative source of targets for Biorad sputters coaters.I had had problems getting targets within a reasonable period of time through Fisons (the last one took six weeks to come). This is not a problem unique to us judging by the response I got on this listserver. David Henriks suggested Testbourne Ltd at Unit 12, Hassocks Wood, Stroudley Road, Basingstoke, Hampshire RG24 0NE, England. I followed this up and they are now able to supply these annular targets (Au or Au/Pd) at price very competitive to Fisons. They have also given us a discount for the little remaining target material on our used targets which we sent them, if you are like us you will have lots of old targets so its good to find a home for them.
Needless to say I have not the slightest commercial interest in Testbourne Ltd.
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
RE: Cryofixation/cryosubstitution of plant material
Many thanks to all who responded to my request. The response has been fantastic. I will report on our efforts as soon as we have got our technique to work.
Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
We have recently started to become more aware of the purity of our glutaraldehyde however a brief excursion into this form of quality control has left me confused. We have analysed four different supplies of 25% glutaraldehyde with the following results: (all stock solutions were kept in the refrigerator at 4deg C)
Stock A: received into the Unit Jult 1993 (500ml bottle of EM grade). Spectrophotometric result:high 235nm absorbance peak with resulting P/M ratio of 3.18 pH 5.16 osmol of 1% solution 119 mosmol/kg
Stock B: received into the Unit May 1994 (500ml bottle of EM grade). Spectrophotometric result:high 235nm absorbance peak with resulting P/M ratio of 3.08 pH 5.24 osmol of 1% solution 127 mosmol/kg
Note: stocks A and B are from the same supplier.
Stock C: received inot the Unit June 1994 (ampule of 25% under nitrogen, 10mls). Spectrophotometric result:low 235nm absorbance peak with resulting P/M ratio of 0.22 pH 3.31 osmol of 1% solution 103 mosmol/kg
Stock D: received into the Unit June 1994 (100 ml bottle) Spectrophotometric result:low 235nm absorbance peak with resulting P/M ratio of 0.18 pH 3.66 osmol of 1% solution 108 mosmol/kg
The P/M ratio is the ratio of polymer ('impurities', absorbance peak at 235nm) to monomer (good stuff, absorbance peak at 280nm).
I am confused because in the literature some say that glutaraldehyde with a baseline P/M ration of 0.2 or more should be rejected (so our stocks A and B should be thrown out). However others report that if the pH drops below 3.5 then it should be discarded.
In our test the two stock solutions with the best P/M ratios have the worst pH readings and vice versa. Do I throw the whole lot out and go fishing?
Brenda Weakley (J of Microscopy, Vol 101, July 1974 p127) reports;1)Low pH of the stock solution is the most detrimental factor in fixation and 2) a high 235nm/280nm ratio gives poor fixation.
I am going to run an experimental trial with these fixatives on tissue however I am interested to hear others' thoughts and opinions on this topic.
Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
A friend in one of the clinical sciences has a tissue section he wants to determine the Pb levels in. Does any one know of a commercial operation that could give him some help?
Thanks.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
I am trying to get my own copy of Electron Microscopy of Thin Crystals by Hirsch et al.(1977 edition) but the book shops I have tried so far have not been very optimistic. Does anyone know of a (specifically) scientific second hand/out of print book shop in the UK, USA or even here in Canada which could help me?
Thanks Ciara Mullan CEMD McMaster University 1280 Main St West Hamilton Ontario L8S 4L7
We are in process of getting a new autoproessor for our EM film.
Currently we have a Labtronix Autoprocessor 1400 which we had purchased from Energy Beam Sciences Inc. 7-8 years ago, and are unhappy with it due to numerous problems and down time.
Are there any out there that are totally automatic in cycle and are made out of stainless steel or some sort of metal?
Any suggestions welcomed.
Raj
Raj-at-Bioimg.umdnj.edu Robert Wood Johnson Medical School Dept. of Pathology (908) 235-4648
Hi, I have been fixing microspheres (100um diameter average) made up of polysaccharides for SEM and I have encountered deformation particularly in the critical point apparatus. To overcome this I put the spheres in a small plastic cylinder that has a removable mesh cover at both ends, so the liquids can flow in and out of it without problems. This small device fits nicely in our polaron critical point dryer and you can purchase them at: SPI supplies PO BOX 656 569 EAST GAY STREET, WEST CHESTER, PA, 19381-0656, USA FAX 1 215 436 5755 PHONE 1 215 436 5400 PRODUCT: MICROPOROUS SPECIMEN CAPSULES # 13215
PELCO INTERNATIONAL PO BOX 492477, REDDING, CA, 96049-2477, USA PHONE 1 916 243 2200, FAX 1 916 243 3761 PRODUCT:CAPILLARY TUBE TISSUE PROCESSING #102, # 6GN200 MICRO TISSUE CAPSULE ASSEMBLY #1025...
Ciara Mullan at Mc Master University was looking for a copy of ELECTRON MICROSCOPY OF THIN CRYSTALS BY HIRSCH ET AL. I have found a number of good books at this location: (I am not affiliated in any way) This place has many books on Electron Microscopes, antique scientific instruments, primarily microscopy. The address is: RThe GemmaryS P.O. Box 816 Redondo Beach CA. 90277 310-372-5969 Hope they have it. Larry Albright
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name
The computer workshop & software exchange went well again this year. We had 10 computer (6 Macs and 4 PC's networked together and available for use from ~ Noon Monday through ~ Noon Friday. 516 people registered at the door and had access to over 500 Mbytes of software which was distributed via the EMMPDL, MASLib and Freeware/Shareware libraries maintained by John Mansfield (U. of Mich) and myself.
Registrants, in addition to bringing their own disks, purchased the entire 500 disk supply brought by students of the American Nuclear Society who were selling them at the workshop for the benefit of participants.
5 New things that occured this year:
1.) Announcement of Anonymous FTP sites for accessing the Software Libraries.
www.amc.anl.gov (ANL) freebie.engin.umich.edu (U of Mich)
2.) Announcment of a World Wide Web Sites for Microscopy & Microanalysis with Society Information.
http://www.amc.anl.gov (ANL) http://www.engin.umich.edu/~jfmjfm/mas_folder/mashomepage.html (U of Mich)
3.) On line computer tutorials on Image processing and telecommunications. These were video taped and will be avaiable through the MSA video tape library at the MSA Business office.
4.) On line Internet connections via a PPP phone link through Tulane University.
5.) Round Robin Grayscale Printer Test.
As proposed by J. Chandler & S. McKernan, we ran a round robin grayscale printer test this year. Two test images were placed on an FTP site (AAEM.AMC.ANL.GOV) and were downloaded by 14 individuals and printed on 15 different printers (with some duplicates).
Thanks are due to the following individuals & 3 commerical firms (in semi-random order) for spending their time and effort to make this round robin test a success.
G. Nord - USGS C. Wood - Dow S. McKernan - Univ. of Minn. J. Neilly - Abbott Labs. M. Bisher - NEC M. Disko - Exxon P. Melvile - ? R. Hermann - ETH Zurich J. Chandler - Colorado State Univ. J. Mansfield - Univ. of Mich N. Zaluzec - ANL Kodak Condonics Polaroid
The following printers were represented:
Laser & InkJet
Apple Laserwriter NTX Apple Laserwriter Pro 630 QMS 815 MR HP LaserJet 4ML HP DeskJet 1200C/PS LaserMaster Unity 1200 XL
These printers ranged from conventional Laser printers & Ink Jets to full blead Dye Sublimination printers or equivalent.
Some pronounced differences were apparent especially in cases where 2 prints from the same printer were contributed from different places but with strikingly different results. The conclusion was that the differences were attributed to the printer drivers used in the computer, rather than to the printers themselves.
We did not conduct a survey of the opinions of the people who inspected the results, however, it was clear (to me) that as expected not all the printers were equivalent. Most of the Laser printers showed a clear dithering pattern. The dye sublimination printers generally showed good tonal response, but some had definite tendancies to show a slight off-color response. For example, some had a slight bluish to green tinge, others did not have good "blacks". Not all the printers showed the ability to reproduce some of the high resolution test images and text output also varied as small fonts were not always reproduced on some models.
In order to continue the test, I have taken all the prints home with me and intent to store them in a file cabinet for the next year. I will bring each of them back to next years workshop (Kansas City) and ask each of the original contributors to send a new fresh copies of the same image (the master image will be kept on file here at the FTP site at ANL). This way we will be able to test for long term stability of the prints.
Next year, if there is still interest we will may take an opinion survey to see what the participants of the workshop think of the output. This years test was just to see what might be done.
By the way, if anyone else still wants to contribute an output from their printer. Download the test images from AAEM.AMC.ANL.GOV and put them in the mail to me. I will store them with the rest for next year.....
If there is still interest we may extend the test to include color.
- Overall it was a busy meeting and reasonably successful workshop -
Cheers - Nestor J. Zaluzec ANL
P.S. I enjoyed touching base with a lot of the Microscopy Listserver subscribers who introduced themselves at the meeting it's always good to associate a real person with the Email messages which come down the line.....
We have got a problem for ES-423E LaB6 filament (style 90-15) bought from The Kimball Physics.
We have a Jeol-2000FX TEM. As our TEM samples are mostly metals, the TEM always works at 200 KV.
We used to use Denka LaB6 Cathode and the emmission of the cathode was 30 micro-Amp.. The life span of the Denka filament was about 500 - 1000 hrs.
We recently changed our cathode with ES-423E LaB6 filament (Style 90-15, The Kimball Physics) which is supposed to last longer than Denka one. The emmission of the filament was kept at 16 micro-Amp.. However, after about 240 hrs, the filament has so seriously run up that we have to change the filament to keep the microscope running. We checked the vaccum system and found nothing wrong. The bias mode and beam current also looks OK.
Is there anyone used the ES-423E filament? We do appreciate any suggestions and comments on this issue.
Thanks
X. Li (Uni. of Wollongong, Australia) P. Renwick (BHP, Australia)
The lifetime of an LaB6 filament depends a lot upon how it is first heared up, as well as design considerations such as the sharpness of the tip. In general, if you heat them up VERY SLOWLY, i.e. over about 15mins to an hour, you should (we do) get lifetimes } 2000 hrs. At this level the sharpness of the tip and its slow blunting due to attack by oxygen etc (which you cannot avoid except with UHV) and sublimation becomes important.
The key issue, SEM or TEM as far as I know is to desorb oxygen (or oxygen containing gases) at low temperatures so as to minimize/eliminate reaction with the LaB6 filament at higher temperatures which produces an oxide layer.
Following along on Prof. Marks comments on saturating a LaB6 in a TEM, does the 15-60 minutes include pre-conditioning the filament. On the surface, these long times for powering up appear to be a waste of money. I don't know what filaments cost, but I would wager a bet that it would not take too many hours of powering up the filament very slowly to have the hourly charge of the TEM exceed by several times any savings seen by stretching the lifetime of the filament by a factor of 3 to 5. Have you done a cost analysis, including down time and pain-in-the-butt factors during filament swaps?
Message-Id: {9408081606.AA04250-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Sample Mounting for Tripod Polishing Orig-Sender: {CINIBUMK-at-ml.wpafb.af.mil}:ddn:wpafb Orig-Author: {CINIBUMK-at-ml.wpafb.af.mil}:ddn:wpafb ----------------------------------------------------------- I've been trying to prepare e-transparent samples of ceramic fibers embedded with epoxy by tripod polishing on diamond impregnated films, a la IBM. As they suggest we have been mounting the samples with super glue to a glass slide, which is then mounted with wax to the SS stub. The problem is that the super glue is debonding from the glass slide. I've tried slightly roughening the glass slide with 600 grit SiC paper and also carefully cleaning the glass prior to bonding. Thisd does not seem to help. The glue always remains bonded to the sample. Does anyone have suggestions for improving the bonding with superglue or any other glue or wax that will hold up and not flake off during the final polishing of thin sections. HAs anyone tried crystal bond? I am new to this technique and welcome the advice of those with more experience in the art.
Mike, if your problem is superglue delamination off of the glass slide, then you are probably just not roughing the slide surface up enough. Don't worry about the slide and grind it until you have a nice translucent surface; the refraction translucence will basically disappear once you have put the superglue on. I went through the same difficulty a few years ago. You can also freely use a larger grit, say 180 or 240 for grinding. Make sure that you grind in multiple directions as well, as I suspect that a large part of the secret is essentially mechanical interlocking.
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
Netters, Richard Easingwood asked a series of interesting questions about purity and qulaity of glutaraldehyde. One of the issues was the pH of the glut. My understanding is that glut has relatively little buffering capacity and so that when you add this aldehyde to your buffered solution, the pH of the solution won't be affected. Is this wrong? Does anybody actually know what the buffering strength of glut is? Thanks, Tobias Baskin
Dear Xin Yang; If you have access to a large chamber SEM with good 5 kv. or lower operating capability, you may find it interesting to examine the LaB6 filament in the SEM. There are several factors that could account for the low life time (240 hrs.) of this filament. Among these are: 1.) Defective when installed, 2.) heated to rapidly, 3.) poor vacuum in gun chamber, 4.) lack of pre heating, and even the possibilty that 5.) a sample exchange was made with the filament saturated.
Although a new filament can be defective, this is very unlikely so one of the other possibilities is the cause. If you can examine the filament in the SEM, you will be able to measure the specified parameters of the filament and determine if the material loss is excessive. A new LaB6 KPI has a diameter of about 320 microns. Check with KPI to verify this and then calculate the loss. Acceptable rates of loss are about 0.1 micron per hour in the worst of cases. You only need a magnification of about 250x lookng straight down at the tip for this measurement. You should also increase the magnification to about 2000x to examine the the tip to see if it still has the correct characteristics.
Especially look for chunks of material missing from the crystal. If there are such defects, this would indicate poor vacuum or a slight vacuum loss while the filament was heated. Do you have a vacuum monitor in the gun chamber? The pressure in the gun should be in the low 10-7 torr range.
If you have access to such an SEM, A convenient holder for the filament can be made from the original base that is used for shipping, by simply cutting off the threaded end. This will fit into a conventional SEM mount like the 32 mm. holder for a JEOL 35 or 820/840 series SEM. Just make sure that you have sufficient Z in the SEM and don't use an chamber interlock if you have one
Hope this helps. If you have an opportunity to try this I would be interested in what you find.
Dear microscopists, Thanks very much to all who responded a week or two ago re my request for peoples' feelings about their print processors. Having read your replies and looked at the Agfa Pro, Agfa Rapiline and the Durst modular unit 'in the flesh' we are tending towards the Rapiline.Unfortunately no one who replied mentioned this model. Several people had reservations about the Ilford 2150 processor (although admittedly some loved it). If anyone has any particularly bad (or good) experiances with the Rapiline I would like to hear from you. Yours faithfully,
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
With reference to the questions asked by Richard Easingwood and Tobias Baskin: Some of the questions regarding the pH of Glut and its buffering capacity were addressed in a paper by Coetzee & Van der Merwe - Some characteristics of the buffer vehicle in glutaraldehyde-based fixatives (Journal of Microscopy, 146, 143-155, 1987). The conclusion reached in this paper was that you need - very approximately - at least 50mM of buffer to adequately stabilize the pH of a 2.5% Glut solution. The problem is in defining what is adequate for your needs. As little as 2% of Glut added to a 0.2M buffer causes a discernable drop in pH. This is true for a wide variety of buffers around pH 6.5 - 8.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Can anyone tell me where I might find a history of EM? I know that Ernst Ruska developed the Electron Lens and recieved the Nobel prize in 1986. I would like some more information on things like: when glass and diamond knifesw were first used. When Epon was developed. When were certain structures first observed like; synapes, microfilaments, or or other things that could not be seen with LM. I am sure that all this information is preserved in different publications but our medline does not allow for searchs back that far. If there is not a "History of EM" some one should write one. Any help on this would be greatly appreciated.
Lary Hawkey That is Larry Hawkey (not Lary) hawkey-at-neuro.duke.edu
Anyone, I am looking for papers which deal with the degradative effects of E beams on cellulose structures. In particular - we are examining single wood fibers under tension inside an ESEM. We are using quite low accel. voltages c. 15KeV. Statistically it looks like the strength of the fibers is unaffected - the tests are over within a minute or so. However, something must be happening somewhere in the fiber wall (hemicellulose maybe) because after exposure to the beam for a little longer we are experiencing some burning. Any body got any words of wisdom (mine is limited) on this. Any comments or better still references, would be appreciated.
Hello, I was wondering if anyone had any experience with x-TEM of 6H-SiC,3C-SiC, on each other and Si. We are trying to look at the interface between SiC-Si and 6H-3C. Any thoughts on preparation (right now we cryo-ion mill) due to the hardness would be appreciated.
Sterling P. Newberry put together a nice history of EM, "EMSA and Its People, The First Fifty Years". 1992 by the Electron Microscopy Society of America, LC#92-72571.
Thanks for all the responses for suppliers of abrasive diamond films. I have compiled a list of the suppliers recommended to me and have included the list with this message. I meet with most of these suppliers while at the MAS meeting in New Orleans, and along with the diamond films they have other supplies that will come in handy for thin foil specimen preparation. Give 'em a call if you don't already have one of their catalogs.
thanks again for all the recommendations, John
replies for suppliers of diamond films:
1. Allied High Tech Products P.O.Box 4608 2376 East Pacifica Place Rancho Dominguez California, 90220 Contact: Clayton A. Smith Phone: 1-800-950-9347
3. Buehler Ltd. 41 Waukegan Rd P.O. Box One Lake Bluff, IL 60044 (708)295-6500 X4546.
4. South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673 TEL: 800-728-2233 714-492-2600 FAX: 714-492-1499
5. Imperial Lapping Films (reference below) 3M Corp. 3M Center St Paul, MN."
The title of the paper is "Recent Developments in The Use of The Tripod Polisher for TEM Specimen Preparation", by John Benedict, Ron Anderson and Stanley J. Klepeis.
Message-Id: {9408101624.AA23224-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: x-SiC TEM Orig-Sender: {rbirkhah-at-uceng.uc.EDU}:ddn:wpafb Orig-Author: {Ronald Henry Birkhahn {rbirkhah-at-uceng.uc.EDU} }:ddn:wpafb ----------------------------------------------------------- Hello, I was wondering if anyone had any experience with x-TEM of 6H-SiC,3C-SiC, on each other and Si. We are trying to look at the interface between SiC-Si and 6H-3C. Any thoughts on preparation (right now we cryo-ion mill) due to the hardness would be appreciated.
Thanks, Ron rbirkhah-at-uceng.uc.edu
---------------------- Replied Message Body ---------------------- To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: x-SiC TEM Orig-Sender: {rbirkhah-at-uceng.uc.EDU}:ddn:wpafb Orig-Author: {Ronald Henry Birkhahn {rbirkhah-at-uceng.uc.EDU} }:ddn:wpafb ----------------------------------------------------------- Hello, I was wondering if anyone had any experience with x-TEM of 6H-SiC,3C-SiC, on each other and Si. We are trying to look at the interface between SiC-Si and 6H-3C. Any thoughts on preparation (right now we cryo-ion mill) due to the hardness would be appreciated.
The UC stands for U. of Cincinnati, so I'm nowhere close to California. But I guess I don't know what is meant by "Tripod polisher". What is it and how does it work exactly? Is it anything like a dimpler?
Maybe someone can help me with my problem. As an electron-microscopist, I do have some troubles with embedding arteries and veins. I've tried to embed this arteries and veins of= =20 human umbilical cords, but didn't succeed completely.=20 The ultrathin sections show several holes and the tissue was damaged by shrinkage. We have tried to embedd the tissues in Unicryl,=20 but it was not satisfactory. Now we are planning to use Lowicryl HM20= .=20 Is there anyone out there who uses Lowicryl HM20 and who is experienc= ed in embedding arteries and veins (pieces). Do we have to use a special protocol? (fixation, cutting in pieces, embedding) Our goal is to look at the endothelial cell layer and the smooth muscle cells, together with the cell-contacts between these cells= =20 (gap-junctions). It will be an immuno-histological study, that's why we will try the Lowicryl HM20 plastic. What do you recomend, Lowicryl HM20 or Lowicryl KM4?
Thanks,=09=09Luc.
=C9=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=D1=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=BB =BALuc Analbers =B3E-mail: Analbers-at-med.ruu.nl =BA =C7=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4= =C4=C4=C4=C4=C4=C4=C5=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4= =C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=B6 =BAUniversity of Utrecht =B3 =BA =BAMedical Faculty =B3 Murphy's Law ??? =BA =BADept. of Medical Physiology=B3 =BA =BA & Sportsmedicine =B3 Well, Murphy was an optimist =BA =BAUniversiteitsweg 100 =B3 =BA =BA3584 CG Utrecht =B3 =BA =BAThe Netherlands =B3 LA =BA =C8=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=CF=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=BC
X400-Received: by mta bottom.ctd.anl.gov in /PRMD=ANL/ADMD= /C=US/; Relayed; Thu, 11 Aug 1994 02:47:52 -0500 X400-Received: by /PRMD=ESnet/ADMD= /C=US/; Relayed; Thu, 11 Aug 1994 02:47:51 -0500 X400-Received: by /PRMD=Switch/ADMD=Arcom/C=Ch/; Relayed; Thu, 11 Aug 1994 02:47:34 -0500 X400-Received: by /PRMD=SWITCH/ADMD=ARCOM/C=CH/; Relayed; Thu, 11 Aug 1994 02:48:31 -0500
John Mansfield North Campus Electron Microbeam Analysis Lab 413 SRB University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 11:10
Date:8/11/94 NC EMAL
Hi, my department (Materials Science and Engineering) would like to purchase a used SEM for the undergraduate teaching lab. It only needs to be a basic SEM, we'd like to get as new an instrument as possible. Budget is tight though and we'd like to pay less than $20K. Does anyone know of any instruments that fit our needs. Thanks.
In response to Steve Bill's concerns about back lighting the sample during Tripod Polishing:
We (South Bay Technology) have made several modifications to the the "L" bracket/Pyrex insert arrangement on a custom basis for customers. We are planning on introducing a variety of different "L" bracket and Tripod Polisher configurations in the next month. These new configurations will allow us to create "custom" packages that will more precisely fit specific applications. One of the first configurations will include an "L" bracket/Pyrex Insert arrangement that allows for easier back lighting of the sample.
I very much appreciate the comments and I encourage anyone else out there who has comments, questions or suggestions for new equipment or modifications to existing equipment to contact me. We are very interested in hearing from you and we will do our best to provide you with what you need. As for some of the more application specific information, I think I will leave that to our many Tripod Polishing experts out there on the Listserver!
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
Neuroscience List {neur-sci-at-net.bio.net} , Microscopy List {microscopy-at-aaem.amc.anl.gov} , Confocal Microscopy List {confocal-at-ubvm.bitnet} , Cell Bio List {cellbiol-at-net.bio.net} Message-Id: {Pine.3.05.1.9408111025.A23368-a100000-at-pauline.sdsc.edu} Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII
Please excuse this post if it is a little off-topic. What are the best Mac programs for compiling scientific references (all prices)? Sorry if this has been asked before. Thanks for all help.
Marc C. Brande, M.S. SD3D Email Discussion List: Live Brain Cell Biology in 3D All aspects of 3D Imaging San Diego 3D Imaging Group To subscribe/unsubscribe, send request to: 3840 Camino Lindo sd3d-request-at-sdsc.edu San Diego, CA 92122 To post a message to list,send message to: Email: BRANDE-at-SDSC.EDU sd3d-at-mailserver.sdsc.edu Voice: (619) 587-4830
If you have an Hitachi H7000/7110 TEM/STEM/SEM and want to use the bulk specimen holder for SEM work, you'll have found out that Hitachi charges $160.00 for each holder (I'm not kinding!) and they have to be special ordered from Japan since Hitachi USA doesn't keep any in stock.
As an alternate you can purchase JEOL 100cx SEM specimen holders for approx. $1.00/ea from Pelco, EMS, Fuller, etc. and modify them to fit the Hitachi bulk specimen rod in about 20 seconds with a pair of needlenose pliers and some wire cutters. The JEOL holders are about 6mm too long for the Hitachi rod and the end pieces are about 2 mm too tall, they are made out of strips of copper and are easily cut and bent.
Does anyone else out there use a Hitachi H7000/7110 for SEM Imaging?
I really love Endnote for storing and generating bibliographies. It is extremely easy to use (particularly if you use Microsoft Word), has powerful database search functions, allows you to put in notes about the reference, and can download reference information from sources such as medline (with the supplementary program, Endlink).
HELLO EVERYBODY WE,(ELECTRONMICROSCOPE UNIT, UNIVERSITY OF PRETORIA SOUTH AFRICA) ARE PARTICIPATING IN A CAMPAIGN TO PROMOTE NATURAL SCIENCES AMONGST CHILDREN (AGE 12-15 YRS) THE MAIN IDEA IS TO MAKE UP A "SCIENCE KIT" CONSISTING OF ITEMS RELEVANT TO THE VARIOUS NATURAL SCIENCE DISIPLINES EG. A SIMPLE CHEMISTRY KIT TO MAKE CRYSTALS. IS THERE ANYBODY OUT THERE THAT HAS BEEN THROUGH SUCH AN EXERCISE,OR THAT HAS SOME IDEAS? NEEDLESS TO SAY THAT THE BUDGET IS TIGHT! I WOULD APPRECIATE ANY CONTRIBUTIONS.
I FORGOT TO SAY THAT WE NEED TO CONTRIBUTE SOMETHING IN THE LINE OF MICROSCOPY TO THIS KIT;IF THAT DID NOT COME THROUGH CLEARLY IN MY 1ST MESSAGE! MY APOLOGIES !
For the last couple of years there has been a poster session devoted to that topic at the Society for Neuroscience annual meeting. I don't have the abstract booklets at my fingertips, but if you check in the programs you should be able to find the relevant abstracts and contact the authors directly. As I remember them from just a brief look, some of the kits and projects were very impressive, and covered a broad age range from elementary through high school.
Cheers,
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
I agree that the book NUMERICAL RECIPES has a very good explanation of the Wiener filter (and many other numerical techniques) -- but has anyone tried to actually use it ?
The occassional non-microscopy posting is not a big deal, however, keep it to a minimum. When I see things getting out of hand I'll speak up.. Remember there are a lot of other venues to get this type of information, but I realize that we all frequently face common problems relative to computers etc.. and it would therefore be nice to see what colleagues are using to solve simple problems (e.g. fonts, references, image formats etc...)
In message Fri, 12 Aug 1994 13:00:56 -0400, "Nestor J. Zaluzec-Argonne Nat. Lab." {ZALUZEC-at-anlemc.msd.anl.gov} writes:
} From: SMTP%"-at-ANLVM.CTD.ANL.GOV:Edbasga-at-USCN.BITNET" 11-AUG-1994 } 11:35:53.58 To: ZALUZEC } CC: } Subj: FEG BSE and EDS advantages? } } Message-ID: {9408111201296D3.ADSK-at-USCN.USCN.UGA.EDU} (UMass-Mailer 4.04) } Date: Thu, 11 Aug 94 12:02:02 EST } From: Edbasga%USCN.BITNET-at-ANLVM.CTD.ANL.GOV } Subject: FEG BSE and EDS advantages? } To: zaluzec-at-anlemc.msd.anl.gov } } We are considering replacing our SEM with a new one. } Our main work is in biological applications for a multi-user } environment at a Medical School. We will be dealing with cells } in culture and molecular biology applications. Dental materials } will also be examined. Since this is an acquisition that we will } have for 10-15 years I feel that a basic instrument with an FEG } would be a more sensible choice than a full blown W/LaB6 set up } (EDS, BSE, and cryostage). If we are locked into a W/LaB6, I feel we } will be unable to pursue low voltage, high resolution applications } especially in the cryo area. } } My major selling point is that the advantage of the higher beam } current density provided by an FEG at low voltages will outshine } any instrument with a conventional emittor. The additional detectors } and cryostage could always be added later to the FEG but a W/LaB6 } cannot be upgraded easily to an FEG. } } I have experience working with several FEG's in SEI mode both at } ambient and cryogenic temperatures. I know an FEG is the best } for high resolution (} 30,000x) for these applications. } } I don't have much experience with EDS or BSE imaging modes with FEG. } I think that the increased beam current density will be an } added advantage for applications such as localizing heavy metal } stained areas of chromosomes and colloidal gold labelled antigenic } sites using both BSE and EDS especially in a cryo environment. } } Will an FEG, in fact, provide better spatial resolution for BSE } and EDS applications for low voltage ( {10kV) and high resolution } imaging. At least one vendor claims there is no advantage } afforded by a higher beam current density for increasing BSE } spatial resolution. } } I need to convince the administration that FEG is worth the } extra cost in the long run. } } Thanks to all of you who respond. } } Ed Basgall, Ph.D. } Medical College of Georgia } Cellular Biology and Anatomy } Augusta, GA 30912-2000 } Ph: 706-721-3524 } FAX 706-721-6893 } ==================== Ed,
See the recent article by Heinzmann et al., 1994, SCANNING 16:, 241-245, and the literature cited there in for discussions on the use of BSE for biomedical specimens with a FESEM.
Thanks to all 18 people who responded to my question about the best mac programs for compiling scientific bibliographies (for journal articles). EndNote ($149) and EndNote Plus ($249) wins hands down (12votes).Also with sister product Endlink you can download refs from medline for integration. They will send you a video if you call for info! From: Niles and Assoc. 800 Jones St. Berkeley, CA 94710 Phone: 510-559-8592
Marc C. Brande, M.S. SD3D Email Discussion List: Live Brain Cell Biology in 3D All aspects of 3D Imaging San Diego 3D Imaging Group To subscribe/unsubscribe, send request to: 3840 Camino Lindo sd3d-request-at-sdsc.edu San Diego, CA 92122 To post a message to list,send message to: Email: BRANDE-at-SDSC.EDU sd3d-at-mailserver.sdsc.edu Voice: (619) 587-4830
Does anybody know of a need for the services of optical microscopy for intrepretation of mine tailings and other environmentally contaminated geological materials? In our lab we are working on a characterization protocol with this emphasis and need to know if it is a viable way to go before investing more time on the project. Thanks Clarissa
Hi, I'm looking for software that will let me design and control experiments on the Macintosh. Basically I'd like to have some kind of c like language that I could "write" the experiment in and have the program "run" this protocol. For example, loop over n trials where each trial consists of displaying 2 images and having the subject select via touch screen the correct image (where correct is defined in the protocol). I have no need to connect to the outside world via TTL's or A/D. I just need a flexible application that allows me to design and run different experimental protocols. Example programs that are on the PC that I'm aware of are Cortex from NIH, Tempo a commercial app from Reflective computing, Mel (not sure who does this one). I'd like to know of any mac programs that do this sort of thing.
thanks Gary Leydon leydon%bruce-at-biomed.med.yale.edu
Regarding the characterization protocol for mine tailings. If you have access to a SIMS, it would probably help you a great deal. Not only can it detect impurity levels down to the ppm or in some cases ppb levels, it can also discriminate isotopes, so if you mean by environmentally unfriendly - radioactive - it may help even further, although the operator probably wouldn`t be terribly excited.
I am personally partial to the Codonics NP-1600, largely because I have one. At $2.00 per shot, it produces prints that rival photographs in resolution, tonal range, and color saturation. It's the only printer I've yet seen that produces true blacks and true whites. The really nifty thing though is its convenience and accessibility. It attaches to an ethernet network and receives print requests via TCP/IP. Its set up as an FTP client, and you just FTP graphics files to it. I've yet to find a format it doesn't know. These features make it software and driver- independent, so its directly available to anyone with an account. Price is about 11K.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
Greetings, I have been involved with elemental analysis of ~10 um diameter calcium carbonate particles. There is interest in looking at the changes in surface charge by some analytical technique. Mass spec, nmr,atomic asorbtion, etc have been ruled out for various reasons. My question to the microscopy group is, would ATM or STM be of any use? If so, by what method could I get somebody to analyze these samples? What would the cost be and how rapidly could it be done? Thanks in advance.
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick (Chuck) * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Anatomy * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-1633 voice * | | * 806 743-1219 fax * | | * Email emccjb-at-lubb.ttuhsc.edu * | | * * __| |__ * * |_______| *************************************************
Subject: Time:3:24 PM OFFICE MEMO German glass coverslips Date:8/16/94
We need to purchase 25mm round German glass coverslips (preferably 1.5mm thickness) for neuronal cell cultures. Can anyone steer us to a supplier? Mike
Mike_Schwartz-at-qm.yale.edu Fax 203-785-5263
Michael Schwartz, Ph.D. Section of Neurobiology Yale University School of Medicine 333 Cedar St. New Haven, CT 06510
} We need to purchase 25mm round German glass coverslips (preferably 1.5mm } thickness) for neuronal cell cultures. Can anyone steer us to a supplier? } Mike
You could try:
Hugh Cartwright 2407 Walter Zimny Drive Posen, IL 60469
(708) 489-1101
Suppliers of 1" round glass slides part# 458 $37.23/100 slides (plain with ground edge)
Ed Vicenzi tel (609) 258-1464 office Princeton University tel (609) 258-1406 lab Princeton Materials Inst. fax (609) 258-6878 70 Prospect Ave. Princeton, N.J. 08540-5211 email: vicenzi-at-phoenix.princeton.edu
Does anybody know of a good book dealing with morphometric calculations? Not having done math (especially calculus) for a number of years, I need to know of a formula for determining the area and volume of plasma membranes in thin sections. Also, if using the formula for the area of a sphere, does the formula indicate the surface area or the internal area? I remember the formula but which area it indicates I've forgotten. Thanks, Phil 8-{)
Our lab (Materials Science and Technology) is looking into the possibility of acquiring a scanning infrared microprobe. Presently, our only capability with respect to infrared analysis is a standard FTIR which is equipped with a diamond cell option for microanalyses. We have considered purchasing an FTIR-microscope attachment to increase our capabilities and to compliment our SEM/TEM and XRD facilities. Recently, we have seen information about a relatively new microanalysis system called a Scanning Infrared Microprobe. This technique apparently provides high resolution molecular microanalysis and morphological mapping of surfaces, bulk materials, etc.
Does anyone know anything about this technique/instrument that they could share with us? And if anyone has purchased one of these systems, are they satisfied or disastisfied, and why? All information is greatly appreciated. Thank you, Eliesh O'Neil Elizabeth (Eliesh) G. O'Neil Research Scientist I GTRI/EOEML Baker 271, 925 Dalney Street Atlanta, GA 30332-0827 (404) 853-0590 (office) (404) 894-6199 (FAX)
In message Wed, 17 Aug 1994 11:50:25 -0400, rutledge phil {prutle1-at-umbc.edu} writes:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) } ============== Martin W. Steer's book UNDERSTANDING CELLSTRUCTURE (1981, Cambridge University press) is a good and easy-to-understand book with many examples and illustrations. M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu
In message Wed, 17 Aug 1994 11:50:25 -0400, rutledge phil {prutle1-at-umbc.edu} writes:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) } ============== Phil, We can either talk about area (a 2 dimensional quantity) or volume (a 3 dimensional quantity) the two are not interchangeable. The surface area of a sphere of radius r is 4*pi*r*r. (four pi r squared). The volume of a sphere of radius r is 4*pi*r*r*r/3 (four thirds pi r cubed). CBB
In message Wed, 17 Aug 1994 11:50:25 -0400, rutledge phil {prutle1-at-umbc.edu} writes:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) } ============== Phil, We can either talk about area (a 2 dimensional quantity) or volume (a 3 dimensional quantity) the two are not interchangeable. The surface area of a sphere of radius r is 4*pi*r*r. (four pi r squared). The volume of a sphere of radius r is 4*pi*r*r*r/3 (four thirds pi r cubed). CBB
My own uses are biological (Centre for Food and Animal Research, Agriculture and Agri-Food Candada) but I have used the SpectraTech instrument for infrared microspectroscopy. The Irus system software is under continuous upgrade, and the company (esp. John Reffner) extremely helpful in working out problems and new applications. They have an instrument installed at Brookhaven National Laboratory (using the National Synchrotron Light Source, which is much more powerful than conventional sources for IR) and are starting to do some very interesting work. I myself am still very much a neophyte at IR microscopy/spectroscopy, but for more info. contact Gwynn Williams at Brookhaven National Laboratory, Associated Universities Inc., Upton, New York, 11973. Have fun, and happy hunting. Shea Miller REs (last line is a typo... sorry!)
Phil- If you want to determine volume (a three dimensional parameter) from a thin section (essentially a two dimensional field) it seems to me you will need stereologic calculations to do the conversion from 2D measures to estimates of 3D parameters. The same would also appear to be true for determining area of plasma membrane. Although it is a 2D parameter, the membrane rarely stays in the plane of section and so its surface area must also be inferred from your thin section measurements. There are a number of good books and articles on the subject. My own favorite is Ewald Weibel's 1979 book Stereological Methods (Academic Press). Volume 1 is a practical treatment of the subject without too much math. Robert Bolender also has a book and computer program on the subject. He is a very readable author. journal articles by either of these two would also be helpful, in particular Weibel's 1970 Int Rev Cytology paper volume 26:235. Papers by Gundersen, Elias, Hyde, DeHoff, Cruz-Orive may also be helpful. Finally Elias and Hyde's 1983 A Guide to Practical Stereology (Karger Publishing- I think?) may be useful.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Wed, 17 Aug 1994, rutledge phil wrote:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) }
Greetings, I'm interested in finding good, contemporary texts on TEM/SEM in French. Is anyone able to point me in the right direction? Many thanks!
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
There have been several good suggestions on this listserver regarding the use of LaB6 cathodes in the JEOL 2000 TEM. As a manufacturer of LaB6 cathodes, FEI has learned a lot over the years about how to best operate LaB6. Much has been covered, but I would like to make one further suggestion that may be helpful. We have found that it is beneficial to recheck your operating point often after start up. In the massive electron guns found in high voltage systems such as the JEOL 2000, it takes a while for the entire gun to reach thermal equilibrium when the cathode is first heated. Therefore, when you first find your operating point, the gun may not have reached thermal equilibrium. As the gun continues to heat up and finally reaches thermal equilibrium, less heat is transfered from the cathode to the gun, and your cathode will over heat. I recommend that the operating point be rechecked every 15 minutes for the first hour of operation. You will probably find that the filament setting needs to be reduced during that first 15 - 60 minutes of operation. This holds true for most high voltage TEMs (200kV and up) of any make, and also to smaller TEMs, to a lesser degree.
An additional option that may be helpful in the use of your TEM would be to try the FEI LaB6 Mini Vogel Mount (MVM) cathode on your next cathode change. We are currently showing good results in the 200 and 400 kV JEOL TEMs, as well as in many others. If anyone would like further details on our LaB6 cathodes, please contact me directly.
Best Regards
Locke Christman FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830 (503) 640-7518 lac-at-feico.com
Although probably not very appropriate for your applications, there is a v. useful book entitled "Initiation a la microscopie electronique par transmission" published by the Societe Francaise de Mineralogie et de Cristallographie, ed. C. Willaime. They are at Tour 16, 4 Place Jussieu, 75252 Paris CEDEX 05.
There has been a few questions & postings recently which tread on the gray area of this server and I've gotten several offline questions and comments on these. Let me share my thoughts with all of you on this.
First, let me remind everyone that ALL comments and question on microscopy and microanalysis are welcome on this server regardless of who posts them. I especially encourage commerical firms to read and if appropriate reply to posting which may need a special bit of information. This is a valuable part of what we are trying to foster. There have been many valuable posting from commmerical firms about solving problems and answers to questions and allow me to thank those that have participated.In the course of these replies it is appropriate to identify oneself and mention if a person or entity is a commerical firm or not especially if a particuliar product is mentioned. So far everyone has very carefully kept to this request and I am pleased.
There have been a few postings which border the gray area and I get occasional off-line questions about these and their nature.
Mostly they are replies for information on questions like: "Does anyone know if a company exists that does/sells XYZ for a fee" since, the request was posed by a subscriber I have no problems with a commerical firm saying "Sure we do/have that, and here's a brief synopsis, contact us off-line for more details" with the appropriate credit lines etc... I feel that this is within the bounds of answering a question and making the information on the existance of a company or service known to the subscribers. Most of the time everything that has been posted has been fine.
There have been the occasional posting which tread the line and I'd appreciate everyones cooperation here. When I see something that is inappropriate I do contact the author and let them know.
You should all also know that alot of the postings from commerical firms first send their reply to me for comment, especially when they mention a product or service, and I (and you should) appreciate their cooperation. Good examples are the recent discussions about FESEM's and LaB6.
So bear with the gray areas and I'll try to keep an eye on things, if you see something which appears strange just Email me and I'll investigate.
Just a reminder to no direct "advertising" of products or services should be conducted. Credit lines should be kept simple and direct to avoid any impression that your trying to sell something. It will make my life alot easier.....
Thanks in advance.... Nestor
P.S. I see no need for further discussion on this issue on the server. If you want to comment just contact me offline at:
Calcium localization in biological samples can be a problem unless the samples are properly prepared. Calcium phosphate or carbonate is very quickly lost under acid or even slightly alkaline fixes. If fixation must be used, try fixing at pH 7.5 - 8. Heavy metals such as osmium and uranium will also solubilize calcium, thus they should be avoided. Calcium also tends to be somewhat mobile during the fixation process and tends to translocate throughout the tissue, giving you localization artefact. For this reason, for calcium localization I routinely snap- freeze the tissue, then freeze-dry and embed in resin. Ultrastructural preservation is of course compromised, but if localization is what you want, this seems to be the best way.
Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
} As a materials scientist I am asking for help with a biological microscopy } problem. } } I recently had a user bring along some sections of biopsies in which he was } interested in finding calcium by EDX in the STEM.. The samples had been } prepared traditionally, namely: } } GTA 2.5%, PFA 2.0% Fix } OsO4 fix } dehydration in ethanol and propylene oxide } Uranyl Acetate en bloc stain } Epon embed } Section } } We found no calcium. I am reminded of a rather similar problem } several years ago when a user was studying embryonic mollusc shells, } and insisted calcium had to be present, but we could not find it. } } Does the traditional method of preparing samples remove calcium?
Yes. Suggest your user that he/she par their rubbish someplace other than your microscope. } Does anyone have a method of preparing samples that would retain } the calcium? } Yes again. Rapid freezing followed by cryoultramicrotomy. In some cases, for qualitative analysis, freeze substitution may be used, instead of cryoultramicrotomy.
} Any help would be much appreciated. } } ********************************************************* } * Anthony J. Garratt-Reed * } * Massachusetts Institute of Technology, Rm. 13-1027 * } * Cambridge, MA 02139, USA * } * * } * Ph: 617-253-4622 * } * Fax: 617-258-6478 * } ********************************************************* } }
Tony Garratt-Reed asked about Ca in biological samples. One problem could be wrong expectations of what is in the tissues. I have run into this problem *a lot*. As much as I explain that for concentra- tions of some millimolar XMA has no trouble with Ca, but for the physiological concentrations in the micromolar range XMA will not detect it, the message does not get through. Embryonic molusk may be mostly chitin or some such material which has no Ca in it. As far as I know, the normal prep methods will remove only the Ca++ solute from the cytoplasm and/or organelles; hydroxyapatite is not affected. Treatment with pyroantimonate will precipitate the Ca and it is visible in the EM, so you have a possible test for whether there is Ca in the sample after all. Also, examination of frozen, hydrated material or material lyophylized in situ in the EM (by heating to -110C for 24 hrs, -100C for 24 hrs and -90C for 24 hrs) which will reduce the mass thickness by about 80%, can el- iminate preparation artefacts for any Ca which is not volatile (essentially all of it). Good luck. Yours, Bill Tivol
In message {Chameleon.940819134408.tonygr-at-emlab.mit.edu} Anthony Garratt-Reed writes: } As a materials scientist I am asking for help with a biological microscopy } problem. } } I recently had a user bring along some sections of biopsies in which he was } interested in finding calcium by EDX in the STEM.. The samples had been } prepared traditionally, namely: } } GTA 2.5%, PFA 2.0% Fix } OsO4 fix } dehydration in ethanol and propylene oxide } Uranyl Acetate en bloc stain } Epon embed } Section } } We found no calcium. I am reminded of a rather similar problem } several years ago when a user was studying embryonic mollusc shells, } and insisted calcium had to be present, but we could not find it. } } Does the traditional method of preparing samples remove calcium? } Does anyone have a method of preparing samples that would retain } the calcium?
Anthony,
I would say that any calcium that was in the bio-systems you mention was washed out from the tissue samples during the above preparation, as most of it was not bound to the tissues in any way.
Alternatives: 1.freezing preparations: a. freeze-dry. b. freeze-substitution; followed by resin infiltration and sectioning.
2. Calcium precipitation in situ using the antimonate method. Tissues are treated with potassium antimonate solutions during fixation to precipitate calcium in situ, followed by the usual embedding techniques. There is always a question about transport of calcium away from its original location during any of these preparations, so keep an open mind.
The cool thing about the antimonate method is that, using EDS analysis, you can check for co-precipitation of Sb and Ca in your sections. Otherwise, you have to assume that any precipitates you obtain in your sections are the ones where the calcium is located; dangerous without the independent confirmation by EDS. Of course, the question about how accurate the localization is, is still there. But hopefully, you can at least precipitate it out within cells, at least, without having all of it washed out.
An uncool thing about this method is that the Sb L-series x-ray peaks lie right smack dab over the Ca K-series x-rays, but actually that just makes the analysis more fun because you get to use that peak deconvolution software that you've got; you've got to strip off the Sb L-series to reveal the Ca K-series beneath - make sure your energy axis is accurately calibrated when you collect these spectra.
Here are two references for the antimonate method. The first also includes EDS analysis of precipitates:
1. An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique, Robert D. Slocum and Stanely J. Roux, The Journal of Histochemistry and Cytochemistry, Vol 30 No. 7, pp. 617-629, 1982. Contains EDS analysis and tannic acid/antimonate procedure.
2. Localization of Ca++ containing antimonate precipitates during mitosis, Susan M. Wick and Peter K. Hepler, The Journal of Cell Biology, volume 86, August 1980, pp. 500-513.
Both of the above have more references to the technique.
3. I have a project in my lab right now doing an antimonate technique on plant cells and they seem to be getting resonable results. Soon I will send you their protocol.
Good luck!
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
I am looking for suggestions to prevent the clumping of cilia during processing for SEM. A buffer rinse prior to fixation didn't seem to help. I have thought of using a dilute Triton x-100 wash to try to remove the mucous layer prior to fixation, and was wondering if anyone has a favorite %. All suggestions welcome.
__________________________________________________________________ Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
In message Fri, 19 Aug 1994 09:04:34 -0400, "Ronald M. Anderson" {ron-anderson-at-VNET.IBM.COM} writes:
} We have a Sorvall MT2-B microtome in need of a complete overhaul. } Is there someone near southeastern New York that anyone can recommend? } } Please direct responses to Phil Flaitz pflaitz-at-vnet.ibm.com offline. } Thank you. We will summarize and post as appropriate. } } 8-) } ========== For Sorvall MT 2B service in NY State try the following:
Bill McGee MICROTOME SERVICE Co. 7568 Florian Way Liverpool, NY 13088 Telephone: (315) 451-1404 M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu
I am having a problem with fogging of histology slides after coverslip mounting. It appears to be the formation of tiny water droplets after mounting, like fine condensation. It appears to be above the focussing plane of the section. This is happening despite 3x3mins. dehydration in 100% ethanol (after 70% and 95% stages), hemo-de, histoclear or xylene clearing agents, and the fact that this is exactly the same protocol I have used for years, and used succesfully with the present lots of clearing agents and mounting medium (Permount) last Fall. Could summertime in upstate New York (increased humidity, although I'm working in a very well air-conditioned lab) cause this level of effect ? (This is a new environment for me.) Has anybody even run into this problem before, and did they find a solution ? Any suggestions would be greatly appreciated.
Patrick D. Reynolds Department of Biology Hamilton College Clinton, N.Y. 13323 Ph. (315) 859-4723 Fax: (315) 859-4807 e-mail: preynold-at-hamilton.edu
Patrick; I possed a similar question a couple of months ago. What kind of slides are you using??? Are they silane coated?? Do you coat your own or buy them pre-coated??? We have reduced the problem quite a bit by using isopropanol for dehydrating but still have the problem at times. It also appeared after years of working but have not found out why, except that we switched suppliers of slides (went from coating our own to usinf pre-coated slides). As can see from my address it is not just a problem of Upstate New York. I worked in Toronto for 7 years and never had the problem. Let me know what you find out and if you can solve it.
Yours Mark Elliott, PhD Pulmonary Research Lab, St. Paul's Hospital Vancouver BC Canada
} We have a Sorvall MT2-B microtome in need of a complete overhaul. } Is there someone near southeastern New York that anyone can recommend? } } Please direct responses to Phil Flaitz pflaitz-at-vnet.ibm.com offline. } Thank you. We will summarize and post as appropriate. } } 8-) } RMC, Inc. bought the old Sorvall, then DuPont, line and still services the MT-2Bs and other ultramicrotomes. I don't have my list of service people in your area here, but the main office in Tucson can be reached at (602) 889-7900. Old MT-2Bs never die, they just need an occasional tuneup!
Tina Weatherby Carvalho Biological EM Facility University of Hawaii
We are currently looking for a laser attachment for a light microscope for single cell ablation. I would like to hear from anyone who is currently doing this to discuss the various systems on the market, and to get your advise and opinion.
Ben August Dept. of Neurology, Tissue Culture Laboratory U of Wisconsin-Madison.
Without having had the experience myself (yet), I would put in a vote for the ethanol being the source of your clouding problem. For "non-critical" uses, we have purchased a cheap, bulk ethanol - 20 gal, presumably 100%. We have used it to rinse glass plates, and found that it leaves a cloudy residue behind - like water spots. Sound familiar?...
I think I'll buy the good stuff from now on. Good luck!
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
The cilia are coated wikth mucopolysaccharides and glycoproteins. You might want to try a protease or other cleaving enzyme. Triton will dissolve lipids, which aren't your problem, but it might help keep any digestion residues on the cilia from adhering to each other.
Let me know what works.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Fri, 19 Aug 1994, randy nessler wrote:
} } I am looking for suggestions to prevent the clumping of cilia } during processing for SEM. A buffer rinse prior to fixation didn't seem } to help. I have thought of using a dilute Triton x-100 wash to try to } remove the mucous layer prior to fixation, and was wondering if anyone has } a favorite %. All suggestions welcome. } } __________________________________________________________________ } Randy Nessler } rnessler-at-emiris.iaf.uiowa.edu } The University of Iowa Central Electron Microscopy Research Facility } } }
I've always used 10- min. changes in the 100% ethanol (3 baths) and 3 baths in xylene, 10 min. each. Change solutions every 2 weeks or 200 slides. Its usually not as humid here in the Pacific NW, but this has always worked. This works for sections up to 40 micron vibratome sections as well as 80 micron celloidin sections.
Incidentially, try DPX from Gallard and Schlesinger, as a coverslipping medium. It won't prevent fogging from residual water, but its nice stuff to work with.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Anthony Garrett-Reed, Given how labile Ca is, I would be surprised if it wasn't lost in preparation. Only the insoluble precipitates like bone can be counted on to hang around during processing. You most likely will have to try freezing and frozen-sectioning of hydrated tissues. Maybe freeze-fracture & examination in an SEM with EDX. There was an excellent paper in the early '80's about doing this with insect tissue for soluble ions (Na, K, etc.), but naturally I forget the ref, and my copy is buried who knows where. Maybe someone else remembers it... Phil Oshel poshel-at-luc.edu
In reply to Anthony Garratt-Reed I strongly agree with Professor Somlyo's response to your query and add a small note of caution about a possible pitfall in the use of pyroantimonate protocols. If you consider the diffusion coefficients of calcium and antimony you may come to the conclusion that the precipitate may tell you more about the point of entry of antimony into the cell or tissue than it does about the normal site of calcium. Further, calcium is not the only biologically important element precipitated by antimony. Dispersive x-ray of cryopreps is the way to go. Best luck! David
David B. Slautterback Anatomy Department 264 Bardeen Labs UW-Madison Voice 608-262-1609 Email dbslautt-at-macc.wisc.edu
Does anyone know whether there is a mailing list/usenet group dedicated to collagen/extracellular matrix. We are looking for a specialist in this field.
Nikon makes digital control backs for their cameras. I think one is available for the F3 (you'll also need a film winder/motor drive). I know they made one for the 8008.
The following query has been forwarded to the group for its consideration
From gsumnich-at-sunstroke.sdsu.edu Tue Aug 23 08:39:41 1994
I am considering purchasing a Cohu 1300 series color video camera to do some video microscopy. I would like to be able to use the integration capabilities of this camera but the one unit I have found to do this (model 440 A Frame Store, Colorado Video Inc.) seems a bit pricey. The unit must control integration time of the camera, capture the video output from the camera, store the image, and send it to the computer at 30 frames/sec. Does anyone know any other packages that will do this?
I have had good success in dissolving mucous on mucosal surfaces using 1% dithiothreitol (DTT) prior to fixation. Its most effective on mucous with many disulfide linkages, as do most vertebrate mucouses.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
The Temple City address and phone number I have for South Bay Technology appear to no longer be valid. Does anyone have a current address and phone for them? Are they still in business?
TIA
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
} The Temple City address and phone number I have for South Bay Technology } appear to no longer be valid. Does anyone have a current address and } phone for them? Are they still in business? } } TIA } } Glen MacDonald } Hearing Development Laboratories RL-30 } University of Washington } Seattle, WA 98195 } (206)543-8360 } glenmac-at-u.washington.edu } } } They moved to San Clemente a few years back. South Bay Technology 1120 Via Callejon San Clemente, CA 92672 714 492-2600 phone 714 492-1499 fax
Edward Goo Associate Professor Department of Materials Science and Engineering University of Southern California Los Angeles, CA 90089-0241 ekgoo-at-mizar.usc.edu (213) 740-4426 phone (213) 740-7797 fax
Thanks for the address, and to all who responded directly to me. This will help someone out looking for a diamond saw. I had an address off a diamond saw that was several years old.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Message-Id: {MAILQUEUE-101.940823135607.448-at-vanlab.paprican.ca} To: *Anatomo Pathologie {anapat-at-reks.uia.ac.be}
Hello, If you do not find a list server to assist you, there is a company in the US. which specializes in finding experts in any scientific area in order to access information from them. They charge a fee and I'm not sure how they would handle a single search situation, but the company name is Teltech and they may be reached as follows:
Teltech 2850 Metro Drive Minneapolis, MN 55425-1566
or
SUPPORT-at-US.TELTECH.COM
Good luck, Laurie
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
I am rebuilding a Denton DV502 evaporator for use in our undergrad EM course. If anyone out there has an out-of service DV502, or any of the following parts available, please contact me directly. Please *do not* reply to the list. -Expanded metal guard for 12"x12" bell jar -Manual lift for 12" x 12" bell jar -Glow-discharge feed-through and ring TIA Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
} Date sent: Tue, 23 Aug 1994 11:44:05 -0500 (CDT) } From: COOK-at-AAEM.AMC.ANL.GOV } To: MICROSCOPY-at-AAEM.AMC.ANL.GOV } Subject: 35 mm time lapse control
} Nikon makes digital control backs for their cameras. I think one is availabl } e } for the F3 (you'll also need a film winder/motor drive). I know they made on } e } for the 8008. }
The multi-function control back for the 8008 and 8008S is NIKON MF-21. Interval-timmer function Long time exposure Auto bracketing Freeze focus Recently purchased for $165.00. Adorama (800)223-2500.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
Embedding adipose tissue Does anyone have any suggestions on how to embed adipose tissue and to retain all the lipid? I want to do some straight foward morphological studies. I have tried a few different methods, and no matter what I do, in at least half of my blocks, the center part has lost the lipid. Also, how easy is it to freeze and do ultracryosections on this tissue? Thank-you in advance. Jeanne
I work with monolayers of differentiated intestinal cells which I grow on glass coverslips. We routinely fix and embed in plastic resins like Epon, JB-4, or LR Gold but I am having trouble getting a good paraffin embedding technique. I tried embedding scrappings of the monolayers in agar prior to paraffin infiltration but was not overwhelmed by the results. any ideas gratefully appreciated.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
The suggestion here in regards to the problem of "Water Dropletts" appearing under the coverslips in recent years, is the use of Re-cycled Xylene in the final xylene rinse and coverslipping step. Re-cycled xylene is fine for the earlier staining & processing steps but just isn't clean enough for the final step, use fresh xyxlene. These are the words of my friendly HT, HTL (Histotech.) Federal guidelines necessitated the use of re-cycled xyxlenes for many lab in the last couple of years (which explains why everything was fine in years gone by.)
It's worked for us, let us know if it works for you.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
My humble replies to several of your queries: 1. I don't think you will ever retain all of your lipids. We have measured phospholipid, cholesterol, and triglycerides before and after fixation (not an easy thing to be sure you are doing right, and probably some artefacts induced) or freezing. In both cases there is selective loss of particular lipid species. Lipids will leach out of intracellular stores in prolonged exposure to glycerol or sucrose as a cryoprotectant as well. This is probably of concern only if you are doing specific anlysis of lipids. For straightforward morphological studies do you really need to retain all lipids, or merely retain the volume and location in an identifiable way (i.e. know where the lipids were?). What is the exact problem with simple fixation with prolonged osmium postfix? Has the central portion of your lipid vacuole collapsed? If not, unless you want to try and measure what species of lipid is present, why worry? It is an artefact most of us crazy enough to study lipids have learned to accept.
2. Ultrarapid freezing followed by freeze substitution is probably the easiest method to preserve the bulk of the lipids in a reasonably similar location and volume. I haven't worked with adipose tissue, but atherosclerotic lesions have a high fat content and this has worked, even for quantitative ultrastructural analysis. the key is good freezing. There are a number of books on the subject and freezing has been discussed in this forum. In my experience, you will probably need to slam freeze if your tissue is of any size at all. Others with more experience than me can probably comment better.
3.Ultracyromicrotomy is a difficult thing (IMHO) and not for the faint of heart or inexperienced. If you go this route, hook up with an expert. Here again, good initial freezing is critical. The comment about getting an expert to help (physically) is probably also applicable to 2 above.
On 24 Aug 1994, Jeanne Barker wrote:
} Embedding adipose tissue } Does anyone have any suggestions on how to embed adipose tissue and to retain } all the lipid? I want to do some straight foward morphological studies. I } have tried a few different methods, and no matter what I do, in at least half } of my blocks, the center part has lost the lipid. } Also, how easy is it to freeze and do ultracryosections on this tissue? } Thank-you in advance. } Jeanne } } }
I suggest phosphate buffered fixatives, and a rapid dehydration schedule with cold acetone. Reference: Mary Williams et al in a pulmonary journal.
On 24 Aug 1994, Jeanne Barker wrote:
} Embedding adipose tissue } Does anyone have any suggestions on how to embed adipose tissue and to retain } all the lipid? I want to do some straight foward morphological studies. I } have tried a few different methods, and no matter what I do, in at least half } of my blocks, the center part has lost the lipid. } Also, how easy is it to freeze and do ultracryosections on this tissue? } Thank-you in advance. } Jeanne } } }
After reading the recent discussion of the LaB6 problem I was still unsure of the exact nature of the cathode failure. Am I correct in assuming that the reason the cathode was taken out of service was that the emission current became too high? Try looking at the base and the sub-base of the cathode. If there is evaporated material between the mount structures at either of these surfaces, you may have an electrical path which will show up as an increased emission current.
I started last year a program of salvaging old computers of no use in the states anymore, by helping nuns of the order of Our Lady of Perpetual Help. These nuns picked me up from the barrios of the Dominican Republic and put me on the right path to chose what I am today.
Old XT and slower machines (primarily MS-DOS based) of no use to us because of newer software demands are still powerful teaching tools for undeveloped countries (e.g., one of the school teaching typing does not have a single electric typewriter; they run 10 or more schools). I brought down with me last year to the Dominican Republic two XT we had here at Tulane in my department, and I am planing to return this fall with other machines. So far I have two electric typewriters and two potential XT 4.7 MHz.
I know firsthand that goods intended for the poor do not reach them first if donations are sent through certain agencies (e.g., my poor parents had to purchase milk and cheese from the soldiers handling donations from the US when I was 12). Thus, I am personally supervising transfer of goods and making sure that those goods stay in the hands of the nuns, and are exclusively used for teaching the kids of the 20 schools they operate throughout the Dominican Republic.
If you know of sources that could donate machines needing very minor repairs additions of floppies, etc. I would be most grateful. I am paying the cost personally for the expenses of repair and shipment, and there is no monetary gain whatsoever on my part from this; just a moral debt payment!
Those with machines to donate can contact me via internet or:
Hi everybody, I'm working with the ultrastructure of the biofilm which have colonised the tubing from dental chair unit. The biofilm consist of bacteria and water organisms which have adhered to the material surface. So if you can see the "biofilm.sea.hqx" and if you know the water organism, please send me his name. Thank you very much,
I would like to know if someone has experience sectioning cerebellum culture specimens. My problem is that this specimen seems to degrades new diamond knives by introducing striations on a new edge. Does anybody know wether cerebellum is inherently rough on the knives or if it may be my embeding techniques at fault. The embeding medium I use is LR white. Any comments would be appreciated.
-I would like to know if someone has experience sectioning cerebellum culture -specimens. My problem is that this specimen seems to degrades new diamond knives -by introducing striations on a new edge. Does anybody know wether cerebellum is -inherently rough on the knives or if it may be my embeding techniques at fault. -The embeding medium I use is LR white. Any comments would be appreciated.
I have cut lots of brain and some of it was cerebellum I have never had any problems. I cut mostly Poly Bed, some of it hard, some of it very soft, never any problem. Are your blocks to soft? You could be getting debris on the knife edge. I would try cleaning it first.
I have had problem with cutting cultured cells. That problem was that someone who changed the culture was using cheap glass pipets that had not been fire polished. Small pieces of glass got in the culture. I only had to hit one to know I had a problem. I alway processed the dishes with plastic pipets, but someone else grew the cells.
I would look for possible sources of contamination at each step. Check to make sure that new plastic items are used whenever possible. Are the grown on glass cover slip? Could something have gotten in the stocks of plastic componates? Look for any source of glass or dust or any like that. Someone else in your lab might be less careful that they should be.
} I would like to know if someone has experience sectioning cerebellum culture } specimens. My problem is that this specimen seems to degrades new diamond } knives } by introducing striations on a new edge. Does anybody know wether cerebellum } is } inherently rough on the knives or if it may be my embeding techniques at fault. } The embeding medium I use is LR white. Any comments would be appreciated. } } B. E. Mesa } 177000.1040-at-COMPUSERVE.COM
I have no experience with LR White, but I know that normal cerebellum, processed with routine methods, will not cause problems. I'd check the plastic to see whether it is too hard or inconsistent. Are you having problems with only a few blocks, or several batches of embedded specimens?
One thing you might try is to section with glass and see whether you get knife marks, and, if you do, maybe see whether they come from the plastic or the tissue. Another thing you might try is to section a blank block of the plastic, even turn the block around and section the opposite end. That way, you'd have blank plastic as close as possible in characteristics as what the tissue is in.
Good luck,
John chandler-at-lamar.ColoState.EDU Fort Collins CO
Hello Paul If you are looking for an basic introduction to dislocations, a book by Hull and Bacon is a good text, titled Introduction to Dislocations, Permagon Press, 1984. If you wish to know about images of dislocations in TEMs and how to characterise them, the monographs by Edington are useful (Practical electron microscopy in materials science, JW Edington, Van Nostrand Reinhold Company) as is Loretto's Electron beam analysis of materials, Chapman and Hall, 1994. Good luck Ciara
In message {199408251930.PAA12250-at-neuro.duke.edu} Larry Hawkey writes:
} B. E. Mesa 177000.1040-at-COMPUSERVE.COM wrote} } } -I would like to know if someone has experience sectioning cerebellum culture } -specimens. My problem is that this specimen seems to degrades new diamond } knives
Larry Hawkey responded:
} I have had problem with cutting cultured cells. That } problem was that someone who changed the culture was } using cheap glass pipets that had not been fire polished. } Small pieces of glass got in the culture. I only had to } hit one to know I had a problem. I alway processed the } dishes with plastic pipets, but someone else grew the cells. } } I would look for possible sources of contamination at each } step.
I only want to underscore what Larry Hawkey said about using glass pipets, as I have seen the same problem with tiny glass fragments carried into the resin and running into them with a knife: Bink! followed by: -at-#%*##&-at-#!!!!!
If you must use glass pipets, make a special set of cleaned pipets to use exclusively for your handling of fixatives, dehydrations series liquids, and embedding resins. I rinse them, inside and out, with distiled water, then ethanol, let air dry.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Message-Id: {9408252103.AA26010-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Re: Dislocation reference Orig-Author: {Ciara Mullan {mullanc-at-mcmail.cis.mcmaster.ca} }:ddn:wpafb ----------------------------------------------------------- Hello Paul If you are looking for an basic introduction to dislocations, a book by Hull and Bacon is a good text, titled Introduction to Dislocations, Permagon Press, 1984. If you wish to know about images of dislocations in TEMs and how to characterise them, the monographs by Edington are useful (Practical electron microscopy in materials science, JW Edington, Van Nostrand Reinhold Company) as is Loretto's Electron beam analysis of materials, Chapman and Hall, 1994. Good luck Ciara
As part of a surplus purchase of SEM specimen boxes, I have a few dozen used aluminum stubs that appear to go with Coates & Welter. Having an ISI/Topcon 'scope, I have no use for them. If you'd like them mailed to you, please contact me directly. Please *do not* reply to the list. Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
To all, re: recycled xylenes-- This problem can also be avoided by the use of Histo-Clear (from National Diagnostics), and a similar compound from Fisher & who else? It's made from essential oils of citrus plants & is non-toxic (& smells like oranges). I've used it a bunch, & it works as well as xylene for histological procedures. [I have no financial interests in Nst. Diag.] Phil Oshel poshel-at-luc.edu
Just a reminder of a discussion that we had several months ago.
DO NOT post images to this listserver. If you want to ask for help about a particuliar image then you should upload the image to an FTP site where individuals (who want) can download the file and try to give you a hand.
I do not want random individuals filling up everones Email boxes with huge unrequested files!
I'm sure there will always be someone who will look at the file but remember in all likelyhood a good fraction of the people on this server may not be interested in your data.
I have sent a copy of this notice to the individual who recently posted an image to the server. I see no need for further discussion.
John Mansfield and I have been considering establishing an upload area on an anonymous FTP site for people who do not have the ability to do this on their own. We will keep you updated as things progress. At the moment the anonymous FTP site both he and I maintain are READ ONLY..
I suggest you get in touch with my colleague Dr. Subash Chandra (e-mail: sc40-at-cornell.edu Phone: 607-255-4137) regarding protocols for preparing plant suspension cells for analysis with SIMS. Dr. Chandra is an expert in the application of SIMS technology to analyse biological material.
Please do not send multiple images over the net, especially on the same day. This will jam the email account if the quota is limited or full fill the floppy disc if you are using Eudora.
Ya Chen ---------------------------------------------------------------------------- ------ | Assistant Researcher/Cryo-SEM Coordinator | Integrated Microscopy Resource (IMR)-- | An NIH Biomedical Research Center | University of Wisconsin-Madison | 1675 Observatory Drive #167
Appearently Phillips no longer supplies the neccesary parts to put a 35mm camera on a Phillips 300. Anyone who has one that is not being used and would like to sell can either e-mail me at cal-at-ssnet.com with the info and I will forward, or contact Dr. Moudrianakis at 410-516-7305. Thanks, Cal
} Embedding adipose tissue } Does anyone have any suggestions on how to embed adipose tissue and to retain } all the lipid? I want to do some straight foward morphological studies. I } have tried a few different methods, and no matter what I do, in at least half } of my blocks, the center part has lost the lipid. } Also, how easy is it to freeze and do ultracryosections on this tissue? } Thank-you in advance. }
I don't know if you want to do optical microscopy or electron microscopy, but if you want optical microscopy, you may fix with 4% paraformaldehyde in 1% calcium chloride and 1% cadmium chloride. The material must be postfixed with 0,5 osmium tetroxide in sacarosis and embedded in glicol methacrilate. Cut the sections with glass knives. Good luck! ============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| r. 268 Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
I am looking for a fluorescence illuminator (or a vertical illuminator that I could modify into fluorescence) for my Olympus Vanox microscope or for the labs' BH-2. If you have a used Olympus illuminator that you would be willing to part with, please contact me directly. Please *do not* reply to the list. TIA Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
As one of the people who has been having the water droplets problem-this is a NEW problem which has just arisen. We have been using Histo-Clear for years with no problem-worked great, as did Hemo-De, the Fisher product. It has only been in the last couple of months that this has arisen, and it happens no matter which clearing agent we use-Histo-clear, Hemo-de or xylene. We used fresh xylene from the manufacturer- we have no access to a still to recycle our xylene. We also changed from using ethanol for dehydrating to using isopropanol, this has helped a bit but we still occasionally get the water droplets. We have increased our dehydrating and clearing times to 5 min in each alcohol and 3x10 min changes in clearing agent which reduces the problem greatly but increases the time it takes for coverslipping. We still do not know why this has started to occur after using standard methods for years.
Mark Elliott, PhD UBC-Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver BC Canada
I am trying to apply an historic stain named "Movats Stain", one of the components in this witches brew is "Wood Stain Scarlet". However no one has any idea who to get it from. The original suppliers got rid of their last batch in 1971! does anyone out there have a musty bottle in their archive, or perhaps know of a supplier or an equivalent dye
Thanks Simon C. Watkins Director SBIC University of Pittsburgh Pittsburgh PA 412-648-3051
Hi, does anyone know of any replacements for OsO4 as a conductive stain for biological specimens. Coating of complex extracted plant tissues for HRSEM with thin (2-3nm) layers of Pt or Cr has not proved enough to avoid charging. This is the case at both low and high kV. As a result I'm having to use OsO4 but would prefer not to, due to its deleterious effect on actin and in masking antigenicity. Ive tried to use uranium but with little success. Any comments or hints? Cheers
Peter
Peter Vesk, E.M. Unit,F09 University of Sydney NSW 2006 phone: 61 2 692 2351 (overseas) fax: 61 2 552 1967 peter-at-emu.su.oz.au
Peter, You might consider binding the OsO4 with thiocarbohydrazide (TCH) to increase the conductivity. See Platt-Aloia, K.A. and Thomson, W.W. 1980. Aspects of the three-dimensional intracellular organization of mesocarp cells as revealed by scanning EM. Protoplasma 104: 157-165. (and references within). Hope this helps.
Mike Postek and I experimented with this procedure about 15 years ago, using variations of the O-T-O (osmium thiocarbohydrazide osmium) technique. Using thiocarbohydrazide as a ligand, ruthenium red worked at least as well, and as I recall, gold chloride also worked. You might try other ligands as well, such as carbohydrazide and hydrazine instead of TCH...its more stable than the other 2, but not as effective. Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
In message Tue, 30 Aug 1994 03:21:13 -0400, peter-at-emu.su.oz.au (Peter Vesk) writes:
} Hi, does anyone know of any replacements for OsO4 as a conductive stain } for biological specimens. Coating of complex extracted plant tissues for } HRSEM with thin (2-3nm) layers of Pt or Cr has not proved enough to avoid } charging. This is the case at both low and high kV. As a result I'm having } to use OsO4 but would prefer not to, due to its deleterious effect on } actin and in masking antigenicity. Ive tried to use uranium but with } little success. Any comments or hints? } Cheers } } Peter } } Peter Vesk, } E.M. Unit,F09 } University of Sydney } NSW 2006 } phone: 61 2 692 2351 (overseas) } fax: 61 2 552 1967 } peter-at-emu.su.oz.au } ============================== Peter,
Try 0.5-1% tannic acid followed by unranium salt solution.
Peter- have you tried using a light tannic acid treatment at the prefix stage to protect actin etc against osmium? Helps both preservation and conductive staining, and may cut down the number of TA/Os cycles later....at low kV I suspect it should be enough by itself if you mount the specimen really carefully. Method in Stowe Fukudome and Tanaka, Cell Tiss Res 1986. Wont help the antigenicity though, I suppose!
Sally ---------------------------------------------------------------------- Sally Stowe | Australian National Univ. Facility Coordinator Canberra, AUSTRALIA ANU Electron Microscopy Unit | Ph 61 6 249 2743 Email stowe-at-rsbs-central.anu.edu.au | FAX 61 6 249 4891 -------------------------------------|-------------------------------- -
I have two questions for which I would appreciate some info back if possible. Also, I think they are applicable to list server goals. 1. Has anyone had any interaction with O 1. Has anyone had any interaction with OSHA or your own safety personnel concerning the interpretation of the prior apporval section of OSHA rule 29 CFR 1910.1450 (the so-called lab standard)? Has this caused anyone any problems?
In our institution, the literal interpretation of prior approval means that it is necessary to have the approval of the department head and the safety office for "any laboratory work with any hazardous substances." Prior approval shall "be required before implementing any new laboratory activities." Some people use the interpretation that prior approval is meant only for "exotic" chemicals. I would be interested in how other EM labs interpret the prior approval rule.
2. Does anyone know of an inexpensive on-line service which provides updated MSDS information? I am aware of a CD ROM service which will provide a CD ROM service on a quarterly basis but I would prefer on-line service.
John C. Wheatley Arizona State University CSSS PSB-234 Tempe, AZ 85287-1704 TEL: 602-965-3831 FAX: 602-965-9004
One of our students is attempting to determine the extent of live cells from the sapwood of tree cores. It has been suggested that 1% aqueous triphenyl tetrazolium chloride may be used to stain for both macroscopic and microscopic determinations of living cells. Is anyone aware of additional staining/microscopy procedures which may be used to make a reasonably accurate quantitative determination of the extent of live cells in sapwood? Richard Crang Professor Plant Biology UIUC (217) 244-3143
One of our students is attempting to determine the extent of live cells from the sapwood of tree cores. It has been suggested that 1% aqueous triphenyl tetrazolium chloride may be used to stain for both macroscopic and microscopic determinations of living cells. Is anyone aware of additional staining/microscopy procedures which may be used to make a reasonably accurate quantitative determination of the extent of live cells in sapwood? Richard Crang Professor Plant Biology UIUC (217) 244-3143
} I am looking for a low cost PC based SLOW SCAN frame grabber } for taking images from an SEM. Any ideas? } Thanks. } } Rob Thomson } EM Facility } Victoria University } Wellington } New Zealand
We are using the ImageSlave System developed by Meeco/Dindema in Sydney Aust. It allows image collection at the photograhic scan rate on our Hitachi S4100 FeSEM. It is also possible to use the memory photograph function via the ImageSlave. The cost is around $3000 (very approximate) AUD. It runs under DOS on a PC.
We have found the system to be very effective.
The address is:
MEECO HOLDINGS 10 Seville St. North Parramatta NSW 2151 AUSTRALIA
Tel + 61 2 630 7755 Fax + 61 2 630 7365
I hope this of use to you.
Colin
##################################################################### ******************************* * Logic is invincible because * 0------* in order to combat logic it * } ---|--- { * is necessary to use logic. * | * P.Boutroux * / \ ******************************* _/ \_
Colin Veitch Tel + 61 (0)52 27 5611 CSIRO Division of Wool Technology Tel + 61 (0)52 27 5891 (dir.) P.O. Box 21 Fax + 61 (0)52 27 5657 BELMONT Vic 3216 Australia
John C. Wheatley wrote} } 1. Has anyone had any interaction with OSHA or your own safety personnel } concerning the interpretation of the prior approval section of OSHA rule 29 } CFR 1910.1450 (the so-called lab standard)? Has this caused anyone any } problems?
We just had a meeting with the Safety Office here at Duke. They are meeting with all Medical Center personnel. This was a two hour session that covered fire safety, Radiation safety, and other things. Nothing was said about use of hazardous chemicals. As far as I know I can use any chemical I want to in my lab. (we are a research dept. not hospital or clinical.) I think that if I want to use radiation, I must clear it with the Radiation Safety office. This is for disposal more than use I think.
Our Safety office does little with training or clearing the use of Hazardous. The only thing they said was that lab personnel have the right to know what they are working with and their supervisor is responsible for telling them. Since I am the only one in the EM lab I guess it is my problem.
I would also like to see some on line service for MSDS information. I would think that some goverment office would provide that for free?
Hello! I am doing some EM on oysters and looking at protists in the tissue. I was wondering if anyone might have a good receipe for a fixative that would be good for fixing the oyster tissue and the protists. I currently use 2.5% glut in 0.2M cacodylate buffer, pH 7.4. Should the fix and the buffer rinses be made up in filtered sea water? What might be the best pH and osmolarity of the fix and buffer? TIA, Phil
} Our Safety office does little with training or clearing } the use of Hazardous. The only thing they said was that } lab personnel have the right to know what they are working } with and their supervisor is responsible for telling them. } Since I am the only one in the EM lab I guess it is my } problem.
If I'm not mistaken, you are also responsible for informing anyone who comes into the lab, especially if they will be doing any work, about hazards they might run into. Some of these issues come down to legal liability, in case of accident or injury.
It's not an easy topic, and I think we all would do well to be aware of the hazards around us, chemical, radiation, electrical, vacuum, etc., and not become too casual.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
I definitely agree that the quality of calibration replicas has decreased over the last 10 years. We calibrate our microscopes every semester and go through many replicas as we have 75 students using them each semester and so many are distorted, of various contrasts, difficult to see, etc. It is almost if there are replicas being made of replicas. I have tried to check them in a calibrated light microscope and they definitely are of different sizes. I would appreciate knowing what the quality assurance is when making them and the statistical deviation of what the measurement should be claimed by the manufacturers.It sure makes for interesting curves!! Perhaps if the manufacturers who make them are listening, they could give us some input. Thanks, Judy M.
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
Microscopy Society of America contact is: Caroline Schooley Box 117 Caspar, CA 95420 707/964-9460 The program is in conjunction with the Lawrence Hall of Science in Berkeley and is in conjunction with the middle schools. judy m
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
I FORGOT TO SAY THAT WE NEED TO CONTRIBUTE SOMETHING IN THE LINE OF MICROSCOPY TO THIS KIT;IF THAT DID NOT COME THROUGH CLEARLY IN MY 1ST MESSAGE! MY APOLOGIES !
Message-Id: {1994Aug26.174159.898881934-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy)
To: microscopy
I definitely agree that the quality of calibration replicas has decreased over the last 10 years. We calibrate our microscopes every semester and go through many replicas as we have 75 students using them each semester. Many are distorted, of various contrasts, difficult to see, etc. It is almost if there are replicas being made of replicas. I have tried to check them in a calibrated light microscope and they definitely are of different sizes. I would appreciate knowing what the quality assurance is when making them and the statistical deviation of what the measurement should be, claimed by the manufacturers.It sure makes for interesting curves!! Perhaps if the manufacturers who make them are listening, they could give us some input. Thanks, Judy M.
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
There have been some very good points made in this thread, especially in Bob Craig's response. Most research labs are far too relaxed on safety and training. Someone in every lab or department, especially where there are multiple users, particularly students, grad students, and clinical students, must be the designated safety officer. Specific issues like fire, chemicals, radiation, biological hazards, etc. can be delegated to individuals iwth expertisew, preferably who are actively using these agents. These people should have some authority along with this responsiblility. If someone is not competent with these agents, they shouldn't be allowed to use them. The University of Washington, at the command of the Seattle Fire Dept., requires that each "Chemical Use Area" i.e. laboratory, have a designated contact person who maintains an inventory of all chemicals stored in that area. The primary purpose is so that the fire dept. knows what to expect in case of emergency. The side effect has been that many labs, including our own, finally realized what they had on their shelves. In our case, many users had been planning experiments and purchasing chemicals without looking to see what was already available. the result was many duplications, outdated chemicals and chemicals that were improperly stored. I weeded the shelves, consolidated chemical purchases and created a Hypercard based inventory system. If anyone is interested in beta-testing the update to this inventory system, reply to me directly.
Further, and it is a pain, but the best first line of defense are procedure manuals. Write a page or two describing proper use, (realistic) hazards, and proper disposal for particularly hazardous chemicals. Include this in your notebooks with the recipes and procedures that use those chemicals. It takes time but each of us is directly in a position of risk from our own actions, as well as everybody else's. I've grouped many of these together for groups of compounds that are related or used for similar purposes. For example, our fixatives share a couple of pages of instruction. Osmium and DAB have their own guidelines.
With 20 or so users in our labs, we found it essential to create a "Main Brain" list. This one page lists various labs, equipment, procedures and reagents and gives the name of someone responsible for training in their use. Even faculty don't touch things until getting instruction from the Main Brain person.
Make it clear to all newcomers that they are expected to read these instructions and contact the Main Brain for whatever they do. Mistakes will still occur, but with less frequency and fewer consequences.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
For those still interested in the earlier question, here is additional information re: EM lab safety, courtesy of one of our labs OSHA safety inspectors/industrial hygenists.
-----------------------------
There is a (relatively, i.e. 1990) new OSHA regulation that applies specifically to laboratory use of hazardous chemicals: 29 CFR 1910.1450 "Occupational Exposure to Hazardous Chemicals in Laboratories." It's primary requirement involves developing and implementing a Chemical Hygiene Plan that sets forth procedures, equipment, personal protective equipment and work practices that are capable of protecting employees from health hazards associated with hazardous chemical use (chemicals that meet the definition of "health hazard" under OSHA Hazard Communication Standard). Employers who might fall under this application should get a copy of the regulation from their regional OSHA office; also available is a small pamphlet "OSHA 3119 - Exposure to Hazardous Chemicals in Laboratories" from the OSHA Publications Office (202) 523-9667. -- David
................................................................. David I. Jacobi (dj5-at-gtri.gatech.edu) Georgia Tech Research Institute/EOEML 022F O'Keefe Building, Atlanta, GA 30332-0837 Phone: 404/894-8089; FAX: 404/894-8275 Elizabeth (Eliesh) G. O'Neil Research Scientist I GTRI/EOEML Baker 271, 925 Dalney Street Atlanta, GA 30332-0827 (404) 853-0590 (office) (404) 894-6199 (FAX)
Maybe one of the manufacture-ers/distributers of slides & coverslips is watching: is there some film now being applied to slides & coverslips, or a change in cleaning methods that might leave such a film? Also: Mark Elliot: are you mounting with Histo-Mount? I found that that makes a real difference when using Hisot-Clear. Phil Oshel poshel-at-luc.edu
John C. Wheatley: Glad to see ASU worries about OSHA [& RCRA?]--you may be unique. For MSDS sheets--Sigma, Baxter, & I think Fisher--by law, everyone who sells chemicals, I think--provides free MSD sheets by call to 800 #'s in there catalogs. Most will fax if mail is too slow. Phil Oshel poshel-at-luc.edu
Phil P., Yes, I've found that 0.2 micron filtered seawater helps much with marine orgs. You may still need fome extra buffering for the pH. Karnovsky's or 4:1 formaldehyde:glut as per Dykstra works well as a fix for molluscs & protista--as a first try anyway. UNESCO published a book on the fixation & preservation of marine animals, esp. zooplankton. You might also try writng marine labs for theirs methods--many have written technique books--like U Alaska Fairbanks & MBL at Woods Hole. Phil Oshel poshel-at-luc.edu
Concerning type of glass used: I am doing cross sectional TEM on vacuum deposited thin films on glass slides, and noticed that several types of glass have a thin surface film structure different in composition than the bulk. For ex, some has a layer denuded of Na (sodaglass), corning glass has a layer denuded of Ba. Layer thicknesses measured about 50nm to 100nm.
Could it be that these layers are present in ordinary glass slides or cover glasses, and has different wetting characteristics ?
Dirk Knoesen Dept Atomic and Interface Physics, Utrecht Univ, Netherlands
Someone replied directly to me re: my slide water droplets comments--Maillot?--but the mssg got erased before I could read it. If this makes sense to whoever sent the note, could they resend it, please? Phil Oshel poshel-at-luc.edu
Yes, chemicals (senus lato.) are supplied with MSDS sheets. And I dutifully read them, catalog them, and inform all my users in the facility of their existance right in the middle of the chmical work lab. However my concern is the really limted amount of information provided by them. Particularly in regards to disopsal of small quantities used in University settings. The MSDS sheets tend to regard spills of tank car quantities not 2 ml lab quantiies, and state that you follow "State and local quidelines". Well state and local quidelines say nothing about 2ml of OsO4 or DAB.
Working with a full respirator and protective suit for 30ml of glutaraldehyde might be very safe, but is a little over kill, and prohibitively expensive, especially when down the hall there are 35 students hunched over several formalin infiltrated corpses in the gross anatomy lab. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
JOURNAL OF MICROSCOPY, VOLUME 175, PART 3, SEPTEMBER 1994
This issue features five invited contributions, originally presented at the 6th European Congress for Stereology, Prague (September 1993).
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 176-185
Stereological analysis of the spatial Poisson-Voronoi tessellation
UTE HAHN & UDO LORZ, Freiberg University of Mining &, Technology, Institut fur Stochastik, Bernhard-von-Cotta-strasse 2, D-09596 Freiberg, Germany
SUMMARY
Stereological model tests and parameter estimators for the spatial Poisson-Voronoi tessellation are discussed. The tests aim to discriminate the Poisson-Voronoi tessellation from more regular or more irregular tessellations. The power of the model tests under some special parametric alternative hypotheses is investigated by simulation. Among the tests considered, the most powerful test is based on the variance of the section cell areas. Various stereological estimators for the model parameter of the spatial Poisson-Voronoi tessellation are compared with respect to their bias and variance by means of a Monte-Carlo study. Formulae are given for variance prediction. An estimator based on vertex counting is found to be best. Robustness is investigated by applying the estimators to Voronoi tessellations generated by other point process models.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 186-194
Second order stereology for pores in translucent alumina studied by confocal scanning laser microscope
LARS KARLSSON & ANDERS LILJEBORG, Royal Institute of Technology, S-100 44 Stockholm, Sweden
SUMMARY
The three-dimensional (3-D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3-D image of the pores. Computer-based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second-order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3-D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 micrometre.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 195-204
Analysis of homogeneity of phase repartition in TiB2-Fe composites using variance and covariance analysis
J. M. MISSIAEN & J. M. CHAIX, Laboratoire de Thermodynamique et, Physico-Chimie Metallurgiques, CNRS URA 29 ENSEEG, BP 75 Domaine Universitaire, F-38402 Saint-Martin d'Heres Cedex, France
SUMMARY
The microstructure of sintered TiB2- Fe material has been studied by image analysis. The dispersion of phases, with particular attention to porosity, is investigated using chord lengths, covariance functions and variance of volume fractions. An extension of the usual point covariance analysis is made by sampling series of 10 contiguous fields, and measuring the covariance in the direction of field alignment. A model of multiscale structure is proposed to interpret the different transitions of the variogram as composition fluctuations at different scales. Boolean models are used to give a geometrical representation of these fluctuations: the dispersion of porosity can be seen either as 20-30-micrometre pore clusters or as a result of TiB2+Fe 30-50-micrometre clusters, probably formed during the elaboration process. Complementary information is obtained from the evolution of measured variance versus field size: a model is proposed to analyse the shape of the experimental curves. At small scales, 'short' integral ranges are determined, also obtained from the covariance. At very large scales (} 300 micrometre), composition fluctuations of about 2% are detected.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 205-213
Modelling of vascular growth processes: a stochastic biophysical approach to embryonic angiogenesis
KONRAD SANDAU & HAYMO KURZ, Mathematik und Naturwissenschaften, Fachhochschule Darmstadt, Shofferstrasse 3, D-64295 Darmstadt, Germany
SUMMARY
In a computer simulation, growth of a capillary network is driven by a stochastic process on a planar hexagonal grid. Starting at a point source, the probabilities for the formation of new capillary elements depend on local biophysical knowledge. This knowledge is mainly derived from the flow theorem of Hagen- Poiseuille and the diameter exponent. The hexagonal grid is visualized as being supported by a cylinder or a sphere. An arterial tree results from the adaptive diameter augmentation, and is considered to have limited fractal properties. The dimension of its border, and the time course of growth and of blood pressure are compared with biological data from the chorioallantoic membrane (CAM) of incubated chicken eggs. The model is discussed with reference to the mechanosensitivity and cell-matrix interactions of endothelial cells, and CAM haemodynamics.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 214-221
Shape processing and analysis using the calypter
ERIC PIRARD, Universite de Liege, Laboratoires de Geologie Appliquee, Avenue des Tilleuls 45, 4000 Liege, Belgium
SUMMARY
After presenting some preliminary criteria that should be respected by any automatic shape analysis technique, this paper focuses on the importance of the binary image encoding method. Most image analysers simply use a raster image to represent a binary object. If, occasionally, a vectorial description is available, it is merely chosen for its performances in data compression. Data compression and shape analysis have different goals and usual methods cannot satisfy both. The calypter is a new descriptor vectorizing the shape as a set of maximal inscribed discs. It is the most efficient means of accomplishing Euclidean mathematical morphology transformations and allows for further developments in binary shape processing. A simple adaptive contour filtering technique is presented. The calypter offers local and global perception of shape characteristics. It permits complete automation of the morphometric roundness charts used in many laboratories and also generates new shape parameters. A case study of three sand populations is presented to show the pertinence of a new 'equivalent roundness' parameter.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 222-228.
Retention of vacuole contents of plant cells during fixation
Z. DONG, M. E. MCCULLY & M. J. CANNY, Department of Biology, Carleton University, 1125 Colonel Drive, Ottawa, Ontario, Canada K1S 5B6
SUMMARY
Changes in the semi-permeability of tonoplast during fixation were studied using beetroot tissue. Using vacuole betacyanin as the indicator, the permeability of the tonoplast was assessed by the leakage of this pigment as determined by changes in the optical density of the solution bathing the tissue. Cryo- analytical scanning electron microscopy was used to monitor the changes in ion concentration in cells during fixation. Fixatives were 3% glutaraldehyde or 4% formaldehyde in 0.025M phosphate buffer at room temperature or on ice. Results showed that glutaraldehyde, especially at low temperature, takes as long as 30h to disrupt the semi-permeability of the tonoplast of beetroot cells, while in formaldehyde on ice, beet cells lose their selective permeability in 15min. This study confirms that the semi-permeability of the tonoplast may not be lost until long after the cytoplasm has been fixed and suggests that this explains why cold fixation in 3% glutaraldehyde for about 12h has become the most reliable standard procedure for the successful preservation of vacuolated plant cells.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 229-237
Central and peripheral nervous structures as seen with the confocal scanning laser microscope
P. CASTANO, A. MARCUCCI, A. MIANI Jr, M. MORINI, S. VERALDI & C. RUMIO, Institute of Human Anatomy, Via Mangiagalli 31, 20133 Milano, Italy
SUMMARY
Central neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi-Cox mercuric impregnation, however, provides specimens which are precipitate- free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He-Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 238-251
Optimizing the performance of confocal scanning laser microscopes over the full field of view
A. ENTWISTLE & M. NOBLE, The Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1P 8BT
SUMMARY
To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view, three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of the plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.
Journal of Microscopy, Vol. 175, Pt. 3, September 1994, pp. 252-265
HREM image simulations for supported particle catalysts
MING-HUI YAO & DAVID J. SMITH, Centre for Solid State Science, Arizona State University, Tempe, Arizona 85287-1704, USA
SUMMARY
The imaging conditions for electron microscope studies of supported ultrafine particle catalysts have been investigated by multislice simulations. Images of Pt and ReO4 particles ranging from 0.4 to 2.3 nm in size were simulated in both plan view and profile view with a rutile (TiO2) support. It was shown that particle visibility varied greatly with the objective lens defocus. Optimum defocus was not favourable for supported particles in plan view since the ultrafine supported particles were the least visible at this defocus. Underfocusing, especially at defoci corresponding to half-spacing fringes in the TiO2 support, led to improved visibility and resolution of the supported particles. Although the structure and shape of supported ultrafine particles should be resolved better with a 400-kV high-resolution electron microscope, their detectability is poorer than with a 200-kV instrument. An ReO4 cluster should be detectable at 200kV on TiO2 supports up to 5nm in thickness, whereas it is only likely to be detectable at 400kV on supports up to 3nm in thickness. The simulations confirmed that optimum defocus is mst favourable for imaging supported particles in profile view. Atomic information for particles as small as a 13- atom Pt cuboctahedral cluster should be resolvable with a 400-kV instrument. The crystalline Ti monolayer observed on surfaces of Pt particles, which could explain the mechanism known as SMSI, was simulated as an example of profile imaging.
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 12:08
Date:9/2/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Hi there, I am about to buy a video printer to replace the majority of our Polaroid prints on our SEM. I want something that will give me about Polaroid size (i.e. 4X5 inch ) but is cheaper. I think Sony and Mitsubishi make printers in this area. Printer needs to be 256 greys and needs to be connected to both an ESEM monitor and also a Mac monitor. Anyone have any suggestions as to the best product? Any to avoid? reply by email please and I will summarize to the net if need be. Please dont send messages saying I should be buying a Dye Sub printer, we have one, I want cheap student notebook type output.
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 12:14
Date:9/2/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Sorry to ask a non microscopy question, but has anyone experience with PC and Mac to S-VHS converters? Actually it is microsocpy related, I want to video tape the Digital Instruments AFM in action (NO, I dont want to point a camcorder at the screen!). I need something that costs less than an arm and a leg and does the job nicely. Any experiences or comments? Email replies please. Thanks. john Mansfield
1.Who can provide me with information on lectin based labeling of carbohydrate polymers (e.g.EPS) for EM (and LM) studies? 2.Does someone have experience in thin film observations of carbohydrate polymers in TEM? 3.Does someone have suggestions (e.g. literature references) for chemical fixation of carbohydrate polymers (EPS) for TEM or SEM observations?
Regards,
Marcel Paques Unilever Research Laboratory The Netherlands Phone:(31)10-4605515 Email: Marcel.Paques-at-URLNL.Sprint.Com
We want to do the same thing as John, so if you would cc: to me as well I would appreciate it. Thanks-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On 2 Sep 1994, John Mansfield wrote:
} John Mansfield } North Campus Electron Microbeam Analysis Laboratory } 413 SRB, University of Michigan } 2455 Hayward, Ann Arbor MI 48109-2143 } Phone: (313)936-3352 FAX (313)936-3352 } jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: } 12:14 } } Date:9/2/94 } URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL } Subject: PC and Mac to S-VHS } } Sorry to ask a non microscopy question, but has anyone experience with PC and } Mac to S-VHS converters? Actually it is microsocpy related, I want to video } tape the Digital Instruments AFM in action (NO, I dont want to point a } camcorder at the screen!). I need something that costs less than an arm and } a leg and does the job nicely. Any experiences or comments? } Email replies please. } Thanks. } john Mansfield } }
Dear Scott, Two referrences for the information you need are: 1) Berger & Seltzer, in Studies in Penetration of Charged Particles in Matter, Pub. 1133, NAS-NRC (1964), available from NAS printing & publishing office, 2101 Constitution Ave, N.W., Washington DC -at-0148. 2) Berger & Seltzer, Stopping Powers and Ranges of Electrons and Positrons, NBSIR 82-2550 (1982) Office of Standard Referrence Data, National Bureau of Standards, Washington DC 20234. As you can see by the second address, these addresses may be outdated. What you need to do is to use the relationships 1/R = w1/R1+w2/R2+... and S = w1S1+w2S2... where the w's are weight fractions. These relationships are good to a few %. The proper units for the range & stopping power are g/cm**2 and MeV*cm**2/g. The earlier ref gives data for Kr & Ag and the later gives data for Kr and Mo. You will have to interpolate the data to get the values for Nb; N is listed in either ref. I can fax you the appropriate pages if you can't get the refs for your- self. I know we have been guarding our copies jealously--they've been here longer than I have. Who knows, NIST or NAS may even have more up-to-date pubs. If so, I'd be interested in obtaining them. Good luck. Yours, Bill Tivol
Regarding grating replicas... just remember Norbert's incredible efforts to make calibration possible with the light microscopes in the last century.\nina allen
We are looking for initial expressions of interest in the following position. I would be grateful if this could be advertised as widely as possible. Any at all interested should send a short CV to my e-mail address below, or fax (61)-9-380-1087, within the next few days.
Many thanks, Andy Johnson, Centre for Microscopy and Microanalysis, University of W.A.
TEACHING AND RESEARCH FELLOW A proposed joint position at The University of Western Australia between the Centre for Microscopy & Microanalysis, the Department of Physics and the Department of Chemistry with the possible involvement of the Department of Mechanical & Materials Engineering.
We are applying for a three year teaching and research position which will have good prospects of continuation for a person with a sound PhD in Physics, Chemistry or Engineering, some research experience involving transmission electron microscopy and electron energy loss spectrometry and an interest in teaching. We envisage the position will combine teaching and research of the latest electron energy loss imaging and diffraction techniques to Honours and Graduate Students with research in that area. The research equipment comprises a Philips EM430 TEM fitted with a Gatan Image Filter housed in the University's Centre for Microscopy & Microanalysis. The Centre also has a wide range of electron microscopes (3 SEMs, a FESEM, ESEM, JEOL 2000FXII TEM) and a microprobe. All are equipped with EDS and modern attachments. The staff of the Centre are actively engaged in teaching and research programs involving electron and confocal microscopy through which the Centre is in close collaboration with the academic departments of the University of W.A., other major universities in Perth and with industry. The fellow will be expected to be actively engaged in similar programs and in the courses of the participating Departments as well as their own research.
At present the University has called for preliminary submissions of the names of potential Fellows who, if successful, will be required to take up their positions by mid 1995.
Some of the important selection criteria are: * applicants will have completed a PhD or equivalent within the previous five years. * the quality of the applicants will be assessed by: their academic record, current & potential performance in research, potential performance in teaching.
This is a wonderful opportunity for a young person to advance their career in a broad academic environment.
Please send your CV, a short version is sufficient at this stage, without delay to Dr Andrew Johnson, Director, Centre for Microscopy and Microanalysis, University of W.A.. email: andy-at-earwax.pd.uwa.edu.au fax 61-9-380-1087
I recently purchase a Reichert Zetopan microscope and am trying to locate certain accessories for it. If anyone has any such items or know of individuals who might, please contact me with descriptions and prices or I will provide a list of desired items. Please contact me directly at:
e-mail: HOWEY-at-UWYO.EDU
OR
Richard L. Howey 703 So. 10 Laramie, WY 82070
Home Tel: (307) 742-3545 Office Tel: (307) 766-2200
You were asking about video printers for an ESEM. We have been using the Mitsubishi model p68 for several years now and are very satisfied. Each of our SEM's (3) has a printer attached which is switchable between the SEM screen and an EDS/MCA display. The Mitsubishi printer takes inputs as RGB (TTL or analog/PC or Mac) as well as regular composite video. We have set our printers up with a switch to toggle the inputs between video and RGB, allowing quick sequential images from either source. The switch connection, across the appropriate DIP switch on the rear of the printer, can be easily installed by a competent technician.
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(61 6) 2493543 Fax:(61 6) 2494891 E-mail:vowles-at-rsbs-central.anu.edu.au
You were asking about video printers for an ESEM. We have been using the Mitsubishi model p68 for several years now and are very satisfied. Each of our SEM's (3) has a printer attached which is switchable between the SEM screen and an EDS/MCA display. The Mitsubishi printer takes inputs as RGB (TTL or analog/PC or Mac) as well as regular composite video. We have set our printers up with a switch to toggle the inputs between video and RGB, allowing quick sequential images from either source. The switch connection, across the appropriate DIP switch on the rear of the printer, can be easily installed by a competent technician.
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(61 6) 2493543 Fax:(61 6) 2494891 E-mail:vowles-at-rsbs-central.anu.edu.au
I recently purchased a Reichert Zetopan microscope and am trying to locate certain accessories for it. If anyone has any such items or knows of individuals who might, please contact me with descriptions and prices or I will provide a list of desired items. Please contact me directly at:
e-mail: HOWEY-at-UWYO.EDU
OR
Richard L. Howey 703 So. 10 Laramie, WY 82070
Home Tel: (307) 742-3545 Office Tel: (307) 766-2200
We have a Siemens electronmicroscope no.302 here, still in good condition and with replacement electronic bulbs and numerous spare parts here.It was produced in 1959. It appears to be of scientific historic interest and might be offered to a technical museum . Morten M.Laane ,Prof.of biology , University of Oslo.
We have a Siemens electronmicroscope no.302 here, still in good condition and with replacement electronic bulbs and numerous spare parts here.It was produced in 1959. It appears to be of scientific historic interest and might be offered to a technical museum . Morten M.Laane ,Prof.of biology , University of Oslo.
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I've gotten a few messages recently asking why messages posted to the newsgroup donot appear to Microscopy and this is the reason why.
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Dear microscopists,
1.Who can provide me with information on lectin based labeling of carbohydrate polymers (e.g.EPS) for EM (and LM) studies? 2.Does someone have experience in thin film observations of carbohydrate polymers in TEM? 3.Does someone have suggestions (e.g. literature references) for chemical fixation of carbohydrate polymers (EPS) for TEM or SEM observations? 4.Does someone have suggestions (e.g. literature ref.) for specific staining procedures of carbohydrates for CSLM observation?
Regards,
Marcel Paques Unilever Research Laboratory The Netherlands Phone:(31)10-4605515 Fax:(31)10-4605671 Email: Marcel.Paques-at-URLNL.Sprint.Com
John- some of the best printers that we have looked at are from: Alden Electronics Inc. (508) 366-8851 40 Washington St. Westborough, MA 01581 they have several models which produce quality images for under $1.00/print. -Mike On 2 Sep 1994, John Mansfield wrote:
} John Mansfield } North Campus Electron Microbeam Analysis Laboratory } 413 SRB, University of Michigan } 2455 Hayward, Ann Arbor MI 48109-2143 } Phone: (313)936-3352 FAX (313)936-3352 } jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: } 12:08 } } Date:9/2/94 } URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL } Subject: Video Printers } } Hi there, I am about to buy a video printer to replace the majority of our } Polaroid prints on our SEM. I want something that will give me about } Polaroid size (i.e. 4X5 inch ) but is cheaper. I think Sony and Mitsubishi } make printers in this area. Printer needs to be 256 greys and needs to be } connected to both an ESEM monitor and also a Mac monitor. Anyone have any } suggestions as to the best product? Any to avoid? } reply by email please and I will summarize to the net if need be. } Please dont send messages saying I should be buying a Dye Sub printer, we } have one, I want cheap student notebook type output. } }
Nestor, General message to microscopy list received at this site, understood, and information filed for future reference. I double checked to make sure had proper address in address book. Jay
Nestor- apologize for cluttering your mail box with last message and this apology. I'm reading this via modem and it sometimes confuses text from one screen with the next screen. Your message was printed out please read and reply....after replying it reprinted text and this time said read and comply. I thought you were a terrible masochist requesting 1300 replies, but who am I to question the sysop. Jay
Anybody out in cyberspace have any experience with Unicryl resin? The ad says it is a hydrophilic LM & EM resin that stains readily with routine polychromatic stains like safranin. I currently use Epon but it requires etching with NaOH saturated ethanol to allow good safranin staining of mucin in goblet cells. if i could skip that step and still be able to use the same blocks for EM, it would be nice. any comments on this resin would be appreciated.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Microscopy Research and Technique will be making its annual changes (rotation) to the Editorial Board for 1995. If you are interested in becoming an Editorial Board Member, please reply to my E-Mail address directly 75022.2723-at-Compuserve.com listing your name, affiliation, mailing address, phone number, Fax number, research interests, and most recent three publications.
Dear Alex, I can take you *back* a decade: Elementary Excitations in Solids by David Pines discusses electrons, phonons and plasmons from what appears to be a non-relativistic quantum-mechanical point of view. There are probably some referrences with QED as their starting point; however, I don't have any. For the main result you need, the stopping-power tabulations in Berger & Seltzer are probably your best beginning--see a previous post of mine. Yours, Bill Tivol
Dear Scott, for number of years I use average Z values calculated from D. Newbury's paper "Fundamentals of scanning electron microscopy for physicists: contrast mechanism" published in SEM 1977/I, IITRI, Chicago, pp 553-568. You can also find the particular formula in my paper in Scanning Vol.3, 4 1980 pp 288-291. To make it easy for you we wrote the programme calculating Z average. This is how you can get it: ftp to lab.csl.utas.edu.au change directory: cd pub/cameca get backsc.ftn quit Although programme is selfexplanatory you should try it on something easy at first such as SiO2----should give you value 10.69(!). Please contact me directly if you need more on the subject. Regards, Wis Jablonski OiC EM/XRay microanalyis at University of Tasmania, Australia Email W.Jablonski-at-csl.utas.edu.au
Dear Scott, For number of years I have been using average Z values calculated from formula by D. Newbury ( Fundamentals of scanning electron microscopy for physicists: contrast mechanism published in SEM 1977/I, IITRI, Chicago, pp 553-568). You can also find this formula with some practical applications in my paper in SCANNING Vol 3, 4 1980 pp 288-291. To make it easy for you we wrote the programme calculating Z average which includes W.This is how you can get it: ftp to lab.csl.utas.edu.au change directory: cd pub/cameca get backsc.ftn quit Although programme is self-explanatory you should try first on something easy such as Si and SiO2 ---average Z 14 and 10.69 respectively. Please contact me directly if you need more on the subject. Regards, Wis Jablonski OiC EM/XRay microanalysis at University of Tasmania, Australia. Email W.Jablonski-at-csl.utas.edu.au
How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment (JEOL 2010) ?
Any suggestion appreciated (inversely as the cost). Thanks in advance.
Armel Le Bail - Laboratoire des Fluorures, CNRS-URA-449, Universite du Maine, 72017 Le Mans Cedex, FRANCE - armel-at-ONE.univ-lemans.fr or lebail-at-naimn2.cnrs-imn.fr
How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment (JEOL 2010) ?
Any suggestion appreciated (inversely as the cost). Thanks in advance.
Armel Le Bail - Laboratoire des Fluorures, CNRS-URA-449, Universite du Maine, 72017 Le Mans Cedex, FRANCE - lebail-at-naimn2.cnrs-imn.fr or armel-at-ONE.univ-lemans.fr
There are other alternatives to Unicryl. LR White and LR Gold are both reasonably good EM/LM resins and are hydrophyllic - allowing fairly easy use of LM stains. JB-4 is an excellent LM resin with excellent LM staining properties, but can't be used for EM work. LR White and JB-4 both cost significantly less than Unicryl (i.e. %25 as much) and there doesn't seem to be additional benifits to the unicryl to justify the cost, specifically for general LM staining, as opposed to immunolabeling (for which I haven't heard of a good justification of the benifit vs. cost either).
You might consider one of these resins instead of Unicryl. But I am interested to here what other people have to say.
(Oh, yes I have no finacial ties with any of the above information or manufacturers)
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
For those of you out there who are tired of hand writing out tinney tiny little penciled labels for embedding with your tissues I have been running tests with HP Laserjet printed labels, with excellent results so far. It seems that the electrostatic deposition of the carbon black pigments (in the genuine HP tonner cartridges anyway) do not disolve in either Spurr's or Quetol 651 resin (I haven't tried any of the other yet) and do not seem to effect the blocks in any manner. You might want to give it a try.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
Several people have asked about apparent missing messages. Due to the volume of mail this server is now handling I've had to restrict the number of attempts which mail tries to be delivered to inactive sites. Delivery is attempted twice for each address at ~6 hour intervals if it is not delivered on the second try then it the attempt to that particuliar address is stopped. I'm trying to find the best compromise in this procedure, since if we wait too long, then the mail queues get huge, but if we don't wait long enough, people who shut off their computers or whose nodes go off line do not receive mail. The 6 hour 2 attempt interval appeared reasonable for a start. I'd like to let it run for a bit longer, before changing it to another value.
Comments, suggestions will be appreciated, but do it off-line and to me direct....
} Date sent: Wed, 07 Sep 1994 10:01:13 +1100 } From: "Richard E. Edelmann" {REDELMAN-at-musom01.MU.WVNET.EDU} } Subject: Embedding Labels } To: Microscopy-at-aaem.amc.anl.gov } Organization: MU School of Medicine } Priority: normal
} } For those of you out there who are tired of hand writing out } tinney tiny little penciled labels for embedding with your tissues I } have been running tests with HP Laserjet printed labels, with } excellent results so far. It seems that the electrostatic deposition } of the carbon black pigments (in the genuine HP tonner cartridges } anyway) do not disolve in either Spurr's or Quetol 651 resin (I } haven't tried any of the other yet) and do not seem to effect the } blocks in any manner. You might want to give it a try. } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } Marshall University - School of Medicine } Huntington, West Virginia } A good description of this method can be found in J. A. Dant and R. E. Kingsley - Laser Printing Labels For TEM. Hints and Tips EMSA (MSA) Bulletin 21(1):67. In the past this lab laser printed large numbers of labels with an automatic numbering system. When using LR White there was always a collection of toner near the tissue but it never caused a problem in our application. In the present we no longer use laser printed labels because of problems encountered over the past two years. It is unclear if the problems we encountered are a result of changes made in toners to make them more environmentally friendly of the switch to micro-fine toner packs used in 600 dpi printers. For teaching labs - At least one student in each section will slip in a set of labels made on an ink jet printer or reduced using a copy machine each with disastrous consequences.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
We've been using labels printed on an Apple LaserWriter for several years with good success in a variety of epoxies, paraffin and Historesin. If you can't get your font small enough, then reduce the document through the print dialog. Photocopied labels will also work.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 7 Sep 1994, Richard E. Edelmann wrote:
} } For those of you out there who are tired of hand writing out } tinney tiny little penciled labels for embedding with your tissues I } have been running tests with HP Laserjet printed labels, with } excellent results so far. It seems that the electrostatic deposition } of the carbon black pigments (in the genuine HP tonner cartridges } anyway) do not disolve in either Spurr's or Quetol 651 resin (I } haven't tried any of the other yet) and do not seem to effect the } blocks in any manner. You might want to give it a try. } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } Marshall University - School of Medicine } Huntington, West Virginia }
I have been doing the same with Epon-Araldite, also with very good results. I use Word for Windows, 6 point type, and print on a Hewlett-Packard Laserjet III.
---------------------------
On Wed, 7 Sep 1994, Richard E. Edelmann wrote:
} } For those of you out there who are tired of hand writing out } tinney tiny little penciled labels for embedding with your tissues I } have been running tests with HP Laserjet printed labels, with } excellent results so far. It seems that the electrostatic deposition } of the carbon black pigments (in the genuine HP tonner cartridges } anyway) do not disolve in either Spurr's or Quetol 651 resin (I } haven't tried any of the other yet) and do not seem to effect the } blocks in any manner. You might want to give it a try. } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } Marshall University - School of Medicine } Huntington, West Virginia }
I've been using computer generated embedding labels for years. Currently using an Apple Laserwriter printer with no problems. If your printer uses ink that bleeds in resin try soaking the labels in ethanol for 30-60min, dry them completely in warm oven and then embed. Anne
Anne Simpson Gomes
EM Unit, F09 "How's it going Eh?!!!"....... Univ of Sydney from NSW 2006 Australia The Compact Canuck!! Fax: (612) 552 1967
Since the topic has taken off, I thought I would add a bit. I have trouble with air bubbles if I push the label down into the well of Polybed. I have found that if\ I put the label on top and place it into the vacuum for a while then sink it, I don't have bubbles in my block.
A few weeks ago I was on the hunt for some histologic exotica, to which I received all the clues needed to track down the reagent. The quest continues: This time the subject is a little known beast named Safran du Gatinais!!!! for makin alcoholic Safran. What alchemy! Any ideas, where to get it from ???
All help deeply appreciated
Simon C. Watkins Director SBIC University of Pittsburgh Pittsburgh PA 412-648-3051
As you are probably aware, "Safran du Gatinais" is French for "Safranin from the Gatinais", which is a region near Paris. You may find that "Safranin O", a common stain available from various sources, might serve as a substitute? A bottle of Safranin O on my shelf, produced by Chroma-Gesellschaft (Stuttgart, Germany), was obtained many years ago from Roboz Surgical Instruments, 810 18th Street, N.W., Washington, D.C. 20006. I think it also appears currently in the Arthur Thomas catalog.
Kent A. Kent Christensen Department of Anatomy and Cell Biology Univ. of Michigan Medical School
----------------------------
On Thu, 8 Sep 1994, simon wrote:
} A few weeks ago I was on the hunt for some histologic exotica, to which I } received all the clues needed to track down the reagent. The quest } continues: This time the subject is a little known beast named Safran du } Gatinais!!!! for makin alcoholic Safran. What alchemy! Any ideas, where } to get it from ??? } } All help deeply appreciated } } Simon C. Watkins } Director SBIC } University of Pittsburgh } Pittsburgh PA } 412-648-3051 } } }
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two stages available for this instrument, one with smaller holders and (the one we have) that accepts the "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder.
Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two stages available for this instrument, one with smaller holders and (the one we have) that accepts the "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder.
Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
} Date sent: Thu, 8 Sep 94 16:48:37 EDT } From: Rajesh Patel {patelr-at-pilot.njin.net} } To: Microscopy-at-anlemc.msd.anl.gov } Subject: Microscopy Society of America
} } I am a member of MSA but it seems I never get any bullitens from them like } the quaterly bulliten and the list of members etc. } } Who do you contact? } The Business Office Microscopy Society of America P.O. Box MSA Woods Hole, MA 02543 Voice: (800)538-3672
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two side entry stages available for this instrument, one with smaller holders and the other (like we have) that accepts "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder as soon as possible. Thanks. Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
Greetings Microscopists, One of our users wants to do LM immunocytochem and standard EM on the same tissue in the same block. Because his antigen is relatively sparse, the ability to "gently" remove any resin would be a plus so that maximum staining could be attempted. Any number of resins come to mind; LR White most prominently. Does anyone else have suggestions that would be better for this purpose? Thanks in advance.
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick (Chuck) * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Biochemistry * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-1633 voice * | | * 806 743-1219 fax * | | * Email emccjb-at-lubb.ttuhsc.edu * | | * * __| |__ * * |_______| *************************************************
I would like to inquire if anyone has a double-tilt side entry specimen holder for the Philips EM300 TEM. There were two stages available for this instrument, one with smaller holders and (the one we have) that accepts the "normal size" specimen holders. We are using this for the current fall semester lab class and would like to borrow/purchase this type of holder ASAP. Thanks.
Bob Roberts Arizona State University Center for Solid State Science PSB-234 Tempe, Arizona 85287-1704 (602) 965-4512
In my dealings with a "new" antigen to localize I always try numerous resins and fixation protocols until I am happy with the results. No one procedure will work all antigens. LR White is a good place to start. Good Luck.
Dear Chuck, you ask about doing LM and EM on the same specimens and blocks. This is by far the best strategy for immunocytochemistry because fixation protocols, antibody dilution and antigen distribution can be easily assesed at the LM level before progressing to electron microscopy. Resins are convenient to use because they are easy to section and store and the morphology is more easily understood by non-microscopists. They can all be used for immunocytochemistry at the light and EM levels but Epon, Araldite and Spurrs have many problems associated with them. Resins developed for immunocytochemistry include the London resins (LR White, LR Gold), the Lowicryls (K4M, K11M, HM20, HM 23) and, more recently, Unicryl. All of the latter can be polymerized by heat or by UV light and, within limitations, all can be used to infiltrate tissues at low temperature. All can be labeled with antibodies and protein A-gold for EM and all have been tested to give positive results (Schwarz,1994, Proceedings ICEM-13, Paris, France pp 225-226; see also Albrecht et al, 1990 Brain Res. 535:49-61; Schwarz et al 1993 Cell Tissue Res 273:417-425). One additional advantage, pointed out by Schwarz, is that antibodies do not penetrate resins, so only the surface is imaged and there is no out-of-focus signal to blur the image (similar to a confocal image). By using the same fixation and sectioning protocols and the same reagents for both LM and EM a labeling reaction is certain to occur for both. The amount of antigen to be detected may also affect the sectioning method to be used. For some antigens, it has been shown that higher labeling efficiencies are produced on cryosections. Frozen biological material, cryoprotected with sucrose, can be easily sectioned for both light and electron microscopy. As with resins, these sections can be labeled with fluorescent antibodies or colloidal gold for light microscopy (silver enhancement will make the gold visible at the LM level) and with colloidal gold for electron microscopy. Always remember, whichever method is used, the amount of signal at the EM level will depend on the amount of antigen present. Small numbers of antigen will result in small numbers of gold particles over the section.
Paul Webster Center for Cell Imaging Yale School of Medicine.
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
Dear Chuck, you ask about doing LM and EM on the same specimens and blocks. This is by far the best strategy for immunocytochemistry because fixation protocols, antibody dilution and antigen distribution can be easily assesed at the LM level before progressing to electron microscopy. Resins are convenient to use because they are easy to section and store and the morphology is more easily understood by non-microscopists. They can all be used for immunocytochemistry at the light and EM levels but Epon, Araldite and Spurrs have many problems associated with them. Resins developed for immunocytochemistry include the London resins (LR White, LR Gold), the Lowicryls (K4M, K11M, HM20, HM 23) and, more recently, Unicryl. All of the latter can be polymerized by heat or by UV light and, within limitations, all can be used to infiltrate tissues at low temperature. All can be labeled with antibodies and protein A-gold for EM and all have been tested to give positive results (Schwarz,1994, Proceedings ICEM-13, Paris, France pp 225-226; see also Albrecht et al, 1990 Brain Res. 535:49-61; Schwarz et al 1993 Cell Tissue Res 273:417-425). One additional advantage, pointed out by Schwarz, is that antibodies do not penetrate resins, so only the surface is imaged and there is no out-of-focus signal to blur the image (similar to a confocal image). By using the same fixation and sectioning protocols and the same reagents for both LM and EM a labeling reaction is certain to occur for both. The amount of antigen to be detected may also affect the sectioning method to be used. For some antigens, it has been shown that higher labeling efficiencies are produced on cryosections. Frozen biological material, cryoprotected with sucrose, can be easily sectioned for both light and electron microscopy. As with resins, these sections can be labeled with fluorescent antibodies or colloidal gold for light microscopy (silver enhancement will make the gold visible at the LM level) and with colloidal gold for electron microscopy. Always remember, whichever method is used, the amount of signal at the EM level will depend on the amount of antigen present. Small numbers of antigen will result in small numbers of gold particles over the section.
Paul Webster Center for Cell Imaging Yale School of Medicine.
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
Looking for a fully automated batch processor for Kodak 4489 or similar TEM sheet film. Something that will handle at least 25 sheets of 3-1/4" x 4" film, preferably 50, from developing through drying. Benchtop unit preferred over freestanding. Anybody know of such equipment?
Larry Thomas Battelle, PNL Richland, WA 99352 tel: 509 376-3785 fax: 509 376-0418 le_thomas-at-pnl.gov
Laurie Marks asks about the appropriate form of the CTF for a tilted sample. Dear Laurie, If you think of a tilted sample as the limit of a sequence of samples consisting of flat segments at different heights, the CTF for a member of the sequence is the sum of the CTF's for each height (i.e. different values of defocus). The limit would be an integral. There may be complications due to the differences in magnification for different heights, but I can't figure out what they would be. Good Luck. Yours, Bill Tivol
We have had some internal disagreements about how thin a metal foil can be mechanically polished prior to jet polishing without inducing dislocations in the final thin section. I realize that this depends, to an extent, on the metal in question, but would like to get a feel for people's opinions/experience and a good reference that addresses this subject. Thank you, Garth B. Freeman, Sr. Research Scientist, Georgia Tech, Atlanta Georgia garth.freeman-at-gtri.gatech.edu
I recently purchased from an estate sale two boxes of chemical microscopy reagents of the type sold by Cargille and McCrone and taken from Chamot & Mason. On the inside cover of both boxes, on the "index" sheet, is printed the term "Shillaber Model." Does anyone know what that means? Who is or was Shillaber? Can someone provide me a reference? I'm just curious?
A postdoctoral position is available employing HREM techniques to surfaces under UHV conditions using a new multi-chamber combined UHV-HREM and surface science facility at Northwestern. Extensive electron microscopy experience is essential; hands on experience with any of MBE growth, XPS, Auger or high-level image analysis would help. Applications with the name of two referees should be sent to: Professor L. D. Marks Department of Materials Science and Engineering Northwestern University Evanston, IL 60208 ldm-at-apollo.numis.nwu.edu
{Pine.3.87.9409130834.A28895-0100000-at-crl6.crl.com} To: "Peter D. Barnett" {pbarnett-at-crl.com} Cc: microscopy-at-anlemc.msd.anl.gov Message-id: {01HH28NE9TZU94DWRU-at-minna.acc.iit.edu} MIME-version: 1.0 Content-type: TEXT/PLAIN; CHARSET=US-ASCII Content-transfer-encoding: 7BIT
Dear Peter,
That must be a reference to Charles Patten Shillaber, Fellow of the RMS and author of one of the best books on the practical use of photomicrography;"Photomicrography in Theory and Practice, John Wiley and Sons, Inc., 1944.
I am looking for an EPMA mineral standard, tugtupite (syn: beryllosodalite). Does anyone know where I can get it (either from requesting or purchasing) ?
Thanks in advance.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
Message-Id: {MAILQUEUE-101.940914140050.416-at-eo.ine.philips.nl} To: ROBERT-at-CSSS.LA.ASU.EDU
DEAR MR. ROBERTS,
WE PICKED UP YOUR EMAIL REGARDING THE SEARCH ACTION FOR A PHILIPS EM300 DOUBLE-TILT SPECIMEN HOLDER AND WE ARE CHECKING VIA OUR CHANNELS IF ANYONE CAN HELP YOU.
AS WE ARE ALSO CHECKING IN SOME OTHER EUROPEAN COUNTRIES, IT CAN TAKE SOME TIME BEFORE WE HAVE A DEFINITE MESSAGE.
IF POSITIVE, WE WILL INFORM OUR SALES ORGANISATION PEI MAHWAH IN USA WHICH ACTIONS HAVE TO BE UNDERTAKEN TO SHIP THE HOLDER TO THE DESIRED LOCATION.
WITH KIND REGARDS,
HANS VAN DOORN
INTERNATIONAL BUSINESS CENTRE PHILIPS ELECTRON OPTICS COMMERCIAL SUPPORT EINDHOVEN - THE NETHERLANDS BUILDING AAE-1 TELEPHONE + 31 40 766385 TELEFAX + 31 40 766786
9/14/94 Frozen tissue to epon?? 10:24 AM Hi, Does anyone have any ideas that this will or will not work; I have some unfixed adipose tissue that is in liquid nitrogen. I would like to look at the morphology of this tissue. I do not need it for labelling. What I was thinking of doing was leaving the tissue overnight in 70%GA/Methanol at -90, then gradually decreasing the temperature and the alcohol concentration, (do the reverse of low temperature embedding), chopping up the pieces a bit, then do the standard embedding . Would it be better to put it in Lowicryl (I don't think so)? What do you think? Thanks, Jeanne
} We would like to get some feedback on these two accelerators. In your } experience what is the shelf life of DMP-30? How much better is the storage } life if unopened? What quantity of BDMA is generally used as a replacement } for } DMP-30? How does it's shelf life compare? Any other rules of thumb? } As you might have guessed, we have had some intermittant problems with } soft embeddments, and suspect that the accelerator might be the culprit. } Thanks. } } Tim Bourett } DuPont Experimental Station } Wilmington, DE USA
Tim: I asked a similar question recently and a summary of the replies follows. I had used DMP30 for 18 years without problem. I generally keep my bottle at room temp in my hood. I never monitored shelf life but I am sure that most bottles lasted over a year. Recently I started having a problem with soft blocks. I switched to BDMA (2x DMP-30 concentration). The problem went away for our routine blocks but one of my grad students still had a weird problem with the interface of free plastic over some cell monolayers not polymerizing. This is with both BDMA and DMP-30. still haven;t solved that problem but I am still unsure if the problem was with the resins or the student. I haven't personally embedded any monolayers in epon since this problem started. I guess I will have to break down and do it myself to see where the problem is. For other tissues (bacteria, intestine) embedded in Epon, the BDMA works fine.
Summary:
On Tue, 12 Jul 1994, Tom Phillips wrote:
} I am tempted to try benzyl dimethyl amine (BDMA) in place of DMP-30 but don't } have the original reference. How much does one use? Anyone have the original } citation? I usually use: } 20 g EmBed812 + } 10 g DDSA + } 10 g NMA + } 0.6 g DMP-30.
I use them at the same ratio. The BDMA has a much longer shelf life.
Rick A. Harris Dept. of Molecular and Cellular Biology Microscopy Facility University of Calif., Davis
I have never tried BDMA, but Hayat gives several recipes for plastic where he shows X% DMP-30 OR 2X%BDMA. (Page 116 Hayat's third edition "Principles and Techniques of Electron Microscopy biological applications") He refered to Mollenhauer, H,H, 1969, Stain Technol., 39:111. Plastic Embedding Mixtures for EM.
Larry Hawkey Duke Neurobiology hawkey-at-neuro.duke.edu
To: tphillips (Tom Phillips)
We make rather large batches, about 233 grams at a time. We normally use 3.44 gm of DMP-30, and were told to use 4.3 gm BDMA. Hope this helps. ___________________________________________________________________ Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
In a "Survey of Embedding Media For Electron Microscopy" by Audrey Glauert in the Journal of the Royal Microscopical Society 1962 Vol.LXXX pp 269-277 she suggests that DMP 30 and BDMA are interchangable, then follows several recipes (Kushida 1959, Finck 1960, Luft 1961) for Epon resin mixes all incorporating BDMA as the accelerant. The trend for some years has been towards using BDMA in preferance to DMP 30 - I'm not sure why but it is much less viscous so that alone makes it more convenient (Glauert mentions precipitates forming when DMP 30 is used in some resin mixes). I started using BDMA years ago and find it works every time. In the same Glauert reference and in her book "Fixation,dehydration and embedding of Biological Specimens" she suggests several epon 812/812 equivalent mixes, I've always found the medium hardness mixture excellent for most of our specimens.It is her recipe we use, more or less. I mix it thus: "mix A" =19ml 812 resin and 31ml DDSA;"mix B"= 26.5ml 812 resin and 23.5ml MNA.(weigh equivalent amounts if you prefer - I've never seen any noticable improvement with the more accurate weighing).
Mix parts A and B together when ready to use and at the same time add 15ul of BDMA per ml of your total resin/DDSA/MNA mixture. Hope this is of use. R Easingwood
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
We use BDMA in our PolyBed 812. We use 2X as much BDMA as we would use DMP -30. Supposedly BDMA is less viscous than DMP-30 and there fore allows for a quicker infiltration of specimens. It also has a slightly longer shelf life than DMP-30 (12 months as opposed to 9 months). We've had no problems since we switched. Hope this helps.
John Aghajanian Worcester Foundation for Experimental Biology
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Its been ages since I worked with BDMA so can't comment on that. but DMP-30 seems ok stored unopened at room temp, in a dark cupboard, for at least 2 years. Once opened, I dispose of the unused accelerator whenever a new bottle of the epoxy resin is opened. Tlhis seems to work and has not given us problem batches. A few years ago, I had many problems with soft blocks suddenly and consistantly appear from a vendor, who shall not be named. Switching vendors for my Spurr's resin eliminated the problems. The vendor blamed the problem on the quality of employees in their region. Never mind that 2 competitors in the same region seemed to have no problems in their quality.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Greetings: I'm attempting to rejuvenate a carbon coater manufactured by a company called Mikros. Inspection of the guts showed a broken relay switch. I'd like to know if anyone knows who might be able to supply parts for this device. The part number I'm looking for is: 115 NO 120T. It is identified in the manual as a "delay relay" and is used in the automatic valving system. N.B. this is an old instrument, circa 1965, so I'm not holding my breath. However, it is exactly like the very reliable instrument I used as a grad student and I'm sure that with a bit of coaxing it could be a decent addition to the lab. Many thanks!
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
Many moons ago I also used a similar Mikros evaporator. The company is no longer in existance therefore you will need the services of a good electronics tech to find replacement parts. Good Luck
Why not freeze substitute. Your specimens will look great.
On 14 Sep 1994, Jeanne Barker wrote:
} 9/14/94 } Frozen tissue to epon?? 10:24 AM } Hi, } Does anyone have any ideas that this will or will not work; } I have some unfixed adipose tissue that is in liquid nitrogen. I would like to } look at the morphology of this tissue. I do not need it for labelling. What } I was thinking of doing was leaving the tissue overnight in 70%GA/Methanol at } -90, then gradually decreasing the temperature and the alcohol concentration, } (do the reverse of low temperature embedding), chopping up the pieces a bit, } then do the standard embedding . Would it be better to put it in Lowicryl (I } don't think so)? } What do you think? } Thanks, } Jeanne } } }
Help! I recently put some tissue into LR White for the first time, and I'm= having a terrible time with it in the beam!! Even on formvar/carbon,= 75KeV, my smallest condensor/objective apertures, the stuff's blowing up. = What's wrong???? Many thanks to all. Grace Kennedy UCSD
} 9/14/94 } Frozen tissue to epon?? 10:24 AM } Hi, } Does anyone have any ideas that this will or will not work; } I have some unfixed adipose tissue that is in liquid nitrogen. I would like to } look at the morphology of this tissue. I do not need it for labelling. What } I was thinking of doing was leaving the tissue overnight in 70%GA/Methanol at } -90, then gradually decreasing the temperature and the alcohol concentration, } (do the reverse of low temperature embedding), chopping up the pieces a bit, } then do the standard embedding . Would it be better to put it in Lowicryl (I } don't think so)? } What do you think? } Thanks, } Jeanne } First of all: How was your tissue frozen - just in liquid nitrogen or in a cryocoolant - ethane, propane or freons ? Good cryofixation is important step in preparastion and the final result - morphology will ,depend on it. I was using Epon and Lowicryl embeddings of the biological materials. In most cases water withdrawal was done by means of freeze-drying at low temperatures.
See: Wroblewski R and Wroblewski J. (1984) Freeze drying and freeze substitution combined with low temperature embedding. Histochemistry 81, 469-475.
Subject: Time:8:00 AM OFFICE MEMO Thanks. Date:9/15/94 I would like to thank everyone who gave me suggestions regarding my question about embedding tissue that has already been frozen in liquid nitrogen. I'm sure that now I will be able to get decent pictures, one way or another.... This listserver is great! Jeanne
At Fernbank Science Cnter in Atlanta, GA we are hoping to hold an independent study class for high school students in various areas of microscopy. We have chemists, bologists, physcists and electron microscopist on staff at Fernbank Science Center. We have on sight a Zeiss 960 SEM, Zeiss 962 SEM with EDX capabilities, monocular and bonocular (binocular) scopes with the capapbilities of changing them to polarizing (simple) scopes. I have had Polarizing Lgith (light) Microscopy training and we have a very activi professional microscopy group in the Atlanta area. We are looking for ideas for exploration in all areas of microscopy using the equipment above. We also a have a Zeis TEM. We hope to begin this course in the winter of 1995. We will have approximately 10 students for 2.5 hurs for 24 sessesions. If you have any ideas for proposals please let me know. I will be happy to share all information as it is developmed for students and teachers. . . . . . . . . . . . . . . . . . . . . . . . . . Staff Fernbank Science Center (phone)404-378-4311 gfs001-at-sol1.solinet.net
Dear Mary, I'd include some electron diffraction in addition to the more usual EM stuff, but that's my field, so maybe I'm biased. ED is a great example of the wave-particle duality for electrons, and since there is only one electron in the column at once (low dose conditions), the electron must interfere with it- self. (Thinking of the particle as a wave packet with narrow Gaussian distri- bution of momenta helps.) Besides, it's really neat to scan the grid in diff mode and to see the ED pattern suddenly appear. Clay minerals have some thin particles which give spectacular patterns. The high school students I've wor- ked with seemed to enjoy this. Good luck. Yours, Bill Tivol
X-Authentication-Warning: sunday.fhl.washington.edu: Host omnicron.fhl.washington.edu didn't use HELO protocol
Hello,
My name is Douglas Hansen. I have been listening in on the microscopy group for a little while. I am looking for several individuals or groups who would be interested in testing and evaluating a sub-micron dimension reference and calibration standard for SEM's. These reference standards are originals, or, if you will, master gratings, and not replicas. Our own evaluations of these reference standards indicate that they are easy to use and have well defined edges. The sample I would provide to those who volunteer will have a calibrated dimension of 0.293 microns. I will provide this sample free of charge and only ask in return that you provide feedback and agree that you will arrange to allow me to use your comments in advertising, etc. if I request it. If the test results are positive, these new standards will be available through most of the microscopy supply distributors within a few months. They are expected to cost about $250 to $300 dollars.
Please respond to me if you have interest in acting as a test site for these reference and calibration standards.
This is Douglas Hansen again. I have had a response to the standard test offer that surprised me greatly. I have had over 20 responses already. Therefore, I will have to withdraw this offer as of Friday Morning. Any e-mail which arrives in my account by 9:00 am Friday will be offered a test sample. All requests which arrive after this time will be sent a letter of regret. I am sorry, but I only have so many beta samples to pass out. However, I want to thank everyone for their interest. If the test results are as positive as our own testing has been, you will be able to purchase these standards through your favorite microscopy supplies distributor within 2 months.
Demo of SEM, AFM, Image analysis equipment by Leica At Greenville Hilton, (Heywood exit off 385 to downtown) Tues, Wed, Thurs, 9/20, 9/21, 9/22 For Further Info, contact 800-248-0665 ext 516 (Mike Webber)
ELECTRON MICROSCOPY FACILITY SUPERVISOR/INSTRUCTOR Miami University is seeking applicants for a full-time, nontenure-track, staff position of Electron Microscopy Facility Supervisor to begin as soon as possible. The successful applicant will hold an M.S. or Ph.D. degree in one of the biological sciences and will be responsible for the operation, maintenance, and supervision of an interdepartmental EM facility containing two TEMs, two SEMs, cryopreservation equipment, EDX spectrometer, five darkrooms, and an image analyzer. Duties include supervision of a full-time EM technician and teaching in microscopy courses. Collaboration on faculty research projects will be encouraged. Please submit curriculum vitae, transcripts of all course work, and three letters of recommendation to: David Pennock, Director, Electron Microscopy Facility, Miami University, Oxford, OH 45056. Application review will continue until the position is filled. Miami University is an Equal Opportunity/Affirmative Action Employer.
I would like some inputs from those already outputing images directly from TEMs. I have a Gatan 673 wide angle camera now outputting images to a SEIKOSHA VP1500 thermal printer from a Hitachi 7100 TEM. I would like to produce TIF or similar fomat images from the camera output, but I do not want to dupplicate the set up I now have in a different room, where a Targa plus 16/32 is already installed for running other softwares.
I need the least expensive video board that would allow this with a decent resolution. The board will be installed on a 486 COMPAQ, and hopefully an Ethernet card will allow me to get my mac to talk with with the PC.
Apology to all who have been trying to get that programme.I have been assured that it will work OK if you follow this: ftp lab.csl.utas.edu.au login name:anonymous password: {email name & address} ftp} cd pub/cameca ftp} dir backsc.ftn ftp} get backsc.ftn ftp} quit Hopefully this should fix it.
ASTIMEX Scientific Ltd. (University of Alberta, Canada) sells Mineral Mount code MINM25-53, diameter 25mm, 53 minerals of the good quality.One of them is tugtupite Na4BeAlSi4O12Cl with the composition as below: BeO 5.36% Na2O 26.50 Al2O3 10.90 SiO2 51.39 Cl 7.58 all in weight % , total 101.7 I purchased the mount about 5 years ago and I have been using it as the source of secondary stds but sometimes as primary stds when NBS stds are not at hand. Regards, Wis Jablonski Uni Tas
Message-Id: {MAILQUEUE-99.940919080550.928-at-eo.ine.philips.nl} To: microscopy-at-anlemc.msd.anl.gov
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Not knowing what time zone you are in I do not know if this will reach you before your 9.00 deadline.However I am the product manager of the Philips Electron Optics SEM activity and as well project of the JESSI project 104 (similar to Sematech) that has as a goal a precise and accurate metrology sem.As such I would be pleased to participate in your test with particular referernce to low voltage metrology.Also I have recently been asked by the ISO organisation to set up a working group to define internationally acceptable standards and procedures for sem metrology.You may be interested in some involvement with this group Please let me hear from you re the test sample and in any case we should stay in contact.You should also contact M Postek at NIST and M Bennet at SEMATECH.Michael has been spending at lot of time on this topic,including a wel defined round robin test of sem's that you would find interesting and Marilyn B is (via TI) producing a mask metrology standard that will be available shortly.
Greetings, For use in vacuum evaporation, we are looking for a source of Tungsten wire that is 1mm diameter. The usual em supply house catalogs seem not to list it in this diameter in their catalogs. We did find some in the TAAB catalog, from England, but I am hoping to find a USA distributor. Thanks in advance for any help.
Try Alfa 800-343-0660 Their catalog # for 1.0 mm dia wire is 10411 Catalog lists $22.80/meter, $84.00/5 meters You may have tough time bending the wire to give any shape as it is usually very brittle.
To: MCI:CENTRAL cc: DRStadden:R_D:Armstrong Subj: Thermal Printers ------------------------------------------------------------------
I recently got word that our Codonics VP-3500 thermal printer, dedicated to an Amray 1820D SEM, was in need of a new print head. I had sent it cross-country to the only Seikosha repair facility, anticipating a $150 charge for cleaning the heads. (I was getting fine, but objectionable, white lines across the images, and couldn't get rid of them by running the cleaning sheet through the paper path, as per directions.) The cost of the new head is $1,350, + a $150 service charge. The 5-year-old unit was $6,700 new.
In terms of technological upgrades, what are my options for, say, under $7500? Of course, color capability is not required. Thanks for any thoughts on this matter.
Dave Stadden Armstrong World Industries, Inc. Lancaster, Pennsylvania
Tobias regarding a source of 1 mm tungsten wire...
A while back I received a brochure from
REFINING SYSTEMS INC. P.O. Box 72466 Las Vegas NV 89170 Tel (702) 368-0579
regarding metal wire, slugs, pellets, etc. I have no experience ordering from them or using their materials, but they did seem to have a range of products that might include the wire of a diameter you need.
please let me know if they have what you need and you liked their service, quality etc. Thanks.
good luck steve ---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
Just a brief note about our ongoing experience that may be related to this thread. This summer I resumed doing light microscopy about a hiatus of about a year. We were consistently getting a water-like reaction when we transferred slides from 100% ethanol to xylene. We changed everything. I thought it was hydrated ethanol...we opened fresh 1 pt bottles of absolute ethanol. I thought my assistant was being sloppy...so I did the staining myself and it still happened. I thought the xylene dishes had been contaminated somehow...so I replaced them with fresh xylene. Our university has a chemical storehouse for frequently used chemicals like xylene. Either it has been a bad lot of xylene from one manufacturer or a generic problem with xylene from that manufacturer (I'd never used xylene with this company before...). The upshot is that we bought xylene from another manufacturer and we aren't having these problems. So I really think we may all be searching too hard sometimes for explanations for aberrations...I thought it was the slides, the coverslips, etc. long before I thought to believe it was bad xylene (another colleague of mine here had the same problem).
-- Nancy L Desmond, Ph.D. Department of Neurosurgery University of Virginia Health Sciences Center, Box 420 Charlottesville, VA 22908 804.924.5607 (voice) 804.982.3829 (fax)
Greetings: I'd first like to extend my thanks to the large number of people who responded to my request for help in locating a part for an old carbon coater I'm trying to revive. Everyone had excellent suggestions and provided addresses and phone numbers. And now...I'd like to find a protocol (that I'd heard of) that would allow me to use bacteria with attached phage for a TEM lab on negative staining. I have easy access to both E. coli and phage, but don't have a source for the technique. Aparently there is a way in which the phage can be induced to attach themselves to the bacteria without subsequent detachment. Anybody out there know about this? Please respond directly. I'll summerize and repost. Many thanks!
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
I require ideas re the cleaning of the following problematic SEM samples, please: Field-collected, museum samples of fly larvae, fixed in 70% eth-OH, very soft and delicate, even after fixation. Detritus and some slime appears to be fixed to the cuticle of some samples. I have tried the following: A weak Photo-flo solution with gentle brushing 100% picric acid etching 0.05% KOH Contrad soap (an alkaline anti-contamination soap)
Setae et al could be damaged by ultra sonic cleaner, so feel that chemical cleaning is the solution, but what???
Any ideas, please? John F. Putterill EM unit, Onderstepoort Veterinary Institute South Africa 0110
} I require ideas re the cleaning of the following problematic SEM } samples, please: } Field-collected, museum samples of fly larvae, fixed in 70% eth-OH, } very soft and delicate, even after fixation. Detritus and some slime } appears to be fixed to the cuticle of some samples. } I have tried the following: } A weak Photo-flo solution with gentle brushing } 100% picric acid etching } 0.05% KOH } Contrad soap (an alkaline anti-contamination soap) } } Setae et al could be damaged by ultra sonic cleaner, so feel that } chemical cleaning is the solution, but what??? } } Any ideas, please? } John F. Putterill } EM unit, Onderstepoort Veterinary Institute } South Africa 0110 } } John: There is a pretty good and simple apparatus you can make that will help clean your critters. It was in: Proc. Entomol. Soc. Wash. 93(1), 1991, pp. 204-205. If you need a copy of this article, send me a fax number and I will fax a copy to you or send me your address and I will mail a copy. Hope it works!
Phil voice: (410) 455-3582 fax: (410) 455-3875 E-mail: prutle1-at-gl.umbc.edu
A few months ago, I offered Field emission reference articles to anyone interested. I recently received a response requesting some literature. However, I lost the message in an unfortunate PC crash. would the person who requested the information please resend the request?
Thanks! Damon L. Heer
FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830
My apologies, I have committed the cardinal sin of sending a request that should have been sent to the listserv address to the list address. Again my humble apologies! John Mansfield.
I am currently preparing a manuscript and for sake of completeness would like to know if anybody is aware of work done using EELS to characterize minor or even trace metals in mineral samples. I've taken a look through the MSA proceedings for the last couple of years but haven't really spotted anything. Can anybody help, please?
Rich Leapman and Dale Newbury published a paper titled "Trace Elemental Analysis at Nanometer Spatial Resolution by Parallel-Detection Electron Energy Loss Spectrometry" last September, 1993, in Analytical Chemistry. Although they did not look at minerals, they did analyze NIST standard glass reference materials.
Hope this helps, John
} I am currently preparing a manuscript and for sake of } completeness would like to know if anybody is aware of work done } using EELS to characterize minor or even trace metals in mineral } samples. I've taken a look through the MSA proceedings for the } last couple of years but haven't really spotted anything. Can } anybody help, please? } } Thanks in advance. } } Greg McMahon } MTL/CANMET } Ottawa, Ontario } Canada
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(616) 2493543 Fax:(616) 2494891 Email:Vowles-at-rsbs-central.anu.edu.au
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 9:53
Date:9/21/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
An number of you have seen me try and set my microscopy to digest mode. I accidentally sent the request to the list and not to the listserv-at-aaem.amc.anl.gov address. When I resent the message to the listserv address I got no reply or confirmation. I therefore assume that the version of the listserver software that Nestor is running does not support digest mode. Nestor is out of the country on business until October 6th and so I cannot ask him. If Russ Cook is monitoring this list maybe he can give some insight. So, for the time being I would not bother trying to set the digest mode. For those of you who don't know it, the digest mode sens you the messages in batches with all of the message titles at the head of the batch. That way you can scan the batch message header and see if there are messages that you need to read.
The best way of reading the mailing list is, in my humble opinion, to use the Usenet News group sci.techniques.microscopy. You only need to download/read the messages that are of interest to you. You can read news with either a local MAc or PC based news reader (e.g. NewsWatcher or Nuntius for the Mac) or from a Unix workstation (using rn, trn or xrn for example) you can read news via Gopher (links vary depending on your location) or via the WWW with NCSA Mosaic or some such other Web browser. The URL for sci.techniques.micrscopy is: news:sci.techniques.microscopy. OK? John Mansfield
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 10:14
Date:9/21/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
We have been trying out the World Wide Web and have set up some web pages that are designed to provide information on our microscopy laboratory and The Department of Materials Science and Engineering here at the University of Michigan. The pages are still under development but you can check them out at URL: http://www.engin.umich.edu/~jfmjfm/emal_info/emal_home_page URL: http://www.engin.umich.edu/~jfmjfm/mse_folder/mse_home_page URL: http://www.engin.umich.edu/~jfmjfm/mseandemalmain.html
Comments and Questions should be directed to me. Thanks John Mansfield
As John Mansfield noted, Nestor is out of town and cannot attend to the microscopy server. Unfortunately, he has not brought me in on the operation, so I cannot help any of you who have questions about its system management.
I would like to sign on to the microscopy e-mail conference group. I am an E.M. technician in neuroscience at U of Penn. Please send further instructions. Thanks. Sally
Applications are invited for a Microscopist in the Light Microscopy Laboratory of 3M's CRL - Analytical and Properties Research Laboratories. The Light Microscopy Laboratory supports the development, production, promotion and protection of all 3M products. This is a state of the art light microscopy laboratory that utilizes more than 30 different light microscopies to solve problems of importance to all the 3M divisions. Applicants should have broad experience in microscopy such as: bright field, dark field, differential interference contrast, phase contrast, polarized light, and interference microscopy; and sample preparation techniques such as metallography, microtomy, ion milling, and etching. Additionally, applicants should demonstrate expertise in one or more of the following areas: confocal microscopy; microspectroscopy (uv/vis, raman, color/appearance); forensic microscopy; image analysis and quantitative characterization of the general morphology of microscopic structures, particles, and defects; quantitative characterization of surface morphologies; and/or fiber and debris identification. This position requires an M.S. or Ph.D. in the physical sciences or engineering.
Job Description -
The successful candidate will apply their knowledge of light microscopy to the solution of problems submitted by 3M research and/or production facilities. This will be accomplished by working with our 3M requesters to identify appropriate microscopy techniques that provide useful answers, carryout the microscopical investigations, and effectively communicate the results. The candidate will be expected to actively pursue the development of new quantitative analytical microscopical techniques or methods. Also, the successful candidate will become a corporate problem-solving resource by sharing their expertise through the appropriate 3M forums.
For confidential consideration please submit resumes to (no phone calls): G. G. Kiperts 3M Center 224-1W-02 St. Paul, MN 55144-1000
Has anyone out there with a GATAN 622 camera attached to their TEM attempted to capture the video into a Macintosh AV? I am considering the purchase of a PowerMac, and would like to hear from anyone who has tried to capture video from the GATAN camera to determine if the AV option is a good investment.
F. Scott Miller Electron Microscope Lab smiller-at-umr.edu University of Missouri-Rolla voice: 314 341 4727 Rolla, MO 65401 fax: 314 341 6934
Sorry to bother you, but my e-mail address changed about a month ago. You may wish to update your directory; mail to the old address does not transfer. allen-at-aaem.amc.anl.gov Thanks and regards. Charlie
======================================== Charles W. Allen Electron Microscopy Center-HVEM-Tandem Facility MSD 212/E211 Argonne National Laboratory Argonne. IL 60439 USA
Sorry to bother you, but my e-mail address changed about a month ago. You may wish to update your directory; mail to the old address does not transfer. allen-at-aaem.amc.anl.gov Thanks and regards. Charlie
======================================== Charles W. Allen Electron Microscopy Center-HVEM-Tandem Facility MSD 212/E211 Argonne National Laboratory Argonne. IL 60439 USA
I am not familiar with the protocol you have in mind but try mixing bacteria and phage together, let sit a few, then fix with glut. This should prevent phages from falling off.
Has anyone out there with a GATAN 622 camera attached to their TEM attempted to capture the video into a Macintosh AV? I am considering the purchase of a PowerMac, and would like to hear from anyone who has tried to capture video from the GATAN camera to determine if the AV option is a good investment.
F. Scott Miller Electron Microscope Lab smiller-at-umr.edu University of Missouri-Rolla voice: 314 341 4727 Rolla, MO 65401 fax: 314 341 6934
Dear microscopists, could someone help me with information about a company and one of its products. How can I come into contact with Citifluor Ltd, London. Does somebody use its Citifluor solution? I It should prevent fluorescence stained objects (in my case aquatic bacteria on Nucleopore filter) from quick fading. Also other suggestions how I can protect DAPI- or AO-stained bacteria from a fast fading of fluorescence are appreciated.
Thanks
Guenter Jost Baltic Sea Research Institute Dept. Biological Oceanography Seestrasse 15 D-18119 Rostock-Warnemuende Tel.: 0381-5197227 Fax: 0381-5197440 jost-at-bio.io-warnemuende.d400.de
X400-Received: by mta bottom.ctd.anl.gov in /PRMD=ANL/ADMD= /C=US/; Relayed; Fri, 23 Sep 1994 02:43:28 -0500 X400-Received: by /PRMD=ESnet/ADMD= /C=US/; Relayed; Fri, 23 Sep 1994 02:43:25 -0500 X400-Received: by /PRMD=dfnrelay/ADMD=d400/C=de/; Relayed; Fri, 23 Sep 1994 02:44:52 -0500 X400-Received: by /PRMD=IO-WARNEMUENDE/ADMD=D400/C=DE/; Relayed; Fri, 23 Sep 1994 02:42:20 -0500 X400-Received: by /ADMD= /C= /; Relayed; Fri, 23 Sep 1994 02:42:20 -0500
Dear microscopists, could someone help me with information about a company and one of its products. How can I come into contact with Citifluor Ltd, London. Does somebody use its Citifluor solution? It should prevent fluorescence stained objects (in my case aquatic bacteria on Nucleopore filter) from quick fading. Also other suggestions how I can protect DAPI- or AO-stained bacteria from a fast fading of fluorescence are appreciated.
Thanks
Guenter Jost Baltic Sea Research Institute Dept. Biological Oceanography Seestrasse 15 D-18119 Rostock-Warnemuende Tel.: 0381-5197227 Fax: 0381-5197440 jost-at-bio.io-warnemuende.d400.de
Since a couple of months I'm now reading the interesting discussions on this server. In the studies of specimen with SAD there is a question that bothers me since a longer time: I'm trying to figure out the precision of the measurement of the lattice parameters in thin, ploycristalline Ni/Ti multilayers (grain size about 10nm) with SAD. The work was done on a Phillips EM430 and Hitachi HF2000 FEG microscope. Did anybody try to calculate whether there is a dependence of the position of the reflection in the diffraction pattern from the position of the grains in the selector aperture due to higher order lens errors? Does there exist literature about this problem?
What tissue will you be labeling? You realize that only the surface will be seen on SEM. BSE is probably the better imaging technique to employ using 20-30nm gold. Need a little more info.
I have used the CitiFluor antifadent for fluorescence and found it to work quite well. The product I used was AFT-10 tablets. One tablet dissolved in 1 ml of 10X PBS, brought up to 10 mls with glycerol (ie 90% glycerol/PBS final). This was to coverslip mounted brain sections using FITC and TRITC double immunofluorescence. The cost was app $110 USD plus tax and shipping for 10 tablets (about 2-3 years ago). Unfortunately, I have lost the FAX number for CitiFluor, but their address and phone follows. Good luck!
CitiFluor Limited The City University Northampton Square London EC1V OHB UK phone (Int): 441-253-4399 ext 3502
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
Subject: Time:10:18 AM OFFICE MEMO re} Kill my feed Date:9/23/94
Dear Erik, Perhaps you do not understand the organization of a list. The "your" which you refer to are other subscribers like yourself which have no control over the list administration. When you send junk mail to the list it is an attack on others like yourself. If you want to be removed from the list I suggest you contact the listserve manager Nestor Zaluzec (Zaluzec-at-aaem.amc.anl.gov) for instructions. Mike Mike_Schwartz-at-qm.yale.edu Fax 203-785-5263 Voice 203-785-4324
Michael Schwartz, Ph.D. Associate Professor Section of Neurobiology Yale University School of Medicine 333 Cedar St. New Haven, CT 06510
--------------------------------------
I want this feed stopped - one of these messages will posted every day until the junk mail stops. This wastes my bandwidth as well as yours but since I have been paying for it for the last 3 months, how could it hurt to overload your systems with junk mail... since no-one will listen to me...
I just got a call from a person at an equipment remarketing company in New Mexico that will be auctioning some equipment from a large government lab, including a Coats and Welter field emission SEM (Model HPS 70). They are looking for good homes for this equipment and the auction takes place TOMORROW, Saturday, Sept. 24. If you want more information, please contact Edith Lewis directly by phone.
Edith Lewis Remarket, Inc. (505) 892-4983
Please don't reply to this list. Thanks.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
Dear John, My Industrial Regional Buying Guide lists Inland Vacuum Industries, Inc 35 Howard Ave., Churchville NY 14428. (716) 293-3330. I don't know their mechanical pump oils, but I've been satisfied with their silicone DP oil. Good luck. Yours, Bill Tivol
Hi, We have been using 49 C melting point paraffin, which we purchased from BDH Chemicals, Poole, UK (Prod. #29839). Now, we are unable to find a source for this wax. Does anyone know of somewhere that we can buy this stuff? Thanks!
PS: Wtih respect to the "kill the feed" demand, I personally find the discussion of other techniques and aspects of EM/LM to be interesting and often directly useful in my research and teaching. I want to thank Nestor for his continued work and all the others who contribute to the discussions for making this a very productive source of information.
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
On my way out of the MSA/MAS meeting in New Orleans, I stopped by the Computer Workshop. I previewed a Macintosh program, Super SEM 1.1 by B.J. Griffin and A. van Riessen from the Center of MIcroscopy and Microanalysis at the University of Western Australia. Unfortunately a floppy disc that I put the ftp information on about this program and the software library was unrecognizable when I returned. Can anyone tell me how to obtain Super Sem?
Thanks, Lou Ross 101 Geological Sciences Bldg. University of Missouri Columbia, MO 65211 (314) 882-4777, FAX 882-5458 e-mail geosclmr-at-showme.missouri.edu
On my way out of the MSA/MAS meeting in New Orleans, I stopped by the Computer Workshop. I previewed a Macintosh program, Super SEM 1.1 by B.J. Griffin and A. van Riessen from the Center of MIcroscopy and Microanalysis at the University of Western Australia. Unfortunately a floppy disc that I put the ftp information on about this program and the software library was unrecognizable when I returned. Can anyone tell me how to obtain Super Sem in the US or do I need to contact the authors directly?
Thanks, Lou Ross 101 Geological Sciences Bldg. University of Missouri Columbia, MO 65211 (314) 882-4777, FAX 882-5458 e-mail: geosclmr-at-showme.missouri.edu
Get a life and stop crying. You sound like my 9-year old daughter when she hasn't had enough sleep!
OK, now for what you need. The following exerpt is from a recent administrative transmission from the listserver to all of us who actively subscribe to the list. The section on unsubscribing when you don't know your original subscription address is obviously relevant to you. If you still have trouble sorting it out after speaking to YOUR SysOp, contact Nestor Zaluzec (the list administrator) at Zaluzec-at-aaem.amc.anl.gov - and leave the rest of us alone!
FROM MISCROSCOPY LISTSERVER 9/5/94:
If you want to unsubscribe you must send a message to
LISTSERVER-at-AAEM.AMC.ANL.GOV donot send it to "Microscopy-at-..."
in the message put the following line.
UNSUBSCRIBE MICROSCOPY NAME-at-HOST (where NAME-at-HOST is your Email address)
and you MUST provide your original subscription address. If you try to unsubscribe from the listserver using an address which is not registered then the message will be ignored (and then forwarded to my Email).
Recently, I have been seeing a couple of individuals trying to unsubscribe mulitiple times. They are sending the message to the correct address BUT the Email addresses which they have been suppling as "their subscription addresses" are not registered on the system.
This most likely occurs, when an individual subscribes from one address then has their mail forwarded to a different computer. Unless the system receives your subscription address there is no way for it (or me if I have to do it manually) to find out who to delete. If you are not sure what address you subscribed with, then try checking the header lines of a recent Microscopy Email message. Usually in that header you will be able to see the route that has occured during your Email transit, particuliarly if your message is being forwarded locally. If you donot know what to do here, then check with you local System Manager.
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
This is Nestor, touching base from France. Up until today I have been out of contact with the listserver.
I have just seen your request however, I cannot unsubscribe you (or anyone else for that matter) unless you supply the address which you have originally subscribed to this server with. THERE IS NO RECORD OF ANY SUBSCRIBER WITH THE ADDRESS YOU HAVE SUPPLIED
SMTP%"anthrax!er298s1146-at-wimsey.com"
I WILL BE MORE THAN HAPPY TO HELP, BUT CANNOT DO IT WITHOUT YOUR PROVIDING THE CORRECT INFORMATION!!!!
To all subscribers, please if you forward your mail remember what your original subscription address is neither I nor the listserver system can read minds!! Especially across the Atlantic!
Reply to: RE} Measures of spatial distribution. Hi Dave, P.J. Clark, F.C. Evans (1954) Distance to nearest neighbor as a measure of spatial relationships in a population. Ecology 35:445-453. Re = 1/( 2 sqrt(N) ) = expected Mean distance between nearest neighbors. Ra = actual Mean distance. Q = Ra / Re Value if Q is: 1.0 for a random distribution. { { 1.0 for clumping or aggregation. } } 1.0 for a more regular or uniform spacing. Qmax: Maximum possible value for Q is 2 * sqrt( 2 / sqrt(3) ) - 2.1491 for a hexagonal (honeycomb) distribution. Notes: 1. See the original paper for derivation of statistics. 2. "Mixed" populations (for example, showing some clumping and some uniform spacing) can result in values of Q that do not tell you anything. In other words, some patterned distributions can result in Q = 1, which would niavely be interpreted as random. 3. Pages 263-295 of the following paper is where I came across the original reference and is my source for the above info: M.J. Potel, S.A. MacKay (1979) Preaggregative cell motion in Dictyostelium. J. Cell Sci. 36:281-309.
Enjoy,
George McNamara Universal Imaging Corporation --------------------------------------
To my image analyst comrades; I am looking for references to the measurements of dispersion. I am working with TEM images of dispersed inorganic materials in polymer blends - I've cooked up a nearest neighbor measure and the distribution thereof but I haven't been able to find any satisfactory (at least to my customer) theoretical workup on that method. Any thoughts on other distribution quality measures would be most welcome too. Thanks
Dave Calvert Eastman Chemical Co. Kingsport, TN Also found at calvert-at-emn.com
Does anyone know of any independents who can perform routine maintenance on a Hitachi H-7000 in the Upstate New York area? Tks in advance! Craig Lending SUNY Brockport, Biology clending-at-acspr1.acs.brockport.edu 716-395-5755
Dear Monica, Your message raises more questions than answers. What is the tissue you are working with? Are you seeking to label surface antigens, or internal ones? Access to the antigen is probably the biggest challenge. Some sort of fracturing may be required (particularly for internal antigens) coupled with some detergent extraction to allow access to the antigen.
In all likelihood you will need to use BEI to find your label. You may subsequently be able to use SEI once you've located the gold. And off hand, I can't think of a reference to immuno on whole tissues, you may wish to read Hodges GM, Southgate & Toulson (1987) Scanning Microscopy 1/1:301-318. Have fun! Cheers
Peter
Peter Vesk, E.M. Unit,F09 University of Sydney NSW 2006 Australia phone: 61 2 692 2351 (overseas) fax: 61 2 552 1967 peter-at-emu.su.oz.au
A member of the Royal Microscopical Society has a complete run of the Journal of Microscopy from 1973 to 1993 which he would like to donate to an Eastern European or Third World Institution or Library which would otherwise be unable to subscribe. This offer is not extended to individuals.
The recepient would have to pay the cost of packaging and delivery to their address.
Interested parties should contact the Royal Microscopical Society BEFORE 10 OCTOBER 1994. Please DO NOT USE EMAIL TO REPLY! Fax or post the name and address of the institution and the name of the person to contact regarding delivery arrangements to: THE ROYAL MICROSCOPICAL SOCIETY 37/38 ST CLEMENTS OXFORD OX4 1AJ UNITED KINGDOM FAX: +44 865 791237.
Please mark your fax/letter: For the attention of Gillian Wilson.
Does anyone out there know a source for barium permanganate? I want to use it as a post stain to enhance the contrast of plant cell walls. Any other suggestions to achieve the same end are appreciated. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
} Does anyone out there know a source for barium permanganate? } I want to use it as a post stain to enhance the contrast of plant cell } walls. } Any other suggestions to achieve the same end are appreciated. } ********************************************************** } * Greg Erdos ** * } * Director, ICBR EMCL ** Phone 904-392-1295 * } * 218 Carr Hall ** FAX 904-392-8598 * } * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * } * Gainesville, FL 32611 ** * } **********************************************************
Pfaltz & Bauer 800-Call-1-PB carries barium permanganate (#B00203)- 50 g for $47.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
There is no place on an open scientific list such as this for rude exchange. Sorry for my rash response to "Kill my Feed!" - it wasn't intended for general distribution (also my mistake), but it was rude nonetheless. I hope at least the info provided will be useful...
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
Several people have posted mssgs to this list regarding used SEMs. Anyone with such info is asked to send it *especially for CHEAP or free scopes--budgets!* directly to:
Several mssgs were posted to this list earlier regarding used SEMs for sale. People with this info **especially for CHEAP or free SEMs--budgets, you know** are asked to send *directly* to:
Does anyone out there have any experience using the Cerius DLS-UI module (from Molecular Simulations). I basically want to introduce particular types of atoms on specific sites within a prototype unit cell and have the program calculate the resulting cell lattice parameters. However, I have not yet been able to get the program to do this and I was wondering if anyone else has. Thus far, responses from the manufacturer's support line have been rather ambiguous.
I have a light-element EDS detecter (ultra-thin window type) which doesn't perform well on light-element detection. For instance, low oxygen and carbon x-ray counts were detected during quartz and graphite analyses, respectively.
I think the problem is due to oil and/or dirt contaminations on the ultra-thin window. Does anyone have cleaned an ultra-thin window before? What kind of procedures I need to follow? Thanks in advance.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
I've used both biological and non-biological and looking for specimen deterioration is one of the best methods. However, I will not allow anyone who has dried their samples with a chemical drying agent such as Hexamethyldisilazane (HMDS) or Pelldri (vendor name) to use my vacuum systems (SEM or Sputter coater). In my experience they contain vacuum volitile compounds which gunk up the vacuum system (as much as the EM supply companies may deny it).
In general well fixed, well dehydrated biological specimens do not present a problem for SEM's. But they may cause a problem for ultrahigh vacuum systems (Cold FEGs, and Auger) and special care needs be taken with insuring specimen dehydration. And I would be very cautious with a windowless EDS detector.
If you need absolute cleanliness for your non-biological work I would stay away from LTSEM stages (Low-Temperature or Cryo SEM of fully hydrated specimens) as they do dirty the vacuum system somewhat.
I haven't had any experience with silicone implants, I don't know if absolutely all the silcone is polymerized or bound up (or even how it is hardened). But possible resultant glass coated aperatures are not cool.
Lets hear what the rest of the peanut gallery out here has to say. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I've used both biological and non-biological and looking for specimen deterioration is one of the best methods. However, I will not allow anyone who has dried their samples with a chemical drying agent such as Hexamethyldisilazane (HMDS) or Pelldri (vendor name) to use my vacuum systems (SEM or Sputter coater). In my experience they contain vacuum volitile compounds which gunk up the vacuum system (as much as the EM supply companies may deny it).
In general well fixed, well dehydrated biological specimens do not present a problem for SEM's. But they may cause a problem for ultrahigh vacuum systems (Cold FEGs, and Auger) and special care needs be taken with insuring specimen dehydration. And I would be very cautious with a windowless EDS detector.
If you need absolute cleanliness for your non-biological work I would stay away from LTSEM stages (Low-Temperature or Cryo SEM of fully hydrated specimens) as they do dirty the vacuum system somewhat.
I haven't had any experience with silicone implants, I don't know if absolutely all the silcone is polymerized or bound up (or even how it is hardened). But possible resultant glass coated aperatures are not cool.
Lets hear what the rest of the peanut gallery out here has to say. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I have a Fisons(Kevex) Quantum Detector which has an ultrathin window. It seems to require cleaning about 3 or 4 times a year. My procedure is to use a plastic pipette to carefully flow ethanol down the detector tube so that it flows over the end of the tube with the window. Do not squirt the window itself with anything!! Use of a small magnifying loop to observe the window helps to determine when it is clean. I forgot to say, remove the collimator first. When you are finished an easy check of the window is a comparison of the peak heights of the copper K and Cu L lines for 20kv excitation. When clean I get a slightly higher L peak. Good luck and pray before doing the cleaning! Jim Kelly
Dear Greg, If you cannot find a supplier and you are willing to try some chemistry with potentially spectacular results, you can try to add Ba(OH)2 and KOH to H2MnO4. Actually, adding BaOH to K2MnO4 might just do the job. If you meant BaMnO4 rather than BaKMnO4, and the K would interfere, you can add concentrated H2SO4 to K2MnO4, carefully add Ba(OH)2, make sure the solution is cold, collect the precipitate--which should be a mixture of BaMnO4 and BaSO4--and acidify until the BaMnO4 dissolves. IF YOU DECIDE TO TRY THIS, BE AWARE THAT H2MnO4 IS **VERY** DANGEROUS. Paper or other organic material explodes on contact with H2MnO4. Let's hope the combination of BaOH and K2MnO4 is what you need. Good luck--really. Yours, Bill Tivol
Dear Robin, I'd be very worried about contamination of the instrument due to vol- atile organic compounds from biological specimens. We do essentially only bio- logical work, and our vacuums, etc. are not anywhere near what you need to have to be sure you get no surface contamination. Cryopreparative methods may give you a better chance--at least the volatiles can condense nearby--but the area the beam hits will still evolve organics. Perhaps replicas can be made of the original specimen--freeze-fracture, evaporate metal onto the specimen, then dissolve the original, leaving a metal shell to examine. I have not done this, but our lab did about 15-20 years ago. Good luck.
The most likely reason for low carbon and oxygen counts is icing of the detector (or window). Most systems are supplied with a de-icing control which warms up the detector while the bias voltage is turned off. This should be tried first before suspecting other contamination.
Richard Leapman National Institutes of Health Bethesda, MD
} Greetings to everyone, } } I have a light-element EDS detecter (ultra-thin window type) which } doesn't perform well on light-element detection. For instance, low } oxygen and carbon x-ray counts were detected during quartz and graphite } analyses, respectively. } } I think the problem is due to oil and/or dirt contaminations on the } ultra-thin window. Does anyone have cleaned an ultra-thin window } before? What kind of procedures I need to follow? Thanks in advance. } } Long Liang } Electron Microprobe and SEM Lab } ARCO Exploration and Production Technology } 2300 West Plano Parkway } Plano, TX 75075 } E-mail : LLIANG-at-is.Arco.COM
Greetings, Just a comment about fluorescent antifade compounds. In the past I have made side by side comparisons of several of these, came to a particular conclusion, and then found a colleague who had done similarly but came to a different conclusion. There are also some discrepancies in the various papers that have been published which make these comparisons. These differences seem to be based on differences in sample and/or fluorochrome. It appears that there is no "perfect" antifade, and that it may be worthwhile to try several for your given application.
In my lab, we use a product called Vectashield (from Vector labs; no affiliation; address available by request) that works great for our applications.
Dear Roy, Two indications that our EDS detector needs to be warmed up and cleaned are the FWHM of a standard peak (Mn K-alpha in our case) and differences in energy calibration when things get really bad. It's not too difficult to run a standard or to look at the FWHM's of a few peaks in the specimen. Comparison with standards taken when the system is known to be clean should tell you when you need to do something. I have to issue the caviat that I don't have light- element capability, and I am working with 1 MV electrons, so your experience may vary. Good luck. Yours, Bill Tivol
Received: from MAILQUEUE by MICROBIO1 (Mercury 1.11); Wed, 28 Sep 94 11:04:15 EST Return-path: {baskin-at-biosci.mbp.missouri.edu} Received: from emoryu1.cc.emory.edu by transporter.microbio.emory.edu (Mercury 1.11); Wed, 28 Sep 94 11:04:15 EST Received: from aaem.amc.anl.gov by emoryu1.cc.emory.edu (5.65/Emory_cc.4.0.1) via SMTP id AA02179 ; Wed, 28 Sep 94 11:02:21 -0400 Return-Path: baskin-at-biosci.mbp.missouri.edu Message-Id: {9409281502.AA02179-at-emoryu1.cc.emory.edu}
Greetings, Just a comment about fluorescent antifade compounds. In the past I have made side by side comparisons of several of these, came to a particular conclusion, and then found a colleague who had done similarly but came to a different conclusion. There are also some discrepancies in the various papers that have been published which make these comparisons. These differences seem to be based on differences in sample and/or fluorochrome. It appears that there is no "perfect" antifade, and that it may be worthwhile to try several for your given application.
In my lab, we use a product called Vectashield (from Vector labs; no affiliation; address available by request) that works great for our applications.
Our labs use Vectashield and / or Fluoromount-G from Southern Biotechnology { (available through Fisher).
The Vectashield works better for most less than ultrapure flourochromes, while the Fluoromount seems to give double the time to fade for the higher grade dyes. Specifically the dyes from Southern Biotech and Molecular probes are the better quality.
These evaluations are purely subjective as we have not done any time studies to varify what we have noticed during observations.
As an alternative, 4%Propyl galate (Sigma) can be prepared in glycerol and used as an alternative mounting medium. This works nearly as well as the Vectashield, IF you maintain a pH near 8.0. We routinely mix 4% PG/Glycerol with an equal amount of PBS for our routine medium. I should add this is for stains that will not be photographed. We save the more expensive material for this purpose. melsen-at-MICROBIO.emory.edu
Ref: Information about electronic enlargers/auto developers
Date posted 9-28-94, please respond within a few days only.
Sometimes ago I saw demonstrated an electronic pinpoint enlarger, but have not since then heard anything about it, nor have I seen it demonstrated at any meeting. I would appreciate some info. about such enlarger (a phone number if possible) and approximate cost. This I need for the preparation of a proposal. Also, what is your preference on automatic resin coated black and white paper developers. At present we use the Kodak machines but it will be replaced soon.
Thanks in advance for your help. ***** ************ ************** *************** *Cesar D. Fermin, Ph.D \||/ Fax (504) 587-7389 * *Tulane Medical School /||\ Answ. Mach.(504) 584-2618 * *Pathology/SL79 \||/ Secretary (504) 584-2436 * *New Orleans, La 70 112 /||\ Lab (504) 5841 * ***** ***************** ************************* ________________________________________________
Does anyone out there with thin window EDS detectors also do a lot of biological cryo SEM? If so are you beset by continual icing problems?
We will soon be replacing an old beryllium window detector with a new UTW (ultra thin window) detector, so this recent thread has been quite timely. We do a lot of cryo SEM on frozen hydrated plant materials and insects, usually for morphology, but also for EDS analysis.
I sometimes have to defrost surfaces with nitrogen gas introduced into the sample chamber, while keeping the sample fully frozen at about -160 C, just enough gas to warm up the surface frost to freeze-dry temperature, especially during Minnesota summers (we do 'poor-persons' cryo, no fancy cryo-transfer devices. Its slick, quick and easy, so not a problem, actually..........until now??)
Not only that, but the in-chamber cryo hook-up acts as a cryo pump, of course (I see slight improved high vacuum during a run) and when that hook-up warms up after shutdown, any trapped oil vapor and moisture is released in a concentrated dose into the chamber.
So am I in for a big headache with high rate of ice buildup on my thin window detector as a result of the cryo work that we do? As long as the sample remains totaly chilled at -160 C, there should not be much moisture released as a result of the e-beam scanning it, should there?
I wish I could park the detector snout in a "garage" when not being used for EDS, but seems like only true windowless detectors come with that option.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
In message "Fermin, Cesar" writes: Also, what is your preference on automatic resin coated black and } white paper developers. At present we use the Kodak machines but it will be } replaced soon. }
In December, 1993, I saved a file of 23 responses to a query on B&W photo paper processors and have added 7 more since then(from mid-summer, 1994). I will send my file to Cesar and if anyone else wants a copy, e-mail your request to me privately and I will get one to you.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
We are currently looking for software package(s) for visualization of stacks of digital images acquired with confocal/wide field microscopes. We know of one good commercial package, running on Silicon Graphics workstatins, but the price is a bit on the "steep" side. If anyone out there uses 3D visualization software (commercial or public domain), we would like to know which one it is, its price tag, the computer platform it runs on and how satisfied you have been.
Thank you in advance for your help. Massimo
_________________________________ Massimo Sassaroli Dept. of Physiology & Biophysics Box 1218 Mount Sinai School of Medicine 1 Gustave L. Levy Pl. New York, NY 10029-6574
Message-Id: {MAILQUEUE-101.940928151439.352-at-vanlab.paprican.ca} To: christoffersen-at-snmail.jsc.nasa.gov
Hi, I have been trying to decide on suitable standard samples for checking EDX detector performance myself and have a couple of suggestions to pass on:
- CaF2 should not produce an oxygen peak but will efficiently excite oxygen in ice which may be present on the crystal. (from Kevex's SuperQuantum brochure)
- CaCo3 (calcite) may be used to monitor the peak heights of carbon and oxygen relative to calcium, as well as to check the resolution of carbon and oxygen and check for the presence of silicon in the spectrum as an artifact produced by the detector.
I'd welcome any comments on the suitability of these two specimens for monitoring detector performance.
Have fun, Laurie
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
Two programs avail. through NIH are: 1) HVEM-3D (PC-based software) high voltage EM lab in Boulder, CO contact Jim Kremer {kremer-at-beagle.Colorado.Edu} he also has some great program for the SGI machine (see his abstract in the 1994 MSA proceedings) 2) SYNU (work station {SGI/SUN} software) microscopy and imaging resource in San Diego, CA contact: Mark Ellisman -at- UCSD or Simon Lee {simon-at-UCSD.edu}
both of these programs are free or close to it, and do everything the $k programs do.
-Mike On Wed, 28 Sep 1994 sassaroli-at-msvax.mssm.edu wrote:
} Hello fellow microscopists, } } We are currently looking for software package(s) for visualization of } stacks of digital images acquired with confocal/wide field microscopes. We } know of one good commercial package, running on Silicon Graphics } workstatins, but the price is a bit on the "steep" side. } If anyone out there uses 3D visualization software (commercial or public } domain), we would like to know which one it is, its price tag, the computer } platform it runs on and how satisfied you have been. } } Thank you in advance for your help. } Massimo } } _________________________________ } Massimo Sassaroli } Dept. of Physiology & Biophysics } Box 1218 } Mount Sinai School of Medicine } 1 Gustave L. Levy Pl. } New York, NY 10029-6574 } } sassaroli-at-msvax.mssm.edu } } } }
Hi I am searching help about the construction and use of the spindle stage. Is this device available from some manufacturer ? anybody can suggest me how constructing one by myself ? I know that exists a book on the subject (Bloss - The spindle stage, Cambridge Un. Press, 1981). Is this book still available ? Thanks
Regarding 3D Visualization software I received mail from a friend of mine, dealing with that subject. Below a copy of his message. Hope it will help you a bit furher,
A new release of the VolPack volume rendering library is now available by anonymous ftp (see instructions below).
VolPack is a software library written in C and intended for use in visualization applications. The library includes implementations of the volume rendering algorithm presented at SIGGRAPH '94 this past summer (see Philippe Lacroute and Marc Levoy, Fast Volume Rendering Using a Shear-Warp Factorization of the Viewing Transformation, Proc. SIGGRAPH '94, pp. 451-458). The algorithm is more than five times faster than any other direct volume rendering algorithm I know of. The library can render a 256 by 256 by 256 voxel volume in roughly one second on an SGI Indigo R4000, running entirely in software, and produces high-quality images.
Here is a brief list of features:
- Renders data sampled on a regular, three-dimensional grid. - Supports user-specified transfer functions for both opacity and color. - Provides a shading model with directional light sources, multiple material types with different reflective properties, and depth cueing. - Produces color (24 bits/pixel) or grayscale (8 bits/pixel) renderings. - Supports arbitrary affine view transformations. - Supports a flexible data format that allows an arbitrary C structure to be associated with each grid point. - Achieves very fast rendering times without specialized hardware.
The principal limitations of the algorithm are that performance is data-dependent (since some of the optimizations are based on coherence), and there are some constraints on resampling filter quality. For a technical discussion of the algorithm and the tradeoffs involved, please see the SIGGRAPH paper (also available by ftp).
A simple Tcl/Tk application based on VolPack is also available. A Tcl/Tk package with a complete set of Tcl bindings for the library routines will be available soon.
The complete VolPack distribution can be retrieved via the Web at URL:
http://www-graphics.stanford.edu/software/volpack
or by anonymous ftp from graphics.stanford.edu (36.22.0.39), directory pub/volpack:
ftp} cd pub/volpack ftp} get README ftp} binary ftp} get volpack.1.0b2.tar.Z ftp} bye
% zcat volpack.1.0b2.tar.Z | tar xvf -
Now look at the README file in the unpacked directory.
The current release is version 1.0beta2. It is a beta release. The distribution includes source code, a tutorial user's manual, man pages for all library routines, and some sample programs with one sample dataset. Other goodies are available at the ftp site.
Contact: Phil Lacroute (lacroute-at-weevil.stanford.edu)
-- Kees van der Wulp TNO - Nutrition & Food Research Institute INTERNET : vanderwulp-at-mbl.tno.nl Division of Toxicology VOICE : +31 15 843101 PO-Box 5815 FAX : +31 15 843989 2280 HV RIJSWIJK (NL) THE NETHERLANDS
Regarding your request for 3D vis. software I received a message from a friend of mine. Hope this will help you.
********************************************
A new release of the VolPack volume rendering library is now available by anonymous ftp (see instructions below).
VolPack is a software library written in C and intended for use in visualization applications. The library includes implementations of the volume rendering algorithm presented at SIGGRAPH '94 this past summer (see Philippe Lacroute and Marc Levoy, Fast Volume Rendering Using a Shear-Warp Factorization of the Viewing Transformation, Proc. SIGGRAPH '94, pp. 451-458). The algorithm is more than five times faster than any other direct volume rendering algorithm I know of. The library can render a 256 by 256 by 256 voxel volume in roughly one second on an SGI Indigo R4000, running entirely in software, and produces high-quality images.
Here is a brief list of features:
- Renders data sampled on a regular, three-dimensional grid. - Supports user-specified transfer functions for both opacity and color. - Provides a shading model with directional light sources, multiple material types with different reflective properties, and depth cueing. - Produces color (24 bits/pixel) or grayscale (8 bits/pixel) renderings. - Supports arbitrary affine view transformations. - Supports a flexible data format that allows an arbitrary C structure to be associated with each grid point. - Achieves very fast rendering times without specialized hardware.
The principal limitations of the algorithm are that performance is data-dependent (since some of the optimizations are based on coherence), and there are some constraints on resampling filter quality. For a technical discussion of the algorithm and the tradeoffs involved, please see the SIGGRAPH paper (also available by ftp).
A simple Tcl/Tk application based on VolPack is also available. A Tcl/Tk package with a complete set of Tcl bindings for the library routines will be available soon.
The complete VolPack distribution can be retrieved via the Web at URL:
http://www-graphics.stanford.edu/software/volpack
or by anonymous ftp from graphics.stanford.edu (36.22.0.39), directory pub/volpack:
ftp} cd pub/volpack ftp} get README ftp} binary ftp} get volpack.1.0b2.tar.Z ftp} bye
% zcat volpack.1.0b2.tar.Z | tar xvf -
Now look at the README file in the unpacked directory.
The current release is version 1.0beta2. It is a beta release. The distribution includes source code, a tutorial user's manual, man pages for all library routines, and some sample programs with one sample dataset. Other goodies are available at the ftp site.
Contact: Phil Lacroute (lacroute-at-weevil.stanford.edu)
From postmaster-at-compuserve.com Thu Sep 29 11:34:48 1994 Received: from frontier.tno.nl by mbimp1.mbl.tno.nl via SMTP (920330.SGI/920502.SGI) for imgp id AA10285; Thu, 29 Sep 94 11:34:48 +0100 Received: from arl-img-1.compuserve.com by frontier.tno.nl (4.1/1.53) id AA15203; Thu, 29 Sep 94 11:27:38 +0100 Received: from localhost by arl-img-1.compuserve.com (8.6.4/5.940406sam) id GAA25988; Thu, 29 Sep 1994 06:33:38 -0400
Re: ? EMDNRM - Mail Delivery Failure. No room in mailbox. 75022,2723 Re: 3D Visualization Software
Your message could not be delivered as addressed.
--- Returned message ---
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Dear Massimo,
Regarding 3D Visualization software I received mail from a friend of mine, dealing with that subject. Below a copy of his message. Hope it will help you a bit furher,
A new release of the VolPack volume rendering library is now available by anonymous ftp (see instructions below).
VolPack is a software library written in C and intended for use in visualization applications. The library includes implementations of the volume rendering algorithm presented at SIGGRAPH '94 this past summer (see Philippe Lacroute and Marc Levoy, Fast Volume Rendering Using a Shear-Warp Factorization of the Viewing Transformation, Proc. SIGGRAPH '94, pp. 451-458). The algorithm is more than five times faster than any other direct volume rendering algorithm I know of. The library can render a 256 by 256 by 256 voxel volume in roughly one second on an SGI Indigo R4000, running entirely in software, and produces high-quality images.
Here is a brief list of features:
- Renders data sampled on a regular, three-dimensional grid. - Supports user-specified transfer functions for both opacity and color. - Provides a shading model with directional light sources, multiple material types with different reflective properties, and depth cueing. - Produces color (24 bits/pixel) or grayscale (8 bits/pixel) renderings. - Supports arbitrary affine view transformations. - Supports a flexible data format that allows an arbitrary C structure to be associated with each grid point. - Achieves very fast rendering times without specialized hardware.
The principal limitations of the algorithm are that performance is data-dependent (since some of the optimizations are based on coherence), and there are some constraints on resampling filter quality. For a technical discussion of the algorithm and the tradeoffs involved, please see the SIGGRAPH paper (also available by ftp).
A simple Tcl/Tk application based on VolPack is also available. A Tcl/Tk package with a complete set of Tcl bindings for the library routines will be available soon.
The complete VolPack distribution can be retrieved via the Web at URL:
http://www-graphics.stanford.edu/software/volpack
or by anonymous ftp from graphics.stanford.edu (36.22.0.39), directory pub/volpack:
ftp} cd pub/volpack ftp} get README ftp} binary ftp} get volpack.1.0b2.tar.Z ftp} bye
% zcat volpack.1.0b2.tar.Z | tar xvf -
Now look at the README file in the unpacked directory.
The current release is version 1.0beta2. It is a beta release. The distribution includes source code, a tutorial user's manual, man pages for all library routines, and some sample programs with one sample dataset. Other goodies are available at the ftp site.
Contact: Phil Lacroute (lacroute-at-weevil.stanford.edu)
Laurie, My only commemnt about these 2 materials is that they both are quite beam sensitive. Using a focused beam (on the EPMA) or high magnification on the SEM will result in an evolved gas from the specimen. If you have a visible light optic system on your machine this process will be easily detectable. Hence, my suggestion would be to use beam currents no greater than 10 nA and relatively low magnifications (will require some experimentation), or a defocused beam of diameter } /=15um if you have that capability. In summary, "go easy on them."
} Hi, } I have been trying to decide on suitable standard samples for } checking EDX detector performance myself and have a couple of } suggestions to pass on: } } - CaF2 should not produce an oxygen peak but will efficiently excite } oxygen in ice which may be present on the crystal. (from Kevex's } SuperQuantum brochure) } } - CaCo3 (calcite) may be used to monitor the peak heights of carbon } and oxygen relative to calcium, as well as to check the resolution of } carbon and oxygen and check for the presence of silicon in the } spectrum as an artifact produced by the detector. } } I'd welcome any comments on the suitability of these two specimens } for monitoring detector performance. } } Have fun, } Laurie
Ed Vicenzi tel (609) 258-1464 office Princeton University tel (609) 258-1406 lab Princeton Materials Inst. fax (609) 258-6878 70 Prospect Ave. Princeton, N.J. 08540-5211 vicenzi-at-phoenix.princeton.edu
Does anyone have experience with high mag. SEM work using mica as a substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a Hitachi S4500 and am having a great deal of difficulty. I have tried coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At higher accelerating voltages (~10 kV) the image is not stable with time. I think I may be destroying the mica at these voltages. At lower accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so low that I have difficulty focusing at mag's above 10k X.
Steve Eppell Facility Coordinator Center for Cardiovascular Biomaterials Case Western Reserve University sje-at-po.cwru.edu
There are basicly two types of spindle stages avilable: 1. detent spindle stage McCrone Accessories & Co. 850 Pasquinelli Dr. Westmont, Illinois 60559-1275 USA Phone: 708-887-7100 Fax: 708-887-7764 The price should be about $40-60 apiece. 2. Supper spindle stage Charles Supper X-ray Co. 15 Tech Circle Natick, Massachusetts 01760 USA Phone: 508-237-2995 Fax 508-655-3913 Catalog # 7058: Crystal spindle stage, $360. Supper spindle is a much better instrument, which allows you to rotate the crystal 360 degrees. However, you will need a x-ray goniometer head to mount the crystal. In the meantime, you will a lot more flexibility to orient the crystal using the two mutually perpendicular arcs on the goniometer head. The standard goniometer head made by Supper costs about $340 apiece. But you may find a cheaper one through other manufacturers. 3. F. D. Bloss' book "The Spindle Stage" is also available through McCrone at about $90. Its catalog number is 455. If you want to use double variation method, you will need a heating cell. Check with Prof. Micky Gunter at University of Idaho to see if he still makes it. His e-mail address is GUNTER-at-IDUI1.CSRV.UIDAHO.EDU. Spindle stage is a extremely powerful technique. Good luck!
Shu-Chun Su Hercules Inc. Research Center 8136-267 500 Hercules Road Wilmington, DE 19808-1599 Phone: 302-995-3498 Fax: 302-995-4135
On Thu, 29 Sep 1994, Giorgio Gasparotto wrote:
} Hi } I am searching help about the construction and use of the spindle stage. } Is this device available from some manufacturer ? anybody can } suggest me how constructing one by myself ? } I know that exists a book on the subject (Bloss - The spindle stage, } Cambridge Un. Press, 1981). Is this book still available ? } Thanks } }
My 2 cents: I've done a fair amount of bio SEM, using CPD, dessicator, freeze, Pel-Dri, & hexamethyldisilizane dried specimens. I've noted very little performance degradation. The major requirement is to make sure the specimens really *are* dry--don't look at them right afte mounting & drying. Yes, for ultra-high vacuum materials science WDX or EDX scopes used for quantitative work, outgassing from biological specimens can be a problem. But then materials science specimens outgass--how many polymers are completely polumerized? their components will also outgass. Besides: 1) most of the contamination from bio samples is acutally from the silver paint/paste--the solvents can take a long time to fully evaporate, eapecially when the mounted samples are in therequired dessicator 2) bio specimens aren't done shrinking until they're completely dry, which takes time! maybe LOTS of time...(I hope your users aren't measuring soft tissues in the SEM/ SEM images). This is my experience with SEMs running at 10-6 & 10-7. If you need 10-9 torr, you may have more problems. Basic cleanliness and back-filling with dry nitrogen (even in a low-humdity environment!) does more for keeping a scope clean. And using turbo-molecular pumps instead of diffusion pumps makes a *big* differnece. EDX detectors: The ice comments are very useful. I did run into a problem once with pump-oil contamination (from a diffusion pump)--the simple fix was a beam collimator over the detector. This not only took care of the oil, by keeping away from the detector (the collimator acts like a cold finger), byt got rid of a spurious Chromium peak. Phil Oshel poshel-at-luc.edu
In message Thu, 29 Sep 1994 09:34:55 -0400, sje-at-po.CWRU.Edu (Steven J. Eppell) writes:
} } Does anyone have experience with high mag. SEM work using mica as a } substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a } Hitachi S4500 and am having a great deal of difficulty. I have tried } coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At } higher accelerating voltages (~10 kV) the image is not stable with time. } I think I may be destroying the mica at these voltages. At lower } accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so } low that I have difficulty focusing at mag's above 10k X. } } Steve Eppell } Facility Coordinator } Center for Cardiovascular Biomaterials } Case Western Reserve University } sje-at-po.cwru.edu } ============ Steve,
I have obtained relatively decent high mag images (X 150,000; upper detector, 7 kV) of Tobacco Mosiac Virus on freshly cleaved mica, coated with 2 nm of platinum (as measured by a quartz crystal thickness monitor; but who knows what the real film thickness on the mica and on the virus is!!), using our HITACHI 4500 FESEM. Rapid focusing to minimize "beam damage" before taking the picture seems to help. In general the specimen as well as the mica appear to deteriorate after they are subjected to just one or two slow scans (photo scan rate). I have found that obtaining high mag SEM images of biomolecules can be very challenging (to put it mildly)!
} Does anyone have experience with high mag. SEM work using mica as a } substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a } Hitachi S4500 and am having a great deal of difficulty. I have tried } coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At } higher accelerating voltages (~10 kV) the image is not stable with time. I } think I may be destroying the mica at these voltages. At lower } accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so low } that I have difficulty focusing at mag's above 10k X. } } Steve Eppell } Facility Coordinator } Center for Cardiovascular Biomaterials } Case Western Reserve University } sje-at-po.cwru.edu
Steve, Looks like you have a problem of thin coating. The SE signal yielding is much higher at low voltage than high voltage.
Ya Chen ---------------------------------------------------------------------------- | Assistant Researcher/Cryo-SEM Coordinator | Integrated Microscopy Resource (IMR)-- | An NIH Biomedical Research Center | University of Wisconsin-Madison | 1675 Observatory Drive #167 \ / | Madison, WI 53706 \ / |------------------------------------------- \/ /--/ | TEL: 608-263-8481 / / / | TEL: 608-265-3083 / /-- { | FAX: 608-265-4076 | Email:YChen-at-macc.wisc.edu | Email:chen-at-calshp.cals.wisc.edu ----------------------------------------------------------------------------
======================================================================= Chris Frethem (612)624-4652 (voice) Cell Biology & Neuroanatomy (612)624-8118 (FAX) U of MN, Minneapolis e-mail: frethem-at-lenti.med.umn.edu
---------- Forwarded message ----------
Steve, Try mounting your proteins on carbon films or carbon coated Formvar membrane on TEM grids instead, and view at higher (10 kV - 30 kV) accelerating voltages. Your grid should be mounted in such a way that you do not generate secondary electrons from a surface below the support membrane; a brass or aluminum chip with a 2.5 mm hole drilled in it, for example. The grid can be simply held over the hole with carbon tape or paint. I'm not familiar with the S-4500 specimen holders so I can't advise how to mount this "holey chip", but make sure that the beam either passes on into vacuum "space" beyond the membrane or that you provide a surface that generates few secondary electrons under this chip. Simply gluing your holey chip to another thin brass or aluminum chip with a thin layer of carbon paint or using double-sided carbon tape should suffice. I have had little experience using mica as a substrate but I'm assuming the instability you're seeing over time at 10 kV is due more to charging than any other factor, especially considering the very thin Cr or Ir sputter coat. The stability you see at 0.5 to 1.0 kV is expected for the same reason. Eliminating the mica should reduce this problem. You will have to search for the ideal kV; too high a kV and your secondary yield may be very low as the beam will blast right through your proteins without creating much topographic contrast. Using Iridium instead of Cr will help at the higher kV's but of course the Ir has more granularity than the Cr, too. Search for the best compromises. We have a Hitachi S-900 and have dealt with some of the issues you bring up in your question, though we can't claim to have solved them all yet! Good luck, and I hope someone else out there can feed you (us) some info too. By the way, how are you preparing these proteins? Air drying from suspension? Crit. pt. drying? Are they fixed? Freeze dried? What are they?
Bye
======================================================================= Chris Frethem (612)624-4652 (voice) Cell Biology & Neuroanatomy (612)624-8118 (FAX) U of MN, Minneapolis e-mail: frethem-at-lenti.med.umn.edu
On Thu, 29 Sep 1994, Steven J. Eppell wrote:
} } Does anyone have experience with high mag. SEM work using mica as a } substrate? I am trying to image proteins (~50 nm X 10 nm X 3 nm) in a } Hitachi S4500 and am having a great deal of difficulty. I have tried } coating the samples with ~1 nm of Cr or Ir in a VCR ion beam coater. At } higher accelerating voltages (~10 kV) the image is not stable with time. I } think I may be destroying the mica at these voltages. At lower } accelerating voltages (0.5 - 1 kV) the image is stable but the S/N is so low } that I have difficulty focusing at mag's above 10k X. } } Steve Eppell } Facility Coordinator } Center for Cardiovascular Biomaterials } Case Western Reserve University } sje-at-po.cwru.edu }
South Bay Technology produces sample preparation equipment for TEM, SEM, metallography and crystallography - so if you want to do the samples on your own we can surely help. If, on the other hand, you prefer to use an outside service, I have the following suggestions of people to contact:
IBM Corp. Contact: Ron Anderson TEL: 914-892-2225 FAX: 914-892-2555
Philips Semiconductor Contact: David Su TEL: 408-991-4798 FAX: 408-991-4801
NREL Contact: Kim Jones TEL: 303-275-3734 FAX: 303-231-1030
I hope this helps! If I can be of any other assistance, please let me know.
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
I am a biologist with limited experience with materials specimens....... a student wants to look at a magnetic sample and I would appreciate any suggestions as to the best way to handle this?
Because of their cell walls, yeast have proven to be difficult to fix and infiltrate successfully. There are a couple of options that I can recommend.
1. If you just need an overall view of ultrastructural organization, use glutaraldehyde followed by potassium permanganate fixation.
2. If you need to do deatiled ultrastructural work or immunolabeling, fix in glutaraldehyde, treat with sodium metaperoidate to make the wall more permeable, and then postfix in osmium. Byers and Goetsch, the first people to look at yeast by EM (as far as I am aware), use an enzyme treatment instead of periodate.
The methods that I use follow in detail. -------------------------------------------------------------------- KMnO4 Technique for Electron Microscopic Study of Yeast Membranes
Robin Wright University of Washington Department of Zoology, NJ-15 Seattle, Wa 98195
Fixation:
Grow cells to early log phase (1 OD/ml) in media. Make up fresh 5X fixative:
Stock Solution Amount Used Final Concentration
20X TBS 6.25 ml 5X 1 M CaCl2 0.125 ml 5mM 1 M MgCl2 0.125 ml 5mM 2.4 M Sorbitol 10.4 ml 1M Distilled H2O 3.1 ml 50% Glutaraldehyde 5 ml 10% Final Volume 25 ml (This is enough to fix 80 ml cells).
Pour 10 ml of 5X Fixative into 50 ml plastic tubes. Pour the 40 mls of cells into the fixative and mix immediately. Let sit at room temperature for 5 minutes. Spin cells down for 4 minutes speed four in the table top centrifuge. Pour off the media into the hood sink with lots of water (this may need to go into a special waste container nowadays). Gently resuspend the pellet in 12.5 ml 1X Fixative (diluted from leftover 5X stock). Let sit at room temperature for 2 minutes then refrigerate overnight 4oC).NOTE: You can get away with fixing many fewer cells with less fixative, or scale down this procedure to fix more samples.
KMnO4 Treatment: Make up 10 mls fresh 4% KMnO4 in water and filter with a 0.2 mm acrodisc. Wash cells fixed with glutaraldehyde four times with deionized water (20mls) using the table top centrifuge at speed 4 for 2-3 minutes. Resuspend in 4 ml water. Aliquot 2 ml into glass conical tube. (You can use the other 2 ml for immuno EM --see LR White protocol). Add 2 mls of 4% KMnO4 and let sit at room temperature for 5 minutes. Spin cells down at speed 4 for 2Õ. Pour off KMnO4. If the cells clump donÕt try to resuspend them completely (clumping is desirable). Add 6 ml 2% KMnO4 and mix with cells by gently resuspending with a pasteur pipet (only trying to get solution under the pellet ). Let sit at room temperature 1 hour. Rinse cells in water twice (or until all the KMnO4 purple color is gone) as before. Cover with 1% uranyl acetyate and let sit ON at 4oC.
Dehydration Series: Dehydrate as follows: (1) The cells are resuspended in 10 ml 50% ethanol and then spun in a conical, glass test tube, in a clinical centrifuge for 5 min. (The use of glass is important for maximizing clumping of the pellet into aggregates of convenient size.) (2) The supernatant is replaced with 1 ml of 70% ethanol. At this point, the cells will form a firm pellet that can be gently dislodged with a pasteur pipette and chipped into small, pepper-size fragments. An aliquot containing 10-20 small chunks is dispensed into glass, snap cap vials containing 70% ethanol. The remaining sample is stored at -20oC in Eppendorf tubes and is available for subsequent processing, if necessary. I have removed aliquots from samples stored in this manner after several months and noted no alteration in ultrastructure or immunoreactivity. (3) The glass vial is capped and placed on a rotating drum for 5 min. Dehydration is completed in subsequent incubations (5 min rotations) in 95% and 3 changes of 100% ethanol. For the absolute ethanol incubations, a new bottle of ethanol is opened to ensure absence of water. I have also used molecular sieves (a teaspoonful wrapped in several layers of lens tissue) to remove traces of water from absolute ethanol. (4) A convenient way to change solutions is to use a pasteur pipette to remove most of the liquid from the vial while it is tipped to allow the sample fragments to gather at one side of the vial. Then, place the end of the pipette flush with the bottom of the vial on the side opposite the sample. The vial is tipped to allow fluid to reach the pipette and the fluid is slowly aspirated. Sample fragments of appropriate size for processing will gather around the bore of the pipette, but will not enter, IF the pipette is properly oriented. This procedure allows solution changes to be relatively rapid and complete.
Resin: Ultra Low viscosity resin is toxic. Be very careful when you handle it. Use only in the hood and wear gloves. Mix resin in a tripour plastic beaker using a tongue depressor. Add in order: 25g vinyl cyclohexene dioxide 52.5g n-hexenyl succinic acid 1.5g DER736 0.2g DMAE Pour mixture into a dispenser bottle. If there is left over resin at the end of your experiment, store it at -70oC. Before you use resin frozen in this manner, allow it to come to room temperature , then uncap it.
Infiltration: (1) Patience is a virtue. The last 100% ethanol is replaced with a solution of 2 parts ethanol to 1 part resin. The vial is capped and returned to the rotator for 1 hr. (2) The resin mixture is replaced with 1:1 resin:ethanol mix and the capped vial rotated for at least 1 hour. (3) The resin is exchanged with a fresh 1:1 mix and the samples are rotated, UNCAPPED, overnight in a hood. The ethanol slowly evaporates, increasing resin concentration gradually. (4) The next day, the residual resin is replaced with fresh 100% resin and the samples are rotated for an hour. The resin is changed and the samples placed under vacuum (20 psi) for 15 min to degas, then returned to the rotator for 1 hour. (5) Small pellet fragments are removed from the vial, using a sharpened applicator stick. The fragment is gently "rolled" across a Kimwipe to remove residual resin and placed into 5 ml plastic embedding cups (Ted Pella, Inc.) containing resin. These cups are placed under vacuum for 1-2 hours.
Embedding: Preheat temperature block or oven to 60oC. Fill out labels with pencil and place into BEEM capsules (make 5 per sample ). Pour 2 drops of resin into BEEM capsules. Transfer a single, good size chunk of cells with an applicator stick (broken so that it has a sharp end) to the capsule. Use a dissecting microscope to help you center the cells and to get rid of the air bubbles (use an unbroken applicator stick). Fill the BEEM capsule to the line with resin. Allow the resin to polymerize in the 60oC temperature block. Section and stain with ReynoldÕs lead citrate. I usually dilute standard ReynoldÕ lead citrate 1:10 with 0.1M NaOH and stain for 1 min.
------------------------------------------------------------------ Processing Yeast Cells for Immunocytochemistry using LR White Resin Robin Wright Department of Zoology, NJ-15 University of Washington Seattle, WA 98195 206-685-3659
We have recently begun to routinely use LR White resin for all electron microscopic studies of Saccharomyces cerevisiae. This resin infiltrates whole cells nearly perfectly, with no special effort, and produces samples that have less lipid extraction than normally seen with epoxy resins. It is easy to section, stains well with uranyl acetate and lead citrate, and produces excellent results upon immunolabeling. In addition, LR White is premixed and much less toxic (non-carcinogenic) than typically used EM resins. The major drawback is that LR White sections are somewhat unstable in the electron beam and it takes a degree of patience to wait until all movement stops before exposing the film.
Use of van Tuinen & Reizman's technique (J. Histochem Cytochem 35:327-333, 1987) involving treatment of fixed cells with sodium meta-periodate was the critical factor in getting consistent results with LR White. This treatment apparently reduces sugar bonds in the wall to allow efficient infiltration of the resin. In addition, we found that the temperature of polymerization must be very carefully regulated and have resorted to using temp blocks (like those commonly used for restriction digestion) specially drilled to contain gelatin capsules. The entire technique is outlined below, drawing heavily on a paper I prepared for Methods in Cell Biology.
FIXATION Gross morphological alterations and changes in the presence and/or location of proteins can be induced by subjecting yeast to centrifugal forces (R. Preston, Carnegie-Mellon University, Pittsburgh, PA; personal communication; L. Pillus, University of California, Berkeley, CA; personal communication). To avoid such changes during fixation for immunofluorescence, the fixative can be added directly to the growing yeast culture (L. Pillus, personal communication). Encouraged by this observation, Chris Kaiser (University of California, Berkeley, CA) and I have tested its feasibility for electron microscopy with excellent results. Since the technique is considerably more rapid than others that require multiple washes prior to fixation, this direct fixation method has become our favored protocol.
(1) 1/10th volume of 10X prefixative solution (10% glutaraldehyde, 2% methanol-free formaldehyde, 0.4M potassium phosphate, pH 7) at room temperature is placed into a centrifuge bottle or tube. (It is good practice to dedicate reusable plastic and glassware for EM, to avoid any possibility of contamination of cultures, etc, with traces of fixatives).
(2) The culture (early log phase) is poured rapidly into the fixative and allowed to sit at room temperature for 5 min. During this time, the medium will turn quite dark (YPD) or yellow (YM).
(3) The cells are pelleted, resuspended in 1/10th volume of ice-cold, 1X prefixative (1% glutaraldehyde, 1% methanol-free formaldehyde in 0.04M potassium phosphate, pH 7), and allowed to complete fixation on ice for 30 min.
(4) Excess fixative is removed by 3 buffer washes (i.e. 0.04M potassium phosphate, pH 7), leaving the cell suspension in each change of buffer for at least 5 min.
(5) Note: Either cacodylate or phosphate buffer gives comparable results and the presence or absence of Ca++ and/or Mg++ (1mM each) does not make obvious differences in results.
PERIODATE TREATMENT
(1) The washed cell pellet is resuspended in 5ml freshly mixed 1% sodium metaperiodate (aqueous) and allowed to incubate at room temperature for 15 min. The sample may become clumpy at this point, an advantage for later steps.
(2) Cells are pelleted and washed once in phosphate buffer.
(3) The pellet is then resuspended in 50mM ammonium phosphate to block free aldehyde sites. No attempt is made to break the clumps of cells, just to ensure that the entire pellet is accessible to the reagent. After a 15 min incubation at room temperature, the cells are washed twice with distilled water.
DEHYDRATION
(1) The cells are resuspended in 10 ml 50% ethanol and then spun in a conical, glass test tube, in a clinical centrifuge for 5 min. (The use of glass is important for maximizing clumping of the pellet into aggregates of convenient size.)
(2) The supernatant is replaced with 1 ml of 70% ethanol. At this point, the cells will form a firm pellet that can be gently dislodged with a pasteur pipette and chipped into small, pepper-size fragments. An aliquot containing 10-20 small chunks is dispensed into glass, snap cap vials containing 70% ethanol. The remaining sample is stored at -20oC in Eppendorf tubes and is available for subsequent processing, if necessary. I have removed aliquots from samples stored in this manner after several months and noted no alteration in ultrastructure or immunoreactivity.
(3) The glass vial is capped and placed on a rotating drum for 5 min. Dehydration is completed in subsequent incubations (5 min rotations) in 95% and 3 changes of 100% ethanol. For the absolute ethanol incubations, a new bottle of ethanol is opened to ensure absence of water. I have also used molecular sieves (a teaspoonful wrapped in several layers of lens tissue) to remove traces of water from absolute ethanol.
(4) A convenient way to change solutions is to use a pasteur pipette to remove most of the liquid from the vial while it is tipped to allow the sample fragments to gather at one side of the vial. Then, place the end of the pipette flush with the bottom of the vial on the side opposite the sample. The vial is tipped to allow fluid to reach the pipette and the fluid is slowly aspirated. Sample fragments of appropriate size for processing will gather around the bore of the pipette, but will not enter, IF the pipette is properly oriented. This procedure allows solution changes to be relatively rapid and complete.
INFILTRATION
(1) Patience is a virtue. The last 100% ethanol is replaced with a solution of 2 parts ethanol to 1 part resin. The vial is capped and returned to the rotator for 1 hr.
(2) The resin mixture is replaced with 1:1 resin:ethanol mix and the capped vial rotated for at least 1 hour.
(3) The resin is exchanged with a fresh 1:1 mix and the samples are rotated, UNCAPPED, overnight in a hood. The ethanol slowly evaporates, increasing resin concentration gradually.
(4) The next day, the residual resin is replaced with fresh 100% resin and the samples are rotated for an hour. The resin is changed and the samples placed under vacuum (20 psi) for 15 min to degas, then returned to the rotator for 1 hour.
(5) Small pellet fragments are removed from the vial, using a sharpened applicator stick. The fragment is gently "rolled" across a Kimwipe to remove residual resin and placed into 5 ml plastic embedding cups (Ted Pella, Inc.) containing resin. These cups are placed under vacuum for 1-2 hours.
EMBEDDING
(1) The sample fragments are then removed, blotted on a Kimwipe, and transferred to labeled GELATIN capsules filled with resin (one fragment per capsule). The longer, narrower portion of the capsule is filled with resin and the wider, shorter portion used as a cap after the sample is introduced. The capsules should have been dried overnight in a 60oC oven.
(2) The sample is allowed to sink to the bottom, positioned in the center of the capsule, and then placed under vacuum for 15 min.
(3) Polymerization is accomplished in a tempblock that has been drilled to contain gelatin capsules. The block is equilibrated at 45 -50oC and polymerization continues for 2 days. A thick pad of aluminum foil is placed on top of the block to maintain heat.
(4) Lois Banta and Scott Emr recommend a 3-day polymerization of LR White at 4oC using UV irradiation, but I have no direct experience with this method.
Grid Preparation and Sectioning
My first immunocytochemistry attempt was a resounding disaster, since the sections floated off all 20 grids during the first wash. That experience underscored the necessity of using a technique that ensures the sections will stay in place during the entire procedure. A formvar-coating technique of Bonnie Chojnacki (Carnegie-Mellon University, Pittsburgh, PA) has proven wonderfully effective for this purpose (see below). Nickel grids (200 - 300 mesh) are used, since they are less "reactive" than the standard copper ones. However, nickel grids readily become magnetized and it will save a great deal of frustration to have non-magnetic forceps on hand for handling them. The "tennis racket" style grids with handles (Ted Pella, Inc.) are highly recommended for the ease of handling they afford.
The hydrophilic nature of LR White requires that the water level in the boat be kept very low to prevent water from leaping onto the blockface as it passes the knife edge. Other special handling is not necessary. For serial sections, Fahrenbach's method (trimming the block to have wide leading and trailing edges and using diluted rubber cement to coat these edges) works amazingly well(Fahrenbach, 1984).
a. Grid Preparation
(1) Nickel grids are placed into a glass vial containing 100% ethanol and sonicated for 1 min. They are then rinsed several times in ethanol, dumped onto filter paper in a glass petri dish, and dried in a 50-60oC oven.
(2) The washed grids are placed in a glass petri dish containing a dilute formvar solution (1 ml 2% formvar in ethylene dichloride into 25 ml 24:1 ethylene dichloride:chloroform). A pasteur pipette can be used to wash the grids into one edge of the dish.
(3) The grids are individually removed with forceps and placed onto clean filter paper to dry. Only the bars are coated with formvar, leaving the entire grid space open for view of the section. "Sticky-bar" grids prepared in this manner are usable as soon as they have dried and are effective indefinitely.
b. Sectioning and Section Mounting
(1) It is wise to prepare a sufficient number of grids for several immunolabeling experiments at one time, taking into consideration all the controls and parameters to be tested. Each grid should contain as many sections as possible. Silicon mats with divided sections (available from any EM supplier) are very convenient for storing grids securely.
(2) LR White sections should not be exposed to chloroform vapors. The hydrophilic nature of the resin spreads the sections to eliminate compression while the section floats on the water surface.
(3) Sections are manipulated into a group using an eyelash glued to a sharpened applicator stick (clear nail polish works well). The dull side of a coated grid is carefully lowered over a group of sections floating in the boat and pressed into the the water surface, without breaking surface tension. The grid is removed and inverted onto a Kimwipe to blot excess moisture. After air drying, the grid is placed onto a silicon mat for storage.
(4) In view of the many grids required to do a complete immunolabeling experiment, I have incorporated Alice Taylor's (University of California, Berkeley, CA) "assembly-line" method. Sections are allowed to accumulate in the boat until a sufficient number have been cut. During the later stages of cutting, grids are loaded into 10 forceps, each of which has been fitted with a narrow piece of tygon tubing. The tubing is pushed down toward the forcep points, holding the forcep closed and keeping the grid in place until needed. The loaded forceps are propped up, ready for use. By having 10 forceps available with grids, the time for loading the sections onto the grids is reduced considerably.
c. Securing the sections
(1) If the sections are to be used immediately, it is essential to secure the sections onto the grid. The dish containing the grids loaded with sections is placed into a 50- 60oC oven for 1 - 2 min. This treatment does not affect immunolabeling or ultrastructure and ensures that the sections do not leave the grid, even under harsh conditions.
(2) Air-drying the grids for 1 - 2 days is also usually effective, but I have lost sections when the heating step is omitted. d. Storing Sections Polymerized resins are surprisingly fluid (Aldrich and Mollenhauer, 1986). Movement of embedded material occurs both in blocks and on sections. For optimal resolution, the sections should probably be used within a few days. In practice, I have detected no noticeable changes in immunolabeling or resolution after 3 months, but the possibility of alterations should be kept in mind.
NOTE: LR White-embedded samples are amenable to immunofluorescence as well as to immuno-gold labeling. Sections should be lifted from the boat using a bacteriological loop. The loop with the water film and sections is then touched to a glass slide that has been coated with a very thin layer of 1% gelatin. (The slide should be dry.) Allow the water to dry from around the sections and then fix the sections in place with 2% formaldehyde in PBS (10 min is sufficient, or you can store the slides in this solution until ready to use.) Rinse in PBS several times and then process as you would for immunofluorescence of whole cells. Use of Coplin jars for washes is recommended.
IMMUNOLABELING
Generation of reagent antibodies of high specificity is of utmost importance for accurate immunolocalization at the electron microscope level. While this requirement cannot be overstated, it is beyond the scope of this article to review the techniques for preparation of the antibody probes. De Mey (1983) is an appropriate initial source for this information. The antiserum should be affinity-purified. While theoretically feasible, immunoadsorption to remove undesired antibodies does not produce a serum with the required specificity for immunocytochemistry. Attempts to utilize preadsorbed sera in collaboration with Johanna Reneke and Jeremy Thorner (University of California, Berkeley,CA) and with Alex Franzusoff and Randy Schekman (University of California, Berkeley, CA) have convinced us of the necessity of affinity purification. Even a miniscule percentage of "contaminating" antibodies left after immunoadsorption can produce severe problems in interpretation. Background staining of the cell wall, vacuole, and nucleus are especially problematic. The time involved in preparation of cells for immunolabeling merits use of the best possible antibody. A prudent investigator would attempt immunofluorescent localization before immunocytochemistry, since it is easier, quicker, and might provide sufficient data without resorting to the more challenging, time-consuming steps of immunocytochemical studies. In addition, immunofluorescence will provide a standard against which immunocytochemical data can be interpreted. LR White sections can be used for immunofluorescent studies. In fact, use of sections provides a much greater intensity of staining than normally seen with whole cell preparations, perhaps due to greater accessibility of the antibody to the antigen. The easiest way to use LR White section for light microscopy is to mount them on solid nickle grids. The sections should be lifted very carefully from below to eliminate folds and "corrugations" in the sections. Obviously, the use of solid grids precludes use of normal phase microscopy, but the sections are so thin (1/50th the width of a yeast cell) that normal light microscopy (including Nomarski) are useless anyway. Preparation of colloidal gold reagents conjugated to Protein A is quite simple (see Roth, 1982 and 1983; Smit and Todd, 1986). Immunoglobulin-congugated gold reagents purchased from commercial sources (Janssen, Piscataway, NJ) give less background in our hands, however. Whether this difference is due to better conjugation techniques or to inherent increases in specificity of immunoglobulin as compared to Protein A is not clear. We generally use Goat-anti-rabbit immunoglobulin-conjugated gold (GARG), purchased from Janssen. We have noticed some problems with clumping and non-specific staining with 15nm GARG particles from this supplier (see results) but reagents with smaller gold particle size were satisfactory.
Controls: Convincing Yourself and Colleagues that the Results are Real For control experiments, the same rules apply to all immunochemical techniques. Labeling of sections in the absence of primary antibody will control for immunoreactivity of the secondary antibody alone. Using preimmune serum in the primary incubations will provide evidence that the labeling observed is dependent on the immune response induced after the antigen is introduced into the animal. In the case of affinity-purification, the pass-through from the affinity column contains those antibodies in the animal that do not react with the antigen and provides a similar control as the preimmune serum and is analogous to controls based on immunoadsorption. In addition to controls based on varying the antibody probe, yeast offers a wealth of possibilities to test whether or not the observations are valid. For example, strains that either overproduce or that lack the particular antigen can be immunolabeled and the patterns compared to that of the wild-type strain. Controls based on reproducibility merit mentioning: conclusions should be based on multiple experiments, including observation of duplicates from a single experiment, labeling of sections from different blocks of a single fixation, and labeling of sections from blocks of cells fixed on another day and/or with a different fixation procedure.
Experimental Procedures Set up of Incubation Chamber:
a. It is very helpful to set up the incubation chamber with labels at the start of the experiment. The chamber consists of a box with a tight-fitting lid, of sufficient size to contain all the grids and all the solution droplets. A padding of paper towels or sponge-cloths (very thin sponges about 6" X 6", from the grocery store) is placed into the bottom of the dish and thoroughly wetted to maintain humidity throughout the labelling steps. The surface should be fairly flat. Paper towels are folded, pressed onto the sides of the dish, and moistened.
b. Onto the wet pad, a length of Parafilm is positioned. Small, colored adhesive-dots ("sticky dots") labeled with the identity of the grid (i.e. strain, fixation variation, etc.) are placed on the left side, at approximately 1 inch intervals down the length of the Parafilm strip. Across the top of the Parafilm, 4 sticky dots are also positioned. The first and third represent the position where droplets of blocker will be placed. The second is the position of the primary antibody (1o) and the fourth is the position of the gold-conjugated secondary antiserum (2o). It is good practice to do duplicates of each incubation, so that a backup is available if technical problems occur.
Reagents: PBST (140mM NaCl, 3mM KCl, 8mM Na2HPO4, 1.5mM KH2PO4, and 0.05% TWEEN-20) is used throughout the immunolabelling protocol, for all washes and as the vehicle for the blocker. Blocker is PBST containing 2% ovalbumin. Glass distilled water of high purity (as for EM) is used and solutions are filtered through a 0.22m filter (Acrodisc) before use. Blocker solution can be prepared, filtered, aliquoted, and stored frozen at -20oC. If the blocking solution has been frozen, it refiltered before use. Both 1o and 2o are diluted into blocker. The dilution factor for the 1o must be empirically determined, using a dilution series. The gold-conjugated 2o should be adjusted to A525 = 0.3 for 15nm particles and to A525 = 0.13 for 5-10nm particles (from Daniela Brada).
Blocking: 20 ul droplets of 2% ovalbumin in PBST are positioned in the appropriate position on the parafilm sheet. The appropriate grid is submerged into the solution and allowed to incubate for 15 min at room temperature. Submerging is preferable to floating, since it will allow labeling of exposed antigen on both sides of the section.
Incubation in Primary Antiserum:
a. After blocking, the grid is removed from the blocker, touched to a Kimwipe to removed excess fluid. For this and subsequent blottings, the forceps tip and grid should be held sideways on the tissue surface, so that fluid between the forceps tips is also removed. This step should be done rapidly, not allowing the sections to dry.
b. The grid is then submerged in a 20ml droplet of diluted 1o antiserum. Length of incubation is probably a matter of convenience. The results presented here were from 2 hour incubations at room temperature. Overnight incubations at room temperature or 4oC have also been successful and may allow a lower concentration of 1o to be used.
Washes:
a. Washes are performed in the wells of porcelain or glass spot plates. The wells are marked and filled with PBST, and the spot plate is placed an orbital shaker.
b. After removal and blotting of excess fluid from the grid as described above, the grid is submerged in the appropriate well. The shaker is adjusted so that the solution is moving as rapidly as possible without spilling out of the well.
c. After 5 min, the grid is removed, blotted, and transferred to the next well. A total of 3 (5 min) washes are performed and the grid is blotted and transferred to a second droplet of blocker.
Incubation in Secondary (Gold-conjugated) Antiserum:
a. After a 15 min incubation in blocker, the grid is blotted and transferred to the diluted 2o. The grid is incubated for 1 hour at room temperature and then washed as above (III.B.5).
b. After the final PBST wash, the grid is washed in distilled water by dipping 10 times with rapid up and down motion in a 5ml beaker of water. This wash removes salts that would crystallize on the section, obscuring the view in the electron microscope. The grid is blotted on a Kimwipe, transferred to a labeled silicon mat, and allowed to air-dry.
OBSERVATION I generally look at the grids prior to staining with uranyl acetate or lead citrate, just to get an overall picture of the labeling pattern. When I am ready to sit down and take pictures, the grids are stained in 2% aqueous uranyl acetate for 1-5 min and in Reynold's lead citrate for 30 sec.
If you encounter any unexpected problems, or if these instructions are unclear, please contact me. I would also appreciate hearing whether or not this technique was successful in your studies and to be updated if you find variations that are important.
If you are going for standard high quality morphology then use 2% glutaraldehyde in 0.1M cacodylate buffer w/ added 0.005M CaCl2. The are growing in some sort of culture medium, so concentrate a sample of them in a tube with a contrifuge then reduce the volume of culture medium such that you can comfortably add an (approx.) equal volume of fixative to the tube. Then resuspend the yeast (gently) in the fix and allow to fix for about 1 hour (I would guess). Centrifuge again and draw off the fix, then wash with rinsing buffer. I've used this method for years on all types of suspended single cell preparations with good results. Good luck and let me know how it goes.
Dan
On Thu, 29 Sep 1994 BARBARA.HARTMAN-at-1773.220.SCHERING-PLOUGH.sprint.com wrote:
} } GREETINGS, } } WHAT IS THE BEST FIXATIVE FOR YEAST? I AM USED TO DEALING WITH ANIMAL } TISSUES SUCH AS LIVER AND KIDNEY SO THIS IS A NEW ONE FOR ME. } } THANKS } } } } BARBARA HARTMAN } SCHERING PLOUGH RESEARCH } FAX 201-579-4211 } PHONE 201-579-4343 } } }
Another substrate you might want to try is silicon wafer, especially if you have a friendly lab doing work on semiconductors nearby. We've found it useful for some imaging of TMV to test coatings. It conducts nicely and yields low background. Otherwise I would agree with the use of TEM grids supporting a carbon coated film as suggested by Chris Frethem.
As regards coatings Cr has a low signal yield due to its low atomic number, so reducing the background is important particularly at high kV. Platinum coatings of around 2nm will give a nice strong signal at the expense of a little resolution. With the size you give for your protein you may find the platinum grains mask detail.
The degrading image will be a combination of radiation damage, contamination and charging, cooling your specimen will help greatly (particularly for the former two) if you have the capability. I wish you luck and patience! Cheers
Peter
Peter Vesk, E.M. Unit,F09 University of Sydney NSW 2006 Australia phone: 61 2 692 2351 (overseas) fax: 61 2 552 1967 peter-at-emu.su.oz.au
JOURNAL OF MICROSCOPY, VOLUME 176 PART 1, OCTOBER 1994 ******************************************************
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 1-7.
Review: The measurement of intracellular antigens and DNA by multiparametric flow cytometry
R S Camplejohn, Division of Oncology, St Thomas' Hospital Medical School, Lambeth Palace Road, London, SE1 7EH
SUMMARY
The aim of this paper is to provide a strategy for measuring intracellular antigens combined with DNA content in cells or nuclei. A series of protocols are included which enable the majority of such antigens to be labelled and further information is provided for cases in which the standard methods prove to be inadequate. The basic principles of cell permeabilization/fixation are described, thus explaining how methods can be divided into three basic categories: (a) alcohol fixation with or without detergent pretreatment; (b) paraformaldehyde fixation followed by permeabilization with alcohol or detergents; (c) permeabilization of unfixed cells. The preparation of nuclear suspensions from paraffin-embedded material is described and the possibilities and problems of staining such suspensions for nuclear antigens are discussed. Examples of results obtained with the detailed protocols are given for staining with antibodies directed against proliferating cell nuclear antigen (PCNA), Ki-67 antigen and KiS1 antigen. Details of published studies of a variety of intracellular antigens are given in two tables. The power of multiparametric flow cytometry in the study of cell proliferation, differentiation and response of cells to damage is underlined.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 8-16.
Rapid estimation of bacterial antibiotic susceptibility with flow cytometry
D J Mason, R Allman, J M Stark & D Lloyd, Division of Microbiology, St Thomas' Campus, Lambeth Palace Road, London, SE1 7EH
SUMMARY
Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15 degrees). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2-5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 17-22.
Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry
A C Pinder & Rosemary G McClelland, Department of Food Biophysics, Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA
SUMMARY
Multi-parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full-fat milk). In all cases, the method was accurate to levels below 10 000 target cells per ml for a total assay time of about 30 min. After 6h non-selective enrichment in the presence of a 10 000-fold excess of competing micro-organisms (Escherichia coli) the corresponding detection limit was about 20 cells/ml. These results suggest that flow cytometry has a significant potential for the detection of pathogenic micro-organisms in the food industry.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 23-33.
Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging
The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the 'Scamper'. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result, almost any desired pattern could be bleached ('written') into fluorescent samples at high definition and then imaged ('read') at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 34-44.
Optical microscopical evaluation of pyrite oxidation of heap leached samples from a Pittsburgh seam coal
Gino A Irdi & Harold B Booher, United States Department of Energy, Pittsburgh Energy Technology Center, PO Box 10940, Pittsburgh, Pennsylvania 15236, USA
SUMMARY
The US Department of Energy and the US Bureau of Monies conducted experiments to determine the feasibility of reducing the pyritic sulphur content of a conventionally cleaned Pittsburgh seam coal by heap leaching. Two identical heaps, one indoor and one outdoor, were constructed, and sprayed with recycled rainwater/leachate for approximately 200 days. Lump samples were selected prior to initiation of heap leaching activities and from both heaps after 5- and 11-month intervals for microscopic (petrographic) examination. These examinations revealed no significant differences in pyritic sulphur removal between indoor and outdoor heaps. Samples from the starting coal were also selected and examined to see how different pyrite morphologies behaved during artificial weathering. Dendritic and framboidal pyrite forms appeared to be the more reactive forms.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 45-53.
A comparison of the analysis of the Fresnel contrast in high and low resolution images for the characterization of the rigid body displacements at a grain boundary
S H Stobbs, D L Medlin, M J Mills & W M Stobbs, University of Cambridge, Newnham College, Cambridge, CB3 9DF
SUMMARY
The Fresnel contrast in high-resolution images of the Sigma=3{112} aluminium twin boundary is quantitatively assessed to determine whether the rigid body displacements might be measured. It is demonstrated that the effects of the shears at the boundary preclude this possibility. By comparison, simulations of low-resolution images of the same boundary show that the Fresnel fringe contrast in this type of image is negligibly affected by the shear displacements as well as being of considerably higher contrast. The reasons why it is thus only the low-resolution images that can provide high-resolution data on the displacements are discussed.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 54-62.
Fractal characterization by frequency analysis. Part III: Effect of noise
Manuel Pancorbo, Eloy Anguiano & Miguel Aguilar, Instituto de Ciencia de Materiales, Consejo Superior de Investigaciones, Cientificas C-3-301, Universidad Autonoma de Madrid, Cantoblanco 28049 Madrid, Spain
SUMMARY
The effect of noise in the fractal characterization by frequency analysis of surface images obtained by scanning tunnelling microscopy (STM), atomic force microscopy (AFM) or profilometry has been studied. The origin of noise and its relationship to the signal is discussed. A procedure to simulate noisy images is presented. From the study it is concluded that the method usually used to characterize noise in STM is not valid and it is shown that fractal characterization of surfaces when noise is present by traditional frequency analysis methods is not possible. A new method to perform both the noise characterization and the fractal characterization of surfaces when noise is present is proposed.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 63-74.
Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher order structures by laser plasma X-ray contact microscopy
Yasuhito Kinjo, Kunio Shinohara, Atsushi Ito, Hisako Nakano, Makoto Watanabe, Yasuhiro Horiike, Yukiko Kikuchi, Martin C Richardson & Kazuo A Tanaka, Radiation Biology Division, Tokyo Metropolitan Isotope Research, Centre, Setagaya, Tokyo 158, Japan
SUMMARY
X-ray contact microscopy with a 300-ps-duration laser-plasma X- ray source has been used to image hydrated human chromosomes. Clearly imaged are individual nucleosomes and their high-order particles (superbeads), elementary chromatin fibrils c. 30nm in diameter and their higher-order fibres of various sizes up to c. 120nm in diameter. The results demonstrate that X-ray microscopy is now capable of opening a new path of investigation into the detailed structures of hydrated chromosome fibres in their natural state.
Journal of Microscopy, Vol. 176, Pt. 1, October 1994, pp. 75-82.
Relocation accuracy on HOME computerized microscopes
James H Tucker, Richard Dye, Joanne Sprey, Christopher Sowter, Evelyn Gray & Gerard Brugal, MRC Clinical and Population, Cytogenetics Unit, Western General Hospital, Edinburgh, EH4 2XU
SUMMARY
A major practical advantage of the HOME (highly optimized microscope environment) computerized microscope is the facility for relocating cells or other microscopic objects. Features can be marked directly on the microscope image using a mouse-driven cursor, and an interactive finder can then be used to relocate the marked features. Tests on a prototype HOME microscope have shown that positions can be relocated with an accuracy of standard deviation (SD) of less than 7 micrometre. The marked features could also be relocated on a second HOME microscope, although with a somewhat reduced accuracy (standard deviations of less than 17 micrometre). The system provides a very user- friendly environment for tasks requiring relocation of microscopic objects.
I have just removed a message of over 500 lines concerning Yeast Fixation. Can I make an appeal to people not to send so much detail straight to the server -- this type of information should go over more private channels directly between interested parties. Yeast fixation may be an important topic, but not everyone is interested is all the details.
Just a small note: In 1986 the lab I was working in ran some experiments on KMnO4 fixation of fungi (yeasts are fungi too) and we found that KMnO4 did not "Kill/Fix" the fungi we were working with. We were growing the fungi in the presence of up to 12% KMnO4! Albiet the growth morphology was very abnormal but the fungi were still growing.
In Robin's protocol for Yeast cell fixation the glut is responsible for the killing/fixation, the KMnO4 is primarily staining. 1 hour in 2-4% glut for SEM is o.k., but for TEM I would recommend 1-2% for 10-20 min max followed by 1% OsO4 2-6 hours. HIghly recommend the use of Na cacodylate buffer for fungal fixation, having tried others, and in reviewing the literature the general concensus is that Na Cacodylate is the best buffer for fungal specimens. (This comes from 12 years of fungal ultrastructural work).
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I realize that this is probably not good equite to post a test here on this listserver, but I have not recieved any mail since the first of September. I just wanted to know if it is working. Thanks for allowing me the use of the listserver. Clarissa
On tues I called Pfaltz and Bauer to check on the current price for BaMnO4, which they gave me. By Wed when I faxed them my order, they had discontinued the item. On monday, which I hope is not too late, I will try ICN. ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Does anyone out there have any experience using the Cerius DLS-UI module (from Molecular Simulations). I basically want to introduce particular types of atoms on specific sites within a prototype unit cell and have the program calculate the resulting cell lattice parameters. However, I have not yet been able to get the program to do this and I was wondering if anyone else has. Thus far, responses from the manufacturer's support line have been rather ambiguous.
Message-Id: {9410011748.AA03062-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: magnetic SEM specimen Orig-Author: {Marcelle A Gillott {magem-at-csd.uwm.edu} }:ddn:wpafb -----------------------------------------------------------
help!
I am a biologist with limited experience with materials specimens....... a student wants to look at a magnetic sample and I would appreciate any suggestions as to the best way to handle this?
Message-Id: {MAILQUEUE-101.941004084856.448-at-bunyip.ph.rmit.edu.au} To: microscopy-at-aaem.amc.anl.gov
on 4 October, Dwight Beebe wrote :
Wtih respect to the "kill the feed" demand, I personally find the discussion of other techniques and aspects of EM/LM to be interesting and often directly useful in my research and teaching. I want to thank Nestor for his continued work and all the others who contribute to the discussions for making this a very productive source of information.
.......I would like to agree that the list serves a very useful function, even though one accumulates a lot of messages during an absence, as I did recently. However, the availability of the wealth of expertise at one's fingertips is invaluable. The unhelpful and silly comments only detract from its value, and if people would take the time to read the information that is available, many of the objections would not be necessary. My thanks also to Nestor and all of the contributors.
The person looking for Super SEM from Griffin and Van Riessen could try Arie at avr-at-uniwa.uwa.edu.au cheers jjm Professor John J. Millar Head, Department of Applied Physics Electron Microscope Unit RMIT, GPO Box 2476V, MELBOURNE AUSTRALIA 3001 +613 660 2602 fax 613 660 3837 email jjmill-at-rmit.edu.au
Help: I'm working with cardiac cell motility in a methyl cellulose matrix. The problem is that methyl cellulose does not lend itself readily to typical specimen protcols for electron microscopy. Does anyone have a fixative or a cross-linking chemical that will stabilize methyl cellulose at room temperatures or cooler?
--
Darryl Krueger Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
ANLEMC is dying..... I've managed to get it back up and running, however, expect that sometime in the next few days that this mail server will have a new home.
Please keep an eye on your mail for the announcement of a new host address. I will have to permanently move the microscopy mailing list so that the problems of the last few days do not repeat themselves.
Sorry for the headaches many of you have had in the last week, it wasn't fun for me either.
Nestor -------------
Nestor J. Zaluzec Argonne National Lab. Materials Science Division
With the few brain cells I have left I have a dim memory of an extended article on lab safety that may have appeared in either the EMSA Bulletin or in the SEEMS Newsletter before either changed their names. If anyone can point me in the direction of this article or a similar one, I would be grateful. As a corallary we would like to have some information on the hazards of elemental Osmium as opposed to OsO4 (Safety dept wants to condemn our refrigerator with the blackened interior. They intend to seal it up and have it buried in a hazardous waste dump in Atlanta) ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Dear Greg, The Handbook of Chemistry and Physics says that bulk osmium is unaffec- ted by air and is bluish-white; whereas the powdered metal reacts with air to produce OsO4--the starting material in your case. I think you can argue that the refrigerator is much safer than the contents. Only the oxide is toxic, and it can be recognized by its smell. Good luck. Yours, Bill Tivol
Message-Id: {MAILQUEUE-101.941004141528.416-at-vanlab.paprican.ca} To: Greg Erdos ICBR EM Core Lab Univers {GWERDOS-at-gnv.ifas.ufl.edu}
There is a recent publication which might be of help to you entitled:
"Electron Microscopy Safety Handbook" Vernon Barber and Joseph A. Mascorro, Editors 1994
published by San Franscisco Press Inc. Box 426800, CA. 94142-6800
ISBN 0-911302-72-7
I have just glanced through it but it had sections on Biological and Materials preparation and X-ray safety as well.
Good Luck, Laurie
} Date: Tue, 04 Oct 1994 15:10:56 -0500 (EST) } From: Greg Erdos ICBR EM Core Lab Univers {GWERDOS-at-gnv.ifas.ufl.edu}
} With the few brain cells I have left I have a dim memory of an extended } article on lab safety that may have appeared in either the EMSA Bulletin or in } the SEEMS } Newsletter before either changed their names. If anyone can point me in the } direction of this article or a similar one, I would be grateful. } As a corallary we would like to have some information on the hazards of } elemental Osmium as opposed to OsO4 (Safety dept wants to condemn our } refrigerator with the blackened interior. They intend to seal it up and have } it buried in a hazardous waste dump in Atlanta)
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
Greg, Osmium compounds are bad, but osmium is probably the most inert (or maybe ruthenium is more inert) metal there is. The reason that osmium compounds are bad is that they want to dump their osmium somewhere, in exchange for whatever you offer. Don't let your bean counters take your fridge, just because it is coated with precious osmium! And for heaven's sake don't let them know that gold compounds are toxic. They will want to bury your wedding ring with the fridge!
I am late to this string, because I was out of town for some time. I agree that long messages cause problems for all, however, in the interest of widest dissemination of information I would suggest also posting a short message to the server indicating that additional information is available directly. Just my HO.
Best-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Fri, 30 Sep 1994, L. D. Marks wrote:
} I have just removed a message of over 500 lines concerning } Yeast Fixation. Can I make an appeal to people not to send so much } detail straight to the server -- this type of information should } go over more private channels directly between interested parties. } Yeast fixation may be an important topic, but not everyone is } interested is all the details. } } Thanks } }
Could anyone help regarding processing of plant material. We have been prcessing plants for SEM by conventional methods with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, osmication, dehydration and critical point drying. We noticed that the plant material, whether leaf, grass or lichen have a tendancy to explode into fragments during decompression at the end of critical point drying. We thought maybe air was still trapped within the palnt tissues and caused the samples to pop. We fix the tissue under vacuum to remove air but still experiance the same problem.
Mark Gould University of Otago Dunedin New Zealand
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
G'day Richard, With regard to your 'exploding' plant material, I would suggest that you slow down the venting procedure at the end of the CPD run. Your specimens could be displaying a botanical version of the 'bends'!!?
As a possible alternative, if this is not the case, we've been having great success recently with a 'low-tech' cold stage procedure. Basically it involves sticking (with dag) a piece of fresh plant specimen onto a 2cmx1cm brass block then plunging the whole thing into liquid nitrogen for about 45 - 60 secs ( this time is important - you don't want the specimen to get too cold). The cold specimen is then quickly transferred to the SEM and you've got viewing times of about 45 - 75mins. I've found that the more hydrated a specimen is, the better it seems to work, i.e. the less charging it displays, possibly because the water is acting as a conductor. This technique is ideal if you're looking at external features and we've even managed to remove cold specimens, fracture them, and then return them to the microscope without the need for transfer devices etc. I've been knocked out with the ease and the results of the technique - try it!
Some reading material along these lines can be found in
Microscopy Research and Technique 1994 vol 28 : 67 - 74
Journal of Microscopy 1993 vol. 172 : 63 - 69
Scanning 1993 vol. 15 : 171 - 173
Cheers, Tony Romeo
Tony Romeo Internet: tony-at-emu.su.oz.au Electron Microscope Unit Telephone: 692 2351 Madsen Building F09 The University of Sydney Facsimlie: 552 1967 Sydney, 2006 Australia
I would agree with Tony Romeo that your main problem may be speed of venting. Our CPD system recommends not faster than 1-200psi per minute and we err on the side of caution.
I note you use an osmium post fix. We normally just glutaraldehyde fix before dehydration for plant material that we have to Critical Point Dry although low temperature observation is much the best for much of our work.
Ian Ian Hallett HortResearch Mt Albert Research Centre Private Bag 92 169 Auckland, New Zealand Fax 64-9-815 4201 Telephone 64-9-849 3660
I have some serious problems dealing with immuno-EM and embedding. We would like to embed endothelial cells (cultured) and afterwards do an incubation (immunogold). First, we've tried Unicryl to embed the cells cultured in polystyrene dishes (Falcon). This couses severe problems because Unicryl melts the polystyrene dishes. I think that Lowicryl will raise the same problems. So we have tried to embed the cells in Araldite. The embedding was succesfull, but we didn't succeed in getting a nice immunolabeling.
IS ANYONE EXPERIENCED IN EMBEDDING CULTURED CELLS FOR IMMUNO-EM? We would like to keep the cell layer intact (and not scraping the cells for suspensions).
Thanks.
Luc.
*************************************************************************** * Luc Analbers * Analbers-at-med.ruu.nl * *************************************************************************** * Utrecht University * LLL * * Medical Faculty * LLL * * Dept. Medical Physiology & * LLL A * * Sportsmedicine * LLL AA AA * * PO-box 80043 * LLL AA AA * * Zip: 3508 TA * LLLLLAAALLLAAALLL * * Utrecht * LLLLLAAALLLAAALLLL * * Tel: 030 - 538911 * AAA AAA * * Fax: 030 - 539036 * AAA AAA * ***************************************************************************
We routinely process intact monolayers for IEM. I early abandoned polystyrene dishes for the same reasons you mentioned. I recommend to all our users to culture cells in Leighton tubes...these are flat-bottomed screw-cap tubes that lie horizontal. The cells grow on an easily-detached plastic coverslip that survives any chemical processing, even propylene oxide. Before embedding, the coverslip can be cut up, flat embedded, and sectioned along with the attached monolayer. It actually sections better than the LR White resin that we normally use. Leighton tubes are available from most of the suppliers of plasticware for cell culture. Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
We routinely embed monolayers growing on glass coverslips in Falcon or Costar 12 well trays using Epon, LR Gold, and JB-4. In the past, I have used Lowicryl K4M and HM20 but I am unsure what type of trays we were using back then. We never have any problem with the trays dissolving. We use only ethanol for dehydration; no acetone or propylene oxide. We have had some problems with LR White doing weird things which I guess (without a whole lot of confidence) is due to differential contraction of the plastic as it polymerizes. Once polymerized, we cut out the bottom of the well using a Dremel moto-tool, cross-hatch the thin layer of plastic above the glass coverslip and slowly immerse in liquid nitrogen. the plastic contracts at a different rate than the glass coverslip and the cells pop off with the plastic in nice little flat squares. we used to re-embed the squares in standard molds but now simply cut them immediately with virtually no trimming needed. it works great. contact me directly if you have any questions. good luck.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Hello microscopists!! I would very much like to order some dyes/stains from Eastman Kodak, but can't seem to find a valid address/phone/fax #. Can anyone out there help me?? Thanks in advance,
Shea Miller Agriculture and Agri-Food Canada Ctr. for Food and Animal Research Rm. 2002, K.W. Neatby Bldg. Central Experimental Farm Ottawa, Ontario Canada K1A 0C6 millers-at-ncccot.agr.ca
You might try Narishige - they are one of the finest sources of micromanipulators, at least for electrophysiologic applications. I don't have a number at hand, but they should be easy to track down - they will be listed in the Science Guide to Scientific Products, and also in the vendor directory in the Program of the Society for Neuroscience meeting (also I imagine FASEB, IBRO and many others). Good luck!
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
In message Wed, 5 Oct 1994 12:55:12 -0400, MILLERS-at-NCCCOT.AGR.CA writes:
} Hello microscopists!! } I would very much like to order some dyes/stains from Eastman Kodak, } but can't seem to find a valid address/phone/fax #. Can anyone out there } help me?? } Thanks in advance, } } Shea Miller } Agriculture and Agri-Food Canada } Ctr. for Food and Animal Research } Rm. 2002, K.W. Neatby Bldg. } Central Experimental Farm } Ottawa, Ontario } Canada K1A 0C6 } millers-at-ncccot.agr.ca } =======================
Kodak Laboraory Chemicals suppliers in Ontario, Canada:
Anachemia Science Anachemia Science 2708 Southview Drive Unit A, 3120 Pepper Mill Court Box Site 31, Box 10 Misissauga, Ontario L5L 4X4 Sudbury, Ontario P3E 4M9 Tel: 416-82804409 Tel: 705-522-5501
Have you tried growing your cells on Aclar plastic film instead of polystyrene dishes? Embedding in Lowicryl HM20 works well. Elaine
On Wed, 5 Oct 1994, Luc Analbers wrote:
} } Hi, } } I have some serious problems dealing with immuno-EM and embedding. } We would like to embed endothelial cells (cultured) and afterwards do an } incubation (immunogold). } First, we've tried Unicryl to embed the cells cultured in polystyrene } dishes (Falcon). This couses severe problems because Unicryl melts the } polystyrene dishes. } I think that Lowicryl will raise the same problems. } So we have tried to embed the cells in Araldite. The embedding was succesfull, } but we didn't succeed in getting a nice immunolabeling. } } IS ANYONE EXPERIENCED IN EMBEDDING CULTURED CELLS FOR IMMUNO-EM? } We would like to keep the cell layer intact (and not scraping the cells for } suspensions). } } Thanks. } } } Luc. } } *************************************************************************** } * Luc Analbers * Analbers-at-med.ruu.nl * } *************************************************************************** } * Utrecht University * LLL * } * Medical Faculty * LLL * } * Dept. Medical Physiology & * LLL A * } * Sportsmedicine * LLL AA AA * } * PO-box 80043 * LLL AA AA * } * Zip: 3508 TA * LLLLLAAALLLAAALLL * } * Utrecht * LLLLLAAALLLAAALLLL * } * Tel: 030 - 538911 * AAA AAA * } * Fax: 030 - 539036 * AAA AAA * } *************************************************************************** } } }
I have some serious problems dealing with immuno-EM and embedding. We would like to embed endothelial cells (cultured) and afterwards do an incubation (immunogold). First, we've tried Unicryl to embed the cells cultured in polystyrene dishes (Falcon). This couses severe problems because Unicryl melts the polystyrene dishes. I think that Lowicryl will raise the same problems. So we have tried to embed the cells in Araldite. The embedding was succesfull, but we didn't succeed in getting a nice immunolabeling.
IS ANYONE EXPERIENCED IN EMBEDDING CULTURED CELLS FOR IMMUNO-EM? We would like to keep the cell layer intact (and not scraping the cells for suspensions).
Thanks.
Luc.
*************************************************************************** * Luc Analbers * Analbers-at-med.ruu.nl * *************************************************************************** * Utrecht University * LLL * * Medical Faculty * LLL * * Dept. Medical Physiology & * LLL A * * Sportsmedicine * LLL AA AA * * PO-box 80043 * LLL AA AA * * Zip: 3508 TA * LLLLLAAALLLAAALLL * * Utrecht * LLLLLAAALLLAAALLLL * * Tel: 030 - 538911 * AAA AAA * * Fax: 030 - 539036 * AAA AAA * ***************************************************************************
I have had similar problems in the past with cell cultures brought to me on plastic microwell dishes or cell-culture flasks. Dehydration with ethanol is not a problem but EVERY resin I've tried- Spurrs, Taab, LR White, HM20 - has melted the plastic. I usually advise those growing the cells to use Thermanox coverslips or polycarbonate filters on which to grow the cells as these can be processed and cut with the cells; thus solving many problems. However, some researchers still find it necessary to use platic (or they alrady have the cells grown before I'm consulted). Thus, I have discovered that by exposing the cells and dish to HM20 for 1 hour at room temperature the dish will melt sufficiently to allow the cells to float off into the resin.(You need an intact monalyer though). I then lift out sections of the cell monolayer and put them in fresh HM20 and polymerize at room temperature under UV light. HN20 will give excellent immunolabelling results with immunogold.
Marilyn Henderson Biological Electron Microscopist CEMMSA The University of Adelaide South Australia
} Hello microscopists!! } I would very much like to order some dyes/stains from Eastman Kodak, } but can't seem to find a valid address/phone/fax #. Can anyone out there } help me?? } Thanks in advance, } } Shea Miller } Agriculture and Agri-Food Canada } Ctr. for Food and Animal Research } Rm. 2002, K.W. Neatby Bldg. } Central Experimental Farm } Ottawa, Ontario } Canada K1A 0C6 } millers-at-ncccot.agr.ca }
=============================================================================== Loling Song Office tel: +31+71-276198 Department of Cytochemistry and Cytometry, +31+71-276200 Leiden University, fax: +31+71-276180 Wassenaarseweg 72, 2333 AL Leiden, The Netherlands email: song-at-RULLF2.LeidenUniv.NL ===============================================================================
Can anyone bring me up to date on an address or phone number for BEEM?. I tried an old one (212-597-3670) but it did not work. I want to find out if their developing tank system is still available.
Thanks
Jonathan Krupp Microscopy and Imaging Lab University of California Santa Cruz, CA 95060 emlab-at-ucsco.ucsc.edu (408) 459-2477
Last year I bought a colloidal carbon paint called TV coat from Ted Pella. This year they no longer supply it. I need some, does anyone know where to get it? regards Mark W. Lund MOXTEK, Inc. Orem UT
X-Sender: UNC900-at-ibm.rhrz.uni-bonn.de Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: Microscopy-at-aaem.amc.anl.gov
Please help, I need to know how to access "The American Ceramic Society". I applied for membership allready some month ago but the only reaction I got was a booking on my mastercard account. Is there any way to access them by email? Their faxmachine seems to be somewhere in the basement.
We have some chalcopyrite material (CuInSe2) I am looking at here using cross sectional TEM and I would really like to focus on the near surface (several monolayers). I suspect that the top few layers would never survive the cross sectional sample preparation process so I was wondering about the idea of putting an ex-situ cap on it (as immediately after growth as possible) of some non-reactive material (SiN would be one example). I realize this might generate some stress in the material, but it is all I can think of at the moment. We can't put anything on it in situ that won't have the possibility of reaction with it (Se4 doesn't stick significantly at room temperature). Any thoughts on this or suggestions as to what might be a good capping layer.
Mark, TV Tube Koat is generally available from electronics supply houses. It is used to maintain conductivity between vacuum tube plugs and the socket. The manufacturer is:
G.C. Electronics Division of Hydrometals, Inc. Rockford, IL 61101
Let me know if you are successful...I need some too.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
Have you tried chemical drying with Pel-Dri or hexamethyldisilizane? After 100% EtOH, go through a 1:2, 1:2, 2:1 series EtOH:chemical then 3 changes in HMDS or P-D then dry: sublimate Pel-Dri in a vacuum dessicator under mild! vacuum (just enough to remove the vapors) below 22 C or dry HMDS in a fume hood overnight or in a fume hood for 2 hr at 60 C neither may be as good as observing frozen-hydrated, but neither do they require a cold-stage. Phil "looking for a job" Oshel poshel-at-luc.edu
Has anyone ever used acrolein? I must use it in a fixative,and I am afraid of it. What special precautions must I take. I am going to use it in a fume hood,and I will wear my standard protective gloves. Will that be good enough or do I need to dress up like an astronaut?
Message-Id: {9410071446.AA09943-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: program to calculate grain misorientation Orig-Author: {pinglu-at-alumina.rutgers.edu (Ping Lu)}:ddn:wpafb ----------------------------------------------------------- Dear microscopists:
I am looking for a program to calculate grain orientation and misorientation at grain boundary, based on electron beam kikuchi pattern. This program is probably available there, I just do not know where to get it. Everyone knows, please let me know.
The Midwest Society of Electron Microscopists (MSEM), RMC of Tucson, Arizona, and Integrated Microscytems are sponsoring a workshop "Cryotechniques in Electron Microscopy" Friday, October 21, 1994 at Chicago State University.
Program: 9:30AM Registration 10:00 AM Gregory M. Becker will speak on glass knife making for cryo-ultramicrotomy and room temperature Ultramicrotomy and cryo-Preparation Techniques including Metal-Block Freezing and Propane Jet-Freezing. 11:00 AM Michelle Wilhite will speak on Practical Aspects of Cryomicrotomy and Immunolabeling 12:00 Noon Luncheon ($7.00) 1:00 to 5:00 PM Hands-on Workshop demonstrating cryosectioning of biological materials, harvesting sections, and demonstration of tools Demonstration of tools and techniques of immunolabeling and postembedding Demonstration of metal-block freequing and other techniques Facility Tour
Do you have any problems now? Bring them in for individual help.
The workshop is free to members of MSEM. Ask for an application. Membership is only $10 per year, $5 for students
To reserve your space call now: Steve Miller FAX 708-696-2541 E-Mail 73150.2217-at-compuserve.com Phone 1-800-388-8801
Sally, I have used acrolein for many years. It is an excellent fixative when rapid penetration or stabilization lipids is required. You should be aware of a few things about it. 1. It is flammable...be sure your hood can handle this and there are no flames or sparks about. 2. It is very volatile...thats what makes it useful as a vapor fixative. 3. It is very irritating to mucous membranes...treat it as you would osmium. In the concentrations that you are likely to use it in working solutions, its really no worse than glutaraldehyde. Using good safety procedures, we have never had an problems with it. It has a very distinctive odor (like burning, rancid, fat), so you always know if you're inhaling it.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
G'day Gib, Thanks for your comments. I would like to make it clear that the technique I had outlined in my earlier note is not mine - it seems to be a procedure which has been around for a while (in various levels of sophistication) but it's value probably hasn't been appreciated as much as it should be. That's certainly true in my case!
As for the kV's that we use, that's been very much a trial and error procedure. On our JEOL 35C we are basically restricted to 10kv or above. This has been OK for the plant material (15kv) and some of the insect stuff (15 - 20kv). I have had to do some higher resolution work on latex particles and for this we went to 20 - 25kv (for images at 40 - 50k). On our Philips 505 we can obviously work at much lower kv's but it's also in use most of the time so I haven't had much of an opportunity to 'play' on it. For the things that I have done on it (latex particles) I've needed higher resolution and so have been working up around the 20kv range.
Hope all that is of some use!
Cheers, Tony Romeo
Tony Romeo Internet: tony-at-emu.su.oz.au Electron Microscope Unit Telephone: 692 2351 Madsen Building F09 The University of Sydney Facsimlie: 552 1967 Sydney, 2006 Australia
Has anyone ever used acrolein? I must use it in a fixative,and I am afraid of it. What special precautions must I take. I am going to use it in a fume hood,and I will wear my standard protective gloves. Will that be good enough or do I need to dress up like an astronaut?
Greetings, I know of two very nice and very inexpensive pieces of software for doing image analysis on PC. Only trouble is, they each require the purchase of a frame grabber card. The frame grabber card which they need costs about 2,000 US dollars. The frame grabber card is the pcvision plus card, and the software is: Morpho.sys and MTV. The software is in the 300 to 500 dollar range, and does stuff like angles, areas, lengths, contours. ehgo.
} From: forbes-at-cip.org.ec (Greg Forbes) } Date sent: Mon, 10 Oct 94 16:28:20 EST } To: microscopy-at-aaem.amc.anl.gov } Subject: Re: Image Analysis Software for PC
} } } Are there any image analysis software packages available for the PC that are } } worth having? I need to do some fairly basic image analysis (such as area, } } circumference etc.) on a PC system. } } } } Thanks } } } } Joe Michael } } Sandia National Labs } } jrmicha-at-sandia.gov } } Joe, } } I understand that there are two which are quite good and quite } expensive: Optimas and Visilog. If you have a large budget and want } addresses let me know. I would also like to know if there are any equal } to these which are more economical. } } Please let me know if you get any answers which are not posted. } } Good luck, } } Greg } } -- } Greg Forbes Telfs. off: +593-2-690990 } International Potato Center fax: +593-2-562286 } P.O. 17-16-129 CEQ, Quito, Ecuador home: +593-2-330971 } Internet: forbes-at-cip.org.ec; } }
I have used both Optimas and Visilog and at the time I was assessing Optimas was much better at basic measurements whilst Visilog was a much more powerful at image processing. We now use Optimas routinely. In New Zealand it cost around one third the price of Visilog (say $US3000-$US4000). We have had a user working with JAVA from Jandel Scientific who was very happy with it for basic measures, this cost less than either Optimas or Visilog. I understand they also produce a Windows image analysis package.
Unfortunately I have not found a satisfactory public domain imaging package for the PC that was any match for NHI Image for the MAC.
Ian
Ian Hallett HortResearch Mt Albert Research Centre Private Bag 92 169 Auckland, New Zealand Fax 64-9-815 4201 Telephone 64-9-849 3660
} Are there any image analysis software packages available for the PC that are } worth having? I need to do some fairly basic image analysis (such as area, } circumference etc.) on a PC system. } I know this sounds negative, but I wouldn't bother. My recomendation would be to by a Macintosh and download NIH-Image from ZIPPY.NIMH.NIH.GOV as this will cost you roughly the same as a 'cheap' commercial package on a PC and in my (admittedly limited) experience is superior. This software is very flexible and gives you virtually direct e-mail access to the author via a user group if you have any problems. The software is also very fast (for a non-hardware analysis solution). For example I tested Image Pro Plus on a 33Mhz 486 PC doing a 'sharpen' filter and this woked at roughly 40000 pixels per second, whereas NIH Image on a PowerPC 8100 (80Mhz) managed about 710000 pix/sec. Incidentally Image Pro Plus under windows has some bugs that I found within 1 hr of starting to try it out, and I managed to crash my system 3 times in 2 hours so I wouldn't recommend it.
Hope this helps ( :-) )
Doug Arrell
+------------------------------------+ | Dr Douglas Arrell | | Mechanical Performance and Joining | | Institute for Advanced Materials | | 1755 ZG Petten | | Netherlands | | {ARRELL-at-JRC.NL} | | Tel. (+31) 2246 5287 | | Fax (+31) 2246 1917 | +------------------------------------+
} } Are there any image analysis software packages available for the PC that are } } circumference etc.) on a PC system. } } } } Thanks } } Sandia National Labs } Joe, } } I understand that there are two which are quite good and quite } expensive: Optimas and Visilog. If you have a large budget and want } addresses let me know. I would also like to know if there are any equal } to these which are more economical. } } Greg } I don't know how many people this might apply to, but it was only yesterday I found out that our University (bristol, England) had a site licence for Visilog. I had been sort of looking for low cost image analysis software (SigmaScan had been brought to my attention) but nothing comes lower in price than something I could get for free from our Computer Centre (manuals will cost 30pounds). The University had paid, maybe, 2000 pounds for the licence but it can then be installed on any number of systems around the University. The most important thing, though, is that the ownership of this wasn't widely advertised so it *could* be worth spending a few minutes on the 'phone seeing if such arrangements exist in your institution.
Lets distinguish between image processing and image measurement, Optimas is good at changing and manipulating images and pretty good at multiple measuring, but if accurate, controlable measurements of images is what you want, let me suggest you look into Morphosys, sold by Exeter sofware 516 689 7838 for $250.00. They sell it for the U of Calif Berkeley regents, it was written by Chris Meacham of the Herbarium and is a very nice program for measurement. I much prefer its simplicity to Optimas or to older prepc systems I have used. I have no connection other than pleased user. Alan Pooley Rutgers Marine science
PS to my previous meassage, as some one reminded me, It is necessary to have a PCVision Board (Cost $2000? ) and a tv camera to use Morphosys, but you can use the same board for Optimas if you need to "up"grade later and will need some sort of image capture hardware for any system. I have had good luck with the PCVision Board, which comes with diagnostic software (if people have trouble, as I did, I can give them a patch using the ofg that will preset the image board for use with Morphosys Alan Pooley Rutgers Marine sci
Recently, I have had an industrial client ask me about methods for obtaining accurate measurement of oxygen in a cobalt alloy in the 100 to 200 ppm range with some reasonable resolution...say microns. The sample is a cross-section and the interest is in oxygen as a function of distance from the surface. I've talked with some local talent with access to a SEM equipped with WDS capability but it doesn't sound too promising. Any suggestions??
Thanks in advance.
James T. Stanley, II Oregon Graduate Institute Portland, OR jstanly-at-mse.ogi.edu
To those medical archivists among you, can anyone out there confirm this anectodal story? I would particularly enjoy any references from texts or articles.
I heard years ago from John Luft that osmium had been used as a 'treatment' for arthritis. Osmium was injected into the painful joint of arthritis sufferers, whereupon it fixed the nerve cells. thereby treating the symptoms. Becuase of its poor diffusion rate and and rapid reduction, it was immobilized at the site of injection and did not travel elsewhere in the body.
(Thank goodness we now have some better treatments.)
thanks in advance
steve
---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
Reply to question about an image measurement program for the PC
I use Morphosys and like it very much, in preference to Optimas at 10 times the price. However Morphosys does no do image processing, ie filters etc nor does it trace/count/etc multiple image objects without individual mouse clicks to direct it. It does a wide variety of geometric steps as well as tracing well contrasted objects amost instantly. (If people have trouble getting good contrast to trace images ie can't get the 'dark field image to follow the edge of the object, I can give some hints about annual light, apertures to define edges and vanes to control fine lighting details, using cardboard and a cheap circular florescent light) The program Morphosys costs $250.00 from Exeter Software 800 842 5892 It is property of U of Calif and written by Chris Meacham of the Herbarium there. I have no connection other that as very satisfied user. It does require the PCVision frame grabber board, at ??$2000.00 800 532 3500 and a tv camera and stand to hold it and the light etc. All output of Morphosys is text and easily edited, rearranged, portions deleted etc before geometric computations done and output put out. alan pooley rutgers marine science
X-Nupop-Charset: English To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Quantitative Oxygen Orig-Author: {Jim Stanley {jstanly-at-mse.ogi.edu} }:ddn:wpafb -----------------------------------------------------------
Recently, I have had an industrial client ask me about methods for obtaining accurate measurement of oxygen in a cobalt alloy in the 100 to 200 ppm range with some reasonable resolution...say microns. The sample is a cross-section and the interest is in oxygen as a function of distance from the surface. I've talked with some local talent with access to a SEM equipped with WDS capability but it doesn't sound too promising. Any suggestions??
Thanks in advance.
James T. Stanley, II Oregon Graduate Institute Portland, OR jstanly-at-mse.ogi.edu
} Are there any image analysis software packages available for the PC that are } worth having? I need to do some fairly basic image analysis (such as area, } circumference etc.) on a PC system. } } Thanks } } Joe Michael } Sandia National Labs } jrmicha-at-sandia.gov
There was some discussion about PC image analysis software a few weeks ago on the NIH-Image mailing list. One of the replies mentioned the existence of a free image analysis program for the PC called UCFIMAGE. The reply stated that it was "developed by Weeks and Mylar at UCF ..... and you can access it via anonymous ftp from IPG.ENGR.UCF.EDU in the /PUB/UCFIMAGE directory".
I have not tried it myself because we use the Mac based NIH-Image program, which serves our needs admirably.
Arthur Day, Electron Microscopy Group Ansto Advanced Materials Program Phone: 61-2-717-3457 PMB 1, Menai (Sydney), NSW, 2234 Fax: 61-2-543-7179 Australia Email: ard-at-atom.ansto.gov.au
In response to the various questions about accessing NIH Image:
The most up to date versions are available on
ZIPPY.NIH.GOV (a shorter name than the one I gave yesterday)
via anonymous FTP.
To find the files look in the PUB/IMAGE directory. In there you will find 4 versions of the software. Version 1.55 is the most stable and is available in floating point or non-floating point version along with the relevant manuals (word 4 mac format) and even the souce code as the compressed files
} There was some discussion about PC image analysis software a few weeks ago } on the NIH-Image mailing list. One of ............... ^^^^^^^^^^^^^^^^^^^^^^
Please, what is the listserver address of this NIH-Image list?
Petr +-------------------------------------------+-------------------------+ | Dr. Petr Schauer | tel.: (+42 5) 41321246 | | ACADEMY OF SCIENCES OF THE CZECH REPUBLIC | fax : (+42 5) 41211168 | | INSTITUTE OF SCIENTIFIC INSTRUMENTS | E-mail: petr-at-isibrno.cz | | Kralovopolska 147, CZ-612 64 Brno | | | Czech Republic | | +-------------------------------------------+-------------------------+
} To those medical archivists among you, can anyone out there confirm this } anectodal story? I would particularly enjoy any references from texts or } articles. } } I heard years ago from John Luft that osmium had been used as a } 'treatment' for arthritis. Osmium was injected into the painful joint of } arthritis sufferers, whereupon it fixed the nerve cells. thereby treating } the symptoms. Becuase of its poor diffusion rate and and rapid reduction, } it was immobilized at the site of injection and did not travel elsewhere } in the body. } } (Thank goodness we now have some better treatments.) } } thanks in advance } } steve
The reference for this work is Chem. & Eng. News 60:8 (April 5, 1982).
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
} I know this sounds negative, but I wouldn't bother. My recomendation } would be to by a Macintosh and download NIH-Image from } ZIPPY.NIMH.NIH.GOV } as this will cost you roughly the same as a 'cheap' commercial } package on a PC and in my (admittedly limited) experience is } superior. This software is very flexible and gives you virtually } direct e-mail access to the author via a user group if you have any } problems. The software is also very fast (for a non-hardware analysis } solution). For example I tested Image Pro Plus on a 33Mhz 486 PC } doing a 'sharpen' filter and this woked at roughly 40000 pixels per } second, whereas NIH Image on a PowerPC 8100 (80Mhz) managed about } 710000 pix/sec. } Incidentally Image Pro Plus under windows has some bugs that I found } within 1 hr of starting to try it out, and I managed to crash my } system 3 times in 2 hours so I wouldn't recommend it.
I am lucky enough to use both NIH-Image and Image Pro Plus for windows, and both programs work perferct on my hardware. I wonder which version of Image Pro Plus Dough used - the one (ver 1.1) I am using seem to be OK.
I do not think it is fair to compare any program running two machines as different as a high en MAC and mid range PC. I guess the difference in your filtering would have been much smaller if you had run Image Pro Plus on a highspeed Pentium based PC. But who really cares about a few milli-seconds - I run most analysis as unatended macros anyway. What counts for most users is that there image analysis program works like the rest of their programs. So - if your a MAC user download NIH-Image. If your a Windows user buy Image Pro plus (or Mocha or...). I am lucky to use both.
We would be interested to hear from users of the Bioquant package, particularly regarding the OS/2 version, and its suitability for doing image analysis, cell tracing and measurements via camera lucida from a light microscope.
Does the package perform well? Is it especially difficult to operate? etc.
Thanks
Lucy Brown brown-at-aecom.yu.edu Dept Neuroscience Albert Einstein College of Med Bronx, NY 10461
I would appreciate any information sent directly to me at *Fermin-at-TMC.Tulane.edu* on guidelines that anyone may have on the operation of a repository for frozen tissues to be used for research an to serve as an intitutional resource. The information may also be faxed to me at the number below. Thanks in advance for any information that I may use to establish guidelines for this important research resource. Yes, imaging is done in some of the tissues, so I am not stretching it too much by posting it here. Sorry!
************************************************************ *Cesar D. Fermin, Ph.D \|*|/ Fax (504) 587-7389 * *Tulane Medical School /|*|\ Answ. Mach.(504) 584-261 * *Pathology/SL79 \|*|/ Secretary (504) 584-2436 * *New Orleans, La 70 112 /|*|\ Lab (504) 5841 * *Fermin-at-TMC.Tulane.edu -} Director of Morphological Services* ************************************************************
I am having difficulty obtaining some 'coaxicon' miniature coaxial connectors to make up a 'nimbin' extension cable to enable some trouble shooting to occur on our Etec scanning microscope. The Australian AMP agents just don't want to know about them. Can any one recommend an AMP outlet in the USA that might have these items? I need 8off partnumber 201143-1, 8off201144-1 and 16 0ff partnumber 328666-0. Thanks, Kerry Gascoigne Research EM Unit, Flinders University South Australia
} Doug Arrell wrote: } } } I know this sounds negative, but I wouldn't bother. My recomendation } } would be to by a Macintosh and download NIH-Image from } } ZIPPY.NIMH.NIH.GOV } } as this will cost you roughly the same as a 'cheap' commercial } } package on a PC and in my (admittedly limited) experience is } } superior. This software is very flexible and gives you virtually } } direct e-mail access to the author via a user group if you have any } } problems. The software is also very fast (for a non-hardware analysis } } solution). For example I tested Image Pro Plus on a 33Mhz 486 PC } } doing a 'sharpen' filter and this woked at roughly 40000 pixels per } } second, whereas NIH Image on a PowerPC 8100 (80Mhz) managed about } } 710000 pix/sec. } } Incidentally Image Pro Plus under windows has some bugs that I found } } within 1 hr of starting to try it out, and I managed to crash my } } system 3 times in 2 hours so I wouldn't recommend it. } } I am lucky enough to use both NIH-Image and Image Pro Plus for } windows, and both programs work perferct on my hardware. I wonder } which version of Image Pro Plus Dough used - the one (ver 1.1) I am } using seem to be OK. } } I do not think it is fair to compare any program running two machines } as different as a high en MAC and mid range PC. I guess the } difference in your filtering would have been much smaller if you had } run Image Pro Plus on a highspeed Pentium based PC. But who really } cares about a few milli-seconds - I run most analysis as unatended } macros anyway. What counts for most users is that there image } analysis program works like the rest of their programs. So - if your } a MAC user download NIH-Image. If your a Windows user buy Image Pro } plus (or Mocha or...). } I am lucky to use both. } } Bo I used version 1.1 also. To get some strange results try doing some pasteing of seperate images into one larger one. By doing this I managed to put part of the image into the information line at the bottom of the window, though how I did it I have no idea!
Doug Arrell
+------------------------------------+ | Dr Douglas Arrell | | Mechanical Performance and Joining | | Institute for Advanced Materials | | 1755 ZG Petten | | Netherlands | | {ARRELL-at-JRC.NL} | | Tel. (+31) 2246 5287 | | Fax (+31) 2246 1917 | +------------------------------------+
} In reading your note to Joe Micheal about the NIH Image - I noted } you used a PowerPC - is there a working native Power PC version of NIH out } yet and / or do you have any comments about operating on the powerPC vs } plain MAC. I just order a 7100 to run a Gatan camera system and frankly } I'm worried about the PowerPC in non native applications. } This message was sent yesterday by Wayne Rasband (the author of NIH Image).
} } } I have uploaded a new PowerPC beta that supports plug-ins. There is now only one missing feature in the PPC native version: you can't open or print documents from the Finder....
--wayne } } } }
As far as I know, non-native applications work fine, just slower than on a top-spec 'normal' mac, though any screen updating is faster. However, what I am telling you is not from personal experience, I have used NIH Image on various macs, but our new PowerPC has not arrived yet. When it does I will give you some info.
Doug Arrell
+------------------------------------+ | Dr Douglas Arrell | | Mechanical Performance and Joining | | Institute for Advanced Materials | | 1755 ZG Petten | | Netherlands | | {ARRELL-at-JRC.NL} | | Tel. (+31) 2246 5287 | | Fax (+31) 2246 1917 | +------------------------------------+
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Several people are worried about NIH image on a Mac PowerPC. So do we, but we need a version of NIH image with FFT ! We would be interested to know if there is a version of NIH image with FFT that can work on a Mac Intosh Power PC (may be a non-FPU version ?). We tried to run an old version of image FFT on a Power Mac and it refused to work asking for a coprocessor...
PS. By the way, why the FFT menu has not been included in the standard NIH image program ?
} Problems with merging two smaller images into one larger sounds } like a memory problem with the PC system. Just not enough there. } I thought so too, but I was working with two 200k images in 8Mb of RAM + a swap file. My system reported 21Mb of free memory, so I don't think that was the problem!
Doug Arrell
+------------------------------------+ | Dr Douglas Arrell | | Mechanical Performance and Joining | | Institute for Advanced Materials | | 1755 ZG Petten | | Netherlands | | {ARRELL-at-JRC.NL} | | Tel. (+31) 2246 5287 | | Fax (+31) 2246 1917 | +------------------------------------+
I'm running NIH-Image on a PPC 8100. The 680x0 code version runs just fine on the PPC. The question of speed is relative. Compared to the MacIIcx I was using, the PPC is a screamer. If you are comparing to the most recent Quadra and Centris machines, you won't be quite so impressed. I've been testing the beta releases of Image in PPC native code and the capability is absolutely amazing. haven't yet tried the latest beta (.30) yet. Tlhe .28 release had problems so don't try it.
As far as low cost image analysis packages for the pc, doesn't Data Translation offer an inexpensive basic package? I thought you could get a price break if bundled with one of their frame grabber cards.
Regards,
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
It is refreshing to see someone present an objective viewpoint. With a few exceptions, most software and hardware products have strengths and weaknesses. Performance is determined by clock speeds, CPU type (CICS - RISC), the amount of RAM and CACHE memory, the BUS type, 16 bit / 32 bit operating system, etc... Trying to compare two very different hardware/software configurations is a very painstaking task that computer magazines spend a lot of ressources trying to do with some kind of objectivity.
We all get used to what we have and use every day. Thanks Bo for not making a recomendation based on your personal preferences.
Patrick Guerin Vaytek, Inc. Fairfield IA 52556 515-472-2227
The program Super SEM, a supercard program for teaching principles of SEM is being developed by the Microscopy Group at the University of Western Australia in Perth. You should contact Brendon Griffin at the following address for more details. Use his UWA address (bjg-at-uniwa.uwa.edu.au) as his mail is forwarded and following him around the world, while he is on sabbatical leave..
Brendon J. Griffin Visiting Research Fellow Until 10th Nov., 1994 Crystallography & Mineral Physics Unit NB email address is always Department of Geological Sciences current University College of London Ph: 071-380-7777 Ext 2406 Gower Street Fax.: 071-388-7614 London WC1E 6BT Email: bjg-at-uniwa.uwa.edu.au
In message Fri, 14 Oct 1994 13:00:07 -0400, gnord-at-mactem.er.usgs.gov writes:
} Hello Microscopists, especially the photon variety. } } I am looking for a light microscope for about $150 for a seven year old } girl very interested in science. Zany-Brainy, a children's learning } center store here in Virginia, has a toy for about $70. I think that I } can do better. Any ideas for new or used light microscopes in my price } range? } Gordon L. Nord Jr. } 959 National Center } U. S. Geological Survey } Reston, VA 22092 } } Office: 703-648-6745 } FAX: 703-648-6789 } } gnord-at-mactem.er.usgs.gov } =============================== Edmund Scientific, 101 E. Gloucester Pike, Barrington, NJ 08007-1380, Tel. order 609 573 6250 (Schools & Ed. pricing 609 573 6270) sells "student" microscopes in your price range.
***************************************************** M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu ****************************************************** e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e- e- e- e- This message was sent using 100% recycled electrons e- e- e- e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-
We have an old JEOL model JSM-2 SEM with a Nuclear Diodes/ EDAX model 508 EDS system. It was built in 1968. Although in good operating condition, we have no use for it and I have suggested that probably no one would want it. I need to somehow verify that no gov't agency, or University would want to acquire it by coming here, packing it up and shipping it. Further that no citizen would bid on it and also come to Mpls to get it. Is there a market or demand for old SEMs or will we have to pay someone to scrap it for metal? Any comments can be sent to me at the e-mail address below. Thanks for your input. Mike Boucher Boucher-at-tcrca.usbm.gov ***************************************************************** Michael L. Boucher Sr. Boucher-at-TCRCA.USBM.GOV Geology-Mineralogy/Chemistry Labs Ph 612-725-4614 Twin Cities Research Center Fax 612-725-4526 U.S. Bureau of Mines Center 725-4500 Department of Interior 5629 Minnehaha Avenue South Minneapolis, MN 55417-3099 U.S.A. *****************************************************************
} I am looking for a light microscope for about $150 for a seven year old } girl very interested in science. Zany-Brainy, a children's learning } center store here in Virginia, has a toy for about $70. } I think that I can do better.
Hello Gordon -
My company imports all types of microscopes. We have a model for your price range that has 40X - 640X, 3 sets of eyepieces, mirror illuminator with aperture disc, wooden box. It is very heavy, and the optics are excellent. An electric substage illuminator is available for a little extra. We have even used this one with video, and it works very well.
I would be happy to discuss your needs. Send me a note for more info.
John Mansfield North Campus Electron Microbeam Analysis Lab University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 12:07 AM
Date:10/15/94 NC EMAL
FACULTY POSITION
THE UNIVERSITY OF MICHIGAN
The Department of Materials Science and Engineering at The University of Michigan is continuing to grow and there are openings for tenure track faculty positions at the level of assistant professor. Candidates at the senior level with records of outstanding accomplishment will also be considered. Applicants must have a Ph.D. degree, be qualified to teach undergraduate and graduate courses in materials science and engineering, and should plan to develop independent and cooperative research programs. A demonstrated research record or potential is required. Preferred areas of expertise include: electronic, magnetic and/or photonic materials; physical metallurgy; materials theory and modeling; ceramics. Women and minority candidates are encouraged to apply. Send curriculum vitae and a list of references to:
Chairman, Search Committee Department of Materials Science and Engineering The University of Michigan 2300 Hayward Street Ann Arbor, MI 48109-2136
Message-Id: {MAILQUEUE-99.941017175027.448-at-eo.ine.philips.nl} To: microscopy-at-aaem.amc.anl.gov
PHILIPS WORKSHOP ON CONVERGENT BEAM ELECTRON DIFFRACTION
March 27-31, 1995
PURPOSE
The purpose of this workshop is to instruct microscopists in the potential of CBED and large angle CBED for the analysis of crystal structures, and to provide hands-on training in the practical techniques as well as example classes dealing with various applications. Although some background in diffraction techniques might help, it is not absolutely necessary and a quick refresher course on diffraction theory will start the week.
TOPICS
The following questions will be answered: 1) What information is contained in a CBED pattern? 2) How are crystal symmetries determined? 3) How are CBED and LACBED done in practice? 4) What are the practical and instrumental limitations? 5) How can crystal defects be analysed?
INSTRUCTORS AND EQUIPMENT
The teachers (from renowned convergent beam groups such as Bristol, UK; Lille, France and Perth, Australia) have a wide experience in organising workshops in this field and will be assisted by Philips application specialists. Invited lectures on more or less advanced CBED applications will be presented by specialists such as Prof. J.P. Morniroli, Dr. D. Cherns and Dr. A. Johnson. A wide range of Philips CM transmission electron microscopes (LaB6 & FEG) will be available to perform all types of convergent beam experiments including energy filtering of the diffraction patterns. Simulations and interpretations of the experiment will be done on stand-alone systems.
MORE INFORMATION
The workshop will be held at the Philips Electron Optics Applications Laboratory in Eindhoven, The Netherlands. Persons who wish to register for the workshop or obtain more information should send a reply. Please enclose your complete postal address and indicate if you want to register and/or receive more information.
With Kind Regards
Eric van Cappellen
FOR MORE INFORMATION CONTACT:
Philips Electron Optics Building AAE, P.O. Box 218 5600 MD Eindhoven The Netherlands Tel. +31 40 766234 Fax. +31 40 766102 E-mail: marcom-at-eo.ine.philips.nl
EUCHEM CONFERENCE ON ELECTRON MICROSCOPY IN SOLID STATE SCIENCE MAY 20-24, 1995 in LUND, SWEDEN
A EUCHEM conference on Electron Microscopy in Solid State Science will be held at Jaravallen, at the seaside 20 km northwest of the university town Lund, on May 20-24, 1995. In the spirit of the EUCHEM conferences, which are European equivalents to the Gordon conferences in the US, there will be a limited number of oral presentations, but plenty of time for discussions. No abstracts or proceedings will be published. The number of participants will be limited to 75 and all participants are requested to present a poster.
Among the topics that will be adressed are: Electron microscopy of new materials. Structure and chemistry of aperiodic features in solids. New microscopy techniques and their application.
For further information, please contact: The Swedish National Committee for Chemistry Wallingatan 24, 3 tr S-111 24 Stockholm Sweden Phone: Int +46-(0)8-4115280, Fax: Int +46-(0)8-106678 E-mail: Anna.Carlsson-at-oorg2.lth.se
If you want to do quantitative analysis of N in high Z materials you better get a standard as close as possible to your "unknowns" the matrix and absorption corrections will be significant. Using Si3N4 as your standard would not be a good idea. Sorry but I can't give you any leads as to standards. Your best bet might be to consult a phase diagram and make a standard.
Geller Microanalytical (508 535-5595) has several nitride standards, including a TaN. I don't know whether or not they are good, but I would hope so since they are advertised and sold as standards. Bastin has numerous articles in the MAS literature regarding N analysis (Microbeam Analysis 1988, p290, for example). I am dabbling with N analyses in volcanic tuffs (appears to be in the form of NH4 in feldspars - much lighter stuff than you are studying). Count rates are poor, to say the least. Good luck.
Jim McGee
James J. McGee email: jmcgee-at-lunatic.er.usgs.gov U.S. Geological Survey Phone: (703) 648-6782 959 National Center Fax: (703) 648-6789 Reston, VA 22092
I have a very old (wrought iron) A/O Spencer paraffin microtome Model 820 with a knife holder for large reusable steel blades. Our lab would like to purchase (or trade) a disposable knife holder that would fit our machine.
Suggestions for where to look would also be appriciated!
I wish to subscribe to the microscopy listserver. I am a structural cell biologist with a major interest in cytoskeleton. My main tools are TEM, various freeze-preparation methods and tissue culture. I am very interested in exchanging ideas on the functional ultrastructure of cytoskeleton. For example, can anyone suggest a suitable 3hr practical class execise for third year BSc students based on microtubules including at least some live material. Thanks!
Does anyone out there care to share their sure-fire way to securely mount LR White 1-2 micron sections to glass slides for immunogold labeling? We have variable success with keeping our sections from bubbling, wrinkling and working their way loose during immuno- incubations. Any secrets you would care to post would be much appreciated.
Thanks in advance.
Dan Possin Ophthalmology EM Lab, Univ. of Washington, Seattle, USA.
Does anyone out there care to share their sure-fire way to securely mount LR White 1-2 micron sections to glass slides for immunogold labeling? We have variable success with keeping our sections from bubbling, wrinkling and working their way loose during immuno- incubations. Any secrets you would care to post would be much appreciated.
Thanks in advance.
Dan Possin Ophthalmology EM Lab, Univ. of Washington, Seattle, USA.
} Hello everyone - } } Does anyone out there care to share their sure-fire way to securely mount } LR White 1-2 micron sections to glass slides for immunogold labeling? We } have variable success with keeping our sections from bubbling, wrinkling } and working their way loose during immuno- incubations. Any secrets you } would care to post would be much appreciated. } } Thanks in advance. } } Dan Possin } Ophthalmology EM Lab, Univ. of Washington, Seattle, USA.
After trying a zillion different coatings (e.g., poly-l-lysine, chrome-gel, albumin), we now use aminopropy-triethoxysilane (Sigma). Dip clean slides in a 2% apts in acetone solution for 30 sec. rinse in acetone, then dH20. dry and store. stable for at least months. this protocol is based on one of Lynne Angerer that she present at a ASCB in situ hybridization workshop. she sites Gottlieb and Glaser (BBRC 63:815-821, 1976) who used apts coated slides treated with glutaraldehyde to adhere single cells. she states that glut post treatment is good for cells but unnecessary for sections. our experience is in agreement with this. Additional advantage: Water beads up on these slides so that when one adds 10-20 ul of antibody, it doesn't spread flat on the slide but stays restricted to a bead.
good luck
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
I've had to resort to slides washed in chromic acid and then subbed with chrome alum-gelatin for some combinations of plastics and mountants. We have a bunch of other slide treatments for specific, and general, purposes. Drop in and try a few. Dale can get some for you if I'n not around.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Tue, 18 Oct 1994, Daniel Possin wrote:
} Hello everyone - } } Does anyone out there care to share their sure-fire way to securely mount } LR White 1-2 micron sections to glass slides for immunogold labeling? We } have variable success with keeping our sections from bubbling, wrinkling } and working their way loose during immuno- incubations. Any secrets you } would care to post would be much appreciated. } } Thanks in advance. } } Dan Possin } Ophthalmology EM Lab, Univ. of Washington, Seattle, USA. }
The nitrogen-K emission line is at 392eV, there are many M zeta lines for elements in the mid-high atomic number range there. Tin for example is at 397eV. These (Mz) lines are however fairly weak!
By interpolating between elements I would say molybdenum ought to have a M-gamma (strongish line) at around the N-k that could cause a problem. This line is not listed for some reason! All I find for Mo M-series is M3-N1 at 331eV (strength listed as 100).
In the tables I use for X-ray emission line identification with EDX there's no listing for indium M-lines. (Johnson and White, ASTM Data series DS46, 1970). My EDX computer system likewise cannot show the M-lines for indium. Below In is Cd, Ag, Pd down to Nb, all of which have M lines with M-gamma the strongest. With the exception of Mo noted. Indium has auger and XPS peaks for M transitions, there's an EELS edge too.
Is there another (better) reference to use for the M-series?
Contributers to our newsletter "Microscopy Today" please note our new eMail address - MicroToday-at-aol.com. Others, including international, who wish no cost subscriptions, have but to supply their complete mailing addresses by eMail - or by fax (608/836-1969)
Some of the lines you listed I never see. One of the best sources for x-ray lines is the Chemical Rubber Co. (CRC) Handbook of Chemistry and Physics. This contains the famous Bearden tables of x-ray wavelengths and energies. You are right about In M-lines. Even in this table (mine is dated 1967) the low energy indium lines are incomplete; perhaps a more up-to-date version would have them.
Basically there are five M-lines that may be observed in the EDS spectrum. We usually only see the two strongest M-alpha and M-beta (typically unresolved). The other three (zeta, gamma, and M2N4) are only about 5% or less of the M-alpha.
I obtained the latter information from Table 3.11 of Goldstein et al., Scanning Electron Microscopy and X-ray Microanalysis, Plenum Press, New York, 1992, p. 127. Chapter 6 on qualitative analysis shows typical spectra for all types of lines from various elements: for example, M-lines from Bi, Ta, and Dy.
Good luck, Charlie
Prof. Charles E. Lyman Department of Materials Science and Engineering Lehigh University, 5 East Packer Avenue, Bethlehem, PA 18015-3195 E-mail: cel1-at-Lehigh.edu Tel: 610-758-4249 FAX: 610-758-4244
} } To:Robert McDonald {robert-at-geology.gla.ac.uk} } From:richard.easingwood-at-stonebow.otago.ac.nz (Richard Easingwood) } Subject:Re equipment list } } } } On another note - do the other folks reading this group think that } } would be useful to compile a list of what equipment we all have } } so that like users could pool info on specific problems etc? } } } } Just a thought and my opinions only. } } } } Robert McDonald robert-at-starav.geology.gla.ac.uk } } I think Robert's idea re equipment list is a good idea but would take an awful } lot of work, compiling, updating etc as I am sure that a list of just the EMs } used by the people who use this listserver would be frighteningly long. } Perhaps it would be better if those who need help with specific pieces of } equipment post their problems on the listserver and just keep the addresses of } respondants for future reference, provided they are happy to be contacted } again for assistance. } I don't deny it would be interesting to see what's out there though. }
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
Message-Id: {1994Oct19.164942.1515942618-at-ms.sjdccd.cc.ca.us} To: MICROSCOPY-at-aaem.amc.anl.gov (MSA list)
MSA, Microscopy Soc of Am does have a list of facilities with their equipment collected by the Tech Forum. They have it on disk. contact Sandy Silvers EM Complex USDA,ARS,RRC PO Box 5677 Athens, GA 30613-6199 706/546-3471 No e-mail address listed.
I collected all the training facilities in the country and in several other countries with all equipment listed, but more emphasis on the course info. This however is outdated since I did this crazy thing in 1982. It was a marvelous amount of information but a fantastic amount of work!!!! Good Luck Judy M
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
} } To:Robert McDonald {robert-at-geology.gla.ac.uk} } From:richard.easingwood-at-stonebow.otago.ac.nz (Richard Easingwood) } Subject:Re equipment list } } } } On another note - do the other folks reading this group think that } } would be useful to compile a list of what equipment we all have } } so that like users could pool info on specific problems etc? } } } } Just a thought and my opinions only. } } } } Robert McDonald robert-at-starav.geology.gla.ac.uk } } I think Robert's idea re equipment list is a good idea but would take an awful } lot of work, compiling, updating etc as I am sure that a list of just the EMs } used by the people who use this listserver would be frighteningly long. } Perhaps it would be better if those who need help with specific pieces of } equipment post their problems on the listserver and just keep the addresses of } respondants for future reference, provided they are happy to be contacted } again for assistance. } I don't deny it would be interesting to see what's out there though. }
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
Brief addition to Charles E. Lyman's suggestion of looking in the rubber book (HB Chem & Physics): I just looked in my 1984-1985 copy and the low energy Indium M lines are not present either.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
Last week, someone asked about LR White sections/semi-thins on slides for= immuno. I have not had this problem with my material, BUT I am meticulous= when I clean my slides before applying any type of slide adhesive. I use= ordinary gelatin/chrome alum subbing solution (0.5% gelatin, ordinary 300= bloom, and 0.05% chromium potassium sulfate). I thoroughly clean the= slides with a good grease-cutting detergent, then rinse with hot tap H2O,= then de-i H20, then dip. I suggest this as a substitute for acid-cleaning,= as it's a little safer. I must caution that the rinsing step is= critical-any soap residue is will render the slides useless (we had a= technician who failed to follow directions and caused a few major= catastrophies...). In closing, I'd like to thank everyone who responded to= my cry for help with LR White thin sections. I think my resin was a little= too elderly. Grace
X400-Received: by mta bottom.ctd.anl.gov in /PRMD=ANL/ADMD= /C=US/; Relayed; Thu, 20 Oct 1994 11:52:58 -0500 X400-Received: by /PRMD=ESnet/ADMD= /C=US/; Relayed; Thu, 20 Oct 1994 11:52:56 -0500 X400-Received: by /PRMD=inria/ADMD=atlas/C=fr/; Relayed; Thu, 20 Oct 1994 11:52:51 -0500 X400-Received: by /PRMD=cea/ADMD=atlas/C=FR/; Relayed; Thu, 20 Oct 1994 11:50:44 -0500
I'm trying to get in toch with Allied Technology but have not yet succeeded in getting their coordinates. If anyone can send their address, telephone and/or fax numbers,e-mail it will be greatly appreciated
Lelia Schmirgeld-Mignot
--------------------------------------------------------------------- SRMP/DECM/DTA Tel : 33-1-69.08.20.68 C.E. Saclay FAX : 33-1-69.08.68.67 91191 GIF sur YVETTE Cedex e-mail : lelia-at-srmp04.saclay.cea.fr FRANCE ---------------------------------------------------------------------
Hi Folks, We have a Nikon Diaphot connected to our Bio-Rad Confocal. The slider on the prism that shunts the laser up or the transmitted light to the oculars is getting very hard to pull. Nikon originally said this is natural and to replace the silicon gel on the prism glides inside the housing, preferably by their own gel. They now say that we should use a high quality oil instead of the gel. Since this is a big deal, hard to do thanks to the confocal assembly, I thought I would ask if anyone else out there has run into this problem. TIA
C. Michael Stanley Molecular Cytology Core Facility Molecular Biology 2 Tucker Hall University of Missouri-Columbia Columbia, MO. 65211 (314) 882-4895 fax= 314-882-0123
Message-Id: {MAILQUEUE-101.941020132043.416-at-vanlab.paprican.ca} To: lelia-at-srmp04.saclay.cea.fr
} I'm trying to get in toch with Allied Technology but have not yet } succeeded in getting their coordinates. } If anyone can send their address, telephone and/or fax numbers,e-mail } it will be greatly appreciated } } Lelia Schmirgeld-Mignot
Hi, I have an address for an E.M. supplier called Allied Hi-Tech Products, 2376 E. Pacifica Place Rancho Dominguez, CA 90220
There numbers are: Phone (800) 950-9347 Fax (310) 762-6808
Hope this helps, Laurie
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
Would you please tell me how to subscribe MICROSCOPY?
I subscribed by command "subscribe microscopy ningx-at-mcmail.cis.mcmaster.ca" the MICROSCOPY IN "LISTSERVER-at-ANLEMC.MSD.ANL.GOV" yesterday, but no respond.
} the oculars is getting very hard to pull. Nikon originally said this is } natural and to replace the silicon gel on the prism glides inside the } housing, preferably by their own gel. They now say that we should use a } high quality oil instead of the gel.
We have had the same problem on all our Diaphots. Nikon claims that their new oil is especially heat resistant and far better than past lubricants. They have cleaned and relubricated the Diaphot we are using with the confocal and it is now much easier to use. We plan to have one of the other Diaphots greased the same way. We have an additional problem with the prism which is looseness on its track, but this is probably unrelated to the new lubricant unless the machining got worn over the years, perhaps when it was tighter. -Michael Cammer
Leon Smuts, I've found that silver paints and pastes *seem* to dry quickly, but actually degass for several hours, or a day or two, even using a vacuum dessicator. I've also found some brands of carbon paint that are contaminated with phosphorus. Don't recall which, off the top of my head, and the formulation have been changed, but it's something that should be checked, especially if you're doing EDX or WDX. Phil Oshel poshel-at-luc.edu
} Subject: Etchant for InGaAs. } } G'day ... } } A colleague of mine needs to find a suitable etchant for InGaAs (indium } content x { 0.3), which will reveal decorated defects inside the material } when the etched surface is examined with Nomarski IC microscopy. } } Any comments or references? } } Stephen Anderson. } .............................................................. } : Stephen Anderson : } : Electron Microscope Unit : } : The University of Sydney Email stephen-at-emu.su.oz.au : } : NSW 2006 Telephone (+61)-2-351 2351 : } : Australia Facsimile (+61)-2-552 1967 : } :............................................................: }
A weak solution of Cl2 bubbled through a beaker of methanol will do it- be careful if the solution becomes saturated,ie a green-yellow colour it may ignite-have some extra methanol beside you if you try this. More esoterically a colleague of mine suggested a solution of KOH: K3Fe(CN)6:H2O in the ratio 6g:4g:50ml. He uses it for SEM imaging of epilayers and reckons that the etchant rate is approximately 2 micrometers/minute at room temperature Good luck Ciara
In message Tue, 18 Oct 1994 16:44:04 -0400, david.rayns-at-stonebow.otago.ac.nz (David Rayns) writes:
} I wish to subscribe to the microscopy listserver. } I am a structural cell biologist with a major interest in cytoskeleton. My } main tools are TEM, various freeze-preparation methods and tissue culture. } I am very interested in exchanging ideas on the functional ultrastructure } of cytoskeleton. For example, can anyone suggest a suitable 3hr practical } class execise for third year BSc students based on microtubules including } at least some live material. } Thanks! } ============== David,
I can suggest a few lab excercises but they would require a fluorescence microscope:
1. If you have monolayer cultured cells such as fibroblasts, you can demonstrate the interrelatioship between microtubules (MTs) and ER organization. The live cells can be stained with DioC6 to demonstrate the normal ER organization and treatment of the cells with nocodazole will result in the disorganization of ER (see Terasaki et. al. 1986, J. Cell Bio).
2. If you have dividing cells, the importance of MTs for chromosomal movement can be demonstrated using MT disruptants. You also can include immunolocalization of the MTs and DAPI staining of the chromosomes as part of the lab using fixed cells.
3. You can use onion scale epidermal cells to demonstrate the importance of F-actin for protoplasmic streaming. Treating the cells with cytoclasin D will stop the streaming. The presence or the lack of F-actin can be determined by rhodamine-phalloidin (Rh-Ph) staining. (For details see on Rh-Ph staining see Parthasarathy, 1987, Plant. Mol. Biol Rep.; Sonobe, S. and Shibaoka, H., 1989, Protoplasma).
I believe MSA has a relatively updated list of facilities through one of their divisions.
On Thu, 20 Oct 1994, Richard Easingwood wrote:
} } } } To:Robert McDonald {robert-at-geology.gla.ac.uk} } } From:richard.easingwood-at-stonebow.otago.ac.nz (Richard Easingwood) } } Subject:Re equipment list } } } } } } } On another note - do the other folks reading this group think that } } } would be useful to compile a list of what equipment we all have } } } so that like users could pool info on specific problems etc? } } } } } } Just a thought and my opinions only. } } } } } } Robert McDonald robert-at-starav.geology.gla.ac.uk } } } } I think Robert's idea re equipment list is a good idea but would take an awful } } lot of work, compiling, updating etc as I am sure that a list of just the EMs } } used by the people who use this listserver would be frighteningly long. } } Perhaps it would be better if those who need help with specific pieces of } } equipment post their problems on the listserver and just keep the addresses of } } respondants for future reference, provided they are happy to be contacted } } again for assistance. } } I don't deny it would be interesting to see what's out there though. } } } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 } }
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: Microscopy-at-aaem.amc.anl.gov
Microscopy Society of America is accepting applications from undergraduate students interested in conducting a research project involving the use of ANY microscopy technique. Applicants must be sponored by a member of MSA. The maximum award is $2500(US). The application deadline has been extended to December 30, 1994. Applications can be obtained by contacting the MSA Business Office at (800) 538-3672, FAX: (508) 548-9053. For more information contact either: Dr. Ralph Albrecht,rma-at-ahabs,wisc,edu (608) 263-3952/4162, FAX (602) 262-7420 ; or Dr. Richard Ornberg, rlornb-at-ccmail.monsanto.com (314) 694-1184, FAX (314) 694-6727.
Greetings, I'd like to know whether anyone knows where I could purchase a copy of the book by G. Meeks: "Practical Electron Microscopy for Biologists". It is out of print and I'd like to find a copy somewhere. If anyone can help, please reply directly to me, not to the list. Many thanks, Dwight
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
I know that the majority of members of the listserver are in the states and hence most of the communication on this subject has been stateside orientated. However as an overseas 'consumer' of the listserver it might be interesting to know of other lists in other countries.
Certainly their is a list available in South Africa which is run by Dane Gernecke and the UCT EM unit for EMSSA (Electron Microscopy Society of Southern Africa).
Are there any others?
Peter _______________________________________________________________
-at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at- -at- -at- -at- Peter D. G. Richards -at- -at- Dept Anatomy and Cell Biology -at- -at- UCT Medical School -at- -at- Observatory -at- -at- 7925 -at- -at- RSA -at- -at- Tel: 021-406 6285. -at- -at- Internet: retep-at-anat.uct.ac.za -at- -at- -at- -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at-
Message-Id: {MAILQUEUE-101.941024182817.320-at-bunyip.ph.rmit.edu.au} To: microscopy-at-aaem.amc.anl.gov
Re : recent topic
Australia has had for many years a full list of instrumentation and personnel in EM maintained voluntarily by people from the national association (ASEM). Te list is updated every two years by the "owners" of the instrumentation and labs in conjunction with the biennial EM conference. The list is a very valuable reference for advice and all sorts of other things. It would be very useful to have an international list, but would take a while to arrange. Maybe this could be started via this server, for all to list their instruments/people/interests in a tight format ?? jjm
Professor John J. Millar Head, Department of Applied Physics Electron Microscope Unit RMIT, GPO Box 2476V, MELBOURNE AUSTRALIA 3001 +613 660 2602 fax 613 660 3837 email jjmill-at-rmit.edu.au
Sender: cemerson-at-kean.ucs.mun.ca {cemerson-at-kean.ucs.mun.ca} Reply-To: cemerson-at-kean.ucs.mun.ca To: MICROSCOPY-at-AAEM.AMC.ANL.GOV Message-Id: {009866A4.2FE243A2.1441-at-leif.ucs.mun.ca}
The Microscopiccal Society of Canada also has a Directory of Electron Microscopy Facilities in Canada, last printed in 1992. We hope to have an update in 1995 including confocal and scanned probe instruments as well Directories are available from the MSC. Call Marie Colbert at 905-525-9140 ext. 22496 or write Marie Colbert, Dept. of Pathology, McMaster Univ. 1200 Main St., Hamilton, Ont. L8N 3Z5. I believe the Directory may also be available on disc.
Carolyn Emerson, Editor, MSC Bulletin, Dept. of Biology, Memorial Univ. St. John's, Nfld, Canada A1B 3X9.
I have seen this microscope, and occasionally buy equipment from Driftwood Associates. I have no other connection with this transaction. Julian Smith III Biology Winthrop University Rock Hill, SC VOX 803 323-2111
One forum that might be used for Instrument Lists is the "Micrsocopy & Microanalysis" World Wide Web server that I also maintain here at ANL. On it is information about some of the ANL Microscopy projects, but also sections dedicated to some of the microscopy/microanalysis societies (MSA, MAS, RMS, MSC, ASEM) I would consider adding updated lists to this site or to the Anonymous FTP site here which services the Microscopy & Microanalysis Software Libraries.
The important thing to remember is that such a listing would only be a subset of any master lists such as Sandy Silver's database, or comparable ones elsewhere in the world.
Can I suggest that those who maintain regional instrument listings contact me off-line. We can then try to set up a "short-form" which could be uploaded to the WWW site (http:/www.amc.anl.gov) or the FTP site (www.amc.anl.gov) This would be a start, and would complement NOT REPLACE each regional listing which would be more comprehensive. Information on how to obtain the complete local listing would, of course, be provided.
Again if your a organizer of such list, contact me at
Zaluzec-at-AAEM.AMC.ANL.GOV
When we have something that is on-line, I'll post the relevent information to the Microscopy Listserver..
I am presently using a Steinheil Optronic Oscillophot M4, which is "wearing out" after 16 years of use. I am looking for a new or used (in good working condition) Polaroid CU-5 camera to mount on my Philips 500X SEM.
Is the CU-5 a good choice, or what other kinds of cameras are people using to record 4x5 inch pictures? My Steinheil has f/stop, enlargement ratio and focusing controls which I like. However, it does not quite cover my 4x5 inch photomonitor screen so there is some loss of detail right in the corners of the pictures.
Any assistance will be greatly appreciated. Thanks.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Goodday! In response to my recent invitation for no cost subscriptions to our newsletter, I am overwelmed with questions. With apologies for the "clutter" to others I would like to address all questions: 1) There is no email version. The 10 yearly issues are available only through the mail so I will need full and proper mailing addresses. 2) There is currently no charge for subscriptions. We may have to charge a modest fee in the future for international subscriptions. 3) The objective of the newsletter, perhaps unlike any other, is to publish material and articles of interest to the working microscopist. It is not a peer review journal and, while we need ad income to publish it free, it is not an advertising catelog. 4) A number of members of this listserver do contribute to the effort and their help is much appreciated. We would greatly appreciate material/articles from others on the list. If interested, please contact me direct and I will provide other guidelines. 5) A new feature in the pub is a series of "Tricks of the Trade" notes - on any microscopy technique and of any length. Authors of each accepted "trick" will be entered in a drawing at next years MSA Conference for a 1 oz gold coin. Do two and you should have between a 1 and 25 to 1 and 50 chance of wining the some $500 worth coin. Regards, Don Grimes, Editor - Microscopy Today
I would like to order some accessories for my EMSCOPE SC650 sputter coater. Does anyone know the address and telephone number of their vendors in the USA ? Thanks in advance.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
Dear Robin, I have just started to look at clay mineral particles on the HVEM, a TEM rather than an SEM. The colleague investigating clay from the bottom of the Hudson river collects a sample of the sediment, washes it and settles it. During the settling, she collects fractions at various times; these are label- led for their nominal sizes-- } 5mu, 2mu, {2mu and { {2mu. I had difficulties getting good dispersal on a carbon-coated mesh grid at first, but I found that if I put a drop of poly-l-lysine on the grid and let it dry, I could then agi- tate the suspension of clay particles and put a 5 mu-liter drop on the grid. This gave me very good dispersion of both the largest and the smallest parti- cles. I must emphasize that this is my first venture with anything like this, so listen to the real experts if any respond. (I forgot to say that the 5 mu- liter drop I put on the grid was just air dried.) I am aware that clay mine- rals' structures change when they dry, but we wanted to try the simplest thing first. Good luck. Yours, Bill Tivol
I would like to know if anyone from this group has ever used anthrancene as a dye or a triplet energy acceptor. If so, I would appreacite any information on its spectral properties, chemsitry, and literature on its triplet energy.
Thank you in advance!
Loling
=============================================================================== Loling Song Office tel: +31+71-276198 Department of Cytochemistry and Cytometry, +31+71-276200 Leiden University, fax: +31+71-276180 Wassenaarseweg 72, 2333 AL Leiden, The Netherlands email: song-at-RULLF2.LeidenUniv.NL ===============================================================================
I would like to know if anyone from this group has ever used anthrancene as a dye or a triplet energy acceptor. If so, I would appreacite any information on its spectral properties, chemsitry, and literature on its triplet energy.
Thank you in advance!
Loling
=============================================================================== Loling Song Office tel: +31+71-276198 Department of Cytochemistry and Cytometry, +31+71-276200 Leiden University, fax: +31+71-276180 Wassenaarseweg 72, 2333 AL Leiden, The Netherlands email: song-at-RULLF2.LeidenUniv.NL ===============================================================================
I think Nestor's idea has a lot of merit. Like most other countries New Zealand has a local directory organised by the newsletter of the NZ Society for Electron Microscopy which could probably be incorporated in some international list
Access to a wider range of instrument users would be very useful as frequently we find we have no one we can ask locally about new or unusual equipment. Ian Hallett HortResearch Mt Albert Research Centre Private Bag 92 169 Auckland, New Zealand Fax 64-9-815 4201 Telephone 64-9-849 3660
R. Crang; I've lost your email address--apologies to the list. The Neutral Red idea I sent you awhile back for looking at wood cells has been stewing in the back of my mind, and it's lately bubbled up that Vaughn was using not Neutral Red, but Rose Bengal. This should also be in Conn's Biological Stains. Phil Oshel poshel-at-luc.edu
I have been trying to immunogold label some kidney tissue embedded in LR White, but after three trys, (30 grids) I have only had three sections remain on the grids, (one section on three grids). I am using gold hex mesh grids, immersion of grids in 30ul drops of reagents. The grids were precleaned with detergent, rinsed with dH2O, rinsed with ETOH, and finally with acetone. Why are the sections falling off??? Any and all comments and suggestions are welcome.
I have read one paper in certain journal about dislocations in Si3N4, about which I forget the names of the journal and authors. In that paper, the Burges vectors of several kinds of dislocations in Si3N4 were determined by TEM. Could you please tell me related information if you wrote or have read the paper. In the paper, it was also mentioned that the dislocations in si3N4 can move only at very high temperature (1700C?).
} Help, } Why are the sections falling off??? Any and all comments and suggestions are } welcome. } } Thanks } Ed Calomeni } Ed, I am informed by that your grids should be formvar coated (prior to putting the sections on), this should help the sections stick and apparently makes no difference to the labelling efficiency (as you might expect given that only one side of the section is exposed to the reagents).
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
We quite frequently have to do SEM of many different kinds of powders. The problem with putting a drop of suspended particles on a stub is that surface tension draws the particles into clumps as the liquid dries. This is the technique that I use: 1. Make a very dilute suspension of the particles in methanol. You may have to determine the concentration by trial and error but it is very low. 2. Set up a vacuum filtration system using 10mm dia. Nuclepore filter membranes. I use filters with a pore size of 0.1um. 3. Vacuum filter a few drops of the suspension through the membrane. 4. Mount the filter on a stub and coat.
By vacuum filtering, the liquid leaves the particles almost immediately so that surface tension doesn't have a chance to form clumps. Nuclepore is the best filter to use because it is a sieve with small holes on a flat background which makes it easier to see the particles.
____________________________________________________________________ John Hudak hudakjm-at-mcmaster.ca I.M.R. - Electron Optics McMaster University, Hamilton, Ontario, Canada
Message-Id: {9410251526.AA25717-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: SEM of powder slurries Orig-Author: {"Griffin, Robin" {rgriffin-at-eng.uab.edu} }:ddn:wpafb -----------------------------------------------------------
Help!
We would like to characterize ceramic powders that are in an aqueous solution. We need to know if the powders are individual powders or agglomerates. Does anyone have any suggestion on SEM sample prep? Is there a drying technique that leaves the particles well dispersed?
Also, what is the typical TEM sample prep techniques used for ceramic powders?
Message-Id: {9410251656.AA26183-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: SEM of powder slurries Orig-Author: {"Griffin, Robin" {rgriffin-at-eng.uab.edu} }:ddn:wpafb -----------------------------------------------------------
Help!
We would like to characterize ceramic powders that are in an aqueous solution. We need to know if the powders are individual powders or agglomerates. Does anyone have any suggestion on SEM sample prep? Is there a drying technique that leaves the particles well dispersed?
Also, what is the typical TEM sample prep techniques used for ceramic powders?
Message-Id: {9410251948.AA27076-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: SEM of powder slurries Orig-Author: {"Griffin, Robin" {rgriffin-at-eng.uab.edu} }:ddn:wpafb -----------------------------------------------------------
Help!
We would like to characterize ceramic powders that are in an aqueous solution. We need to know if the powders are individual powders or agglomerates. Does anyone have any suggestion on SEM sample prep? Is there a drying technique that leaves the particles well dispersed?
Also, what is the typical TEM sample prep techniques used for ceramic powders?
Dear Microscopists we have a problem with SEM of cyanobacteria filtered onto Nucleopore membrane filter. Perhaps somebody has a good suggestion for gently removing the mucopolysaccharide (?) film around the filamentous cyanobacteria to get good pictures of the cell surfaces. Any suggestions will be very much appreciated!
Guenter Jost Institute for Baltic Sea Research D 18119 Warnemuende Germany e-Mail Jost-at-bio.io-warnemuende.d400.de
The Royal Microscopical Society has set up two new special interest groups in Digital Imaging and Three-Dimensional Microscopy. The purpose of the groups is to organize meetings, workshops, seminars and courses with the support and backup of the RMS. It is anticipated that, unlike the Sections of the RMS, these special interest groups will 'wax and wane', or form new permanent Sections, as the occasion demands.
If you would like to be added to the mailing lists for these groups, please send your name and address (and email address) to the Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, UK. Email RMS-at-VAX.OX.AC.UK. Please indicate which group you would like to receive information about.
If you have any suggestions/input for these groups, please contact the group convenors.
DIGITAL IMAGING GROUP CONVENOR: Dr Dominique Jeulin, Centre de Geostatistique, ENSMP, 35 rue St Honore, F-77300 Fontainebleau, France. Email: JEULIN-at-CG.ENSMP.FR
3-D MICROSCOPY GROUP CONVENOR: Dr Alan Entwistle, Ludwig Institute for Cancer Research, Courtauld Building, 91 Riding House Street, London W1P 8BT, UK.
We have had several LaB6 cathodes which have not quite stopped emmitting but were close. Check out the mount, these are usually made of tungston or carbon both of which will decay faster than the LaB6 crystal will. We have tried sending them back to the maker for warranty replacement but to no avail. Best Bet--buy a new one or switch back to tungston filaments.
Message-Id: {1994Oct26.090311.1567169635-at-ms.sjdccd.cc.ca.us} To: microscopy-at-aaem.amc.anl.gov (MSA list)
To eliminate the problem of the sections falling off, for years we have used "grid glue". Take a 1 inch piece of double sticky tape and put it in 20 ml. of ethylene dichloride. Wait a few min. until the adhesive comes off and then remove the piece of tape. In a closed bottle this stays good for months. We dip the cleaned grids in the grid glue, let dry and use the grids for picking up sections. We never use formvar coated grids for sections, but always do use grid glue. Prior to use we clean all of our grids in a small amount of acetone in an ultrasonic cleaner for a minute or so. There is some sort of residue on the grids when they are new which is then removed. We have used all EM supply houses, and found cleaning necessary before picking up sections. Actually we clean all of our grids for any use, i.e. whole mounts, or sections. I believe I published this little trick some years ago, 1982 in an SEM paper on Mounting materials where we used the grid glue for a different purpose. Hope it works for you also. I should add that we use a variety of resins, so don't think it is resin dependent necessarily. We have used LR White, Embed, the old Epon, Spurrs, Araldite, etc.
Good Luck Judy M.
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
for sections falling off grids, try dipping your (clean) grids in Butvar a 0.10% solution (in chloroform), then allow to dry on a clean piece of glass or a fiberless/ashless piece of filter paper. store in a clean dry place. both surfaces of the section will be available for immuno reagents, since the butvar is only on the grid bars
Hello, I've worked with LR White and immunogold, and have not often had this problem even with days of incubations.
1. The grids, I use Ni, 200 hex. I use only alcohol to clean them. In fact, I store the grids in 100% EtOH. I suspect that the acetone could be a problem if it is not washed off very very well. We've found that acetone leaves a residue on our grids, that is why we use only ethanol.*** Also be sure , just prior to cutting, that you wash the grids off with about a ml of water, then dry right up to a 75 watt bulb. We do this to be sure the EtOh is gone, but also because of "static" problems we have in the lab.
2. If the antigen site is not too heat sensitive, imediately after wicking the grids to " just damp", I hold the back side of the grid up 2mm away from a bare, 75 watt lamp. This is done only 5 seconds longer than all visable moisture is gone and the grid looks dry. ** This has kept our sections on and flat, even with " jet" washing from a water bottle.
3. How thick are your sections, very thick sections will fall off of the grid more easily. I cut LR White at 65-80nm. If this is hard to cut, go back to the embedding to improve the block. **** when I finish polymerizing the LR White, I put it into a desicater jar, and put it under vacuum for 3-4 days before sectioning. Storage is also under vacuum. For some reason, this seems to help a lot. Good Luck! Lou Ann --------------------- Lou Ann Miller U of Illinois College of Veterinary Medicine 217-244-1566 lmiller-at-ux1.cso.uiuc.edu
The Midwest Society of Electron Microscopists (MSEM) presents a Free* workshop: Scanning Probe Microscopy of Materials and Phenomena
Place: Northwestern University, Norris Center Room 2A 8:30 Registration 9:00 - 12:00 Speakers: Dr. Z. Zhang, Universiity of Wisconsin-Madison Mr. Ian Smith, Park Scientific Instruments Dr. Jerry Zajac of Amoco Dr. Carla Alves from Topometrix Dr. John B. Ketterson, Northwestern University 11:45 - 1:00 Lunch (Norris Center Cafeteria) 1:00 - 5:00 Panel Discussion: "Where to from Here" Vendor displays and demonstrations Student Poster Presentations Facility tour
For reservations and further information call: Joyce Craig or Jeffrey Schmelz E-Mail BAFPJEC-at-UXA.ECN.BGU.EDU fAX 312-995-3759 pHONE 312 995-3800 FOR INFORMATION ABOUT THE STUDENT PRESNTATIONS CONTACT MEETING ORGANIZER: VINAYAK P. DRAVID e-MAIL VPDRAVID-at-CASBAH.ACNS.NWU.EDU 708 467-1363 708 467 7798 fax 708 491-7820
*The workshop is free to MSEM members. Ask for an application. Membership is only $10 per year, $5 for students.
To keep sections on the grid, I usually clean my grids just before picking up the sections. I use a squirt bottle with 10% acetic acid followed by a distilled water rinse and then an acetone rinse. I do this by gripping the grid in my tweezers and squirting the grid with the different solutions and then putting the grid down on a piece of #50 Whatman filter paper. I never have a problem with the sections falling off. The grids I use mostly are the standard copper grids. I use the same procedure for nickel grids except after the acetone wash I do a second distilled water rinse. Hope this helps. Phil 8-{)
Message-Id: {m0r0GIS-0007KIC-at-stjohns.ohsu.edu} Message-Version: 2 } To: Microscopy-at-AAEM.AMC.ANL.GOV
Mark your Calendars
Portland area's Pacific Northwest Electron Microscopy Societies mini-meeting will be held on Tuesday, November 1, 1994. The meeting agenda includes a buffet at Shang Hai Nobel House restaurant a short PNEMS business meeting and a presentation by OPTIX Inc.. The restaurant is located at John's Landing 5331 S. W. MacAdam Ave, just south of downtown Portland.
The business meetings topics include the spring '95' PNEMS meeting, MSA 1999 national meeting, and miscellaneous items. The presentation will cover imaging topics from archiving large digital files to 3-dimensional reconstruction. Buffet begins at 6:00 PM and the presentation will conclude around 8:00 PM.
Since the Society will be providing the buffet please RSVP to Bob Kayton (503)494-2504 by October 27. Please contact Bob Kayton if you have any questions. Any interested microscopists are encouraged to attend.
Message-Id: {m0r0HMx-0007KEC-at-stjohns.ohsu.edu} Message-Version: 2 } To: microscopy-at-aaem.amc.anl.gov
Mark your Calendars
Portland area's Pacific Northwest Electron Microscopy Societies mini-meeting will be held on Tuesday, November 1, 1994. The meeting agenda includes a buffet at Shang Hai Nobel House restaurant a short PNEMS business meeting and a presentation by OPTIX Inc.. The restaurant is located at John's Landing 5331 S. W. MacAdam Ave, just south of downtown Portland.
The business meetings topics include the spring '95' PNEMS meeting, MSA 1999 national meeting, and miscellaneous items. The presentation will cover imaging topics from archiving large digital files to 3-dimensional reconstruction. Buffet begins at 6:00 PM and the presentation will conclude around 8:00 PM.
Since the Society will be providing the buffet please RSVP to Bob Kayton (503)494-2504 by October 27. Please contact Bob Kayton if you have any questions. Any interested microscopists are encouraged to attend.
Two questions concerning X-ray analysis from a desperate Masters student. 1. Has anybody worked with the ZAF program QUAD2 developed by Farthing et al and presented at the Int. Congr. X-ray Optics and Microanalysis, Manchester, 1992. 2. How and what is an Electron Probe Microanalysis. We have our EDX on an SEM and I understand how that set-up does it's compositional analysis but I don't understand how standards are used. Let me explain that I understand most of the equations in the ZAF analysis and I have read the standard texts but I think I'm missing something on the operations. Replying to me directly can be done at henne-at-sfu.ca Thanks in advance. Cheers. Dan Henne Simon Fraser University Vancouver, Canada
Message-Id: {m0r0HjT-0007KFC-at-stjohns.ohsu.edu} Message-Version: 2 } To: microscopy-at-aaem.amc.anl.gov
MEETING
Hi,
The Portland area's Pacific Northwest Electron Microscopy Society is having its first mini-meeting on Tuesday November1, 1994. The meeting agenda includes a bufet at Shanghai Nobel House restaurant a short PNEMS business meeting and a presentation by OPTIX Inc. The restaurant is located at John's Landing, 5331 S.W. MacAdam Ave., just south of downtown Portland.
The business meetings' topics include the spring '95' PNEMS meeting, MAS 1999 national meeting, and miscellaneous items. The presentation will cover imaging topics from archiving large digital files to 3-dimensional reconstruction. Buffet begins at 6:00 PM and the presentation will conclude around 8:00.
Since the Society will be providing the buffet please RSVP to Bob Kayton (503)494-2504 by Nov. 1. Please contact Bob Kayton if you have any questions.
Just to let you all know I have noticed duplicate and sometimes triplicate messages on the net. Looking at the headers they appear to be coming from the originators, the majority of the time. There are some that are mirrored by the system if the computer crashes during a session, but that has become less frequent with the new system. All I can say is sorry :-( for the traffic. If anyone is not sure about how to send a message touch base with me off-line. Remember the mail queue sometimes gets very long and if you post a message it may not appear on the net for several hours, or sometimes not until the next day. Just be patient, it will get there!
Just for the record the Microscopy Listserver has just passed it's first anniversity/birthday. We started operation on Oct 1, 1993 and the system has long ago passed the point of it's 1,000,000th Email message delivery. To date we've delivered over 2500 postings/messages to over 1500 subscribers (plus an unknown number of readers via the SciTechnqiues Microscopy Newsgroup) 1M is conservative since by simple math 2.5Kx 1.5K = 3.75M, but not every subscriber has seen every message (except, of course, for me). Not bad for our first year of operation. I don't actually know who got the 1Mth and I doubt if I could find out. In any case just bear with the glitches and things will work out in the long run.
Does anyone know if there is an 'off the shelf' fluorescent drug or compound analogous to FITC-phalloidin, but which will react with microtubules? Thanks for your help. David.
Does anybody out there do flat embedding of tissue embedded in LR-White? I know the embedding mold has to be covered during the curing to keep the air out but do they make a special mold for doing this? All I have are the standard embedding molds for flat embedding. Can these be used somehow? Thanks in advance, Phil 8-{)
In message Thu, 27 Oct 1994 09:08:40 -0400, rutledge phil {prutle1-at-gl.umbc.edu} writes:
} Does anybody out there do flat embedding of tissue embedded in LR-White? } I know the embedding mold has to be covered during the curing to keep } the air out but do they make a special mold for doing this? All I have } are the standard embedding molds for flat embedding. Can these be used } somehow? } Thanks in advance, } Phil } 8-{) } *************
Yes, Ted Pella (USA 1-800-637-3526; Canada 1-800-243-7765) supplies teflon molds (cat# 10506) and ACLAR film (cat# 10502) that provide an oxygen-free environment for the polymerixation of LR White & Lowicryls.
***************************************************** M.V. Parthasarathy Professor of Plant Biology; Director, EM Facility Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu ****************************************************** e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e- e- e- e- This message was sent using 100% recycled electrons e- e- e- e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-e-
Phil, I have been using silicone embedding molds for years for embedding LR White. After filling the molds, I place it into an airtight container which is flushed with an inert gas (dry nitrogen, argon, freon, all work). The cover is installed and the container placed in the embedding oven. I use a 1 pint paint can with a hole in the top for introducing the gas. Once flushed, the hole is covered with a piece of tape. This system works just fine.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
=From: TOWER::GWERDOS "Greg Erdos ICBR EM Core Lab Univers" =To: IN%"prutle1-at-gl.umbc.edu" =CC: GWERDOS =Subj: Re: LR-White = =} From: IN%"prutle1-at-gl.umbc.edu" "rutledge phil" =} Subj: LR-White =} =} Return-path: {prutle1-at-gl.umbc.edu} =} Received: from AAEM.AMC.ANL.GOV by gnv.ifas.ufl.edu (PMDF V4.3-10 #3240) =} id {01HIRLP5TFJ48XUR11-at-gnv.ifas.ufl.edu} ; Thu, 27 Oct 1994 09:37:58 -0500 (EST) =} Date: Thu, 27 Oct 1994 09:08:40 -0400 =} From: rutledge phil {prutle1-at-gl.umbc.edu} =} Subject: LR-White =} X-Sender: prutle1-at-umbc8.umbc.edu =} To: microscopy {microscopy-at-aaem.amc.anl.gov} =} Message-id: {Pine.SGI.3.90.941027090342.4522A-100000-at-umbc8.umbc.edu} =} MIME-version: 1.0 =} Content-type: TEXT/PLAIN; charset=US-ASCII =} Content-transfer-encoding: 7BIT =} =} Does anybody out there do flat embedding of tissue embedded in LR-White? =} I know the embedding mold has to be covered during the curing to keep =} the air out but do they make a special mold for doing this? All I have =} are the standard embedding molds for flat embedding. Can these be used =} somehow? =} Thanks in advance, =} Phil =} 8-{) =####################################################### =Phil, = For LR White etc. where polymerization is oxygen sensitive. I punch out =circles of unexposed but cleared EM film with a paper punch. I put these under =resin in a polypropylene microfuge tube. They settle part way down the taper =and form a flat platform for tissue. The tube can be nealy filled with resin, =closed and successfully polymerized. Then you cut the tube down the side, =remove the solid plastic and break it at the film exposing your tissue at a =flat surface ready for sectioning. = Send an address or a FAX number and I can provide more details and =references. =********************************************************** =* Greg Erdos ** * =* Director, ICBR EMCL ** Phone 904-392-1295 * =* 218 Carr Hall ** FAX 904-846-0251 * =* University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * =* Gainesville, FL 32611 ** * =**************************************************************** ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-846-0251 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * ****************************************************************
I flat-embed regularly in LR White, between 2 sheets of Aklar film from Ted= Pella: I infiltrate my sections (60-80 microns, vibratomed) as usual, them= immerse them in a small puddle of something like araldite or eponate, etc.= on the bottom piece of Aklar, then place a second sheet of Aklar on top. = Bake as usual. Incidentally, this is NOT the material that I had trouble= with in the beam--that stuff was actually a regular tissue block. I= finally decided, thanks to the many kind responses I received, that my LR= White is just too old. Grace
We would like to use recordable CD-ROMs for archiving images from a Gatan system we will be purchasing. I would be interested in hearing from anyone using CD-ROMs for storage.
I just saw an ad for a Ricoh CD-ROM unit for approx.$3000.00. It is both multi-session and multi-platform. Has anyone used this unit?
To whom it may concern: We have an old Coates & Welter Cwikscan FE-SEM. While talking to someone about our instrument it came to light that there was a message on this bulletin board about another Cwikscan which someone was trying to find a user for. I am interested in finding out more information about this SEM. Any help is appreciated. Thanks, Jerry Allsman Rolls-Royce Inc. Atlanta, GA
I've flat embedded items in Historesin using Peel-a-Way paraffin molds and ;bottle caps that are carefully covered ;with a layer of mineral oil or melted paraffin. Also handy is to make some non-stick microscope slides by coating them with silane. Then lay down a 100-300 micron slice of infiltrated tissue and cover ;with methacrylate. lay another coated slide on top, being careful to not entrap air bubbles. Now paint the edges with paraffin and allow to polymerize. Sandwiching between slides is a standard method in our lab for methacrylate embedding of slices.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Thu, 27 Oct 1994, rutledge phil wrote:
} Does anybody out there do flat embedding of tissue embedded in LR-White? } I know the embedding mold has to be covered during the curing to keep } the air out but do they make a special mold for doing this? All I have } are the standard embedding molds for flat embedding. Can these be used } somehow? } Thanks in advance, } Phil } 8-{) }
We currently have an old KEPCO Model NTC 2000 power supply in need of repair. Attempts to call the company at the number listed on the manual that came with the supply were fruitless.
Does anyone know if this company is still in business and if so, how I may contact them. Alternatively, is there a company that can repair the power supply?
Please direct all responses directly to me at baltrus-at-orion.petc.doe.gov
Thanks for your help!
John Baltrus US Dept of Energy/Pittsburgh Energy Technology Center
Greetings Microscopists, I'm looking for info on magmetic shielding of microscope labs. Specifically, we are building a new materials science building, and would like to make absolutely certain that magnetic fields are minimized. We are mostly interested in keeping fields from outside the labs (from machine shops and the like) from causing problems. Any thoughts or advice on this matter would be appreciated.
Has anyone out there got any experiance/ideas re root nodules for TEM?
We have prepared clover root nodules for TEM and have had problems getting good sections - the problem is that regions in the middle of the sections of the plant cells fall out.. I'm not sure whether the fixation of the centers is poor or whether its a resin problem. I have found that Agar 100 works better (has fewer holes) than Spurrs which I initially used. I fix under a slight vacuum for 2 hours to aid infiltration of the 3% glut 0.2M cacodylate into the 1-2mm diameter nodules, plus slice a portion off the nodules to open them up a little but to no avail.I use 1% Oso4 in same buffer as postfix. The regions which are intact look good however. Any ides?
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
MMS FALL BUFFET DINNER & IMPORTANT BUSINESS MEETING
UNIVERSITY OF MINNESOTA, ST. PAUL CAMPUS STUDENT UNION, CHERRYWOOD ROOM, THURSDAY, NOVEMBER 10, 1994, 5:00 - 9:00 PM
SPEAKER: JAN HINSCH, LEICA INC.
TOPIC: "BUSMAN'S HOLIDAY"
MMS will hold its sixth annual Fall Buffet Dinner on November 10, 1994, at the University of Minnesota's Cherrywood Room located on the 2nd floor of the St. Paul Campus Student Union, 2011 Buford Avenue, St. Paul Campus( NOTE: This year's Buffet Dinner is NOT at the Campus Club as in previous years). We hope to provide a pleasant evening during which microscopists will be moved to renew or begin their membership in MMS, MSA and MAS. A wine, cider and cheese social from 5:00-6:00 in the Cherrywood Room. will kick off the evening. The buffet dinner follows from 6:00-7:00. The dinner entre will be Halibut Steak with Chicken Strips (Cacciatore Style), Caesar Salad, Rice Pilaf, Fresh Broccoli Spears, desserts and beverages. The total cost is more than the $10.00 fee but MMS is picking up about 40% of the tab as a courtesy to our membership. The dinner affords an excellent opportunity to meet microscopists from many disciplines, talk shop, and to have a pleasant time together. The program is from 7:00-8:00 in the Cherrywood Room. Parking is available behind the Union and at other St. Paul Campus locations.
Our featured speaker, Jan Hinsch, from 1978 - Present, has been Director of LEICA's WILD-LEITZ Laboratory for Applied Microscopy in Rockleigh, New Jersey He is a Fellow, New York Microscopical Society, an Honorary Member and recipient of the Outstanding Microscopists award from the State Microscopical Society of Illinois. Jan has had numerous photomicrographs and articles published in a wide variety of journals - ie. "Industrial Microscopy", 1979 Industrial Research & Development; "Critical Focusing in Low Power Photomicrography", 1979, American Laboratory; "Essentials of Light Microscopy and Photomicrography", 1983, Pathology Annual; "Refined Approaches to Microscopical Light Management", 1988, The Microscope. Here is Jan's description of his talk, "Busman's Holiday": "The vacationing microscopist faces a dilemma. If this is to be a time to conquer the unknown should not one leave the microscope and any thought of it at home? And yet, this is also a time of opportunity to collect samples of many kinds and, inspired by the natural phenomena around, to meditate on the forms and illustrations of the action of light and its significance to us microccopists. This is the account of someone trying to have it both ways." We will hold a short business meeting just before the talk. The Buffet Dinner is $10.00 per person payable at the door. (non-member $20.00, includes new membership), $7.00 for current students(non-member student $12.00, includes new membership). In order for us to provide an accurate head count to the Cherrywood Room, please make a phone reservation by calling Mike or Ev.
Mike Coscio(612-569-1331, E-MAIL: mike.coscio-at-medtronic.com) Ev Osten(612-736-0104, E-MAIL: efosten-at-mmm.com) - Buffet Coordinators
--
Gib Ahlstrand, MMS Newsletter Editor Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.crl.umn.edu
We have a had similar problems. We found that air bubbles in the root nodules, lung, ... prevented the fixation through embedding to work properly. Try pulling a vacuum on the tissue in the fix after cutting a small hole in one end. If the tissue floats, clip a staple or small paper clip to one end to act as a weight. On more difficult samples, we need to repeat the vacuum a second time when in 100% ETOH (because of the surface tension in H2O) to get rid of smaller bubbles. Good luck Mike
--------------------------------------------------------------------------- | Michael Dunlap | lab (916) 752-0284 | | Facility For Advanced Instrumentation | fax (510) 422-2282 | | University of California | mrdunlap-at-ucdavis.edu | | Davis CA, 95616 | | ===========================================================================
Well I have seen some other solutions to your flat embedding woes and thought I might as well throw in my 2 cents worth too. At the very least its another option to try.
Look up my technical paper Flat Mold Embedding with LR White and Lowicryl K4M. Simpson Gomes,A and Simon G.T. J. of EM Technique 13:266-267 1989.
Note: this journal is now called J. of Microscopy Reseach and Technique.
Have fun, looks like you have several good ideas to work with.
Anne
Anne Simpson Gomes
EM Unit, F09 "How's it going Eh?!!!"....... Univ of Sydney from NSW 2006 Australia The Compact Canuck!! Fax: (612) 552 1967
The following info comes from my boss Dr Maret Vesk. She has done extensive work on clover root nodules.
"Some possible causes: Air in intracellular spaces = no resin infiltration = holes in sections. This is very common in plants and may be helped by evacuating roots in distilled water before fixation. They should sink, not float. 0.2M buffer wiil definitely cause plasmolysis, most workers use 0.025-0.05M buffer with phosphate rather than the toxic cacodylate being the preferred buffer. Use very slow infiltration of resin (Spurr's is perfectly fine), adding drop wise over a matter of days if necessary. Is your dehydration long enough (2 x 30min)? Have had no problems with either clover nodules or wheat paranodules"
Maret (via Anne)
Anne Simpson Gomes
EM Unit, F09 "How's it going Eh?!!!"....... Univ of Sydney from NSW 2006 Australia The Compact Canuck!! Fax: (612) 552 1967
The EM-unit here at Utrecht Univ, Biology Dept is using a CD-ROM system for recording images. They are writing it with a Kodak DCD Writer 200 Plus unit, multisession. I am saving XTEM images with it, which I read here in my lab on a Panasonic CD-Rom driver. I am using a 486, 66MHz PC and either the CorelDraw or PhotoShop packages to analyze the images, primarily for morphological details, and it works fine.
Dirk Knoesen, Debye Institute, Dept Atomic and Interface Physics, Uuniversity Utrecht.
I just wonder if anyone knows about immersion oils that transmit 340 nm light well. Zeiss and Nikon immersion oils attenuates this wavelength heavily, and the attenuation is dependent of the thickness of the oil layer, leading to unstable calibrations from time to time and where the focus is in the sample. I now use glycerol which transmits nice in 340, but this is not optically optimal because of its lower refractive index.
============================================= Mikael Gustafsson MD, PhD Dept Med. Microbiology and Dept Internal Medicine, Cardiology section University of Linkoping SWEDEN
I just wonder if anyone knows about immersion oils that transmit 340 nm light well. Zeiss and Nikon immersion oils attenuates this wavelength heavily, and the attenuation is dependent of the thickness of the oil layer, leading to unstable calibrations from time to time and where the focus is in the sample. I now use glycerol which transmits nice in 340, but this is not optically optimal because of its lower refractive index.
============================================= Mikael Gustafsson MD, PhD Dept Med. Microbiology and Dept Internal Medicine, Cardiology section University of Linkoping SWEDEN
} } To:Bob Keller {keller-at-micros.mrd.bldrdoc.gov} } From:kleifer-at-cimesg1.epfl.ch (Klaus Leifer) } Subject:SAD precision 2 } } Hallo Bob, } thanks for your comment which I received one month ago on the microscopy } server with respect to precision of evaluation of electron diffraction } patterns. The system, I'm working on has the following properties with resp= ect } to diffraction. } - Ni/Ti multilayers on Si substrate } - grain sizes about 3 to 15 nm (in both lateral and orthogonal direction wi= th } respect to the interfaces) } - texture with respect to interfaces (with an angular deviation of +-5=B0) } } I need a better precision of SAD to measure strains, absolute average latti= ce } plane spacings and their variations. } Another reason is that the opinion I get from people with whom I discuss is } that the general limit of SAD is 1%. If I then regard the fact that most of } the people I know (me included) are still evaluating their diff. pattern wi= th } a ruler (perhaps the precision of human eye is not so bad, but numerical fi= ts } of the pattern similar to X-ray patterns are not yet very current) I think } that an enhancement of the 1% precision is still possible. } This was shown i.e. by Y. Le Page (1992, Microscopy Research and Technique, } Vol23,No3) } } SAD presents the advantage, that I do have the calibration of the Si substr= ate } directly on the same diffraction pattern. } CBED on single grains would be possible with respect to evaluation of discr= ete } spots positions(-} I'll try this on a Hitachi HF2000 with 1nm FWHM of the } probe). The difficulty is that I lose Si calibration on the same } pattern.Changing from diff. to image mode and back to diff. again gives me } typical deviations of the position of the Si spots of about 0.5%!! } The HOLZ lines in only 5nm thick cristallites don't have enough intensity. } } I was thinking and calculating to get a possible error for the SAD } measurements. The probable errors, I found are: } - distortions of the pattern due to lens spiral and barrel distortions (I t= ry } to exclude this } taking for calibration the Si spots close to the spot I evaluate) } - precision of the measurement of the peak maximum (well, I'm using a ruler= ) } - form of the grains together with the inclination angle of the grains with } respect to the } beam may change the position of the reflection in between 5e-4 (in = a } calculation I } made taking grains that are only limited in size in beam direction) } and more than 1% } (for grains which are limited in size only in one direction } orthogonal or inclined to } the beam. I don't observe such some grains but cannot exclude the } influence of } stacking faults or twins on the diff. pattern). } - !! one error about which I have no idea is the following: is there a } correlation between } the position of a grain in the SAD aperture and a probable change = of } the position of } its reflections in the diffraction pattern due to higher order } abberations of the } objective lens which depend on the position of the object? Do there } exist } calculations? } What do you think about this? } Best wishes } Klaus } } } } Klaus, } } } } There is some discussion of using SAD for lattice spacing measurements in } } Reimer's TEM book, chapter 9. Generally, that's not a good approach if yo= u } } would like to determine lattice constants with better than a 1 percent err= or } } or } } so. This is a significant error if you are concerned with elastic strain } } measurements or phase identification. In practice, unless you use a } } calibration } } standard, the camera length will not be known accurately and further, ther= e } } is } } also some distortion in the pattern caused by the projector lens. } } } } It is not possible to use SAD to determine lattice constants in the direct= ion } } of } } the incident beam. CBED will give you such info. since HOLZ reflections c= an } } be } } excited. Using a STEM unit, it becomes possible to produce CBED spot size= s } } around 5 nm dia. fairly routinely, which would allow probing of individual } } grains in some multilayer systems, presuming you can get single grains } } through } } the foil thickness. } } } } I recognize from your email address that you are at the same institute as = Dr. } } P. } } Stadelmann. He has written some very nice simulation software that may al= low } } you to experiment with how sensitive spot patterns are compared to CBED HO= LZ } } patterns for lattice constant measurement. } } } } Regards, } } } } Bob Keller } } NIST }
Subject: Time:11:28 PM OFFICE MEMO RE: MagShields for labs Date:10/28/94
It is virtually impossible, and highly impractical, to build an effective magnetic shielding system for an entire laboratory room. About the best that you can do is to locate the room in which the electron microscope will be housed as far as possible from potential sources of large alternating magnetic fields, such as: power lines that carry large currents; transformer substations; building switch boxes and junction boxes; elevator motors; etc. It is also very important to be sure that all metal water and sewer pipes, and metal heating and ventilating ducts in the neighborhood of the instrudment room are individually grounded some distance before they enter the region of the lab, and that the electrical contractors are alerted to the problems that can arise from large 'ground loop' currents flowing through such items. Ground loops through steel structural members of the building itself can also produce large magnetic fields. You need to talk to the university architects and plant design engineers to be sure that every possible precaution is taken to avoid them, too. You will have to be very persistent about these matters, because most of the time construction people are not concerned with them. It is also a very good idea to have a separate ground rod installed in each instrument laboratory, so that each instrument has a good electrical ground that is free from outside effects. Good luck!
The strain of the substrate (we measure macrosopical stresses in the order of 1e9 dynes/cm2), when calibrating the distances on the diffraction pattern with the Si substrate reflections , surely is a problem in the case of my specimen. Do you perhaps have citations concerning the relation between substrate-film strain and corresponding lattice parameter variations of the substrate?
Your idea to measure the distortions from high resolution images can be an interesting alternative to the diffraction analysis.
Concerning the variations of 0.5% of spot positions in the diffraction image: I first acquired a diffraction pattern, then changed to image mode to look at the specimen. Afterwards I changed back to diffraction mode again and made a second photo of a Si diffraction pattern. The evaluation of both patterns showed deviations of the Si reflection positions between both patterns of about 0.5% (comparing of course the same Si reflections, not having touched diffraction focus and having defocussed the beam in both cases the condensor until the end). I measured differences of 0.2-0.6% between the reflection positions of the two negatives. All deviations went in the same direction and the Si spots are sharp.
Concerning the calculation: I wanted to know an order of magnitude of the deviation of the reflection position, when one starts to tilt cristallites. In the the first calculation I made two weeks ago I used the following basic geometry:
I I e-beam directio= n I I
______________________________________ I I I foil thickness d ______________________________________ So the grain here has a thickness d in electron beam direction and is infinitisemally long. I calculated then with the program of Pierre Stadelmann the position of the diffraction spot position with respect to tilt angles of the grain ( you also could calculate this analytically) and the distance between the knot in the reciprocal space and the Ewald sphere. From this distance (supposing a two beam case) I calculate the intensity of the diffraction spot for every tilt angle of the grain. This gives me a curve 'position of reflection/intensity' which is roughly a (sinx)2/x2. So this curves would represent the form of a peak of a Dedye Scherrer diagram with random orientation of grain with the shown geometry. =46rom the width of this peak I can see, at which tilt angle reflections of = a grain are still visible and the corresponding shift of the reflection position. I made this calculation for 1,2,5 and 10 nm thick grains and in this geometry the possible deviation of the reflection positions from the untilted grain was for 1nm thick grains was around than 1.5e-3, for 5nm thick grains about 4e-4. Grains that are tilted by higher angles, this means which show higher deviations in the reflection position are for a factor 100 lower than the reflection at 0=B0tilt. Of course, there would still be missing a treatment of grains with a realistic form! Did somebody calculate this? Best wishes Klaus
I noticed that while discussing SAD accuracy, noone mentioned the fact that dynamical diffraction can change the position of spots: see Hirsrch et al 1965; Hashimoto et al Phil Trans Roy Soc London 253 (1961) 459, or for a very short summary Marks, Ultramicroscopy 12 1984 237.
Scientific sessions start on Tuesday, April 18, 1995. Original contributions will be presented in the following areas:
Confocal microscopy
* theory of confocal imaging * scanning confocal designs: point / slit arrangements, beam scanning, direct field scanning (bilateral, tandem) * high resolution optical 3-D Microscopy * two photon and time resolved fluorescence imaging * transfer functions and deconvolution
Applications
* confocal microscopy in-vivo: approaches, problems and prospects * 3D imaging for agricultural research * fluorescence 3-D imaging in cell biology and neurobiology * fluorescent probes, in-situ hybridization * 3-D cytometry * microstructures of materals, polymers and thin films * application in environmental sciences
Near-field microscopy
* near-field scanning techniques (NSOM, STM, AFM) * high resolution DNA - imaging * spectroscopy and surface modification * combined near-field and confocal designs
Optical tweezers and scalpel
* instrumentation * applications
Electron Microscopy
* cryo-microscopy * low-voltage SEM * electron beam tomography
X-ray microscopy
* theory and instrumentation of x-ray microscopy * x-ray sources
3-D imaging processing
* 3-D reconstruction of histological, optical and tomographical sections * visualization models in 3-D and 4-D microscopy * supercomputing in microscopy * analysis of serial section images, 3-D scene recognition * 3-D image restoration and image quality
CONTRIBUTION AND PUBLICATION OF EXTENDED ABSTRACTS
Papers are invited for oral and poster presentation from the fields indicated by the scientific program and related areas. Deadline for submission of abstracts is December 31, 1994. An extented abstract (minimum 700 words and maximum 1500 words) is required for each presentation, and will be published as a suplement issue of Zoological Studies (ISSN 1021-5506). All text will be typeset by the publisher. Authors are encouraged to submit their manuscript in electronic forms. Files created from the following word processos are acceptable, otherwise, please submit your manuscript in ASCII form (both Mac and IBM-PC format). Manuscript can also be submitted by e-mail to: elepcc-at- ubvms.cc buffalo.edu, however, a hardcopy has to be sent by mail (or faxed) to the address in USA. A hardcopy is required accompany the electronic form.
Mac Word, Wordstar, Word Perfect, Microsoft Word, Ventura, ASCII
Figures and photos are permitted, however they have to fit the following format specified in the photo and diagram format guide for direct photoreproduction. Original photographs (both B&W and color) and line drawings are required. If it is possible, authors please provide a FAX number to facilatate the transmission of galley proof in early 1995. The organizing committee will make a selection of the abstracts for oral presentation. By the end of January 1995 authors will be notified about acceptance and the final program will be mailed to all registrants.
ACCOMMODATION
The Howard Plaza Hotel provides subtantially reduced room rates for conference participants of Focus on Microscopy '95. Hotel Accommodation at the Howard Plaza Hotel is offered on a first come, first served basis. Please refer to Focus on Microscopy '95 for qualifying the reduced room rates at booking: (refer to Registration form)
Howard Plaza Hotel 160 Jen Ai Road, Sec. 3 Taipei, Taiwan, Republic of China
Phone: 886-2-700-2323 FAX: 886-2-700-0729
The room rate includes 10% service charge, welcome wine, fruit basket, newspaper and the use of health club facilities including sauna. Major credit cards (American Express, Visa, Mastercard, JCB, Diners Club) are accepted at the hotel.
REGISTRATION & CONFERENCE FEE
The conference fee is US$220, which includes, documentation, abstract book and refreshments during breaks. A preregistration fee of US$180.00, is available when postmarked before January 31, 1995.
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China Airlines is the official air carrier of Focus on Microscopy 95, special discount airfare is available through CAL's world wide branch offices.
INFORMATION
Registration, abstract forms and enquires:
N. America and Europe:
Focus on Microscopy '95 c/o Dr. P. C. Cheng Advanced Micrscopy and Imaging Laboratory Department of Electrical and Computer Engineering State University of New York at Buffalo P.O. Box. 84 Getzville, NY 14068 USA Tel and Fax: 716-645-3868 e-mail: elepcc-at-corn.eng.buffalo.edu
Other nations:
Focus on Microscopy '95 c/o Dr. J. L. Wu Institute of Zoology Academia Sinica Nankang, Taipei, Taiwan 11529 Republic of China Tel: 886-2-789-9500 Fax: 886-2-789-9503/886-2-785-8059 e-mail: zojlwu-at-ccvax.sinica.edu.tw
ORGANIZERS
C.P. Chen (Taipei) G.J. Brakenhoff (Amsterdam) A.Kriete (Giessen) C.H. Chou (Taipei) P. C. Cheng (Buffalo)(Chairman) C.J.R. Sheppard (Sydney)
P.P. Hwang (Taipei) C. Cogswell (Sydney) D.M.Shinozaki (London,Cana da) W.Y. Lee (Taipei) M. Gu (Sydney) E.H.K. Stelzer (Heidelberg ) H.K. Wu (Taipei) V. Howard (Liverpool) T. Wilson (Oxford) J.L. Wu (Taipei) H. Kim (Rochester) W.L. Wu (Taipei)
THE CONFERENCE IS JOINTLY O RGANIZED AND SUPPORTED BY
The Society for 3-D Imaging Sciences in Microscopy, Amsterdam Institute of Zoology, Academia Sinica, Taiwan, R.O.C. Electron Microscopy Society of China, Taipei, R.O.C. Life Science Research Promotion Center, NSC, R.O.C. AMIL, State University of New York at Buffalo, U.S.A.
I completely agree with the message of Wil Bigelow that shielding a whole building is not the right way if it would work at all. If you will shield your measuring and recording equipment you can place them together with the object you measure in a so called cage of Faraday. There should be guidelines on how to build such a cage and of what material, but it might be fairly expensive. In that case you might have a good shielding.
If you want to make precautions during construction time, not only look for good grounding points seperated from the mains but also try to avoid high temporal (far less than a second e.g. starting mercury arc lamps etc.) current 'pulses' enter your room by using a saturated kind of transformer. This kind of transformer (1:1) will block electrical pulses that may disturb your measurements. I hope I made clear what I meant because I don't know the english technical term.
If you use a number of wall plugs take care that this supply points all have the same electrical phase (as you may know 220 volts is one of the 3 phases), because different phase mains plugs can cause unwanted electrical signals if you connect your instuments to them.
Hope you can use this information.
Regards -- Kees van der Wulp TNO - Nutrition & Food Research Institute INTERNET : vanderwulp-at-mbl.tno.nl Division of Toxicology VOICE : +31 15 843101 PO-Box 5815 FAX : +31 15 843989 2280 HV RIJSWIJK (NL) THE NETHERLANDS
A few years ago a company had set up a stand trying to sell magnetic shielding at one of our local microscopy conferences. We were setting up new labs, so I asked them about it. They were selling "boxes" generally used for keeping high intensity fields in, rather than low intensity fields out, for enclosing NMR machines etc. The dimensions in their illustrations were not much larger than the size of an SEM, although I suppose they could build bigger boxes. The walls were pretty thin-looking and when asked they said it was a "special material" :-). Somehow I don't think that putting one of these around a microscope would do much good, since low intensity 50-60 Hz magnetic fields are not really easy things to block.
Their price for a "box"? A mere $45,000 AUD. :-)
Arthur Day, Electron Microscopy Group Ansto Advanced Materials Program Phone: 61-2-717-3457 PMB 1, Menai (Sydney), NSW, 2234 Fax: 61-2-543-7179 Australia Email: ard-at-atom.ansto.gov.au
I can't add any suggestions to what others have said about magnetic shielding for labs. However, the need for shielding reminds me of several years (1971-78) I spent at Temple University School of Medicine, located on Broad Street in north Philadelphia, Pennsylvania. One of the city's main subways went underground up Broad Street. I remember someone's EM facility at ground level in a small building. Every time a subway would come by, the EM beam would undergo rather striking excursions on the screen. They tried various approaches (Faraday cage, Gaussian ring, etc.), but I don't think it was ever fully solved.
Our EM facility on the 6th floor of the medical school building seemed all right. However, an EM newly installed in another department on our floor had terrible intermittent beam shifting problems. It was hard to see how it could be due to the subways, since our EMs seemed OK. After a few bewildering weeks (both for the company and for the investigator) it was discovered that the EM had been installed near an old (but still operational) elevator that had an extremely heavy iron counterweight, that traveled up and down all day long. Whenever the counterweight passed the 6th floor, the EM beam drifted off to the side, then drifted back. Who would have thought of anything like that?
As a small comment, it can be just as effective to shield only critical parts of the column using Nu-metal. Using one graduate student, a pair of metal cutters and a local company for Hydrogen annealing of the pieces you can perform miracles for $200-$300.
It may be of interest to know that at least one company (Link, through Oxford) offers a magnetic shielding system which is an active feedback system to compensate for generated fields in the laboratory. I am not sure whether it is just for 50/60 ac or dc or both, and I have no experience of the system, but would be mildly interested to hear anyone's experience. I agree with the comments regarding prior checks and appropriate location rather than shielding. Cheers jjm Professor John J. Millar Head, Department of Applied Physics Electron Microscope Unit RMIT, GPO Box 2476V, MELBOURNE AUSTRALIA 3001 +613 660 2602 fax 613 660 3837 email jjmill-at-rmit.edu.au
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As part of a surplus purchase of SEM specimen boxes, I have a few dozen used aluminum stubs that appear to go with Coates & Welter. Having an ISI/Topcon 'scope, I have no use for them. If you'd like them mailed to you, please contact me directly. Please *do not* reply to the list. Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
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From uucp Mon Oct 31 15:59:42 GMT 1994 remote from attmail } From AAEM.AMC.ANL.GOV!MICROARCHIVE Mon Oct 31 15:59:17 +0000 1994 remote from internet Message-Version: 2 } To: rratleng!internet!rratleng1!rratlengp0!allsman by with SMTP ID 0r1z8f.11yLRI; Mon, 31 Oct 1994 15:59:17 +0000 MTS-Message-ID: {internet3041559470} UA-Content-ID: {941031110037.712-at-AAEM.AMC.ANL.GOV} End-of-Header: EMail-Version: 2
Hello everybody, I am a regular reader of all the mails that are in Microscopy. I really admired people willing to help each other. Now I have a problem I need some input from all of you experienced guys out there. I am working on mechanically alloyed materials and it has become really hard to make samples out of it on quick basis It usually takes a long time to embed it in epoxy and mechanically polish and then Ion mill.
Now we have decided to explore a different path and are trying to press the powder in a die till about 30000lbf. We have started to work on Copper so we can optimize the technique and use it for other materials as well. I am experiencing difficulties keeping the samples powder in tact and it keep breaking on me when I try to cut the sample out of pressed material. If somebody has worked on it or has some knowledge about the process I would really appreciate any help.
Since your building plans are not final, now is the time to make the appropriate measurements for magnetic fields. To do this job thoroughly, you should lay out the proposed areas in a grid and then measure the horizontal magnetic fields in 3-dimensions. This procedure will enable you to site your electron columns properly or, if the fields are too large everywhere within the grid, take preventative action before the building plans are approved and the contracts are signed.
Could someone teach me how to convert the unit mV (millivolt) to mG (milligauss)? Recently we asked one service engineer to survey our new EPMA/SEM lab. He wrote down the readings of AC magnetic fields in mV.
Thanks in advance.
Long Liang Electron Microprobe and SEM Lab ARCO Exploration and Production Technology 2300 West Plano Parkway Plano, TX 75075 E-mail : LLIANG-at-is.Arco.COM
} Date sent: Fri, 28 Oct 94 10:05:55 EDT } From: dfalcon-at-VNET.IBM.COM } To: microscopy-at-aaem.amc.anl.gov } Subject: EMSCOPE address
} I have an EMSCOPE model SC650 large sample sputter coater that needs } some repair parts. The phone number for the manufacturer in England that is } on the paperwork that came with the unit is no longer a valid number. Does } anyone know if the company (EMSCOPE LABORATORIES LTD--KENT,ENGLAND) is still } in existence and what their present number is---and/or any North American } representatives?? I suspect they may have been bought by another company } and may have a new name and number now. } Thanks, } Doug
Doug
Your suspicion is correct and EMSCOPE no longer exists, they were bought out some years ago by Biorad (Polaron) who later sold most of their microscopy business in the UK (and elswhere) to VG Technologies and Fisons. They may still suport some of the old EMSCOPE equipment. Another alternative is to contact a firm called EMITECH which was founded by some ex members of the EMSCOPE staff and they may be able to help you with your problems.
US address: EMITECH INC, 3845 FM, 1960-West, Suite 345, Houston, Texas 77068. Fax 713-893-8443. Phone 713-893-2067 or 800-444-3137 toll free for sales and service.
UK address: 11 Enterprise Centre, Newtown Road, Ashford, Kent, TN24 0PD. Fax 0233 640744 Phone 0233 646332
I have no connection to any of the above firms.
Ian
Ian
Ian Hallett HortResearch Mt Albert Research Centre Private Bag 92 169 Auckland, New Zealand Fax 64-9-815 4201 Telephone 64-9-849 3660
I've checked mine with a narrow-range paper--seems to be pretty close, about 11.8 or so. I tried adjusting it, but saw no difference in staining. Grace Kennedy, UCSD
Hello Glenn, We had a fearful problem with 150Hz (3 phase) VLFI in our laboratory. Partly due to the fact that the laboratory was located adjacent to a major power transformer substation for the university. I spent some years working on the problem trying various solutions. First I got a search coil and plotted the fields. There is a commercial one made here called a "Gauss Maus" You can get them cheaply now there is paranoia about health effects of VLFI. A big surprise was that that the radiation was maximal up against the wall of the substation but it did not fall away as the square of the distance inside our buiding. Instead it was high near the walls, vertical columns and cross bearers in our steel framed concrete building. We did an experiment turning power off to all sections of the faculty a bit at a time and found that field was proportional to total power use and not to any one section of the building which would have implicated particular cabling runs. My conclusion was that the field was being induced in the frame of the substation which was welded into the frame of the main building so they were magnetically coupled. Then the frame of the whole building and every bit of reinforcing joined to the frame had the field induced in it. I found almost the same field throughout the building no matter how far from the substation. Of course you can also get local fields generated in heavy cables but A. live and return cables are usually routed side by side and the fields cancel each other B. the field falls off with square of distance so unless the cable goes right over your column the field is negligible. You can also get fields induced in airconditioning ducts water pipes etc. and you might need to insert non metallic sections to isolate sections in the e.m. lab. A local firm RFI Industries 54 Holloway Drive Bayswater Melbourne VIC 3153 Australia built a very effective fully screened room for the E.M. in a nearby hospital. They got good attenuation using quite cheap sheet metal and not the really pricey magnetic shields like hipernoom. By the way many engineers imagine it only takes chicken wire to shield a room. This is OK for radio frequencies but entirely useless for 50/60 Hz. We tried local shielding of the column but it made no difference. All columns have shielding built in already. And unless you make a full metal box which completely surrounds the column you really cant keep VLFI out. It just sneaks in the holes you have to leave to work the controls! We tried using a cancelling field which worked fine in trials but couldn't cope in the microscope itself. AC VLFI fields are complex multi directional 3D interference patterns and to compensate them fully you need compensating fields on 3 axes. Also the sensor detecting the field to generate the reverse phase feedback needs to be as close as possible to the volume you wish to be compensated. Since the microscope itself generates fields you will pick up e.g. the SEM scan signals and fed them back so as to neutralise them. There is a book in the Series " Practical Methods in Electron Microscopy" Ed. Audrey Glauert. Volume 4, "Design of the Electron Microscope Laboratory" by R.H. Alderson. North-Holland Publishing Co., American Elsevier, N.Y. ISBN 0-444-10807 6 PUBLISHED 1975. Which deals with this and other problems in a very practical way. In the end we solved our problem by moving to a low field building. Hope you manage better! Mel Dickson.
Having processed some frog skin for TEM, looking at the thick sections I noticed the tissue looks like small balls. Can anyone tell me what this might be? Looking at normal skin such as human tissue this looks completely different. Is there a good book that might describe what these structures are? TIA, Phil 8-{)
We routinly use 4% paraformaldehyde, 2% glutaraldehyde in 0.2M sodium cacodylate buffer at pH 7.2. We probably need a mininium of 500 ml of fixative to perfuse the rat. After perfusion, store whole organs in 3% glut or dice up tissue and store in buffer. Good Luck.
We are in the process of having installed a new JEOL TEM and the service engineer suggested we use an "on-demand" gas regulator to control the dry nitrogen vent gas for the column. This type of regulator allows the pressure in the column to reach 1 atmosphere but no more to prevent over pressuring the column and blowing out vacuum seals, thin windows, etc. I know I have seen such regulators but I cannot find a source. If anyone is using this type of regulator could you please supply a model number and vendor.
Thanks in advance.
Norman Elliott | E-mail: nee-at-lanl.gov Los Alamos National Lab | Fax: 505-665-2104 MST-7 MS E549 | Voice: 505-667-1587 Los Alamos, NM 87545 |
We could not find an inexpensive regulator from gas supply houses, etc. so we use regulators designed for scuba tanks. Still not cheap, but less expensive than the alternaives we found. They have worked well without problem for about 8 years now.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Tue, 1 Nov 1994, Norman Elliott wrote:
} Dear listreaders, } } We are in the process of having installed a new JEOL TEM and the } service engineer suggested we use an "on-demand" gas regulator to control } the dry nitrogen vent gas for the column. This type of regulator allows } the pressure in the column to reach 1 atmosphere but no more to prevent } over pressuring the column and blowing out vacuum seals, thin windows, etc. } I know I have seen such regulators but I cannot find a source. If anyone } is using this type of regulator could you please supply a model number and } vendor. } } } Thanks in advance. } } } } } Norman Elliott | E-mail: nee-at-lanl.gov } Los Alamos National Lab | Fax: 505-665-2104 } MST-7 MS E549 | Voice: 505-667-1587 } Los Alamos, NM 87545 | } } }
I recently replaced the Wehnelt aperture in my Philips CM12 electron gun. The original aperture had a hole diameter of 0.6 mm and I replaced it with one that has a 0.8 mm dia. A 0.4 mm hole is also available in my column kit.
I know from my experience on an SEM that increasing the hole diameter will increase the beam current output from the gun (which I did to get more counts for x-ray microanalysis).
What else is affected by the hole size - resolution, coherence, or what? How should one decide what diameter to use for this aperture on an SEM or a TEM?
--
Gib Ahlstrand, MMS Newsletter Editor Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.crl.umn.edu
Reply... RE} EM shielding/ M. Rich Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
Opinions and experiences here are my own.
We have a field problem in our JEOL 4000EX room. Before all the microscopes were installed the JEOL engineers did a field survey in each room. All was fine. They then installed a 2000FX in one room and got it up and running and then in the adjacent room installed the 4000. Now both reached spec without problem. But, as many of you may know, JEOL had some fearful problems with the guns of the 4000 series. We had really bad filament image flickering and it never seemed to be totally cured (in spite of extensive glow discharging, oops! I mean conditioning). Tracing the problem and trying to blame everything except the gun, lead our engineers (and there were several groups of them!) to test for fields and they notice that there was a substantial field about 5 feet from the scope in the floor. It was below the required level at the objective lens, but that didn't stop them from mentioning it (many times). Turns out that the supply for the 2000 ran under the floor of the 4000 room and the phases were sufficiently unbalanced to produce this field. Now, the 4000 scope had been installed after the 2000 was up and running and so we had taken the resolution tests with that field present and so we didn't think it was a problem. This did not prevent the engineers trying to say that our gun problem was related to that field. The alternative at this point was to admit that the gun was bad and replace it. After a good deal of discussion the gun was replaced. By this time the scope had been down so long that I had asked JEOL Service for a rebate on my service contract since they had not maintained the scope in operating condition for a reasonable portion of the period covered by the contract. The moral of this story is. When you build your scope facility, make sure that you specify where everything goes. And I mean everything, water, power, ground lines, light locations (fluorescent lights can interfere with the scopes, the chokes have quite a field)., everything. If we hadn't had the field in the floor we would not have had to argue about it! It's easier if you don't build in problems for yourself! Don't let the builders or the bean counters change the plans after you have approved them. Our plans were amended and we now have building air instead of our own system and I always have toasting microscopes in the fall and spring when the A/C is in the process of being turned on or off! As I say just my opinion and experiences. I am not trying to get at JEOL, it is just that it took a long time to convince them that it was their gun and not our facility.
The following is an abbreviated version of an offline email dialogue between myself and Paul Nolan of Queen's University regarding his "LaB6 Fix" question. Paul, Nestor and I agreed that this would be a good dialogue to share with the listserver. If anyone has any questions regarding these issues, feel free to contact me.
Best regards, Damon L. Heer. ***************************************************************
I'VE GOT A LaB6 THAT STOPPED EMITING. LOOKS GOOD UNDER THE SEM EXCEPT FOR THE TUNGSTEN BALLS ALL OVER IT! GOING TO TRY TO CLEAN IT IN MY CARBON EVAPORATOR. THINK IT WILL WORK? ANY OTHER IDEAS? (CAN'T AFFORD A NEW ONE)
THANKS, PAUL ----------------------------------------------------- Dear Paul:
You noted that it looks good in the SEM. Do you mean that the crystal still appears to have a reasonable emitting surface remaining? how long did it run before it stopped emitting?
You also noted a build up of tungsten balls on it. Is this build up on the mount or the crystal? I've never seen this kind of contamination on a tip. this is because I mainly deal with our LaB6 cathodes which don't have tungsten in the structure. However, if you are using a Denka cathode, the cement that is used to keep the crystal in the mount may be causing this contamination.
To clean it, we sometimes rub the crystal with a q-tip with a little isopropyl alcohol. If this doesn't remove it, try lightly rubbing it with the wooden stick portion of the q-tip, again with IPA. This basically applies a rather non-abrasive scrub to the crystal. The difficulty here is that both Denka's and Kimball's LaB6 mounts are very fragile and may break under any contact.
Unfortunately, Ed Calomeni is very correct in saying that your best bet is buying a new one.
Good luck! Damon L. Heer ------------------------------------------------------ Damon. There seemed to be no structural damage to the filament ie cracks or defects but the tungsten balls cover the whole filament.
The filament has been in our TEM for about 2 years but has not had too many hours of use . It is a Kimball filament so i dont know if there is any W in the mount or not. I will give it a wipe and look at it in the sem again and see what has happened. In your opinion is this W going to stop the crystal from emitting? (There appears to be no other damage and there is continuity between the mounting posts) ...
Cheers, Paul ------------------------------------- Hi Paul,
In my opinion, if there is no contamination on the cone and flat of the crystal, it should work fine. When an electron microscope is saturated properly, the emission only comes from the flat area.
You noted that the cathode stopped emitting while in use. The reason this usually happens is the emitting flat at the tip of the crystal cone has evaporated to a point. If there still is a flat left on the emitter, I imagine that the flat did become contaminated, but is unnoticeable in the SEM.
I know that a large amount of time is necessary for changing tips in a TEM, so the following recommendation should be weighed against that.
If there remains a reasonable amount of the flat on the crystal, and the W can be cleaned away from the crystal cone and flat, it may be possible to drive off any remaining contamination by operating the cathode at about 0.2 amps above the normal operating filament current for a short period of time. This may recover the cathode.
You mentioned that the flat on the filament was the important part to have clean, well it seems there is no definite flat part on the tip. I was unaware because of my inexeperience with LaB6 that there should be a flat, i assumed it should be pointed (like mine seem to be) Is this filament dead?
cheers paul ----------------------------------- Hi Paul,
Kimball, as well as FEI, uses a "truncated tip" for LaB6 emission. The truncated tip consists of a cone with a flat top that is normal to the optical axis. The "flat" is oriented with the {100} crystal plane, which has been shown to be the best orientation for electron microscope applications. The cone angle and the flat diameter are chosen by the type of application it will be used for. FEI's standard cone angles and flat diameters (and I believe the same goes for Kimball) are as follows:
SEM and Low Res TEM 90 degree full cone angle, 16 micron flat diameter
Hi Res TEM 60 degree full cone angle, 5 micron flat
When the flat is gone, the emission comes from the side of the cone, which isn't the {100} plane and is not normal to the optical axis. The result is poor emission down the column.
If there is no flat left on your tip, I'd say it's dead.
Coincidentally, a colleague and I just wrote an article for our semi-annual newsletter, the FEI Focus, regarding crystal tip issues. If you wish, send me your address and I could send you a copy of this. A reprint also appears in the most recent issue of Microscopy Today.
Cheers, Damon
FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830
Last year, we installed a JEOL JEM 1210 and were advised to purchase the demand flow regulator for the dry nitrogen. We ended up with a Matheson Model 3421 vacuum regulator which meets JEOL's specs...in fact its one that they recommend. Its pricey at about $400, and is available from any of the industrial gas suppliers, ie AIRCO, etc. However, its maximum inlet pressure is 50 psi, meaning that you can't couple it directly to 3000 psi nitrogen tank. You still must buy a general purpose, dual stage nitrogen regulator, then attach the demand flow regulator to this. The whole setup will end up costing about $600. Incidentally, if you are installing a TEM, then you know that you will have to buy a cylinder of sulfur hexaflouride and a regulator for this. A 100 lb bottle of sulfur hex is about $400, but you can use a nitrogen regulator for its installation if you buy a $15 adaptor. I can supply this information if you need it. Good Luck.
-=buddy=-
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
I have someone interested in the fracture surfaces of choclate. I want to use some type of replication process, as I don't want to put chocolate in my SEM. No, I don't want ESEM, or cryoSEM. I am worried that the solvents of formvar or similar plastics might distort the fracture surface. I need low mag shots, so high resolution replicas are not necessary. thanks in advance, Randy Nessler rnessler-at-emiris.iaf.uiowa.edu
I deleted the original post on this subject, but from what I remember, I think the original poster was looking for a pressure relief valve that tripped at 1 atm, instead of a regulator that would supply 1 atm. We used to have a little pop-off valve that came with a thin window x-ray detector. It was supposed to be installed in the line that bleeds the chamber, so as to prevent a positive pressure from forming in the SEM specimen chamber. A positive pressure ran the risk of rupturing the window. You might call Kevex/Fisons, since this is who supplied the pop-off valve for us. Randy Nessler rnessler-at-emiris.iaf.uiowa.edu
Can anyone point me in the right direction for techniques for making silicon monoxide and aluminum oxide films. A discussion of the resultant surface prop- erties would also be helpful, if such info is written down someplace.
Dear Gib, The wehnelt aperture size and the fields produced in and around the filament and wehnelt determine the locus of points from which electrons emitted by the filament end up being accelerated and sent down the column. The larger the aperture size, 1) the more electrons go down the column, 2) the less cohe- rent the beam, 3) the larger the crossover size, and 4) the more the anode is heated by electrons extracted at angles significantly different from 0 degrees. The best wehnelt aperture size depends on what the instrument is used for. The largest possible size is that for which anode heating becomes a pro- blem. For people like me who do low-dose work and diffraction and whose EDX detector gives high dead time for more intense beams, a smaller aperture is better; for those who are fighting to increase intensities all the time, larger is better. Yours, Bill Tivol
In article nee-at-lanl.gov (Norman Elliott) writes: } } Dear listreaders,
} We are in the process of having installed a new JEOL TEM and the } service engineer suggested we use an "on-demand" gas regulator to control } the dry nitrogen vent gas for the column. This type of regulator allows } the pressure in the column to reach 1 atmosphere but no more to prevent } over pressuring the column and blowing out vacuum seals, thin windows, etc. } I know I have seen such regulators but I cannot find a source.
} Hi Norman. The regulator you need is available at a cost of about $200 from dive shops selling scuba gear. They are just breathing demand valves which allow gas to flow when you suck on the valve but not otherwise. Its important to always have gas available on the pressure side or the microscope vacuum will suck the valve diaphragm to destruction! We have such a system feeding our SEM from the bled off gas in out 175Litre Liquid nitrogen tank, which is the driest gas you can get. If you have a UTW window on your EDS you might install a relief valve on the line so pressure in the microscope won't ever go over atmospheric.
Sulfur hexafluoride is the insulating gas used in high voltage tanks. In the early days, all tanks were filled with dielectric oils, and some of them contained PCB, forcing the manufacturers to find another type oil. As accelerating voltages increased, the manufacturers switched to Freon as a superior insulatant. Of course recently, the EPA has banned the use of most Freons, so now most manufacturers have switched to sulfur hex., which is supposed to be completely inert. The HV tanks don't come pressurized with it, so you have to supply it for the service engineer. As I understand it, SEMs with their lower accelerating voltages still have oil in the tanks.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
} I recently replaced the Wehnelt aperture in my Philips CM12 electron gun. The } original aperture had a hole diameter of 0.6 mm and I replaced it with one that } has a 0.8 mm dia. A 0.4 mm hole is also available in my column kit. } } I know from my experience on an SEM that increasing the hole diameter will } increase the beam current output from the gun (which I did to get more counts } for x-ray microanalysis). } } What else is affected by the hole size - resolution, coherence, or what? How } should one decide what diameter to use for this aperture on an SEM or a TEM? } } -- } } Gib Ahlstrand, MMS Newsletter Editor } Electron Optical Facility, University of Minnesota, Dept. Plant Pathology } 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 } 612-625-9728 FAX, giba-at-puccini.crl.umn.edu } } We also have a CM12 with LaB6 filament. The general "rule of thumb" is to multiply the Wehnelt distance, ie. distance you screw back the filament into the anode, by 2 1/2 to obtain the Wehnelt aperature size you require. I back off the filament by 200 um. Therefore I require a 500 um. aperature. The lifetime of the filament is just under 1000 hrs.
Hope this gives you some assistance.
Fred Pearson Electron Optics Facility Institute for Materials Research McMaster University Hamilton, Ontario voice (905) 525-9140 x24609 fax (905) 521-2773 eoptics-at-mcmail.cis.mcmaster.ca
} I have someone interested in the fracture surfaces of choclate. I } want to use some type of replication process, as I don't want to put } chocolate in my SEM. No, I don't want ESEM, or cryoSEM. I am worried } that the solvents of formvar or similar plastics might distort the } fracture surface. I need low mag shots, so high resolution replicas are } not necessary. } thanks in advance, } Randy Nessler } rnessler-at-emiris.iaf.uiowa.edu
What will most probably work in your case is one of the dental replicating silicones. These are formulated to make quick, accurate replicas of tooth surfaces and are used in the preparation of ceramic (and other) crownwork. Any dentist should be able to help. We have used these silicones in replicating leaf surfaces, as well as human skin, and the resolution is OK up to at least 1000x mag. There are other (cheaper) types of dental impression materials available, but these water-based formulations give low resolution and are of course not suitable for direct exposure to the SEM vacuum. Have fun.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Message-ID: {MAILQUEUE-101.941102083619.1088-at-anat.uct.ac.za} To: microscopy-at-aaem.amc.anl.gov
In answer to Phil's question regarding frog skin:
What area of the skin are you looking at? If it is only the epidermis, the 'balls' are probably the epidermal cells which have shrunk (a common occurence) during fixation. If it is dermal then you may well be looking at glands situated in the dermal region. If the balls are within the cells then the possibility is that they are the frog equivelant of melanin.
I am unaware of any decent book on frog skin per se. I do have some references which you may find useful if you wish to have them.
O.k., I'll ask for us ignorant folks. Why is a tank of sulfur hexaflouride needed for the installation of a new TEM as was mentioned in responding to the low cost regulator series?
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
I am interested to get into contact with those who use an Astromed liquid nitrogen CCD camera, type 3200, on top of any type of microscope to quantify fluorescence images. I would like to exchange experience and test images.
Please mail me directly at : vanderwulp-at-mbl.tno.nl
Hope to hear from you.
PS. Does anybody know of a mailing list used by astronomers because this equipment is frequently used by them.
Thanks for any comment. -- Kees van der Wulp TNO - Nutrition & Food Research Institute INTERNET : vanderwulp-at-mbl.tno.nl Division of Toxicology VOICE : +31 15 843101 PO-Box 5815 FAX : +31 15 843989 2280 HV RIJSWIJK (NL) THE NETHERLANDS
Reply... RE} Sulfur hexaflouride? Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
On a realted note, how are people managing the switch from Freon to SF6? According to our JEOL Service engineers, the upgrade of our 4000 to SF6 is a freebie as part of our maintenance contract, but they want to stiff us for $20K to upgrade the 2000FX. I have been hoarding a few large cylinders of freon to make sure we can continue to run and service our 2000 for a number of years with freon. What are other folks doing? Jfm.
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O.k., I'll ask for us ignorant folks. Why is a tank of sulfur hexaflouride needed for the installation of a new TEM as was mentioned in responding to the low cost regulator series?
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
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Our JEOL 4000 is affected by magnetic fields due to any TV monitor in the room that is not very new and highly shielded. This includes Mac monitors and the TV monitor for our TV rate camera.
The filament appears to flicker at 3-5 Hz and we are unable to measure a magnetic field at that frequency even near the monitor let alone the microscope! We do measure the 50-60 Hz field at the monitor and the stronger this field the larger the effect on the beam. The effect is greater the closer the monitor is to the scope and the closer to the objective lens.
For best images we turn off the monitors before exposing film. There is an obvious difference!! For some seeing was believing.
Roseann Csencsits Argonne National Laboratory Argonne, Illinois 60439 USA 708-252-4977
this is a reposting- our mail program has not been sending out all correspondence and that which it sends out has had the wrong return address. Sorry for cluttering cyberspace if you've seen this before.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
---------- Forwarded message ----------
We could not find an inexpensive regulator from gas supply houses, etc. so we use regulators designed for scuba tanks. Still not cheap, but less expensive than the alternaives we found. They have worked well without problem for about 8 years now.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Tue, 1 Nov 1994, Norman Elliott wrote:
} Dear listreaders, } } We are in the process of having installed a new JEOL TEM and the } service engineer suggested we use an "on-demand" gas regulator to control } the dry nitrogen vent gas for the column. This type of regulator allows } the pressure in the column to reach 1 atmosphere but no more to prevent } over pressuring the column and blowing out vacuum seals, thin windows, etc. } I know I have seen such regulators but I cannot find a source. If anyone } is using this type of regulator could you please supply a model number and } vendor. } } } Thanks in advance. } } } } } Norman Elliott | E-mail: nee-at-lanl.gov } Los Alamos National Lab | Fax: 505-665-2104 } MST-7 MS E549 | Voice: 505-667-1587 } Los Alamos, NM 87545 | } } }
Our lab is considering building, or purchasing (new or used) an Optical Diffractometer. Does anyone have a list of suppliers, a used instrument, or wishes to share their design? Comments on manufactured models are welcomed. Email directly if you wish to avoid biased opinions.
Thanks Fred Pearson fax (905) 521-2773 McMaster University Hamilton Ontario, Canada eoptics-at-mcmail.cis.mcmaster.ca
I need advice, recommendations, information about video microscopy systems; I want to buy a system to use in my undergraduate classes (gen. zool., invert. zool.) to observe small, live animals. I especially need specific brands, models that others have used successfully in similar situations. The students themselves will be using the system and making and editing videos--so the equipment needs to be fairly sturdy yet enable good quality images. At this time I have price quotes from a number of sources (Carolina Biological, Fisher, Nikon, Olympus) but of course each salesperson recommends his/her system! Thanks for any advice. P.M. Biesiot Dept. Biol. Sci. Univ. Southern Mississippi Hattiesburg, MS 39406-5018 pbiesiot-at-whale.st.usm.edu
} } O.k., I'll ask for us ignorant folks. Why is a tank of sulfur } hexaflouride needed for the installation of a new TEM as was } mentioned in responding to the low cost regulator series? } SF6 is used in only some new TEMs. Hitachi uses SF6 in the guns of all TEMs that go to 200KV and over. And as of yet only in the HV tanks of the FE-TEMs. Its used as an insulator, instead of oil or Freon. Cal-Hitachi service
For years we used a basic optical bench with a low power laser and lenses we scrounged from microscopes and enlargers and cameras. The best idea is to buy two 2 meter benches and use an optically flat mirror (astronomy supply house stock) to bend the pattern back so you can view the pattern as you move the negative around. The best viewing/recording device is an old 4x5 camera of some sort. You don't even need one with a working shutter or light tight bellows as you will be using the diffractometer in the dark. You can then easily use polaroid 45 materials for hard copy of the patterns. BUT you will have to be cautious about viewing the pattern and always e.g. have the zero order beam blocked with a metal patch. I have the scars to show how careful you need to be.
email me for further information. Mel Dickson } Thanks } Fred Pearson} fax (905) 521-2773} McMaster University} Hamilton Ontario, Canada} eoptics-at-mcmail.cis.mcmaster.ca
The "small balls" structures might be mucous glands. See fig. 5 in Hauser F et al 1990. Expression of spasmolysin (FIM-A.1): an integumentary mucin from Xenopus laevis. Exp Cell Res 189: 157-162 - it shows a phase constrast micrograph of Xenopus skin with mucous glands that might be interpreted as "small balls".
On Tue, 1 Nov 1994, rutledge phil wrote:
} Having processed some frog skin for TEM, looking at the thick sections I } noticed the tissue looks like small balls. Can anyone tell me what this } might be? Looking at normal skin such as human tissue this looks completely } different. Is there a good book that might describe what these structures } are? } TIA, } Phil } 8-{) }
Hello, I have been approached by a first grade teacher at Pond Cove Elementary School who wants to instill an early love for microscopy in the children. Are there any free or low-cost dissecting microscopes out there?? Thanks, Ada Ada Olins Univ. Tennessee-Oak Ridge GSBMS Biology Division, ORNL P.O. Box 2009 Oak Ridge, TN 37831-8077
Michel et al., I have used methylamine tungstate quite successfully for seing some very fine filaments on the surface of tooth decay bacteria. I follow Wyatt et al., 1988. Oral Microbiol Immunol 3:162-168. If this journal is not widely available outside dental schools just send a psotal address or fax # and I can get a copy to you.
Being a scientist doesn't insure good spelling, any more than being in a company insures courtesy.
A. K. Christensen
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On Thu, 3 Nov 1994, John Mardinly, 5-2346, Page 322-6490, SC2-24 wrote:
} Folks; } What I want to know is why there are so many of you out there claiming } to be scientists yet can't get the spelling of fluoride any better than } "flouride"? } } John Mardinly } Intel MATTEC }
A few weeks ago a person posted a note about having a SuperCard stack about SEM's and offered the FTP site for it........I replyed to him and asked for the site name but never got any reply.....could somebody help me out? It would be great for teaching and training.....
Thanks
--
E.Todd Voiles *** Facility Manager Central Facility for Electron Microscopy University of Nebraska tvoiles-at-unlinfo.unl.edu
Our Hitachi has a "branch" in the vent line with a pressure relief valve which then allows us to use a standard regulator - would that be a suitable solution to the problem?
We have used a Polaron optical diffractometer for several years. It came as a complete package: bench, lenses, laser, holder for negatives, etc. The addresses were:
Polaron Equipment Ltd. 53-63 Greenhill Crescent Watford Business Park Watford Hertfordshire WD1 8QS
Polaron Instruments, Inc. 2293 Amber Drive Line Lexington Industrial Park Hatfield, PA 19440 (215)822-2665
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
Good Friday Morning, Ada Olins (Univ. of TN) asks for (inexpensive) microscope sources suitable for young students. In this broad area, I would like to introduce the "DiscoveryScope" to readers. It is "an inexpensive, exceptionally durable viewing system for exploring small things. It's 1-inch focal length lens produces beautiful wide field, high resolution views of the small invertebratres, tiny plants, protist, and inorganic subjects. Special chamber and holders are provided for viewing both terrestrail and aquatic organisms. Not just for beginners, DiscoveryScope makes a useful "spotting scope" for any microscopist who collects samples from the field, particularly if macro photography is intended." Discovery Scope Inc PO Box 607 Green Valley, AZ 85622 Tel/Fax: (800)398-5404 I have no financial interest in this product (unfortunately) - and but submit that it is a nifty device! Don Grimes, Microscopy Today
Microscopy & Microanalysis Public Domain Software Library site in a few weeks. Brendon Griffin will be visiting ANL in November and will be bringing a copy along. The FTP site is:
WWW.AMC.ANL.GOV
It currently hosts the EMMPDL, MASLIB, Test Images, and PDL Software all for Microscopy&Microanalysis.
Your Friendly Neighborhood SysOp (who can sometimes spell)
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 14:24
Date:11/4/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
I currently have a working copy that Brendon sent me and I will make it available when he gives me the OK. Warning, it is an 11 megabyte file and requires at least 5 megabytes to run. It runs only on a Mac.
Does anybody have a suggestion for a shutter for a Nikon Diaphot light source? We're looking for the following requirements: - easy to mount on upper (halogen) or lower (mercury) lamp input - computer controlled via NIH-Image (or IP-Lab Spectrum) - cheap!
The two main applications are: - time lapse w/ fluorescence - collecting random fields w/ fluorescence
At this point we don't need selectable filters, just a shutter, although we wouldn't mind getting various filter positions as part of the deal.
anybody know an FTP site with an MSDOS program for displaying TIFF files? Archie and veronica turn up lots of pictures but no programs.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
Jay Jerome asked about TIF viewers for PC machines...
There are currently NO TIFF viewers in the ANL Library for PC's. but the ANL FTP site has GIF viewers for PC machines (both DoS and WinDoze) the are in the following path:
ANLAEM Software Library ANL Shareware Library Freeware Shareware PC Viewers
The site address is: WWW.AMC.ANL.GOV The programs are called: VGIF.exe, VUGIF.exe, WINGIF.exe, CSHOW.exe
The PC commerical program PhotoStyler certainly opens TIFF files, and so do a lot of other commerical programs (PhotoShop etc...).
As a note of warning. Some PC TIFF reading programs will not read TIFF files that were created on a MacIntosh. Most Mac programs that I've used, on the other hand, are intelligent enough to recognize the differences and translate appropriately. Also not all TIFF readers can handle the full TIFF specification. Many are coded assuming that 8 bit gray scale and/or 24bit (8x3) color is the end product. If you have 12,16 ... bit scientific data many (but not all) will not display properly. Make sure you take this into account if your looking at something other than photographic quality images.
There are also the usual suite of Translator programs which will convert between the different image formats. Those programs are usually cheaper than the full blown image processing/editing programs. By purchasing one of these translator programs you could then use the Shareware Viewers....
We have been using a 286 PC-based Kontron IBAS-II with IBAS 2.0 software for image analysis for several years with only middling success.
We am now assisting some researchers with semi-quantitative analysis of the silver grains from their in situ hybridization studies. They are interested in purchasing a Macintosh system to run the NIHIMAGE program you have developed. Their interest primarily stems from the following paper: Lucas, L.R., Mize, R.R. and Harlan, R.E. Semiquantitative analysis of in-situ hybridization results using IMAGE software: a rapid method for counting reduced silver grains over mRNA-positive cells. J. Neuroscience Methods 52:101-109 (1994). They have asked us to help them determine what computer and video hardware they need to set up their system.
Is anyone willing to suggest the required hardware for an adequate Mac system to run NIHIMAGE? We want neither a Volkswagen nor a Cadillac, just a good level system with a Macintosh they can also use for other purposes. Do you recommend a Quadra or a PowerPC? Which model? We assume that 8 MB RAM is minimum and 16 MB is better. Do you have any particular video camera and frame grabber your recommend? (We currently have a DAGE MTI CCD72 video camera). Our computer network manager has a nubus VideoSpigot frame grabber available if it would be of use. We assume it is best to run the program using two monitors. Any recommendations for the video monitor?
We've noticed from reading some of the information on the microscopy usenet newsgroup that a PowerPC version of NIHIMAGE is being developed. Is the PowerPC version tested enough for a "novice" user? We have about 5 years of image analysis experience on the IBAS system so we know much of the terminology and the steps involved in image analysis. We just son't have a clue about the Mac...
Any assistance you can provide would be greatly appreciated.
Aloha, Tina Weatherby Carvalho Marilyn F. Dunlap Biological EM Facility Pacific Biomedical Research Center University of Hawaii at Manoa 1993 East-West Road Honolulu, HI 96822 (808)956-6151 or (808)956-6251 or FAX (808)956-4768 e-mail: dunlap-at-ahi.pbrc.hawaii.edu tina-at-ahi.pbrc.hawaii.edu
} } { {Michael Rich, rich-at-egr.msu.edu } { {Michigan State University asks about cheap magnetic shielding.. Michael, there is no such thing because of the physics of magnetic fields. Radio frequency interference CAN be excluded by making a Faraday cage - a complete box - using copper mesh or even fine chicken wire mesh. BUT to excludelow frequency magnetic fields you must use solid sheets of some ferromagnetic material. Any gaps between sheets must be covered over by strips of similar material. To entirely shield a room the six faces of the room must have the shield applied (yes that includes the floor and ceiling). The material must either be a high permeability material like the alloy hipernom or it may be ordinary steel sheet but much thicker. To shield a complete room costs $40 - 50,000. Much of the cost lies in the fact that the construction should be done just right to obtain best shielding. Try looking up the yellow pages for a shielding supplier. I have an old address for one: Amuneal Manufacturing Corp, 4737 Darrah St. Philadelphia Pa 19124.
Recently I have been using PVP (polyvinylpyrrolidone) in various techniques but to date I have had no luck. Firstly I have tried PVP with LR Gold resin (as recommended by the LR technical blurb), I have also tried adding it my fixative to increase osmotic pressure (as recommended in the paper I have been following to localise the enzyme Thiamine Pyrophosphatase) and finally I have tried it when infusing tissue with cryoprotectant for cryoultramicrotomy (as recommended by Tokuyasu)
In these papers they talk of "dissolving" the PVP in the various solutions but my PVP will not dissolve!!! I am left with a fine white suspension which quickly settles out when stirring is stopped.
Question: Should the PVP dissolve (and perhaps leave a clear solution) or is the fine suspension I have normal? (It blocks up canulas when perfusing, it makes it difficult to see the tissue when processing).
Note: The Merck index states that PVP is soluble in alcohols and water - have I got the wrong one?
Many thanks in anticipation.
Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
} anybody know an FTP site with an MSDOS program for displaying TIFF } files? Archie and veronica turn up lots of pictures but no programs.
Jay, Try Graphic Workshop (gws70c.zip), which is located for example at the ftp.uni-mannheim.de (134.155.50.51): /disk2/systems/msdos/graphics
Regards,
Petr +-------------------------------------------+-------------------------+ | Dr. Petr Schauer | tel.: (+42 5) 41321246 | | Head of Electron Microscopy Laboratory | fax : (+42 5) 41211168 | | ACADEMY OF SCIENCES OF THE CZECH REPUBLIC | E-mail: petr-at-isibrno.cz | | INSTITUTE OF SCIENTIFIC INSTRUMENTS | petr-at-mrkev.vabo.cz | | Kralovopolska 147, CZ-612 64 Brno | | | Czech Republic | | +-------------------------------------------+-------------------------+
We used tons of sulfur hexafluoride when I supervised a lab testing image intensifiers. We would flood bare tubes with it, so breathed a lot of it. It is non-toxic, and can only make you pass out if it excludes the oxygen. Sulfur hex actually quenches sparks as it decomposes in the arc. As for fluorine remaining in the tank this is highly unlikely, since it would react with anything it came into contact with.
As for spelling, I have come to the rule of thumb for fluorine, fluorescence etc. that if it spells flour it is wrong. I still have fond feelings for a professor who gently pointed this out to me.
The balls seen might be 'membrane blisters' as written up in scanning vol 1 p 166-173 1978 E. Shelton & Mowczko the basic idea is that OsO4 sometimes creates '? blebs 'like little balloons on the surface of cells, these were up to 6 microns long but mainly 1-2 microns or sometimes smaller *(they say .2 to .6) in a variety of cells and conclude that glutaraldehyde a lone is best foir fixation. Since many people have not found this, it may only be certain tissues that do this in most people's routine handling of fixation. Alan Pooley Marine science rutgers nj
Concerning spelling errors etc (flouride vs fluoride etc) may I suggest to all the gentlemen and lady scientists that we quietly suggest corrections of errors ti individuals and avoid blaring them over the net. ( I expect an individual correction for my mistyping of 'to' in the previous l Alan Pooley marine sci sem lab rutgers nj
Hey folks: A reply to Allen Mitchell -- I think you're problem might lie in the molecular weight of the PVP you're using, or the temperature you're trying to dissolve it at, or both. We routinly use PVP with a m.w. of 40,000 (Sigma cat. PVP-40) in fixatives for in situ perfusion of rat livers -- 2% w/v takes a while (several minutes) to go into sol'n, but it does go in. I also make a Tokuyasu-style infiltration medium for cryosectioning -- that uses PVP m.w. 10,000 (Sigma PVP-10) but it is dissolved into NaCO3-phospate (100g of PVP into 30ml of liquid!) and must be done by alternating between a 60C oven and a sonicator full of hot water. Feel free to E-mail me (gmartin-at-welchlink.welch.jhu.edu) for particulars of preparing this stuff (it's a real witch's brew). I can't speak as to the particulars of PVP added to LRGold -- I've always used LRGold straight-up -- but I would advise against putting you're specimens into a sol'n cloudy with undissolved PVP.
Take Care, Greg Martin Dept Cell Biology and Anatomy Johns Hopkins School of Medicine
On Mon, 7 Nov 1994, Richard Easingwood wrote:
} A question from Allan Mitchell } } Recently I have been using PVP (polyvinylpyrrolidone) in various techniques } but to date I have had no luck. } Firstly I have tried PVP with LR Gold resin (as recommended by the LR } technical blurb), I have also tried adding it my fixative to increase } osmotic pressure (as recommended in the paper I have been following to } localise the enzyme Thiamine Pyrophosphatase) and finally I have tried it } when infusing tissue with cryoprotectant for cryoultramicrotomy (as } recommended by Tokuyasu) } } In these papers they talk of "dissolving" the PVP in the various solutions } but my PVP will not dissolve!!! I am left with a fine white suspension } which quickly settles out when stirring is stopped. } } Question: Should the PVP dissolve (and perhaps leave a clear solution) or } is the fine suspension I have normal? (It blocks up canulas when perfusing, } it makes it difficult to see the tissue when processing). } } Note: The Merck index states that PVP is soluble in alcohols and water - } have I got the wrong one? } } Many thanks in anticipation. } } Allan Mitchell } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 } } }
} Reply to: RE} PVP solubility } } Richard, } Could you send me the reference for the use of PVP, i.e. the Tokuyasu ref? } } Thanks---Mike } } Mike_Schwartz-at-qm.yale.edu } Fax 203-785-5263 } Voice 203-785-4324 } Michael Schwartz, Ph.D. } Associate Professor } Section of Neurobiology } Yale University School of Medicine } 333 Cedar St. } New Haven, CT 06510
Mike, the reference is Tokuyasu, K.T.(1989) Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy.Histochemical Journal 21, 163-171. I will fax a copy to you if you like.
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
One last (?) word on spelling errors: "It's a damn small mind that can think of only one way to spell a word." - Andrew Jackson (seventh President of the U.S.)
} A question from Allan Mitchell } } Recently I have been using PVP (polyvinylpyrrolidone) in various techniques } but to date I have had no luck. } Firstly I have tried PVP with LR Gold resin (as recommended by the LR } technical blurb), I have also tried adding it my fixative to increase } osmotic pressure (as recommended in the paper I have been following to } localise the enzyme Thiamine Pyrophosphatase) and finally I have tried it } when infusing tissue with cryoprotectant for cryoultramicrotomy (as } recommended by Tokuyasu) } } In these papers they talk of "dissolving" the PVP in the various solutions } but my PVP will not dissolve!!! I am left with a fine white suspension } which quickly settles out when stirring is stopped. } } Question: Should the PVP dissolve (and perhaps leave a clear solution) or } is the fine suspension I have normal? (It blocks up canulas when perfusing, } it makes it difficult to see the tissue when processing). } Note: The Merck index states that PVP is soluble in alcohols and water - } have I got the wrong one? } Many thanks in anticipation. } } Allan Mitchell } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301
Dear Allan, We make up sucrose/PVP as a cryo-protectant using the following technique. It finallly goes into solution after being stirred overnight. Dissolve 30g PVP in 80ml water at approx 60C, add sucrose (79g) and 20 ml 0.5M PBS. Stir in coldroom overnight. Store at 4C. The solution is a clear yellow. Much like a good Hunter Chardonnay. Hope this helps. Regards, Gerald Little.
Dr Gerald J. Little | Ph (61 49) 215618 The Neuroscience Group | Discipline of Anatomy | Fax (61 49) 216903 Faculty of Medicine and | Health Sciences | The University of Newcastle | Email ANGJL-at-medicine.newcastle.edu.au Australia, 2308 |
MR-Received: by mta ISHTAR.MUAS; Relayed; Tue, 08 Nov 1994 05:49:55 -0400 (EDT) MR-Received: by mta ISHTAR; Relayed; Tue, 08 Nov 1994 05:49:55 -0400 (EDT) Disclose-recipients: prohibited
We have been using several different fluorescein based dyes to measure pH gradient within epidermal cells of roots from maize seedlings in situ. Treatment of the tissue with snarf-am under the appropriate conditions leads to a rather uniform labeling of both the vacuole and cytoplasm. Therefore, the snarf in these cells should be in pH environments ranging from pH 5 in the vacuole to almost pH 8 in to cytoplasm. We would like to be able to measure this pH gradient. The pH dependence graph of snarf in the Molecular Probes book makes one think it should be able to detect pH changes throughout this range. We have a calibration curve using a ratio of two excitation wavelengths of 420 and 475 nm at a constant emission wavelength of 580 + 30 nm. However, this ratio varies only between pH 7 and 9. We have experimented with another calibration curve using a ratio intensities with excitation and emission wavelengths of 420 nm and 580 nm compared to that obtained at exciation and emission wavelength of 480 and 620 nm. With standard solutions, this ratio shows pH-dependent changes from pH 5 to 9 but the majority of the change is between pH 7 and 8. However, the calibration does not look as clean when one tries to do the calibration by clamping cells at various pH by various means. Any suggestions would be appreciated. Thank You for Your Reply in advance, Dave Brauer plant Physioogist dbrauer-at-arserrc.gov
If your simply interested in viewing Tiff images (and many other formats) on a PC I would suggest Norton Desktop for Windows (by Symantec). The Norton Veiwer does offer limited image manipulation such as zooming. If your like me and have basically been forced from DOS to the Windows environment for $90 Norton Desktop greatly improves windows and has alot more to offer than just viewing and shuffling files (though as a replacment for Windows Filemanager alone I would recomend it). I don't know if the lastest version of Norton Commander has added Tiff viewing compatablity - but I wouldn't doubt it - and it runs under DOS without windows.
If you need additional image manipulation or another "viewer" WordPerfect 6.0 ($130 - educational discount) handles Tiff format with no problem, and allows limited image manipulation (zooming, cut, pasting, contrast,brightness) and printing. I would assume that most other current word processors should have similar features.
But for "real" image processing you need to spend much more money for the dedicated image processing programs. You might want to check out the article "High-end Image Editing Software" in Computer Shopper, October 1994, 14: 548-558, and the article "Graphical Voodoo" in the same issue p.610-622 which offers an excellent review of the various imaging formats pros and cons.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
Position available A postdoctoral position is available within the Structural Biology Imaging Center of the University of Pittsburgh Medical School. The focus of the position will be to study the interactions/role of spectrin like proteins in muscle, brain and other tissue systems. Specifically dystrophin and alpha actinin. The principal approach will be to use a variety of high resolution light and electron microscopic techniques (confocal, Immuno-EM, etc) to define where, when, and in association with which proteins these specific molecules and isoforms of these molecules are found. The applicant should have experience/interest in modern microscopic methods and be willing to learn other techniques as needed.
If you are, or know of someone who may be interested please contact the director of the lab listed below Simon C. Watkins Director SBIC 840 Scaife Hall University of Pittsburgh Pittsburgh PA 412-648-3051 email Swatkins-at-Pitt.edu
Its alright, Bill. In the spirit of academic freedom & creativity I'm perfectly willing to accept your spelling of "Copland". Or to put it to you as a quote from our oatmeal poet-lorryate[sic], Bob Dylan, "Don't think twice, its alright......Babe".
In message {9411072352.AA03570-at-MOLE.mmm.com} writes: } One last (?) word on spelling errors: } "It's a damn small mind that can think of only one way to spell a word." } - Andrew Jackson (seventh President of the U.S.)
Gib
P.S. - to follow soon. Must go aline the CM12 for 120kV.
I agree with Scott, Adobe Photoshop is the best way to view/manipulate images, but for the Mac, there is a shareware program called GIFconverter 2.3.2 which will open any picture file and convert to any known format you can think of.
"You can't help respecting anybody who can spell TUESDAY, even if thy don't spell it right; but spelling isn't everything. There are days when spelling Tuesday simply doesn't count." A.A. Milne. from The House at Pooh Corner.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
John F. Mansfield North Campus Electron Microbeam Analysis Lab. 2455 Hayward Ann Arbor MI 48109-2143 Tel: (313)936-3352 Email: jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html Time: 12:59 Date: 11/8/94
Graphic Converter is better & supports more formats.
--------------------------------------
Ron
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I am looking for feed back from anyone out there listening.
I am wondering how UNIX, Windows NT and OS/2 compare overall but especialy how the latter two compare with UNIX. I am looking into upgrading operating systems. In general the hardware platform will be a 586/90MHz PCI/EISA Bus, 16 Meg RAM, 1 GB diskspace, eithernet, etc.. I would like to go with UNIX (Novell or SCO), but I am concerned about possible problems with running the vast array of software written for the windows world in the X-windows environment and if the windows software would run better under Windows NT (Or OS/2).
Does anyone out there have any experiences to share? General preferences? Considerations or comments?
If you E-mail directly I'll try and coallate the info and either post it as one message or make it available to anyone who'd like it.
Thanks in advance.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
Message-Id: {9411082037.AA25076-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Re: format converters, was Orig-Author: {"John Mansfield" {John_Mansfield-at-mse.engin.umich.edu} }:ddn:wpafb ----------------------------------------------------------- John F. Mansfield North Campus Electron Microbeam Analysis Lab. 2455 Hayward Ann Arbor MI 48109-2143 Tel: (313)936-3352 Email: jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html Time: 12:59 Date: 11/8/94
Graphic Converter is better & supports more formats.
Dear Peter, I actually run a screen recoating service from the E.M. Unit here at very reasonable rates. If you are interested in any further information contact me direct.
Cheers,
Tony Romeo
Tony Romeo Internet: tony-at-emu.su.oz.au Electron Microscope Unit Madsen Building F09 Telephone: +61 2 351 2351 The University of Sydney Facsimile: +61 2 552 1967 Sydney, NSW 2006 Australia
Message-Id: {1994Nov08.163926.3183459-at-mail.magic.ca} To: microscopy-at-aaem.amc.anl.gov
Hey Ron, I think you have the programmes mixed up, GraphicConverter v2.0 by Thorsten Lemke converts most any graphic file. In addition you can do a little image processing.
I'm trying to embed plant material is some type of matrix that will allow me to make fairly thick sections, say 25 - 50 um.
I've used paraffin and the tissue starts to fracture when section thickness gets greater than 20 - 25 um. Frankly besides embedding the tissue in plastic and then grinding it down I'm not sure what else to do.
Bob, I don't know about Graphic Converter that John was talking about, but most images that are passed around these days (that I know of) are either in TIFF, GIF, or JPEG format. This program will convert between those and a a handful of others. I think i pulled it off of an Apple shareware site. I think the U. of Texas has one. If you can't find it, I could try sending it to you compacted. But check out the one Jahn Mansfield was talking about first.
We use Adobe Photoshop (both PC and Mac) and have found it very powerful and useful. It is however expensive (at least in Australia). JASC produce a Windows product called Paint Shop Pro which is not as expensive (around $70 USD) but can read and write many (around 40) image formats including most of the more common TIFF formats. It also has contrast/brightness controls, gamma correction and some filters.
I think, though I'm not sure, that there is a shareware version available.
According to the manual the address and 'phone number is:
JASC Inc. 10901 Red Circle Drive, Suite 340 Minnetonka, MN 55343 (612) 930-9171
I have no interest in the company, I just use the software!!
Other programs which may be of use are Image Alchemy (PC) or NIH Image (Mac).
Good Luck!!
##################################################################### ********************** * Between the idea * 0------* And the reality * } ---|--- { * Between the motion * | * And the act * / \ * Falls the Shadow * _/ \_ * T.S. Eliot * ********************** Colin Veitch Tel + 61 (0)52 27 5611 CSIRO Division of Wool Technology Tel + 61 (0)52 27 5891 (dir.) P.O. Box 21 Fax + 61 (0)52 27 5657 BELMONT Vic 3216 Australia
There have recently been several items posted concerning magnetic shielding of EM rooms. Several people have pointed out that this is an expensive and generally impractical thing to attempt, and they are right.
Something that is often not appreciated is that you don't actually "shield" magnetic fields in the sense of "blocking" them. Instead you "shunt" the fields around the area you are trying to protect. The commonly used shielding materials have extremely high initial permeability which means that magnetic fields will travel through them in preference to other routes. In other words, you don't build a "dam" in the way of the field, but instead you provide a "sponge" to channel it around the area to be shielded.
This sounds fairly simple until you realize that the high permeability of the material means that you can also easily channel magnetic fields INTO the area you are trying to shield if the enclosure is not a properly designed magnetic circuit. Just sticking a sheet of "mu-metal" in the way of the field is likely to make things worse. Holes or open joints can also create "lensing" effects.
Even in the best of cases, all of the magnetic flux won't flow through the path provided by the "shield" -- some amount of field always fringes out into the surrounding area. So at best, all you can do is attentuate the fields (not eliminate them entirely). The more efficient the circuit you provide for the fields to travel in, the greater the attenuation. However, as enclosures increase in size, more of the field lines will find it advantageous to take a "short cut" across the enclosed free-space region. For a simple cylindrical field, the attenuation varies as the permeability times thickness of the shield, divided by its diameter. In practical terms, this says it's pretty easy to shield a small region, but to get comparable shielding of a room, for example, the shield has to be quite thick (or consist of multiple isolated layers). Obviously, it is best to shield the smallest possible volume, which is why you want the shielding designed into your EM, rather than applied as an afterthought.
By way of reference, the initial permeability of a typical 80/20 (Ni/Fe) shielding material will be in excess of 20,000, as compared to several hundred for "soft" iron (free space is 1.0). Oriented silicon steel, as used in transformers, has IP of about 1000 and is often a good shielding material.
The above remarks apply principally to the case of lower frequency fields (such as the omnipresent 60 Hz). For high frequency fields one gets substantial eddy-current shielding just by using a good electrically conductive enclosure, such as aluminum.
There is real art to designing a good shield, and for all but the simplest cases, one is well advised to seek the assistance of someone who does it routinely. I'm not such a person, but have had good luck in the past working with Advance Magnetics (219) 223-3158, whose catalog also contains a lot of helpful application information (I have no connection to this firm). There are also a number of other good companies in this business.
By the way, I have been looking for years for an inexpensive PC-based program which would be useful for designing magnetic shields -- ideally something in the freeware/shareware realm. If someone out there knows of such a thing (or knows of someone who might), I'd appreciate hearing about it.
Another good shareware alternative for viewing TIFF, or just about anything else, is Paintshop Pro. It claims to handle three or so types of compression for TIFF files, up to 32 bit colour depth etc. I don't know whether it will cope with _all_ TIFF files but it works for everything I have tried it with.
Good Luck
Doug
+------------------------------------+ | Dr Douglas Arrell | | Mechanical Performance and Joining | | Institute for Advanced Materials | | 1755 ZG Petten | | Netherlands | | {ARRELL-at-JRC.NL} | | Tel. (+31) 2246 5287 | | Fax (+31) 2246 1917 | +------------------------------------+
I have been questioned as to the validity of my inquiry of PC Operating systems on this BBS, let me explain.
I am setting up a local network system to intergrate my EM Facility. This platform will be the backbone server for the exchange of data (Images from LM, LSM, TEM, SEM, EDS, etc), such that users can manipulate their images at a variety of sites. It is being designed to be networked directly to microscopes which use DOS, Windows, and X-Windows. This net will also allow for sellection between a variety of hardcopy output devices. It is also the intention that this system serve as an internet node allowing for the exchange of images across the net.
The platfrom will also be used for tasks such as E-mail, word processing, statistical analysis.
The microscope/networking end is covered. However I was interested in collecting further info for the operating system to function under. Since the users of this BBS are very familar with computer systems and complex software (i.e. high end imaging software) I thought this was a good place for this question.
I am sorry if you disagree.
Yes, this type of a system could be purchased from a vendor out there for however many $10k's (and for those of you who can afford it great) but the I know many labs out there are like mine in which we need to do everthing we can to save cost, and still get the best we can afford.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
A number of people have commented recently on how difficult it is to shield stray fields, suggesting that one needs to shield a whole room at the cost of } $10K. I would like to point out that simple, and cheap shielding with a little Mumetal has, EXPERIMENTALY, worked in at least two cases I know of: a) The old Cambridge HREM where a small shield was introduced in the column. (Ask Dave Smith now at ASU for further details.) b) Our own HREM here at Northwestern.
True, such a simple shield may not completely cure the problem, but for a few hundred dollars it is well worth the attempt! A very simple test that my students devised was to wave a small magnet near the microscope to test what regions were sensitive.
I should also comment that most people build small (cheap) shields around LEED systems, and most surface science systems operating with low energy electrons use shields with holes in them (for flanges). The VG STEMs also have a shield above the objective which has holes in it.
} Dear All, } } Anyone out their re-coat their phosphor screens? } } How does one go about it? Are the supplies available in South Africa? } } The screen I am proposing to coat has an alluminium base, about 12 cm } diameter, for a Hitachi TEM. } } Many thanks for any advice. } } Peter Richards. } } _______________________________________________________________ } } } -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at- } -at- -at- } -at- Peter D. G. Richards -at- } -at- Dept Anatomy and Cell Biology -at- } -at- UCT Medical School -at- } -at- Observatory -at- } -at- 7925 -at- } -at- RSA -at- } -at- Tel: 021-406 6285. -at- } -at- Internet: retep-at-anat.uct.ac.za -at- } -at- -at- } -at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at--at- } Peter: Recoating screens is a fairly simple thing to do. I used to recoat mine all the time using a technique used by a now defunct company called AEI. I used to use a phosphor put out by JEOL. Always got beautiful screens. Their phosphor gave me a screen with good contrast levels. If you can give me a fax number, I will fax a copy of the technique to you. I still have some of the phosphor and could send some to you if there isn't any problem with the mail system getting it to you. Let me know. Phil
This is to add on to the message posted by Colin Veitch: } } JASC produces a Windows product called Paint Shop Pro which is not as expensive (around $70 USD) but can read and write many (around 40) image formats including most of the more common TIFF formats. { {
This is a very nice product. It is shareware and can be downloaded from Compuserve, AOL, etc. The registration fee is $69. It lists at least 20 discrete formats (GIF,TIF,JPG, etc) and seems to handle them all smoothly, including converting between.
JASC also sells a companion Windows product called "Image Commander" which allows you to setup "catalogs" of images and view them as a page of thumbnail pictures or select a particular one for viewing. It has provisions for organizing the catalogs, annotating the images, etc., and would look like a very useful thing for anyone handling lots of digitized micrographs. This is also shareware, and the registration is $39.
I have no interest in the company, but have had good experience with both products.
Message-Id: {1994Nov09.134725.3200862-at-mail.magic.ca} To: microscopy-at-anlemc.msd.anl.gov
S
Hey Ron, I think you have the programmes mixed up, GraphicConverter v2.0 by Thorsten Lemke converts most any graphic file. In addition you can do a little image processing.
I am gathering and storing away much of the information on this bulletin board regarding networking of microscopes and image storage systems. I, for one, appreciate the inquiries of people such as Richard Edelmann regarding computers and operating systems for microscopy imaging. Also, I have noticed in the last week that my spelling has improved vastely.
Nancy Smith Director, EM Lab CSU,Hayward Hayward, CA 94542
Although you may end up using it inelegantly, e.g. via telnet and ftp, probably the way to go is: o Unix machine with tons of disk space as a server when images are stored temporarily o stand alone PCs (or Macs, etc.) have their own removable media and users MUST purchase their own disks for storage.
We have found that attempting to network PCs (some DOS, some windows, some OS2), UNIX, OS7, and Macs is nearly impossible. The only platform that appears to be compatible with all the other platforms is UNIX.
Subject: Time:5:05 AM OFFICE MEMO Re:Spelling Fluorine Date:11/10/94
Like the professor who pointed out that if the first five letters spelled 'flour' it was wrong, I had a biochemistry professor who told us to split the word into two syllables, and if it came out 'flo-urine', it was wrong.
Our department has available, for the cost of moving, a Hitachi HS-8 TEM. It is a one owner instrument and has been maintained on manufacturer's service contract since installation. It is currently fully operational. We will be moving this microscope very soon.
Please contact me directly for more information.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
I recently spend 4 weeks in the Netherlands learning microscopy for yeast samples-only morphology and immunogold techniques. Now I am trying to set up our lab in America to do these techniques. I have three major problems-limited experience, no one at my institute can help me, and having learned in Europe I have no knowledge of good American suppliers for EM stuff.
Here is what I need: Right now we have a sorvall MT2-B "Porter-Blum" ultra-microtome. We are looking for a table for it (vibration resistant), as well as for a new ultramicrotome.
I also would like to know a good general supplier (other than EM sciences) for things like grids, grid holders, capsule holders, etc....
I also would like to know where people get good quality formaldehyde, glutaraldehyde, potassium permanganate, and ethanol ( in other words the basic chemicals needed).
Thanks for your help. Kim Wilson-Oregon Graduate Institute
In article {MAILQUEUE-101.941108141914.288-at-anat.uct.ac.za} , RETEP-at-anat.uct.ac.za writes: } Dear All, } Anyone out their re-coat their phosphor screens? } How does one go about it? Are the supplies available in South Africa? } The screen I am proposing to coat has an alluminium base, about 12 cm } diameter, for a Hitachi TEM. } } Many thanks for any advice. } } Peter Richards.
Dear Peter, We routinely recoat our screens, and, if the phosphor is available, the rest of the supplies will be easy. The steps are: 1) Make } 500 ml 0.5% gel- atin solution, 2) Filter with type GS 0.22 mu millipore -at- ~30 C, 3) Suspend 10.6 g (for ~45 g/cm**2 and ~170 cm**2 screen size, your mileage may vary) phosphor in 500 ml gelatin soln, 4) Warm and degas the suspension while, 5) Clean screen blank, 6) Glow discharge blank for 15 min, 7) Place blank in funnel so that it is horizontal and so that there is an area between the edge of the blank and the wall of the funnel (our screen blank is not completely circular, but has two straight sides cut along chords, you may have to build a frame to achieve this geometry), 8) Attach a hose to the bottom of the funnel, and clamp it off, 9) Fill the funnel with 50% ethylene glycol to a level just above the bottom face of the blank and make sure there are no air bubbles trap- ped beneath the surface, 10) Pour the phosphor suspension down a glass rod and move the rod around to avoid pileup on any one area, 11) Cast a film of collo- dion diluted 1:100 in n-butyl acetate on the surface of the liquid by deposi- ting drops with a Pasteur pipette until the entire surface is covered, 12) When the n-butyl acetate has evaporated, sweep the film aside with a wedge of filter paper; repeat until the liquid surface is free of debris, 13) open the clamp on the hose and allow the liquid to drain slowly--~2 drops/sec--until the level is below the bottom surface of the blank, 14) Place a clean sheet of paper over the top of the funnel and draw air through the hose and past the screen; leave overnight, and 14) Remove the screen from the funnel, check for evenness of coating and lack of foreign objects (eyebrows etc.), then bake at 60 C for 24 hours. Good luck, it takes a little practise, but the blanks can, of course, be reused. Yours, Bill Tivol
Fluorine being on the scene, I take my chance once more to ask again a question stayed without any response since two months :
"How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment (JEOL 2010) ? Any suggestion appreciated (inversely as the cost)."
The question may be formulated in another way : Has somebody ever worked with some success on inorganic fluorides by TEM ?
An element of response is in "The electron microscopic observation of phase transition in KFeF4, RbFeF4, and RbVF4," by R. Deblieck, J. Van Landuyt & S. Amelinckx, J. Solid State Chem. 59, 379-387 (1985). The problem with inorganic fluorides is electrical conduction : "After washing in benzene, in order to remove tape residues, the thinned plates are mounted on 3-mm copper grids by means of silver paste to ensure good thermal and electrical contact. In spite of these precautions electrical conduction remains a problem and substantial charging phenomena still occur under the electron bombardment for observation. Moreover, all materials are subject to damage by electron irradiation..... The investigated compounds prove to be sensitive to decomposition under electron irradiation, probably through temperature-driven evaporation of fluorine."
Other experiences on inorganic fluorides by TEM ? Suggestions ?
Microscopists do not like fluorine, this may be why there are some spelling problems.
Armel Le Bail - Fluoride Lab - armel-at-ONE.univ-lemans.fr
Message-Id: {9411101344.AA06245-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM and inorganic fluorides Orig-Author: {lebail-at-cnrs-imn.fr}:ddn:wpafb ----------------------------------------------------------- Hi,
Fluorine being on the scene, I take my chance once more to ask again a question stayed without any response since two months :
"How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment (JEOL 2010) ? Any suggestion appreciated (inversely as the cost)."
The question may be formulated in another way : Has somebody ever worked with some success on inorganic fluorides by TEM ?
An element of response is in "The electron microscopic observation of phase transition in KFeF4, RbFeF4, and RbVF4," by R. Deblieck, J. Van Landuyt & S. Amelinckx, J. Solid State Chem. 59, 379-387 (1985). The problem with inorganic fluorides is electrical conduction : "After washing in benzene, in order to remove tape residues, the thinned plates are mounted on 3-mm copper grids by means of silver paste to ensure good thermal and electrical contact. In spite of these precautions electrical conduction remains a problem and substantial charging phenomena still occur under the electron bombardment for observation. Moreover, all materials are subject to damage by electron irradiation..... The investigated compounds prove to be sensitive to decomposition under electron irradiation, probably through temperature-driven evaporation of fluorine."
Other experiences on inorganic fluorides by TEM ? Suggestions ?
Microscopists do not like fluorine, this may be why there are some spelling problems.
Armel Le Bail - Fluoride Lab - armel-at-ONE.univ-lemans.
If necessary, you can add additional NuBus cards to your Mac by adding a NuBus expansion chassis. I have one made by Second Wave which gives me an additional three slots (as well as a power supply and connections for an internal hard disk drive) on my Mac IIci. There are also six slot chasses available.
Second Wave can be contacted at: 9430 Research Blvd., Echelon II, Suite 260 Austin, TX 78759-6541 512-343-9661 512-343-9663 (FAX) Applelink: D0864
I have no connection to Second Wave, I'm just a satisfied customer.
Carl Henderson Electron Microbeam Analysis Laboratory University of Michigan 2005 C.C. Little Bldg. Ann Arbor, MI 48109-1063 USA ----------------------------------------------------- (313) 936-1550 (voice) **** Next time, **** (313) 763-4690 (FAX) *** take the *** chender-at-umich.edu (e-mail) **** train! **** ----------------------------------------------------- On Thu, 10 Nov 1994, Steve Bill 5355/5128/5642 wrote:
} } We are currently using a HP Laserjet 4M to output TEM images from our Gatan } Slow-scan camera (mac based). What I'm looking for is an enhancement that will } improve the output of images from this printer. I've heard of some grey-scale } sofware upgrades and some add-in cards that are supposed to boost the resolution } or improve the grey-scale rendition of the HP laser printers but I don't have any } specific information or pointers to any of them. } } The Mac by the way has no NUBUS slots available (all three are taken) so anything } we consider must either be software-based or hardware added to the printer itself. } } Thanks, } } Steve Bill }
Dear Armel et al., My experience with fluorides is limited to organics, but the problem of radiation damage is still serious with these fluorides as well. The best procedure IMHO is to use as low a dose as possible--especially for focusing, etc., where the electrons are causing damage, but not being recorded. We use our fairly crude, but sensitive, video system to scan grids for areas of interest, focusing, etc., and LoDose or SO163 for image recording. I have found that if the lens column is aligned well, the wobbler can be used to focus quickly within one click of the fine objective control (=.99 micron). A subsequent through-focus series should yield good images. This works at mags up to 200,000, and for low dose images, I have had my best luck with somewhat lower mags than this. I use a 30 micrometer C2 aperture with the wehnelt bias on the lowest setting and the C1 lens maximally excited. This produces a beam current in the picoamp range, where charging, heating, etc. are minimized (it helps to use 1.2 MV electrons if you have them). Remember, alignment of the lens column is critical--check and correct it the same day you take the pictures. Good luck. Yours, Bill Tivol
In response to: "How to avoid the almost systematic decomposition of inorganic fluoride compounds under the electron beam during a TEM experiment...?"
You might try cryo EM, that is lowering the temperature of the specimen towards that of liquid nitrogen or liquid helium. This preserves visible structure for a time in biological specimens, presumably due to a decreased vapor pressure of the products of radiolysis.
We are struggling with imaging droplets of liquid fluorinated hydrocarbons, by freezing the drops on a substrate and viewing them with the low-temperature scanning electron microscope at -180 C. At low temperature, large (1mm diameter) drops are resistant to the 10kV beam, small (1 m) droplets sublime. Interestingly, even after a ca. 100A gold coat the fluorocarbon drops appear brighter than adjacent aqueous structures, perhaps due to charging or the differing secondard electron coefficients of fluorocarbon and water.
Jacob Bastacky, MD 1-116 Lawrence Berkeley Laboratory EMail: sjbastacky-at-lbl.gov University of California Phone: (510) 486-4606 Berkeley, California 94720 Fax: (510) 486-4750
Steve Bill 5355/5128/5642 {sbill-at-asdg.enet.dec.com}
Steve,
Information about LaserPrinter Enhancements:
PC based controllers are available from the following companies;
LaserMaster Corporation - WinJet Product Line (Windows Only) Talltree Systems- JLaser? Greylaser? (? if still selling) DPTech - ? (not sure if they are still selling units) XLI Corp - ?(not sure of Name) Should be selling controllers only No complete products including engine.
Mac Based products; I am currently not aware of any in a controller or software only basis.
Complete Printer subsystem for High-Res output -- PC & MAC & Unix*
LaserMaster ImagePrinter 1800 DPI (#2 Version) Appletalk, Eithertalk, TCP/IP, Novel Netware, Parallel (HS), Serial communications on unit. 24 meg Ram 66 Mhz Multi-Tasking Pipeline Processor 240 Meg Hard Drive internal Pipeline Assoc. Postscript Level 2 interpreter & PCL 4 Languages 8 1/2 X 11" to 12.5 X 19" paper size on line.
*unix is only for TCP/IP standards on limited System OS
This is only 1 of our printers avaiable. I do work for LM in the Scientific and Medical industries and can help you if you would like. Contact Greg Begin LM Imaging at 1-800-950-6363 Ext: 3207 or Mail address of gregb-at-sales.LMT.com I will be out at the American Heart Assoc. meeting next week and then the Radiological Society of North American durring the last week of Nov. Greg Begin LM Imaging Div. LaserMaster Corporation /\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\
} Date sent: Thu, 10 Nov 94 09:59:20 EST } From: Steve Bill 5355/5128/5642 {sbill-at-asdg.enet.dec.com} } To: microscopy-at-aaem.amc.anl.gov } Copies to: sbill-at-asdg.enet.dec.com } Subject: Laser printer enhancements?
} } We are currently using a HP Laserjet 4M to output TEM images from our Gatan } Slow-scan camera (mac based). What I'm looking for is an enhancement that will } improve the output of images from this printer. I've heard of some grey-scale } sofware upgrades and some add-in cards that are supposed to boost the resolution } or improve the grey-scale rendition of the HP laser printers but I don't have any } specific information or pointers to any of them. } } The Mac by the way has no NUBUS slots available (all three are taken) so anything } we consider must either be software-based or hardware added to the printer itself. } } Thanks, } } Steve Bill }
After so many requests for the procedure, it is a little cheaper to put it out on the microscopy board. So, here goes for those who want it. I get my phosphor from JEOL, cat.#423-011(green) or 423-010(yellow). These are old cat. numbers, they might have changed. You can call the parts dept. at (508) 536-2342 and ask for Maria Ramos.
1. Withdraw the old screen from the microscope, taking care to avoid depositing any loosened phosphor in the viewing chamber. 2. Remove the old coating from the screen plate by washing in acetone. The cleaned plate must be free from all particles of matter and the surface must be free from blemishes and scratches. 3. Prepare a sufficient volume of 4% (w/v) suspension of phosphor powder in acetone containing about 1% of collodion. To make collodion use: 4grams parlodion, 25ml 100% ETOH, 75ml ether., Any grade of collodion that is suitable for specimen preparation can be used. The total volume must be sufficient to fill the selected dish with liquid to a depth of about 1cm above the surface of the screen plate in position on the bottom of the dish. 4. Agitate the suspension vigorously then pour it rapidly into the dish. 5. Wait about 5 seconds to allow large particles to settle and violent swirling to cease. 6. Slide the screen smoothly into the liquid, preferably without scraping the bottom of the dish. 7. Cover the dish with a piece of plexiglass with a small (1/4"-1/2") hole drilled in it and leave it to settle (I usually let it sit overnight). When the remaing liquid is clear, draw off the liquid by inserting a suction tube through the hole in the lid. It is extremely important not to disturb the screen or the liquid above the screen in any way as this is being done. Draw off the liquid steadly and slowly then remove the suction tube. 8. Leave the screen to dry without any further disturbance of any kind. Do not lift the lid to inspect the screen until the powder is quite dry because a slight change in drying conditions can produce a visible mark on the damp surface. 9. When the screen is dry, remove the screen and wipe off any excess phosphor from the back and sides of the screen plate. 10. Put screen back into the microscope.
Note: A newly-coated screen will de-gas for a short time when it is first placed in the microscope. Pumping times may therefore be longer than normal at first.
A very small particle size is desirable for high resolution screens and it may be found advantageous to agitate the phosphor suspension ultrasonically before putting it in the dish. Screen resolution is also affected by the thickness of the phosphor layer and by light scattering within it.
It may take a few tries to get the hang of it, but when I had to recoat my screens this technique worked rather well. Fortunately, I no longer have to recoat my screens.
According to a OSHA publication (1978), liquid containing Osmium tetroxide should be absorbed in vermiculite, dry sand or similar material and then disposed off in a sealed container in a secured sanitary landfill.
Greetings, There was an interesting paper in the Recent Microscopy Today (october) in which different ways of neutralizing liquid osmium waste/spills are tested. They find corn oil-soaked kitty litter to be the best treatment of all, with pure corn oil not far behind.
Return-Path: ert-at-leka.hut.fi Received: from leka.hut.fi (leka.hut.fi [130.233.224.22]) by santra.hut.fi (8.6.8.1/8.6.7) with ESMTP id QAA07915 for {listserver-at-anlemc.msd.gov} ; Thu, 10 Nov 1994 16:19:57 +0200 Received: (from ert-at-localhost) by leka.hut.fi (8.6.8.1/8.6.7) id QAA110591 for listserver-at-anlemc.msd.gov; Thu, 10 Nov 1994 16:19:44 +0200
dear Nestor,
Microscopists, I will like to participe in the network of the microscopists.
My email: eero.ristolainen-at-hut.fi
Address: Helsinki University of Technology Center for Chemical Analysis FIN-02150 Espoo, Finland
Michael, Have you considered: 1) freezing sectioning on a sliding (=sledge) microtome or 2) a vibratome (I've had my best luck with 10% gelatin fixed 16-20 hr in 10% formaldehyde) Phil Oshel poshel-at-luc.edu
With budgetary restictions on the sciences these days, many of us are no longer able to attend the National Microscopy Society of America (MSA) meeting. We are limited in the ability to present our work, share our ideas and research, or meet with our colleagues due purely to financial limitations.
Shams Ghoniem of the Iowa Microscopy Society has suggested that MSA consider awarding grants to professional and scientific staff similar to the program currently in place for students. This recommendation, put forward by Peter Ingram, LAS/MSA president, was met with a lukewarm response by the MSA council. I believe that MSA would consider such a proposal if enough support by the MSA membership is provided. MSA would benefit by increased attendance at meetings, more incentive to join MSA, as well as in supporting education and professional development of their members.
I am requesting therfore that letters be sent fo Peter Ingram in support of this proposal. We need letters from across the country including letters from faculty who are willing to match an MSA award. These letters need to be sent by the first of January to be influential at the next MSA committee meeting.
His address is: Peter Ingram LAS Director Research Triangle Institute Box 12194 3040 Cornwallis Road Research Triangle Park, NC 27709
E-mail: ingram-at-rcc.rti.org
I thank you for your attentive ear and for your letters!
I understand that there are sources for particularly sharp AFM tips. The normal AFM tip has a radius on the order of 10's of nanometers and isn't very useful for "deep" contours. Does onyone out there know of a source for sharper tips.
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
Group - Preface: I find myself "semi-retired" (meaning that I have more free time than most of you), yet interested in microscopy (our newsletter "Microscopy Today") and rather alert when it comes to data bases. Result: I am considering building the equipment listing data base that has been discussed. My THOUGHT: To build a data base with two sections: Section 1: A simple listing of members - names, companies, addresses, tel. & eMail address, etc. To include all interested, not necessarily only those identified with equipment. Hard copy of this section could, of course, be easily provided. Section 2: A listing of equipment categories. I.E., I would expect SEM, ESEM, etc. to be categories. Each category to be a "field" in which we c ould list individual products - which can be searched on. Then, perhaps, to provide the data base on disks for those interested. Perhaps there is a way that one can access the data base by eMail? Before I consider getting serious, I have two questions: 1) Would the above solve the need as discussed? Or is there other and/or dif ferent detail that should be included? Applications? 2) I happen to use "Q&A" as my data base software. In Q&A, items can be separated in a keyword field with semicolons and then searched on. I can export to any other data base software. I do not know if other data base software will allow this keyword search? Rather than swamping the listserver with comments from the whole membership, may I suggest that only those who have previously commented on this subject publish their response to the full readership. Others might just send their opinions to me. Assuming that the concept is OK, or close, the next step is to ask your help in defining categories. I will ask for that assistance should the project be a "go". Regards, Don Grimes, Microscopy Today
My computer address at Cambridge has been changed. It is noe pe13-at-cus.cam.ac.uk Will you please make the necessary alteration at your end so I may continue to get all the good stuff on the MSA file server.
unsubscribe eliesh.oneil-at-gtri.gatech.edu Elizabeth G. (Eliesh) O'Neil Research Scientist I Georgia Tech Research Institute Electro-Optics, Environment, and Materials Laboratory Materials Analysis Center 925 Dalney Street, Baker 271 Atlanta, Georgia 30332-0827 Phone: (404) 853-0590 FAX: (404) 894-5073 Email: eliesh.oneil-at-gtri.gatech.edu
Please do not forget the User Facilities and equipment lists and DATABASE that Sandy Silvers has compile and distributes via her local society. It is called the MSA TECH FORUM FACILITY DIRECTORY. The last update that I received from Sandy was dated 7/94. This is a working DATABASE with the following information...
Facilities ] Phone Lists State Listings Equipment Listings.
The contact phone number listing is : 706-546-3471
We have called Vincent and Associates at (800) 828-6972 regarding their products. We are waiting for specifications and pricing via snail mail. Has anybody experience with operating one of their shutters (e.g. D122 or iD123) from a serial port, specifically from within NIH-Image? They make it sound very simple, but we thought we'd find out whether anybody has experience.
The Ludl system, at first glance, seems far more expensive. Is it significantly better? And the same question as above regarding the Vincent shutter.
We're waiting to hear from Scientific Imaging Syatems too.
Group - In response to my recent "offer" to develop an appropriate data base, I note the comment from Nestor where he recommends the data base as developed by Sandy Silvers. I accept that as a "non-recommendation" to work with me. I should point out that the "meat" of Sandy's data base is two listings: 1) An "equipment listing" including type/model, year installed, whether under a service contract, and user charge rates. 2) An "ancillary equipment listing" including quantity, manufacturer, type, model #s, etc. There is no way (I BELIEVE) to do a search on specific instruments or applications - and come up with a list of others with a specific interest. I should also point out that Sandy's system has sections for: 1) Type of Facility (biology, material science, core univ., etc. 2) List of personnel and users. 3) Other services provided 4) Number of users per year. Kindly understand that I have NO INTEREST in competing against Nestor and his wishes - we (including I) owe him too much. And, considering the amount of work involved, I have no interest in accomplishing a proper, searchable, data base without the full support of the "management" of the listserver. As a result, I withdraw my offer - with thanks to you all that have expressed interest and support. Don Grimes, Microscopy Today
I know of one case where sheilding was successful. The Field emmission sem at cell biology, Yale Medical school had all the usual problems until a layer of (I thought it was brass sheet but am not sure now) metal was applied on all 6 surfaces of the room at ???? cost. I don't have his email but Phillip Male (pronounced Mal) at cell biology yale university is the one to contact, the shielding DID work! alan pooley marine sci rutgers univ nj
Does anyone have names and phone numbers for suppliers of standards for my EDX analysis. I need to know the compositions to see how well my setup is doing it's work.
Cheers from Sunny Vancouver. Dan Henne Simon Fraser University henne-at-sfu.ca
Sorry If I gave the wrong impression. I have no problems with either Don or Sandy running a DATABASE on equipment. I just wanted to make sure that Sandy's work over the last few years was remembered.
Don, your more than welcome to continue to use this forum for suggestions and/or comments. I apologize if I gave the wrong impression....
As I Mentioned on the net earlier, we have an old JEOL model JSM-2 SEM with a Nuclear Diodes/ EDAX model 508 EDS system, built in 1968. It has a manual vacuum valving system and the power supply is vacuum tube based. It was refurbished in the mid 80's and the EDS was repaired. Although in good operating condition a year ago when last used, we have no further use for it. We have permission to donate it to a non-profit charitable institution or University. Anyone wanting to acquire it would have to come here to Minneapolis, pack it up and ship it at their own expense. JEOL will not be able to supply parts for it, so you're on your own there. Only qualified organizations may apply; no individuals or for-profit businesses. Any requests can be sent to me at the e-mail address below. If you're seriously interested, please contact me ASAP. We must move it by the end of the year. By the way, thanks for all the advice given to me earlier.
Mike Boucher Boucher-at-tcrca.usbm.gov ***************************************************************** Michael L. Boucher Sr. Boucher-at-TCRCA.USBM.GOV Geology-Mineralogy/Chemistry Labs Ph 612-725-4614 Twin Cities Research Center Fax 612-725-4527 U.S. Bureau of Mines Center 725-4500 Department of Interior 5629 Minnehaha Avenue South Minneapolis, MN 55417-3099 U.S.A. *****************************************************************
I need some expert advice on the preparation of positive-image transparencies. I typically use 35 mm slides in my own presentations, but one of my collaborators insists on overhead transparencies. I have seen composite overheads which appear to have positive images on film attached to them. So my questions are:
Are these made by exposing light-sensitive film on an enlarger?
What type of film is used? (Manufacturer, Stock No., Sizes available)
What are the common exposure times and f-stops on the enlarger?
What are the developer/fixer chemical ratios and developing/fixing times?
A prompt response would be appreciated since this project was thrown into my lap on short notice with an even shorter deadline. BTW, I tried a laser xerox of a print (HRTEM image) onto a transparency and the results were unacceptable.
Cheers,
David A. Howell MSM Dept. Michigan State University E. Lansing, MI howelld-at-egr.msu.edu
Color laser copiers working in B/W mode give very good quality reproductions, and are far more convenient than printing methods. Most Kinko's have a copier -- you may have to work with them to make sure that they give good quality results, and many Universities also have an appropriate copier.
Very often when I am cryo-sectioning , the sections curl so badly that they are useless. This was the reason for purchasing the static-line ionizer. This really did not help the curling problem, although it served other purposes. My sections are mostly polymer-elastomer blends, primarily nylon based. If you have any suggestions I would be eternally greatful. Also if anyone has suggestions on how to pick up the samples off of the cryo knife that would also help. Right now we are using a very fine sewing needle, but something even thinner yet ridgid would be better
Thank you
Andrea Monisera. --------- End of Original Message ---------
I have seen the eyelash hairs of Dalmatians (dogs) used for this. they are forked at the end and work very well for picking up cryosections
You can take your slide to a digitizing scanning device and then feed that image to print it or to have it printed on an overhead transparency. This is done in some photo departments.
Nina Allen.
} } I need some expert advice on the preparation of positive-image } transparencies. I typically use 35 mm slides in my own } presentations, but one of my collaborators insists on } overhead transparencies. I have seen composite overheads } which appear to have positive images on film attached to them. } So my questions are: } } Are these made by exposing light-sensitive film on an } enlarger? } } What type of film is used? (Manufacturer, Stock No., Sizes } available) } } What are the common exposure times and f-stops on the enlarger? } } What are the developer/fixer chemical ratios and developing/fixing } times? } } A prompt response would be appreciated since this project was } thrown into my lap on short notice with an even shorter } deadline. BTW, I tried a laser xerox of a print (HRTEM image) onto a } transparency and the results were unacceptable. } } Cheers, } } David A. Howell } MSM Dept. } Michigan State University } E. Lansing, MI } howelld-at-egr.msu.edu }
} I am in search of a US supplier for polyester wax. This product is } produced by a company in the UK. } } Please respond directly to my e-mail address } } TIA } } Skip } melsen-at-MICROBIO.emory.edu } I expect there might be enough interest for a general reply to the list, if anyone knows, since there is an article in the new Biotechnoques [17(5):846-848] by L.L Richardson and M. Dym about polyester wax embedding for LM level immuno. They cut at 5-10 micorns at 4 degrees in a cryostat and ethanol dehydrate and air dry to get the sections to adhere to slides. The supplier they list is BDH, Poole, UK. I've never cut the stuff, but their pictures look decent. I have heard of cutting polyester wax on a sliding microtome with a funnel of dry ice above the block so the cool vapor keeps the sections from melting. If anyone has experience with cutting this wax or info about U.S. suppliers, I'd be interested in hearing about it.
------------------------------------------------------------------ |Glenn Holm *mime mail ok* Internet:karuzis-at-wccf.mit.edu | |M.I.T Dept. of Brain + Cog. Sci. This VAX doesn't do NeXTmail | |Cambridge, MA 02139 "Real Neuroscientists don't do gels!" | ------------------------------------------------------------------
Reply... RE} MSA staff support Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
This is a reply to the original message posted to the Microscopy List Server that is included below. These opinions are mine own and do not necessarily relect those of my employer.
As a University employee who has to watch every dollar, I frequently find it is expensive to go to conferences. However, I do not think I should get handouts from MSA. I think that MSA money is better spent encouraging students (grade school, high school and both undergraduate and graduate university students) to get interested in various aspects of microscopy. The outreach programs in schools, the MSA scholoarships and bursaries for attending our conferences are much more important and have an impact on a greater number of people than sending a few senior people to the meetings. There are too many people wanting to milk money out of MSA for their own benefit, rather than the good of the community as a whole. As I say, just my "humble" opinion. John Mansfield.
--------------------------------------
Dear fellow microscopists,
With budgetary restictions on the sciences these days, many of us are no longer able to attend the National Microscopy Society of America (MSA) meeting. We are limited in the ability to present our work, share our ideas and research, or meet with our colleagues due purely to financial limitations.
Shams Ghoniem of the Iowa Microscopy Society has suggested that MSA consider awarding grants to professional and scientific staff similar to the program currently in place for students. This recommendation, put forward by Peter Ingram, LAS/MSA president, was met with a lukewarm response by the MSA council. I believe that MSA would consider such a proposal if enough support by the MSA membership is provided. MSA would benefit by increased attendance at meetings, more incentive to join MSA, as well as in supporting education and professional development of their members.
I am requesting therfore that letters be sent fo Peter Ingram in support of this proposal. We need letters from across the country including letters from faculty who are willing to match an MSA award. These letters need to be sent by the first of January to be influential at the next MSA committee meeting.
His address is: Peter Ingram LAS Director Research Triangle Institute Box 12194 3040 Cornwallis Road Research Triangle Park, NC 27709
E-mail: ingram-at-rcc.rti.org
I thank you for your attentive ear and for your letters!
Kathy Walters secretary, Iowa Microscopy Society
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Reply... RE} } Linking pc to Mac Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
I think the easiest way of linking a PC to a Mac over Ethernet would be to put ethernet cards in both machines (I think you have a Quadra with built in E-net and so you are all set there. Get a thin net connector for the Quadra and configure the board for the PC for thin net. If you have an EISA bus PC I would suggest getting a NE3200 (I think it is) 32 bit card that should give you the fastest throughput. However if you are on a really tight budget then you can get a 16bit card from 3Com for less than $200. You can just connect the two computers with one strip of thin net cable and terminate both ends. Then I would use public domain or shareware TCP/IP software for each machine. The Mac OS System 7.5 comes with MacTCP and you can use Fetch, NCSA Telnet or XferIt (or the commercial program Versaterm) to run ftp on the Mac and NCSA telnet to run ftp on the PC (there may be other better implementations of the TCP/IP stack for the PC that are shareware, I am not sure.) Fetch is really nice, you could put the PC into server mode (run ftpd) and use the nice interface of Fetch to put or get your files. If you are using only those two machines you can use pretty much any IP address you like for the two machines. If you put them on the Internet though, you will need to get IP addresses allocated by your local network administrator. Hope this helps. John Mansfield --------------------------------------
} cytogen-at-tigger.jvnc.net wrote: } } } Hello- } } I have a pc-based system to read in digital medical image data. I transfer } } these as TIFF or Interfiles to a Quadra 950 for processing and display and } } eventually for submission to the FDA. At the moment I use MacLinkPC to } } transfer the files over a serial line. At the rate of 57,600 baud, 100Mb } } of image data takes all night to transfer. What is the EASIEST way to take } } advantage of the Quadra's built-in Ethernet capability to speed up this } } transfer? I would prefer a solution which did not include taking a lengthy } } network administrator's course. Any suggestions will be appreciated. } } } } John G Wolodzko } } (jwolodzko-at-cytogen.com) } } Cheapest and fastest might be to use a hard-disk cartridge drive on both } the pc and Mac, then use sneaker-net! } } Roger Pyle
I've used the Syquest cartridge/sneaker-net route and it may be the fastest. A less expensive solution may be the ethernet and "PhoneNet PC" software (sold as part of Timbuktu from Farallon). I have an Asante EN2000+(thin-net) ethernet card on a PC running PhoneNet and a "friendly-talk" thin-net adapter on the Quadra. I create a "server" share folder using system 7 Appleshare on the Quadra and use PhoneNet to make the PC a client on the Quadra's share folder.(You can't make the PC a server using PhoneNet). With the proper priveliges set up on the Quadra you copy your files into it's share folder. For easiest running I would call Farallon and ask them what their default ethernet card is and buy that with the Timbuktu software. Installation and setup is then straightforward without "taking a lengthy network administrator's course". Lots of Luck, Fran Tanzella Sr. Chemist, SRI International
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I haven't used the negative reversal procedure in a few years to make sheet film into transparencies. It wasn't cost effective to keep it up and never gave me the quality that I liked.
The method I now use is to use a dye-sublimination printer to write directly to "transparency" film. This produces photographic quality output. Of course this means you need to find someone with a dye-sub printer. Typical cost/print is about $2-$3 for an 8x10 transparency. The printers themselves run about $10K.
If you have a good laser-printer (at least 300 dpi) and a graphics/image processing program that does reasonable dithering (Adobe Photoshop for example) then you can print to "laser printer transparency" film. This give marginally acceptable prints but I wouldn't want to use these unless there was no other alternative. The better laser printers support a "photo-grade" mode (the name varies depending upon the manufacturer) which greatly improves the gray-scale dithering pattern, but it still won't compete with dye-subs (in my opinion).
The very high end printers (~2700+dpi) like the Linotype Imagesetters give results which compare with dye subs. These are typically found in graphic arts departments. If you look with an eye piece you can still see the dither pattern but it's pretty small.
There are also the ink-jet printers which depending upon the graphic do okay jobs. I tend not to use these for continuous tone images but prefer them on solid fill graphics. Mainly due to their lower cost/print ~$1 for an 8x10.
But all of this assumes that you have the images/slides already in some computer and ready for printing. If not then you'll have to go across to your graphic arts department and have them shoot an 8x10 negative and do the reversal for you. Last time I checked the price for this (here at ANL) was about $12/transparency.
This likely doesn't solve your problems but at least you can ask around and see if the printers are handy. I would be surprized if there were none on your campus.
Sorry Subscribers, I'm still having a system problem. You will likely continue to get doubling of some messages that donot clear the queues before a crash happens. I'm looking into the problem.
We are looking for a good home for a fully operational Philips 300G TEM. New HT tank stable operating at 20,40,60,80, and 100 kV. Both biological stage and goneometer stage. Several specimen holders (single-tilt, double-tilt, tilt-rotate). Planning on surplusing soon (Univ. of Wash., Seattle, WA).
Interested parties contact by email: merock-at-u.washington.edu
} I am in search of a US supplier for polyester wax. This product is } produced by a company in the UK. } } Please respond directly to my e-mail address } melsen-at-MICROBIO.emory.edu
Try Polysciences, Warrington, PA or Aldrich Chemical Company, Milwaukee, WI. I haven't used polyester in years, but both stocked it as of 1988. Aldrich sold 5 gal and up quantities and a wide range of mol wts; I had gotten two 1-gal samples from them that lasted me years. (See also my paper - 1987, JEM Technique 6:35-41). --Jon
Jon L. Norenburg {norenbur-at-onyx.si.edu} Invertebrate Zoology, National Museum of Natural History Smithsonian Institution, Washington, DC 20560 Voice 301-238-3508, Fax 301-238-3361
Return-Path: {dieguez-at-iris1.fae.ub.es} Received: from oxmail.ox.ac.uk by vax.ox.ac.uk (MX V4.1 VAX) with SMTP; Wed, 16 Nov 1994 00:39:56 +0000 Received: from aaem.amc.anl.gov by oxmail.ox.ac.uk with SMTP (PP) id {12213-0-at-oxmail.ox.ac.uk} ; Wed, 16 Nov 1994 00:39:34 +0000
Has anyone had experience rebuilding an SEM electron gun?
Our Hitachi S-570 SEM has had emission current fluctuations which I traced to the gun cable-gun connection inside the gun. Trouble is, the connections are surrounded by a white, opaque (silicone?) potting compound. When I press down on the potting compound, my emission current troubles disappear, thus I know that the connection is shaky inside.
I'm guessing that I can carefully cut away the old potting compound, expose and repair the cable to gun connections, and finally repot the gun. Not knowing exactly what I am getting into, I thought I would ask for some net wisdom before the first slice.
BTW, the instrument is 9 years old and a new gun and cable assembly runs about $5K from Hitachi. There's my incentive!
Thanks,
Carl
Carl Henderson Electron Microbeam Analysis Laboratory University of Michigan 2005 C.C. Little Bldg. Ann Arbor, MI 48109-1063 USA ----------------------------------------------------- (313) 936-1550 (voice) **** Next time, **** (313) 763-4690 (FAX) *** take the *** chender-at-umich.edu (e-mail) **** train! **** ----} (While you still can!) -----------------------------------------------------
} Does anyone have names and phone numbers for suppliers of standards } for my EDX analysis. I need to know the compositions to see how well } my setup is doing it's work. }
Another source is: Cannon Microprobe 1041 NE 100th St. Seattle, WA 98125 (206) 522-9233 Attn: Bart Cannon
They can make a SEM mount with your choice of standards from a list of hundreds of minerals/materials. The prices are also reasonable.
Disclaimer: I have not purchased the standards, nor do I have any association with Cannon Microprobe. Just passing along some info I recently came across.
Carl Henderson Electron Microbeam Analysis Laboratory University of Michigan 2005 C.C. Little Bldg. Ann Arbor, MI 48109-1063 USA ----------------------------------------------------- (313) 936-1550 (voice) **** Next time, **** (313) 763-4690 (FAX) *** take the *** chender-at-umich.edu (e-mail) **** train! **** -----------------------------------------------------
Thank you for putting forward your comments, so that I may effectively clairify my position.
I strongly feel that University Professional and Scientific staff (P&S) are often overlooked for their contributions to microscopy. Being a part of the University community, I can only speak from that perspective. I do not know how easy or difficult it is for staff from industry or goverment receive support from their employers. I do know that University P & S staff as a rule have very little control over use of grant money for travel, and also have much smaller salaries with which to fund themselves to go to meetings. I do not suggest that MSA pick up the tab for all expenses, but funding from a $10,000 source could go a long way towards improving someone's chances of obtaining matching funds from their employers or other university sources. As an example, there could be twenty-five $400 awards. I may be accused of thinking small, but what I originally had in mind was $2000 total for the program (perhaps five $400 awards) with a simple waiving of registration fees for another 5 people. Why couldn't we try this as apiolet program for one year? If no one applies it hasn't cost MSA a cent.
As Microscopists we cannot afford to be stagnant. There has to be continuing education so that we are able to provide meaningful assistance to our employers, and the many opportunities available at the annual MSA meeting are the best source for this nessecary growth in ourselves.
Return-Path: {dieguez-at-iris1.fae.ub.es} Received: from oxmail.ox.ac.uk by vax.ox.ac.uk (MX V4.1 VAX) with SMTP; Wed, 16 Nov 1994 02:35:10 +0000 Received: from aaem.amc.anl.gov by oxmail.ox.ac.uk with SMTP (PP) id {16027-0-at-oxmail.ox.ac.uk} ; Wed, 16 Nov 1994 02:34:50 +0000 !temhelp {microscopy-at-aaem.amc.anl.gov}
When cutting cryo-sections it is necessary to have an anti-roll plate on the knife. The best probe to use to pick up frozen sections, is an eye lash stuck to an applicator stick. An anti static gun may help to reduce the charge build up in the block-section-knife region. If all else fails read the appropriate chapter in "Low Temperature Microscopy and Analysis" by Patrick Echlin, Published by Plenum in 1992.
Good luck with one of the most frustrating activities in cryomicroscopy
Patrick Echlin Director, Multi-Imaging Laboratory School opf Biological Sciences CambridgeOn Mon, 14 Nov -1 kayton-at-ohsu.edu wrote:
} ------------ Original Message ------------- } } } } Very often when I am cryo-sectioning , the sections curl so badly } that they are useless. This was the reason for purchasing the static-line } ionizer. This really did not help the curling problem, although it served } other purposes. My sections are mostly polymer-elastomer blends, primarily } nylon based. If you have any suggestions I would be eternally greatful. Also } if anyone has suggestions on how to pick up the samples off of the cryo } knife that would also help. Right now we are using a very fine sewing } needle, but something even thinner yet ridgid would be better } } } Thank you } } Andrea Monisera. } --------- End of Original Message --------- } } I have seen the eyelash hairs of Dalmatians (dogs) used for this. they are } forked at the end and work very well for picking up cryosections } } } Bob Kayton } OHSU } Portland , Oregon } } E-mail address kayton-at-ohsu.edu }
Nestor writes: * * The method I now use is to use a dye-sublimination printer * to write directly to "transparency" film. This produces photographic * quality output. Of course this means you need to find someone * with a dye-sub printer. Typical cost/print is about $2-$3 for * an 8x10 transparency. The printers themselves run about $10K. * There is a dye-sub printer (PrimeraPro) available from: Fargo Electronics 7901 Flying Cloud Dr. Eden Prairie, MN 55344 (800) 258-2974 which lists for $1895.00 with 8X10 prints costing about $3-$4 each. There is also a 4X5 format available at a little over $1 per print, you just have to switch the ribbon and feed tray. I have tested this printer for wax thermal transfer, dye-sub prints and dye-sub transparencies. Although it may not be the same quality you will find form the $10K printer, it appears to me to give you } 95% of the performance for less than 20% of the price.
Keith A. Kelley Miles Research Center kelley-at-mrc.com
There is a software package written by David Bright called MacLispX which might be what your thinking about. I've never heard of Isaac. MacLispX is available downloadable from the EMMPDL FTP site (WWW.AMC.ANL.GOV) check out the ANL Shareware/Mac Shareware/Imaging/MacLispX area
For information about the ISSAC system, contact Michael Postek at NIST, Gaithersburg. 301-975-2299
John
} } } There is, I think, a software package from NIST called ISAAC. } It is supposed to handle images with more pixels and more bits } than other programs for acquiring SEM images. } 1 Does anyone know where to get more information on this? } 2 Does anyone have experience using it? } } If you want a giggle, read the piece in New Scientist (November 5 1994, } page 21), which is where I heard about it. } } Alwyn Eades } Alwyn Eades } Center for Microanalysis of Materials } University of Illinois
ABSTRACTS FOR THE JOURNAL OF MICROSCOPY, VOLUME 176 PART 2, NOVEMBER 1994. **************************************************************************
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 85-98.
High performance X-ray detection in a new analytical electron microscope
C E Lyman, J I Goldstein, D B Williams, D W Ackland, S von Harrach, A W Nicholls & P J Statham, Materials Science & Engineering, Lehigh University, Whitaker Laboratory, 5E Packer Avenue, Bethlehem PA 18015-3195, USA
SUMMARY
X-ray detection by energy-dispersive spectrometry in the analytical electron microscope (AEM) is often limited by low collected X-ray intensity (P), modest peak-to-background (P/B) ratios, and limitations on total counting time (t) due to specimen drift and contamination. A new AEM has been designed with maximization of P, P/B and t as the primary considerations. Maximization of P has been accomplished by employing a field- emission electron gun. X-ray detectors with high collection angles, high-speed beam blanking to allow only one photon into the detector at a time, and simultaneous collection from two detectors. P/B has been maximized by reducing extraneous background signals generated at the specimen holder, the polepieces and the detector collimator. The maximum practical t has been increased by reducing specimen contamination and employing electronic drift correction. Performance improvements have been measured using the NIST standard Cr thin film. The 0.3 steradian solid angle of X-ray collection is the highest value available. The beam blanking scheme for X-ray detection provides 3-4 times greater throughput of X-rays at high count rates into a recorded spectrum than normal systems employing pulse-pileup rejection circuits. Simultaneous X-ray collection from two detectors allows the highest X-ray intensity yet recorded to be collected from the NIST Cr thin film. the measured P/B of 6300 is the highest level recorded for an AEM. In addition to collected X-ray intensity (cps/nA) and P/B measured on the standard Cr film, the product of these can be used as a figure- of-merit to evaluate instruments. Estimated minimum mass fraction (MMF) for Cr measured on the standard NIST Cr thin film is also proposed as a figure-of-merit for comparing X-ray detection in AEMs. Determinations here of the MMF of Cr detectable show at least a threefold improvement over previous instruments.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 99- 109.
The preparation of cryosections from plant tissue: an alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals
Dennis B Lazof, Jack G Goldstein, Thomas W Rufty, Cynthia Suggs & Richard W Linton, USDA-ARS, Crops Research Laboratory, PO Box 1168, Oxford NC 27565, USA
SUMMARY
A method involving cryostat sectioning (10 micrometre thickness) and freeze-drying is presented for the preparation of plant tissue for microanalytical studies. The method is well suited for semi-quantitative imaging by secondary ion mass spectrometry (SIMS) and offers significant advantages over bulk freeze-dried or freeze-substitution preparations. Segments of corn or soybean root (5mm) are quench-frozen, embedded externally, sectioned in a cryostat (10 micrometre), pressed onto ultrapure Si and slowly freeze-dried. Images of these sections with secondary electron microscopy and SIMS indicated good morphological preservation. It was possible to section tissues of a wide developmental range, as well as roots varying sixfold in diameter. SIMS images are presented which demonstrate the ability to detect and localize nutrient tracers, such as Rb+, following brief exposures (10 min) to the intact plant. Likewise, a toxic metal (Al) was localized in root tissue after brief exposure ( {1 day) of the intact plant root to micromolar external concentrations. Elemental redistribution during processing was minimal, as demonstrated most explicitly by the lack of movement of loosely bound Ca from the outer cell walls into the adjacent embedding material. Preservation of compositional differences between cellular content and cell wall was supported by a semi-quantitative treatment of SIMS images.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 110- 120.
Vitrification of aqueous suspensions from a controlled environment: an improved plunging device
B J Battersby, J C W Sharp, R I Webb & G T Barnes, Department of Chemistry, The University of Queensland, Queensland 4072, Australia
SUMMARY
In the process of vitrifying aqueous suspensions for cryo- transmission electron microscopy, water is solidified without crystallization. Vitrification can be achieved by rapidly plunging an aqueous thin film into a liquid cryogen. The preparation of aqueous thin films prior to vitrification must be performed in an environmental cabinet at controlled temperature and humidity in order to prevent evaporation and temperature- induced phase changes in the thin film. The device described here incorporates several important features which make the apparatus simpler and more convenient to use than similar devices described in the literature. One of these features includes the use of a totally enclosed environmental cabinet in which the grid, sample, micropipette and absorbent paper are equilibrated before thin- film preparation. Other features include a cryogen dewar on a swing arm for easy refilling, a guillotine shutter which is used to trigger the plunger electrically and a semi-automatic system which facilitates rapid transfer of the vitrified specimen from liquid propane to liquid nitrogen for storage and reduces handling of the specimen. To demonstrate the utility of the device, results showing the influence of temperature on the morphology of phospholipid vesicles are presented. A commercial cryotransfer apparatus (which is used for the transportation of the vitrified specimen to the electron microscope cold stage) has been modified to reduce the possibility of reversion of the vitreous phase to the crystalline ice phases.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 121- 131.
An atomic force microscope for cytological and histological investigations
Tullio Mariani, Antonio Musio, Carlo Frediani, Isabella Sbrana & Cesare Ascoli, Istituto di Biofisica - CNR, Via Lorenzo 26, 56127 Pisa, Italy
SUMMARY
An atomic force microscope (AFM) specifically designed for cytological and histological studies and able to operate on the same scale of the highest optical magnification is described. The AFM is a non-invasive instrument; it operates on samples which do not require any kind of treatment and it can produce information that supplements and completes the information given by traditional microscopical methods. The apparatus has been used to image fixed human chromosomes and to investigate the action of trypsin during the staining for banding. First results showed that banding patterns very similar to G-banding pre-exist to staining and to trypsin treatment in human metaphase chromosomes, and that the trypsin treatment induces a structural collapse in the chromatin. The instrument was also used on thin sections of plant tissue and gave promising results. Experience confirmed that AFM is a suitable tool for this kind of investigation, and proved the importance of developing AFMs specifically designed for routine use in cytology and histology, conceived for non- specialized users.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 132- 142.
Unstained and in vivo fluorescently stained bacterial nucleoids and plasmolysis observed by a new specimen preparation method for high power light microscopy of metabolically active cells
Eduard Kellenberger & Cornelia Kellenberger van der Kamp, Institut de Genetique et Biologie, Microbienne (IBGM), Universite de Lausanne, Rue Cesar Roux 19, CH-1005 Lausanne, Switzerland
SUMMARY
Microscope slides were coated with a layer of gelatin, the thickness of the gelatin increasing linearly along the long axis. The bacterial suspension is applied to the dried gelatin and covered by a coverslip. The medium is absorbed by the gelatin and thus the cells applied against the coverslip. By this method, cultures of concentrations below 100 000 000 cells/ml provide statistically relevant numbers for observation without prior concentration steps. It is easier to apply than the existing methods for the observation of bacterial nucleoids by phase contrast imaging. Because the cells are maintained in growing conditions the method is useful for the vital fluorescence DAPI- staining of various bacterial species and for observations of plasmolysis and its reversal at different physiological conditions and extracellular osmolalities. The previously generally assumed view that the plasmolytic changes of the cell morphology are immediate upon the hyperosmotic shock and are rapidly repaired when the cell is able to metabolize actively was confirmed; this is in contrast to some recent claims.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 143- 151.
Fibre optic scrambling in light microscopy: a computer simulation and analysis
Frederick B Reitz & Len Pagliaro, Center for Bioengineering WD-12, University of Washington, Seattle, Washington 98195, USA
SUMMARY
Optical fibres bent in two mutually perpendicular planes have proven useful for randomizing illumination in light microscopes. These optical scramblers can increase the resolution and/or contrast obtained with several modes of light microscopy. Here computer simulations are used to investigate several factors affecting light randomization in curved optical fibres in order to further the theoretical basis for scrambler design. Light passing through 90 degree bends of optical fibre of varying radii of curvature was modelled by ray tracing in two dimensions, and scrambling mechanisms were observed. The effects of varying the position and angle of entry of light on the phase and direction of propagation of emergent light were determined. It was found that (a) thorough scrambling does not necessarily require high numerical aperture entry of light into the fibre, (b) considerable order persists after a single 90 degree bend of an idealized fibre and (c) a higher degree of scrambling (at the cost of transmission efficiency) is achieved in more tightly curved fibres. The pathlength variations introduced by scrambling proved smaller than typical laser coherence lengths, requiring temporal scrambling (vibrating the fibre).
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 152- 157.
Edge detection, three-dimensional cell boundary reconstruction and volume and surface area estimation from differential interference contrast images
C M Kenyon, M Yanai & P T Macklem, Meakins Christie Laboratories, McGill University, Montreal, Canada H2X 2P2
SUMMARY
Three-dimensional (3-D) cell morphology is important for the understanding of cell function and can be quantified in terms of volume and surface area. Differential interference contrast (DIC, or Nomarski) imaging can enable cell edges to be clearly visualized in unstained tissue due to the slight difference in refractive index between aqueous media and cytoplasm. DIC is affected in only one direction - the direction of the optical shear. A 1-D edge detector was used in that direction with a scale length equal to that of an in-focus edge to highlight cell boundaries. By comparison with the signal from the edge detector on an out-of-focus slice, the in-focus slices could be segmented and, after noise suppression, cell outlines obtained. A voxel paradigm was used to calculate cell volume and differential geometry was used for surface area estimation. We applied this approach to obtain 3-D information by optical sectioning of motile Amoeba proteus.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 158- 166.
Nuclear diffuseness as a measure of texture: definition and application to the computer-assisted diagnosis of parathyroid adenoma and carcinoma
A J Einstein, J Barba, P D Unger & J Gil, The Lillian & Henry M Stratton -, Hans Popper Department of Pathology, Box 1194, One Gustave L Levy Place, New York NY 10029, USA
SUMMARY
A measure of texture, the nuclear diffuseness, was formulated for use in biological classification, and specifically to characterize quantitatively chromatin texture. Nuclear diffuseness corresponds to the amount of local intensity variation in the digitized image of a nuclear profile. As a setting in which to test the efficacy of nuclear diffuseness as a diagnostic tool, the identification of parathyroid adenoma and carcinoma was considered. Digitized images of sections of parathyroid chief cell nuclei were obtained from 16 biopsies, and the nuclear diffuseness, as well as other morphometric descriptors, were computed. With just the average nuclear diffuseness and average nuclear profile area, jackknife (leave- one-out) classification using an artificial neural network was able to diagnose correctly and unambiguously the condition (normal, parathyroid adenoma, or parathyroid carcinoma) in 15 of 16 cases. In one case, the neural network assigned a higher weight to the correct diagnosis, but was unable to distinguish between normal and adenoma conclusively.
Journal of Microscopy, Vol. 176, Pt. 2, November 1994, pp. 167- 177.
Estimation of the transport properties of polymer composites by geodesic propagation
R Bremond, D Jeulin, P Gateau, J Jarrin & G Serpe, Centre de Geostatistique, Ecole Nationale Superiere des Mines, 35 Rue Saint-Honore, F 77305 Fontainebleau, France
SUMMARY
The purpose of this study is to estimate the transport coefficients (diffusion, permeability) of polymer composites from two-dimensional images of these media (polyethylene/polyamide alloys) obtained by the scanning electron microscope. The main characteristics of the media investigated are a wide morphological variability (nodular and lamellar media) and transport properties that are very different according to the phase. The computation tools used must accordingly have a wide range of validity. The idea of the proposed algorithm is to calculate the distance from an edge of the image, with a modified distance according to the phase crossed. The results obtained are compared with those obtained by finite difference integration of Fick's laws, and with permeability measurements of hydrocarbons in the materials.
Tim Prusnick asked in a post to sci.techniques.microscopy about info on EM. Dear Tim, For technical aspects on how EM's work and as a referrence, I reccom- mend Principles of Electron Optics by P.W. Hawkes and E. Kasper from Academic Press (St Edmundsbury Press Ltd. in the UK). Yours, Bill Tivol
Tony Romeo Internet: tony-at-emu.su.oz.au Electron Microscope Unit Madsen Building F09 Telephone: +61 2 351 2351 The University of Sydney Facsimile: +61 2 552 1967 Sydney, NSW 2006 Australia
Tony Romeo Internet: tony-at-emu.su.oz.au Electron Microscope Unit Madsen Building F09 Telephone: +61 2 351 2351 The University of Sydney Facsimile: +61 2 552 1967 Sydney, NSW 2006 Australia
I would to know if there are any training courses specifically for the preparation of biological materials for examination in a SEM and TEM.
Sorry, I cannot be specific about the type of samples, I just need to know about general techniques (embedding, fixing, staining etc).
Are there any held by the RMS for example?
As I am in materials characterisation centre, the staff (including me!) have little experience of the preparation techniques. In particular hands on training would be a must.
Keith Moulding
Materials Characterisation And Preparation Centre, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Our interests are to descriminate the interdiffusion of methacrylate = resins into mineralized substrates using TEM. At present, we are unable = to distinguish resin interdiffusion depth versus epoxy embedding media = following decalicification. Ideas are to find specific stains for the = resins, or, maybe label the methacrylate with a reactive colloidal gold. = Any suggestion on how to descriminate these phases would be appreciated = very much. Help needed,,,thank you
A member of our organization has been asked to make a presentation related to EM in education. Knowing that this is a topic near to the hearts of many on this list, we thought we might ask for some input.
(1) As part of the presentation, we intend to list some of the principal areas where EM has made important contributions. Specifically, we're trying to identify areas where a discovery of large societal significance owes principally to EM. We have a few ideas, but are looking for others. Suggestions ?
(2) We're looking for the names of people who might have had experience in using EM in educational programs (as opposed to research) at the secondary and/or college levels, or have a strong interest in promoting same.
People with suggestions can contact me directly or via this list-server.
Our interests are to descriminate the interdiffusion of methacrylate resins into mineralized substrates using TEM. At present, we are unable = to distinguish resin interdiffusion depth versus epoxy embedding media = following decalicification. Ideas are to find specific stains for the = resins, or, maybe label the methacrylate with a reactive colloidal gold. = Any suggestion on how to descriminate these phases would be appreciated = very much. Help needed,,,thank you
In article YBerta-at-ms-mail.chemse.gatech.edu writes: } } } We need to replace the gold target on our ISI sputter coater; SPI is out of } the targets right now and Electron Microscopy Sciences doesn't carry targets } for ISI. }
Any competent local gold worker can make you a target much cheaper than the specialist suppliers. Many of Sydneys jewellers have manufacturing capacities and can easily make me a target any specified diameter and thickness. Better still, they will accept an old perforated target and add gold to restore a solid sheet. Gold palladium however is too hard and has too high a melting point for local manufacturers to rework.
We just had a 100 lb tensile stage delivered from Ernest Fullam to fit on our Leica/Cambridge S360. Is there any one out there who has one fitted on an S360? We need some feedback on practical fitting. The main problem is the plate that adapts the drive motor to the left side port has no flat ground on its edge to clear the door hinge. Our workshop can easily grind the flat, but I'd like a clue where the (eccentrically located) motor should be located. Looks as if 12 O'clock to 3 O'clock might suit. Any other feedback on operation will be welcome as the manual is a minimalist document.
An oldi, but very nice article is written by Naziri et al. 1972: In situ superplasticity experiments in the 1 million volt electron microscope. J. Microscopy, 97, 229 - 238
Hopes this helps.
Cheers, Timon
} Hello! } } I wonder if anyone can help me: } } I just got the title of a lecture I need to give in } connection to my Ph.D. 'defence' in 14 days..: } } "In Situ Experiments in the Transmission Electron Microscope" } } I have found some references in the ICEM-94 proceedings } and some late numbers of Ultramicroscopy.... } } -Does anyone have some other references? } } -Are you working with this, I am very interested in getting } some ideas!!! } } -What are the main problems? -What can be studied? } } etc... } } You may respond directly to me or through the news-group! } } I am waiting.. } } Randi. } } email: randih-at-imf.unit.no
------------------------------------------------ Timon Fliervoet, Timonf-at-earth.ruu.nl, Faculty of Earth Sciences, Department of Geology, Utrecht University, P.O. Box 80.021 3508 TA Utrecht, the Netherlands. tel: ++31 30 - 535054, fax: ++31 30 - 537725
Randi, A recent issue of the Materials Research Society bulletin (MRS Bulletin,June 1994,Volume XIX, No.6) is devoted almost entirely to materials science research now being done using in situ electron microscopy.
________________________________________________________________ Tom Fellers Florida State University Center for Materials Research and Technology Tallahassee, FL 32306-4000 (904) 644-6559 fellers-at-phy.fsu.edu
Does anyone know of a video board for the Mac which will allow acquisition of the next video field after an external trigger? The boards I've seen (Scion LG-3 and Neotech) will allow display of a single field, but even or odd has to be specified. What I need is a board which will acquire the next field whether it is the even or odd field. I understand the early image grabber boards did work this way. My problem is that I need to acquire an image which is illuminated by a single srobe flash (on the order of 10 nsec duration). Both Illumination and acquisition are triggered from an external source which is not controlled. I've tried synchronizing the strobe to flash twice (once at each video field), but this results in to much motion artifact. If I grab and display an entire video frame, I get an excessive amount of flicker. If I grab an entire frame but display only one field, about half of the time I display non-illuminated images. If I wait for the next even or odd field, I can have up to a 30 msec delay, which is unacceptable.
Any information about video boards or other solutions to this problem will be appreciated. At this point I'm open to almost any suggestion. Thanks.
Dave DeFily defily-at-tamu.edu Medical Physiology, Texas A&M
There is no room on the microscopy forum for crude jokes of this type. It is, in my opinion, very unprofessional to send jokes of this type across a public forum of this calibre. We should try to keep all information and questions related to microscopy and not to jokes that will offend a lot of individuals. Let's try to act professional from now on! Phil 8-{(
I didnot find the joke posted by Calvert either funny or appropriate. He is now history and has been removed from the listserver. I do not want to start a moderated system because I don't have the time, however I will not hesitate to stomp on things like that.
I also agree that those jokes ARE NOT appropriate for this server. They had absolutely nothing to do with microscopy to say the least.
Peling
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
Several years ago, Leitz was purchased by Cambridge, which in turn was purchased by someone else, etc. The company that includes what was Leitz is now called Leica. An address from 1992 is: Leica Inc., 111 Deer Lake Road, Deerfield, Illinois 60015, Tel: 708-405-0123, Fax: 708-405-0147. The world headquarters for microscopes is (again, from some time ago): Leica Mikroskopie und Systeme GmbH, Ernst Leitz-Strasse, P.O. Box 2040, W 6330 Wetzlar 1, Tel: 64 61-29-0, Fax: 64 41-29-33 99. You can probably find a more convenient source in the Washington D.C. area.
--------------------------------
On Mon, 21 Nov 1994, Mark O. Walderhaug wrote:
} Gentle List Readers, } I have recently been given a Leitz Laborlux 11 Microscope. } It presently only has one objective lens and I'm interested } populating the turret with more objectives. I'm having a } hard time finding any information about Leitz. Am I looking } in the wrong place, or has it been swallowed up by another } manufacturer? } Would some kind person point me in the right direction to } find out about getting objectives for this microscope? } Thank you for you patience and kindness. } } Mark O. Walderhaug voice: 202 205-4682 fax: 202 401-7740 } Microbial Ecology Branch HFS-517 Food and Drug Administration } 200 C St. S.W., Washington, DC 20204 USA BITNET: MOW-at-BFD } Internet: mow-at-vm.cfsan.fda.gov or mow-at-bfd.ssw.dhhs.gov }
On Mon, 21 Nov 1994, Mark O. Walderhaug wrote: } Gentle List Readers, } I have recently been given a Leitz Laborlux 11 Microscope. } It presently only has one objective lens and I'm interested } populating the turret with more objectives. I'm having a } hard time finding any information about Leitz. Am I looking } in the wrong place, or has it been swallowed up by another } manufacturer? } Would some kind person point me in the right direction to } find out about getting objectives for this microscope? } Thank you for you patience and kindness.
Gentle List Poster,
Altho' I am not entirely too kind, I will answer your question... Yes, Leitz has been swallowed up "buy" another. Cambridge Instruments and Wild Leitz "merged" in April of 1990. Their new name, at least in the US, is Leica. You may have luck at, according to the letter they sent me:
Leica Inc. 111 Deer Lake Road Deerfield, IL 60015 [may have changed to 60090??? sorry, don't know...] (708) 405-0123 fax: (708) 405-0147 [or: (708) 405-0030???]
The different info within the [..] are from my American Laboratory (February 1994) Buyers' Guide. If anyone desires to subscribe (free to qualified individuals) to American Laboratory, they can be reached at:
Question: is this Laborlux 11 you received a newer or older model of the Laborlux S [1989 model]? Just curious...
You are quite welcome, and hope this helps you. -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /\v/\ | | Metallographic Lab. | Missouri Speleological Survey /\v/\ | | Rolla Research Center | Bat Conservation International /\v/\ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /\v/\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
Two years ago I was fortunate enough to attend the International Conference on Current Trends in Immunomicroscopy at the George Washington University Medical Center, where Dr.Gwen Childs gave a great talk on In Situ Hybridization. At that time her email address was "GCHILDS-at-UTMBEACH". I'm sure she'd be of help. References at that time were:
Childs et al, Molecular Endocrinology 1:926-932 (1987). Hoeffler et al, Histochem J 18:597-604 (1986). Childs et al, Endocrinology 130:335-344 (1992). " " " " 131: (July,1992)
You might need to read this book: "Dynamic Experiments In The Electron Microscope",E.P.Butler,K.F.Hale; Practical Methods in Electron Microscopy Vol 9 (ed. A.M.Glauert); North-Holland P.C.;1981. In general, in situ experiments could reveal oriented phase transition (topotaxy....) and structural disorder (domain, superstructure, modulation..).
Mr-Received: by mta PETVAX.MUAS; Relayed; Tue, 22 Nov 1994 07:15:55 -0400 Mr-Received: by mta PETVAX; Relayed; Tue, 22 Nov 1994 07:15:56 -0400 Mr-Received: by mta SRVR01; Relayed; Tue, 22 Nov 1994 07:16:57 -0400 Disclose-Recipients: prohibited
} Perhaps Mr. Calvert should have spent more time and energy composing his } "real message". } } Perhaps Mr. Calvert simply typed the wrong address for this posting.
Another option might be that he left his machine unattended and it wasn't him at all. Applications such as Eudora do not require a password to _send_ messages (well, it doesn't on our system, anyway) and, theoretically, _anyone_ could send messages purporting to be me (or anyone else).
Thankfully, we don't seem to have anyone that puerile here :-)
If it was indeed an attempt at humour, then it is to be condemned, but the whole thing seems so _unlikely_ that I, at least, am not jumping to hasty conclusions.
Anyway, may as well add another tuppence worth while I'm here...
Concerning in-situ experiments: One suggestion might be to contact Gatan (say) for a brochure of available TEM stages. This may provide a range of heating, cooling, straining, whatever stages with suggested and actual experiments. There are innumerable heating/cooling/straining studies out there.
Slightly more off the beaten track these days - considerable radiation damage research has been performed in-situ: One of the most elegant (and difficult!) being by TJ Black, ML Jenkins, MA Kirk (and possibly IM Robertson, CA English ?) in the early 1980's in which disordered zones created by cascade collapse during ion bombardment were imaged in weak(ish)-beam dark-field using a superlattice reflection of initially-ordered Cu3Au in-situ in the Argonne Tandem/HVEM. This was done using a liquid He stage.
Ahh.. found these refs.. TI: COLLAPSE OF DEFECT CASCADES TO DISLOCATION LOOPS. AU: Kirk_MA Robertson_IM Jenkins_ML English_CA Black_TJ Vetrano_JS JN: Journal of Nuclear Materials 1987 Vol.149 No.1 pp.21-28
TI: DISPLACEMENT CASCADE COLLAPSE IN Cu//3Au AT LOW TEMPERATURES. AU: Black_TJ Jenkins_ML Kirk_MA JN: Institute of Physics Conference Series 1984 No.68 pp.343-346
There will be earlier ones, but BIDS doesn't go back before 1983 and I don't have the relevent file to hand. See the above for refs. I seem to remember there was a Proc. Roy. Soc. article on this (?), and it wouldn't surprise me if there was something in Phil. Mag. A as well...
Hope this helps - you certainly have a large field to cover/select from!
-------------------------------------------------------------------------- Dr. S.L. King Dept. Electronics and Electrical Engineering, University College London Torrington Place, London WC1E 7JE England
We are attempting to remove the aluminum disk from beneath the magnetic layers on a hard disk. Given the problems with polishing aluminum mechanically, the decision was made to try chemical methods first. I realize that there are plenty of sources on chemical thinning and electropolishing, but what I'm looking for is good advice on what will work readily to eliminate 100 microns to 1 mm of alloyed aluminum and leave the NiP and higher layers alone. We have tried heated gallium with no great success (perhaps too low of Al solubility for our mass). Any assistance would be appreciated.
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
Help! We just bought a Sony UPD-7000 dye-sub printer for our computer facility, and don't have the printer driver for the Mac. Does anyone have it and can share, if it's not illegal, or have any suggestions as to where to get it? I've tried all the Sony reps I can find, to no avail. We have the printer, so I think we would have the rights to the driver as well. I'm interested in either a Chooser document or a Photoshop export module. thanks
Judging from the answers posted, it is not clear what type of in situ experiments are in question. I personally posted a book title on immuno-in situ directly to the user asking question. Please be specific when theme is ambigous. Never assume that only users of your own background are reading the message! Thanks.
************************************************************ *Cesar D. Fermin, Ph.D \|*|/ Fax (504) 587-7389 * *Tulane Medical School /|*|\ Answ. Mach.(504) 584-261 * *Pathology/SL79 \|*|/ Secretary (504) 584-2436 * *New Orleans, La 70 112 /|*|\ Lab (504) 5841 * *Fermin-at-TMC.Tulane.edu -} Director of Morphological Services* ************************************************************
I am looking for some advice on methodology to use to study the internal structure of a paperboard laminate which will undergo edge wick, permeation experiments. In this experiment, four fresh cuts are made on a square sheet of a paper laminate which is then placed into an aqueous bath. Theoretically, the fluid should be equally absorbed by the laminate assuming its fiber matrix is randomly oriented -- this does not seem to be the case with one of our samples. We must identify why this one sample performs differntly.
We will use microtomy to prepare cross-sections of this laminate and our FEG SEM to evaluate its internal structural orientation. From my experience working with paper samples, I know the visual artifacts may be subtle without the help of a staining system. Does anyone have any recommendation of a (heavy metal/or other type-cellulosic) stain which could be used to track the degree of permeation either optically or with the the backscattered electron detector on our SEM?
Any other thoughts about evualting the morphology of our laminate would be greatly appreciated.
Happy Holidays,
Dr. Lisa Detter-Hoskin Sr. Research Scientist Materials Analysis Center Georgia Tech Research Institute Atlanta, GA 30332-0827
Message-Id: {m0rA1ZK-0007KIC-at-stjohns.ohsu.edu} Message-Version: 2 } To: Microscopy-at-AAEM.AMC.ANL.GOV
Has anyone else out there had a problem with little bubbles showing up on their 0.5 to 1 micron sections from Spurrs blocks? These are very small, and only appear after I coverslip my slide. The most frustrating characteristic is that the bubbles are not consistantly present. Suggestions are appreciated.
Dear Daniel, Since aluminum is complexed by OH-, a mild base may do the job. I'm not sure what is alloyed with the aluminum on a hard disk, but maybe it could be complexed as well--if it is magnesium, a phosphate will probably do the job. Good luck. Yours, Bill Tivol
We also have been having major problems with holes appearing in our ultrathin sections of Spurr resin embedded specimens (nematodes). We're not sure we've solved this yet but the problem may be due to the resin dissolving material from some plastic containers or if the blocks are polymersied uncovered some of the components volatalise off, resulting in the holes. Has anyone any other suggestions?
____________________ David Wharton Department of Zoology P.O. Box 56 Dunedin Tel (064) (03) 479 7963 Fax (064) (03) 479 7584
I am looking for some advice on methodology to use to study the internal structure of a paperboard laminate which will undergo edge wick, permeation experiments. In this experiment, four fresh cuts are made on a square sheet of a paper laminate which is then placed into an aqueous bath. Theoretically, the fluid should be equally absorbed by the laminate assuming its fiber matrix is randomly oriented -- this does not seem to be the case with one of our samples. We must identify why this one sample performs differntly.
We will use microtomy to prepare cross-sections of this laminate and our FEG SEM to evaluate its internal structural orientation. From my experience working with paper samples, I know the visual artifacts may be subtle without the help of a staining system. Does anyone have any recommendation of a (heavy metal/or other type-cellulosic) stain which could be used to track the degree of permeation either optically or with the the backscattered electron detector on our SEM?
Any other thoughts about evaluating the morphology of our laminate would be greatly appreciated.
Happy Holidays,
Dr. Lisa Detter-Hoskin Sr. Research Scientist Materials Analysis Center Georgia Tech Research Institute Atlanta, GA 30332-0827
} Judging from the answers posted, it is not clear what type of in situ } experiments are in question. I personally posted a book title on immuno-in } situ directly to the user asking question. Please be specific when theme is } ambigous. Never assume that only users of your own background are reading the } message! Thanks.
Agreed, when I first saw the words in-situ I thought of hot stage experiments or on-line inspection. Experiments typical in the materials testing arena or even materials processing. Please provide more detail regarding in-situ experiments, it is a very broad term. . .
P. Joyce
Peter J. Joyce Graduate Research Assistant - Materials Science & Engineering University of Texas at Austin (512) 471-5723
From the other post in this thread, I thought the problem was coverslip media bubbles. I must not have read it carefully enough. My best luck for infiltrating specimens has been to leave the specimens in a 50%ETOH/50% Spurrs mixture overnite. The next day, I change to 100% resin, about four times in eight hours. It doesn't seem to matter how they are polymerized, in Beem capsules that are open or closed. Randy Nessler rnessler-at-emiris.iaf.uiowa.edu
On Wed, 23 Nov 1994, David Wharton wrote:
} We also have been having major problems with holes appearing in our } ultrathin sections of Spurr resin embedded specimens (nematodes). We're not } sure we've solved this yet but the problem may be due to the resin } dissolving material from some plastic containers or if the blocks are } polymersied uncovered some of the components volatalise off, resulting in } the holes. Has anyone any other suggestions? } } } ____________________ } David Wharton } Department of Zoology } P.O. Box 56 } Dunedin } Tel (064) (03) 479 7963 } Fax (064) (03) 479 7584 }
Reply... RE} Coating screens Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
This response is a little late, but we send our screens to the same people that JEOL do (or at least did). I believe the company is: Grant Scientific Corporation Electron Microscopy Products Division 1385 Rock Island Road Gilbert, South Carolina 29054 803-892-2841 Contact: Dunkelberger, Dana We have had good luck with them and get our 2000, 4000, and EM420 screens recoated there. Hope this helps.
} On Tue Nov 22 12:12:01 PST 1994, Robert Kayton,MAC,CROET wrote: } } } Has anyone else out there had a problem with little bubbles showing up on } } their 0.5 to 1 micron sections from Spurrs blocks? These are very small, = and } } only appear after I coverslip my slide. The most frustrating characterist= ic } } is that the bubbles are not consistantly present. =7FSuggestions are } } appreciated. } }
If there are bubbles in the resin from mixing (likely) it is also possible to remove them by centrifuging. This avoids the problems of placing uncured resin in a vacuum system (messy and the hardener generally is more volatile and you may pump sufficient hardener off to prevent full curing).
Norman Elliott | E-mail: nee-at-lanl.gov Los Alamos National Lab | Fax: 505-665-2104 MST-7 MS E549 | Voice: 505-667-1587 Los Alamos, NM 87545 |
-Thank you all for answers conserning in-situ experiments!
I have my background in materials science, and I guess that was what my committee thought about when giving me the title 'in situ experiments in the transmission electron microscope', but it has been interesting to see that 'in situ' can mean different things (hybridisation) as I also saw in my litterature seach.. I need to mention something about this..
It was exactly the point to find out what people thought about when seeing the title above.. (I am free to say whatever I want under the given title, and need to pick out something..)
Due to heavy construction work adjacent to our EM facility, that is expected last for the next three years, I am looking into the possibility of installing vibration isolation platforms for 2 or 3 of our instruments. I am aware of TMC (Technical Manufacturing Corporation, Peabody, MA) that manufactures such platforms. Are there other manufacturers of vibration isolation platforms for EMs? Also, I would be grateful to hear comments from persons who have installed the plaforms in their facilities. Thanks!
My friend and I are trying to do simple indexing of some TEM electron diffraction patterns we obtained from an aluminum alloy. On asking around, we were refered to this group for info on NIHimage software. We are graduate students of physical metallurgy. So, if any good soul out here knows how we can get NIHimage or any other user-friendly and affordable software to help us complete our work, we shall appreciate the assistance.
} We also have been having major problems with holes appearing in our } ultrathin sections of Spurr resin embedded specimens (nematodes). We're not } sure we've solved this yet but the problem may be due to the resin } dissolving material from some plastic containers or if the blocks are } polymersied uncovered some of the components volatalise off, resulting in } the holes. Has anyone any other suggestions? } } } ____________________ } David Wharton } Department of Zoology } P.O. Box 56 } Dunedin } Tel (064) (03) 479 7963 } Fax (064) (03) 479 7584 } Dave: have you tried vacuum infiltration with Spurrs? On difficult to infiltrate specimens, I leave the specimen in 3:1(Spurrs:Propylene Oxide) overnight under vacuum then I leave it in pure resin under vacuum until I get ready to embed it that afternoon. I then cure it in a vacuum oven for 8 hours(I use receipe A in the Spurrs handout for the resin). Hope this helps! Phil
PS: I leave the caps off of my specimen bottles when I leave them in 3:1 overnight.
I am sorry to hear that some people have had trouble with delays in filling their orders for my book 'Vacuum Methods in Electron Microscopy'. I have checked with the publisher, Portland Press, and have been assured that orders placed with the following should be handled promptly: 1. In the U.S.A. and Canada: Portland Press, Ashgate Publishing Co., Old Post Road, Brookfield, VT 05036-9704 Ph: 802-276-3162 Fx: 802-276-3837 2. In the UK and Europe: Portland Press, Commerce Way, Colchester, CO2-8HP, UK Ph: 0206-796351; Fx: 0206-799331 3. In Japan: OBK Marketing Services, T's Building 3F, 1-38-11 Matsubara, Setagaya-ku, Tokyo 156 Ph: 03-5300-1658 Fx: 03-5300-1615
I will be very much interested in having comments on the book from any of you that buy it, to see how well you think I have succeeded in presenting the information electron microscopists need about their vacuum systems. Wilbur C. Bigelow
Back in the 1960s Professor L. O. Brockway and several of his graduate students carried out a series of rather elegant experiments in which they recorded the nucleation and growth of oxide particles on single-crystal thin films of several metals in a heated gas reaction stage inside a JEOL JEM-6A TEM using a cine camera. Here are references to some of their papers: J. Electrochem. Soc. Vol 121, No. 11, Nov. 1974, p. 1534 (1974); ibid, vol. 119, p.899 (1972); "Proc. Symp. Funds. of Gas-Surface Interact", p. 147, Academic Press,(1967); J. Appl. Phys. Vol 37, p. 2703 (1966); ibid, Vol 34, p.921 (1963); "Single Crystal Films" M. H. Francombe & H. Sato, Eds., Macmillan (1964). Good luck with your talk - hope you pass your exam successfully.
Regarding your problem of tiny bubbles in Spurrs.....
You dont say if the bubbles are all over, or only in the blank resin or the tissue....
- the mixing process can introduce bubbles: After mixing the resin, centrifuge it - try 5-10 min at high speed on a table top centifuge using 15 or 50 ml conical tubes, depending on the volume of resin you are preparing
- If the problem is only within the tissue, try infiltrating under vacuum
Studies by transmission electron microscopy certainly made a unique, and extremely important, contribution to elucidating the microstructural changes that occur during the heat treatment of steels. This was a matter of fundamental importance in the field of physical metallurgy, because it served as a foundation for understanding metallurgical heat treating processes in general, and lead to the improvement of these processes and the production of improved metal and alloy products in many instances. Much of this work was carried out by Subcommittee XI, of Committee E4, of the ASTM in the early 1950s, and is summarized by a report by Bill Grube which was published in "Revue Universelle des Mines", Series 9, Vol. XII, No. 10 (1956) and in the "Proc. of the ASTM" Vol. 56 (1956). I have a copy of the first of these articles that I can loan you, if you are interested in seeing it. Wilbur C. Bigelow (bigelow-at-umich.edu)
In response to the apology offered to the listserver by Dave. Apology accepted, at least by me.
Bummer it had to happen to you, but these sort of fopas are a part of the system. And we all need to get used to the limitations of this new form of communication. We all need help SPELLING, and we all say things which may be offensive (at least to some group in our society), and now that our communication is being recorded and disseminated via the information superhighway we better brace ourselves for some high speed cyber-collisions. After all how many of us know enough about this INTERNET system to be confident that Dave was the person who sent the jokes? there are a lot of hidden doors in the NET.
Let's all be a little more understanding of our human counterparts, someday we may ask for the same.
Last night there was a news story on hackers changing a doctors medical records, several PAP smear tests were reversed via the INTERNET, some kind of practical joke (not so funny to those patients "misdiagnosed").
} Due to heavy construction work adjacent to our EM facility, that is } expected last for the next three years, I am looking into the possibility of } installing vibration isolation platforms for 2 or 3 of our instruments. I am } aware of TMC (Technical Manufacturing Corporation, Peabody, MA) that } manufactures such platforms. Are there other manufacturers of vibration } isolation platforms for EMs? Also, I would be grateful to hear comments from } persons who have installed the plaforms in their facilities. Thanks! } M.V.: At one time I had 2 AEI 801 E.M.s and the department decided to redo the department from the ground floor(where I was) up. I put rubber pads with a hardness of 80 durometers under the scopes and had no problem with vibration. They had to jackhammer a pit in a room next to the microscope and I still could use the scope while they worked. Of course, I did this only if it was a diagnostic path. case. For routine research using the EM I used the scope whenever they weren't digging. The noise would drive you batty. So, I found other uses for my time. This may help or you may have a condition where you have to have these platforms. I try the least expensive (the pads were 4"x4" and cost me at the time less than $10.00) way to do things like this first. Good luck! Phil 8-{)
Hi, sorry about the blank post: fat fingers. Here's the real one.
Greetings! =09I am teaching a TEM course this Fall and am having considerable=20 difficulty with two areas:1) the students are finding it very hard to=20 stigmate the objective lens properly, and 2) we are having very poor=20 success with a negative staining protocol using bacteria and T-even=20 bacteriophage. I=D5m hoping that those of you who have taught others to=20 properly align a TEM might send me input on their stigmation=20 technique/instruction. We are using a JEOL JEM 100S, stigmating at 50K=20 (the mini-lens assembly is defective, preventing stigmation at higher=20 mags). The resulting negatives clearly show that the astigmatism has not= =20 been corrected. I have worked individually with the three students and,=20 while I seem to be able to stigmate the lens correctly, they remain very=20 frustrated by their inability to do so. I now wonder if there are other=20 (better) teaching techniques that I might try. In brief, I stigmate the=20 lens using a holey grid with the beam spread to obtain the most coherent=20 illumination, working at the interface between focus and just=20 over-focus. I find the black fringe inside the hole easier to=20 visualize. Once I find the point of focus, I bring the image to just=20 over-focus and determine which of the two (X,Y) stigmators seems to have=20 the greater effect. Then, one at a time, I adjust the stigmators to=20 yield an even fringe within the hole, as I shift back and forth from=20 focus to just over-focus. I encourage the students to be patient and=20 take their time, and to work with the room and panel lights off, so that=20 the image can be viewed distinctly. Any and all comments will be=20 appreciated. =09I won=D5t go into detail regarding the negative staining protocol,=20 as I=D5m not sure that it=D5s necessary or relevant to many of the readers.= =20 However, our problem seems to be that we are not obtaining the negative=20 image that one expects (with either 2% PTA, pH 7, or 2% UA in Bacitracin,= =20 filtered). We have tried freshly prepared solutions, new bacterial=20 cultures, variations in application of the stains (drop vs. flotation)=20 and still seem to have little success. Anyone out there using these or=20 similar techniques on bacteria or virus? Again, I=D5d appreciate comments= =20 from all who have insight or experience with these techniques. I have=20 read the appropriate texts and articles and can=D5t seem to pin down the=20 likely source of our problems. =09Finally, I have been given an Ilford Ilfoprint Mark II Super 12=20 wet print processor and now would like to find a few parts for it. =20 Specifically, chemicals (activator and stabilizer) and the two bottles=20 that function as solution reservoirs. Can=D5t afford to buy the dry to dry= =20 version. Anybody able to point me to a source? Kodak Polycontrast paper= =20 is used for the majority of the prints from our darkroom. =09Thanks for your time and assistance! =09=09Dwight
Dwight Beebe IRBV, Dept. de sciences biologiques=09=09beebed-at-ere.umontreal.ca Universite de Montreal=09=09=09=09Voice:514-872-4563 4101, rue Sherbrooke est=09=09=09FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
} grammatical, and word-omission errors. Not to beat a man while he is } down, but there is no reason not to take advantage of one of the greatest } benefits of writing in a text editor: the chance to view one's work, } read it over, and modify it if necessary for clarity. This is especially } true in a public forum.
Don't forget that while some people might be writing brilliant prose on Windows or Mac machines beautifully ethernetted to the World, others (like me) are struggling with ancient Unix editors (vi, pico) hoping to get a message finished and sent off before the software decides to insert spurious characters or the modem throws a wobbly, kicking us off line, or someone else in the office picks up the 'phone, inserting a series of odd characters which might, or might not, mean something to the computer, including, perhaps, even posting something. I hope no-one judges the contents of the message purely on its presentation - very much a symptom of the eighties and nineties (at least in this country, it seems).
this message is for an annoucement to microscopists in materials sciences, concerning a set of programs, "SHRLI-SIMPLY" ((c) T. EPICIER and M.A. O'KEEFE), running on PCs, and which is available on FTP.UNIV-LYON1.FR, in PUB/DOS/HRTEM (usual login - anonymous - and binary transfer). As can be expected from its title, this package is running SHRLI ((c) M.A. O'Keefe, 1980), which allows HRTEM simulations to be conducted (multislice method, 128x128 FFT). But is allows more than this : building cells /supercells (crystalline or 'non-crystalline' nature), drawing/modifiying structures, calculating 'geometrical' reciprocal lattice sections and/or dynamical diffraction patterns, helping the user to index S.A.D. patterns, plotting Pendellosung curves, calculating CTF, numerical diffractograms (including astigmatism defect), comparing calculated and digitized images,.. Although the programs are running under DOS, a graphical user-interface makes any operation (calculation, screen display, files selections, print,...) very easy. This version of S-SIMPLY is freeware, and has limitations ; however, all options are correctly (hopefully) working. Further information is available in the README.TXT and INSTALL.BAT files, accompanying the SIMPLY1.ZIP and SIMPLY2.ZIP files. Obviously, feel free to contact me for any comments and/or criticisms regarding these programs....
P.S. recent publications illustrating (in a very limited way) the use of S-SIMPLY are : - "Superlubricity of molybdenum disulphide", J.M. MARTIN et al, Phys. Rev. B, 48, 14, 10 583 - "Quantitative HREM analysis of potassium incorporation into cordierite", T. EPICIER et al, Proceed. ICEM 13 (Editions de Physique, Paris), 1994, p. 391 - "Benefits of HREM for the study of metal-ceramic interfaces", T.E. and C. ESNOUF, J. Phys. III France, 4, (1994), 1811. ______________________________________________
Thierry EPICIER GEMPPM-502, INSA de Lyon, 69621 VILLEURBANNE, France tel : (33) 72 43 84 94 (83 85) FAX : (33) 72 43 85 28 Email : EPICIER-at-CISMSUN.UNIV-LYON1.FR
this message is for an annoucement to microscopists in materials sciences, concerning a set of programs, "SHRLI-SIMPLY" ((c) T. EPICIER and M.A. O'KEEFE), running on PCs, and which is available on FTP.UNIV-LYON1.FR, in PUB/DOS/HRTEM (usual login - anonymous - and binary transfer). As can be expected from its title, this package is running SHRLI ((c) M.A. O'Keefe, 1980), which allows HRTEM simulations to be conducted (multislice method, 128x128 FFT). But is allows more than this : building cells /supercells (crystalline or 'non-crystalline' nature), drawing/modifiying structures, calculating 'geometrical' reciprocal lattice sections and/or dynamical diffraction patterns, helping the user to index S.A.D. patterns, plotting Pendellosung curves, calculating CTF, numerical diffractograms (including astigmatism defect), comparing calculated and digitized images,.. Although the programs are running under DOS, a graphical user-interface makes any operation (calculation, screen display, files selections, print,...) very easy. This version of S-SIMPLY is freeware, and has limitations ; however, all options are correctly (hopefully) working. Further information is available in the README.TXT and INSTALL.BAT files, accompanying the SIMPLY1.ZIP and SIMPLY2.ZIP files. Obviously, feel free to contact me for any comments and/or criticisms regarding these programs....
P.S. recent publications illustrating (in a very limited way) the use of S-SIMPLY are : - "Superlubricity of molybdenum disulphide", J.M. MARTIN et al, Phys. Rev. B, 48, 14, 10 583 - "Quantitative HREM analysis of potassium incorporation into cordierite", T. EPICIER et al, Proceed. ICEM 13 (Editions de Physique, Paris), 1994, p. 391 - "Benefits of HREM for the study of metal-ceramic interfaces", T.E. and C. ESNOUF, J. Phys. III France, 4, (1994), 1811. ______________________________________________
Thierry EPICIER GEMPPM-502, INSA de Lyon, 69621 VILLEURBANNE, France tel : (33) 72 43 84 94 (83 85) FAX : (33) 72 43 85 28 Email : EPICIER-at-CISMSUN.UNIV-LYON1.FR
this message is for an annoucement to microscopists in materials sciences, concerning a set of programs, "SHRLI-SIMPLY" ((c) T. EPICIER and M.A. O'KEEFE), running on PCs, and which is available on FTP.UNIV-LYON1.FR, in PUB/DOS/HRTEM (usual login - anonymous - and binary transfer). As can be expected from its title, this package is running SHRLI ((c) M.A. O'Keefe, 1980), which allows HRTEM simulations to be conducted (multislice method, 128x128 FFT). But is allows more than this : building cells /supercells (crystalline or 'non-crystalline' nature), drawing/modifiying structures, calculating 'geometrical' reciprocal lattice sections and/or dynamical diffraction patterns, helping the user to index S.A.D. patterns, plotting Pendellosung curves, calculating CTF, numerical diffractograms (including astigmatism defect), comparing calculated and digitized images,.. Although the programs are running under DOS, a graphical user-interface makes any operation (calculation, screen display, files selections, print,...) very easy. This version of S-SIMPLY is freeware, and has limitations ; however, all options are correctly (hopefully) working. Further information is available in the README.TXT and INSTALL.BAT files, accompanying the SIMPLY1.ZIP and SIMPLY2.ZIP files. Obviously, feel free to contact me for any comments and/or criticisms regarding these programs....
P.S. recent publications illustrating (in a very limited way) the use of S-SIMPLY are : - "Superlubricity of molybdenum disulphide", J.M. MARTIN et al, Phys. Rev. B, 48, 14, 10 583 - "Quantitative HREM analysis of potassium incorporation into cordierite", T. EPICIER et al, Proceed. ICEM 13 (Editions de Physique, Paris), 1994, p. 391 - "Benefits of HREM for the study of metal-ceramic interfaces", T.E. and C. ESNOUF, J. Phys. III France, 4, (1994), 1811. ______________________________________________
Thierry EPICIER GEMPPM-502, INSA de Lyon, 69621 VILLEURBANNE, France tel : (33) 72 43 84 94 (83 85) FAX : (33) 72 43 85 28 Email : EPICIER-at-CISMSUN.UNIV-LYON1.FR
} In response to Dave Calvert's apology, I would to add my two cents about a } common problem in electronic correspondence: lack of editing...
Also, many mail programs have cancellation commands which, if used soon enough (e.g. before the letter gets out of the spool), will allow you to kill the thing before it gets out the door. This won't save you from "next morning regrets," but it can help you out of that feeling you get when you hear the door slam and you realize you've just left your keys in the car...
A comment was recently posted regarding the appropriatness of the listserver as an avenue for a "fast literature review". I just wish to put in my two cents worth on the subject. I think first of all, a few facts about myself are appropriate. I'm 31 years old, and recently received my Doctoate in Germany (6 months ago, to be exact). I am also a real fan of doing proper literature reviews, and when somebody approaches me with a problem, and for one reason or another I know they haven't done a thorough literature review, I get quite upset. This said, I see absolutely nothing wrong with using the listserver as a means of obtaining information for such an exercise. After all, I think the main point behind these comprehensive type examinations is to teach the student how to make best use of his or her time to solve a specific problem on short notice. Therefore, I think the use of the listserver is a very clever idea. It also has obviously brought whole new insights into in-situ experiments. As a materials scientist, I would never have come to the idea that such experiments can also play a significant role in biological research, as has now been pointed out to me. Furthermore, I believe that most scientists, as a result of the changing times, are finding themselves involved in many different projects (upon completion of their university theses). I think very few of us, if any, have the time to do complete literature reviews on all of them, even though all of us would dearly love to such. A number of years ago, this situation might have been different (I admit, I can't say for certain), when scientists at laboratories perhaps had only one or two major projects to work on.
I think it would be very interesting to hear what other scientists with more experience think about this. I would also like to kindly ask Nestor to step in if he believes this is inappropriate for the listserver.
I'm afraid my recent message about "SHRLI-SIMPLY" was sent 3 or 4 times.... My apologize for that, it was not a marketing strategy !!! ______________________________________________
Thierry EPICIER GEMPPM-502, INSA de Lyon, 69621 VILLEURBANNE, France tel : (33) 72 43 84 94 (83 85) FAX : (33) 72 43 85 28 Email : EPICIER-at-CISMSUN.UNIV-LYON1.FR
Message-ID: {MAILQUEUE-101.941126114645.320-at-eye1.eye.ufl.edu} To: microscopy-at-aaem.amc.anl.gov
Dear Phil: We process retinas for histology, TEM, and SEM: frog, chicken, mouse, rat, rabbit, pig, cow and human. We alter our processing techniques slightly for the species and size, and for what we want to end up with (immunolabelling, EDS, straight morphology, etc.). You are welcome to contact me at: eclausnz-at-eye1.eye.ufl.edu Evelyn Clausnitzer U of Florida Ophthalmolgy
Message-ID: {MAILQUEUE-101.941126115206.352-at-eye1.eye.ufl.edu} To: microscopy-at-aaem.amc.anl.gov
Does anyone have a good procedure for the fixation, processing and mounting of whole mammalian retinas? TIA Phil
Dear Phil: We process retinas of several species and for a variety of purposes. Please contact me at eclausnz-at-eye1.eye.ufl.edu for details. Evelyn Clausnitzer U of Florida Ophthalmolgy
I'm looking for anyone that has parts for a Philips 500 series SEM system. I'm in the process of modifying the secondary electron detector system and am looking for as much as possible of a second SEI detector system (grid, scintillator, pmt...) to save the cost of building a complete new detector. If you've got any of these items and no longer need them can you please touch base with me off-line.
} Dwight Beebe } IRBV, Dept. de sciences biologiques=09=09beebed-at-ere.umontreal.ca } Universite de Montreal=09=09=09=09Voice:514-872-4563 } 4101, rue Sherbrooke est=09=09=09FAX:514-872-9406 } Montreal, PQ H1X 2B2 Canada
Hi Dwight. We have a lot of experience in teaching students both microscopy and preparation. First can I suggest you think what is most important for the class you are taking. Virus morphology, preparation or microscope operation? For our virology class, we make a very heavy concentration of T4 phage with a sucrose density gradient centifugation so they deal with a suspension known to have ample virions. (And we check it before hand). We use 1 p.c PTA with bacitracin. I use parlodion filmed grids coated with carbon, then exposed to ion discharge within a few hours of use to guarantee wettability. The students apply the phage suspension to the filmed grids and allow adsorption for say 5 minutes. Then they wash away the sucrose by touching the grid on three drops of distilled water ( 1% sodium acetate if you want to add some ions). Then they add a drop of PTA. For best negative staining they should blot off the stain almost at once as with prolonged contact you get some positive staining as well.
We let each student put in one of their grids and look around. We try to let them experience some of the excitement of seeing something they don't understand first, then understand it by say turning up the magnification or finding a virion. They get prompted with questions like "what do you see? describe it to us? what do you think it is? etc. They take a picture (or two) each. I don't bother getting them to do astigmatism as its only a 4 hour class (all the microscopy you can learn in 4 hours!).
I only teach astigmatism correction when the students are intended to become independent operators. This occurs in one on one coaching in microscope use. I TELL students about astigmatism in introductory lectures but I dont expect them to absorb much except the name and that its an ever attendant problem while focussing. In fact I introduce the stigmators as being additional focus controls for two cylindrical lenses to correct defects in the main lens. I use the model of human astigmatism being corrected with additional lenses.
I gave away using holes for correction about 25 years ago. Even moving from one hole to the next on a grid can change the environment enough to introduce a little astigmatism and I assert that changing grids is not acceptable. Putting in a separate grid was all right when you moved the aperture by thumping the column with a rubber hammer, but not now.
I teach astigmatism correction by observing the structure of the phase granularity you can see near focus. For REALLY GROSS astigmatism (most often introduced by someone giving the knobs a good old wind) I use a bit of the edge of the grid and correct on the fringes there. A TV system on the column is great for demonstrating these effects.
For phase granularity correction; wind the objective focus to and fro and watch the background structure. If it goes from streaks one way to streaks the other, there is astigmatism. Find the objective focus that gives the "best" focus. Use ONE astigmatism control like a focus control; wind it to and fro and find the "best" focus. Repeat with the other astigmatism control. Repeat the whole cycle, starting with the objective until the background structure no longer makes streaks, but makes circles that vary in size as the focus is changed. With experience you can make nearly perfect correction and even recognise a required amount of defocus. At real focus the granularity becomes vanishingly small (phase contrast at a minimum) and everything looks a little fuzzy.
Please send feed back on how useful you find these ideas.
all the best in casting microscopic pearls of wisdom
Practice makes perfect, that is the only recommendation I have for astigmating the microscope. In my many years, I have always prefered using a 1% ammonium molybdate solution for negative staining. It is not a dense as PTA or UA but it seems to be a better "overall" stain. What are your samples in? If in a sucrose gradient (for virus) the sucrose interfers with the negative staining, also are your bacteria in a growth media? After adhereing the samples to a carbon-coated grid, try a brief rinse in dH2O, than stain for 10-15 sec. If lack of sample on grids is the problem, try using a glow discharged grid. No help for you with the print processor parts.
Forwarded message: } From dast-at-msc.cornell.edu Wed Nov 23 18:56:00 1994 } Reply-To: dast-at-msc.cornell.edu } From: dast-at-msc.cornell.edu } Message-Id: {199411232355.AA11766-at-jordi.msc.cornell.edu} } X-Originated-From: jordi.msc.cornell.edu } X-Msc-Version: IDA Client - Sparc } Subject: Re: InGap on GaAs (fwd) } To: hunt-at-msc.cornell.edu } Date: Wed, 23 Nov 1994 18:55:49 +0000 (GMT) } Cc: dieguez-at-iris1.fae.ub.es } In-Reply-To: {199411231425.AA25253-at-borg.msc.cornell.edu} from "hunt-at-msc.cornell.edu" at Nov 23, 94 09:25:08 am } X-Mailer: ELM [version 2.4 PL21] } Mime-Version: 1.0 } Content-Type: text/plain; charset=US-ASCII } Content-Transfer-Encoding: 7bit } Content-Length: 2130 } } Angel, } } See comments below - in CAPS } } } } } Forwarded message: } } From vaytek-at-INS.INFONET.NET Sat Nov 19 05:53:29 1994 } X-Originated-From: lynx.msc.cornell.edu } X-Msc-Version: IDA Server } Date: Fri, 18 Nov 1994 16:47:23 CST } From: vaytek-at-INS.INFONET.NET } To: microscopy-at-aaem.amc.anl.gov } Message-Id: {00987A8C.867A916E.336-at-INS.INFONET.NET} } Subject: InGap on GaAs } } From: MX%"dieguez-at-iris1.fae.ub.es" 16-NOV-1994 18:20:14.00 } To: VAYTEK } CC: } Subj: } } Return-Path: {dieguez-at-iris1.fae.ub.es} } Received: from AAEM.AMC.ANL.GOV by INS.INFONET.NET (MX V4.1 AXP) with SMTP; } Wed, 16 Nov 1994 18:20:11 CST } Date: Wed, 16 Nov 94 07:48:46 +0100 } From: dieguez-at-iris1.fae.ub.es (Angel Dieguez Barrientos) } Message-ID: {9411160648.AA09675-at-iris1.fae.ub.es} } Subsject: CTEM - Need information about lines parallel in a layer } Apparently-To: microscopy-at-aaem.amc.anl.gov } } } My question is about a group of parallel lines that I have } observed in a sample of InGaP on GaAs, where the substrate is } misoriented 10 degrees. } } SOUNDS LIKE (001). USUALLY MISORIENTED TOWARDS 110 BUT NEEDS } TO BE SPECIFIED. } } The lines observed appear more clearly } when I tilt the sample about 30 degrees and I see in the 111 } direction (I am talking about a cross-section). Due that the } thickness of the layer is 2 microns, I think that the lines are } not due to moiree fringes provided for the superposition of the } substrate and the layer. On the other hand, because of the layer } is clearly observable, I think that the lines not correspond } to fresnel fringes due to thickness variations. } If it can help you, this system is ordered by not much (CuPt } structure). The lines are parallel to the substrate in a 011 cross- } -section and tilted about 12 degrees in a 0-11 cross-section. } } } COULD IT BE MISFIT DISLOCATIONS ? WHAT IS THE COMPOSITION OF THE } INGAP ? IS IT A LATTICE MATCHED COMPOSITION ? IF YES, HAS THE } COMPOSITION BEEN CHECKED WITH SOME OTHER METHOD (RBS, KNOWLEDEGABLE } X-RAY ? ) } } } } } } I need your help!!!!. } Please help me!!!!!!. } } Sinceresly yours A. Dieguez. } } } ieter, } I thought you might be able to suggest something. } This is from the microscopy listserver. } John Hunt }
I'd be curious to know how many of you that are experiencing problems with= Spurr's have had these difficulties (or any others) since the additive was= changed in the NSA of American manufacture. You might remember that I= requested help with problems using Quetol 651 last year--it appears now= that the NSA modification is the culprit: I changed to FLUKA NSA and have= just produced blocks identical to what I had prior to June of 1993. I= can/will provide anyone interested in details a complete workup and= explanation as to how I finally solved the mystery. Grace Kennedy UCSD
Dear Dwight, I, too, have trouble with stigmating our HVEM. Some of this comes from lower contrast at higher voltage, and some is just lack of skill. I have found it necessary to shoot sets of photos, first for the coarse adjustment (which usually does not change--unless someone has done something major) and then for the angle and magnitude of the fine. A significant help has been using our crude-but-sensitive video system in conjunction with a small condenser aperture and a large (admits 0.3 nm) objective aperture. Using the small beam gives high coherence--which you also found useful--but the image is too faint to cor- rect the astigmatism from the phosphor screen. Using the large obj. aper. im- proves the contrast without disturbing the fringe (we only get ~0.5 nm resolu- tion, so a 50 mu aper. is big enough for us; your mileage may vary.). Under these conditions, I am able to adjust the stigmator *reasonably* well. Obviously, these procedures might not be appropriate for your instrument and/or purpose. Good luck; I will be interested in any good ideas which come out of this discussion. Yours, Bill Tivol
EMS sent out a letter earlier this year explaining that the formula for the= additive in NSA which keeps Spurr's blocks light colored has been changed. = Althought Stacie K. from EMS swears that the change is quite recent, and my= Quetol problems started in June of 1993, I nevertheless purchased NSA from= FLUKA--My Quetol problem appears to be completely solved. I just ran 4= test blocks with two different accelerators with identical results--perfect= blocks. In the course of the last year and a half, I have tested/run down= virtually everything you can think of to solve this problem, with no= progress until now. I was just wondering if the Spurr's bubble problem= could be related to this change in additive; after all, Spurr's contains a= component remarkable similar in structure to Quetol. Any ideas???? Grace
In addition to Grace Kenndy's comments about NSA, I was wondering how many people are having trouble with section contamination in the last year or so. I've personally been having trouble and no of a few others which have been getting uniform peppering on their sections. The tourble doesn't seem to be the urnayl acetate, nor lead citrate (a variety of recepies and batches yield the same results). But resins using MNA instead of NSA do not seem to have this problem. Could the new NSA formulation be the source of the problem?
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
Teaching them how to stigmate. How ambitious. You seem to be doing the right thing and have the key words,"time" and "patience". Aren't those two of the most important concepts in teaching EM, from embedding and sectioning through photography?
Teaching EM can be frustrating, as it is sometimes difficult to get students out of the mindset that all they need do for a course is read, listen, and write. Now they are in a situation where they have to plan ahead, apply concepts to real situations, and realize that the outcome is dependent on hundreds of techniques, chemicals, physical situations, and equipment conditions, any one of which can be screwed up with or without the students' knowledge.
It is rewarding when students return (as they occasionally do) and acknoledge that later success in graduate school, medical school, and jobs was aided by having taken EM. Some specifically use EM in research and work, while others no longer use it as a technique, but still have improved their situations in less tangible ways because of it.
In recent weeks I've had occasion to find that two of the labs I work with have been using formaldehyde stock solutions that were 3-4 years old. I would like to put together a memo to all the other labs I work with alerting them to the need to use fixatives of a more "recent" vintage. I have looked in several histology texts to see if I can find a guideline for when formaldehyde (either 37% stock, or 10% buffered) become too old to use, but they haven't been much help. Does someone have a reference they can refer me to or could you pass on what your guidelines are?
My experience is primarily in TEM and I know that concentrated glutaraldehyde tends to polymerize over time and we never let our 3% glut. exceed 4 months in age. I've learned that formaldehyde gets more acidic over time, but when is it too far gone?
Thanks for you assistance.
Douglas W. Cromey, M.S. Cell Biology and Anatomy Arizona Health Sciences Center 1501 N. Campbell Ave. Tucson, AZ 85724 (602)626-2824 dcromey-at-ccit.arizona.edu
The same discussion has been going on in our lab with regards to 4% paraformaldehyde. We have noticed a definite change in as little as one week after fix preparation, and marked changes after 2 weeks. Our fixatives are generally kept no longer than 4 weeks. The changes we notice are that with transcardiac perfusion, the body gets very rigid within 5 min. when using fix less than 1 week old. It takes longer for body stifeening after 1 week and athe body remains semi-limp after two weeks, but the brain histology still looks fine after overnight immersion. So it might be that there is some diminuition of fixative strength, but not so much that early postmortem changes are not arrested, and it takes longer for tho9rough fixation to occur. Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Formaldehyde that is prepared from paraformaldehyde powder is normally made fresh just before use since it very poor shelf life. However, it is a relatively pure form of formaldehyde and is used usually with glutaraldehyde as a fixative for EM studies. Commercially available 37% formaldehyde solution on the other hand has impurities including methanol and therefore is not good for EM studies. But it can be readily used for studies at the light microscopy level. It is quite stable compared to the formaldehyde generated from paraformaldehyde powder. I had the opportunity a few years ago to compare results obtained from cells fixed with a new and a 2 year-old stock of the 37% formaldehyde (for the fluorescent-localization of F-actin with Rhodamine/Phalloidin). I could not detect any difference.
Message-Id: {m0rCdbG-0007KOC-at-stjohns.ohsu.edu} Message-Version: 2 } To: Microscopy-at-AAEM.AMC.ANL.GOV
RESEARCH ASSISTANT
Faculty Research Assistant Electron Microscopy-1.0 FTE. Requires BS in biological science (MS preferred),, training in biological lab instrumentation and protocol. Experience in EM tissue preparation is required. Responsible for histotechniques. Send Letter, curriculum vitae and names, addresses, and phone numbers of three references to :
Benita J. Pinz, Executive Assistant College of Veterinary Medicine OSU Corvallis, OR 97331-4801
Whenever possible we only make enough fixative for that day. That goes for glut., paraformaldehyde or glut/para mixes. If we need to store it before use we keep it frozen. We have not done any longevity test but it seems to be good at least one month. We usually use it up before any longer time. We do the same with 8% glut vials taht we have opened and not used completely. We seal the top with parafilm and then place the vial in a screw top jar with a good seal.
} Whenever possible we only make enough fixative for that day. That goes for } glut., paraformaldehyde or glut/para mixes. If we need to store it before use } we keep it frozen. Greg, You keep the glutaraldehyde frozen? I had understood that that was inappropriate, but I would be happy to be convinced otherwise. I've not researched this but I also have never run into a lab where this is done. I do TEM on a very infrequent basis, but often need to do fixation on very short notice. So, any improvement in shelf-life of reagents would be a real boon to me. Another win for the "net"! :-) --Jon
Jon L. Norenburg {norenbur-at-onyx.si.edu} Invertebrate Zoology, National Museum of Natural History Smithsonian Institution, Washington, DC 20560 Voice 301-238-3508, Fax 301-238-3361
To members interested in fixatives, formaldehyde solution in particular.
Here are some additional informations about storage of commercial form- aldehyde. The principal changes which may take place in formaldehyde on storage are as follows (listed in their order of importance from a practical standpoint): (1) Polymerisation and precipitation of polymer. (2) The Cannizzaro reaction, involving oxidation of one molecule of form- aldehyde to formic acid and reduction of another to methanol. (3) Methylal formation. (4) Oxidation to formic acid. (5) Condensation to hydroxyaldehydes and sugars. The changes are detrimental to product quality but may be avoided or kept at a minimum by maintenance of proper storage conditions. With optimum conditions of storage, commercial formaldehyde will remain unimpaired for long periods of time. In general, proper storage involves avoidance of temperature extremes and the use of storage in glass bottles, inert to corrosion by the mildly acidic solution. Low temperature favor polymer precipitation, high temperatures accelerate the reaction leading to chemical loss of formaldehyde. At improper storage temperatures, a form- aldehyde solution gradually becomes cloudy and eventually solid hydrated polymer separates as a precipitate. Much more could be said about this highly interesting fixative which possesses many unusual characteristics. ---------------------------------------------- * Sverker Enestr|m * * Department of Pathology, Link|ping, Sweden * ----------------------------------------------
Since an aqueous solution of formaldehyde (even made fresh from paraformaldehyde) exists as an equilibrium between methylene glycol and formaldehyde (skewed heavily towards the m.g.), covalent linkage of formaldehyde to tissue molecules is very slow , reaching equilibrium in 16 h -at-37 C (eg., reviewed in Fox et al. J Histo Cyto 33:845-853, 1985). This argues that BRIEF fixation in formaldehyde (as in immuocytochemistry preps) induces artifacts because of the high concentration of methylene glycol, and that little actual crosslinking occurs. Why use formaldehyde as a fixative for EM?
(((((((((((((((((((((((((((((())))))))))))))))))))))))))))) R. Howard Berg Biology Department University of Memphis, Memphis, TN, 38152
If you are really interested in reading about fixatives and how they work there is a chapter devoted to this in the book by G. Griffiths. The ref. is "Fine Structure Immunocytochemistry" 1993 published by Springer Verlag, Heidelberg.
Let me add a note for those workinf on mineralized tissues after formaldehyde fixation. Small larval bivalve shells (80 to 250 mis microns) dissiolve quite ras rapidly in even buffered formaldehyde 10% solution. If fixation is required in aldehyde, use glutaraldehyde by preference, or use formaldehyde the minimum time and replace with 70-95% ethanol. or better yet (if only mineralized material will be studied, use 95% ethanol as sole fixative. Shells last for years o in ethanol alan pooley marine sci sem lab rutgers univ
I have an enquiry about any paper published on virus ultrastructure which uses FESEM images. I've checked the obvious Journals but one of you out there might be able to drop a note with a reference for me. No. I'm not writing a thesis (term paper, literature review). Thanks,
as an additional measure to all those already mentioned ;-) for keeping formaldehyde solutions as long as possible, I seem to remember that, before those days when we began preparing our formaldehyde immediately before use, we kept it in bottles with a lot of calcium carbonate on the bottom. This took acidification into account, but did not avoid polymerisation, of course ;-) HTH John
*********************************************************** * Jean C. Desclin (John), Associate Prof. of Histology * * Laboratory of Histology - Faculty of Medicine * * Brussels Free University (U.L.B.) * * e-mail: desclinj-at-ulb.ac.be (internet) * * snail mail: route de Lennik 808 * * B - 1070 Brussels Belgium * ***********************************************************
Hi, You may add to the in-situ experiment list some work we did here:
Work from M. Flueli thesis HREM dynamic observation of gold nanocrystals (same as J.O. Bovin, D. Smith,...) HREM dynamic observation of sintering of gold nanocrystals induced at room temperature by the electron beam (M. Flueli et al., Surf. Sci. 202 (1988) 343-353)
Work from D. Ugarte HREM in situ transformation of fullerenes into bucky onions (MRS Bull. XIX no 11 nov. 1994,39-42; Europhys. Lett. 22 (1993) 45-50
If there is some interest, I can provide a video movie (about 10 min. at total for these 3 items, at present with the european PAL standard, but NTSC may be available too).
B. Hall and D. Reinhart do electron diffraction of silver clusters produced in a supersonic He or Ar stream. They showed the co-existence in various concentrations of fcc cuboctaheras, decahedras and icosahedras. (B.D. Hall et al, Phys. Rev. B43 (1991) 3906-..., B.D. Hall et al., Rev. Sci. Instrum. 62 (1991) 1481-1488, B.D. Hall et al., Z. Phys. D20 (1991) 457-... and D26 (1993) S73-...)
There are also two others teams using in-situ in Lausanne (conventional TEM). One (R. Gotthardt) in the field of in-situ straining of metals at room, low or high temperature. The second one (G. Gagnon) has a video movie of thermal fatigue, up to crack generation, for an Al based alloy reinforced by oxides particles. I don't have the details right now. If these authors have not answered to your call themselves, I am ready to find more if anybody is interested.
Yours
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Centre Interdepartemental de Microscopie Electronique Address: EPFL-CIME, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 Fax: +41(21)693 44 01 (Central European Time) E-mail: philippe.buffat-at-cime.uhd.epfl.ch // pabuffat-at-cimesg1.epfl.ch ______________________________ Eudora F2.1 ___________________________
Hi, You may add to the in-situ experiment list some work we did here:
Work from M. Flueli thesis HREM dynamic observation of gold nanocrystals (same as J.O. Bovin, D. Smith,...) HREM dynamic observation of sintering of gold nanocrystals induced at room temperature by the electron beam (M. Flueli et al., Surf. Sci. 202 (1988) 343-353)
Work from D. Ugarte HREM in situ transformation of fullerenes into bucky onions (MRS Bull. XIX no 11 nov. 1994,39-42; Europhys. Lett. 22 (1993) 45-50
If there is some interest, I can provide a video movie (about 10 min. at total for these 3 items, at present with the european PAL standard, but NTSC may be available too).
B. Hall and D. Reinhart do electron diffraction of silver clusters produced in a supersonic He or Ar stream. They showed the co-existence in various concentrations of fcc cuboctaheras, decahedras and icosahedras. (B.D. Hall et al, Phys. Rev. B43 (1991) 3906-..., B.D. Hall et al., Rev. Sci. Instrum. 62 (1991) 1481-1488, B.D. Hall et al., Z. Phys. D20 (1991) 457-... and D26 (1993) S73-...)
There are also two others teams using in-situ in Lausanne (conventional TEM). One (R. Gotthardt) in the field of in-situ straining of metals at room, low or high temperature. The second one (G. Gagnon) has a video movie of thermal fatigue, up to crack generation, for an Al based alloy reinforced by oxides particles. I don't have the details right now. If these authors have not answered to your call themselves, I am ready to find more if anybody is interested.
Yours
__________________________________________________________________ Philippe Buffat Ecole Polytechnique Federale de Lausanne (EPFL) Centre Interdepartemental de Microscopie Electronique Address: EPFL-CIME, Batiment MX-C, CH-1015 Lausanne, Switzerland Phone: +41(21)693 29 83 Fax: +41(21)693 44 01 (Central European Time) E-mail: philippe.buffat-at-cime.uhd.epfl.ch // pabuffat-at-cimesg1.epfl.ch ______________________________ Eudora F2.1 ___________________________
} Greetings from downunder } } I have an enquiry about any paper published on virus ultrastructure which uses } FESEM images. I've checked the obvious Journals but one of you out there } might be able to drop a note with a reference for me. No. I'm not writing a } thesis (term paper, literature review). Thanks, } } Mel Dickson } m.dickson-at-unsw.edu.au } fax +612-385-1067
We got some very promising results for using FESEM to image of the reovirus.
Centonze, V.E., Chen, Y., Borisy, G.G. and Nibert, M.L. (1994) High-resolution imaging of reovirus particles by cryo-scanning electron microscopy: steps in virus disassembly visualized without image refinement. Science (Submitted)
Chen, Y., Centonze, V.E., Nibert, M.L. and Borisy, G.G. (1994) Cryo-Scanning electron microscopy of reovirus structure. In: 52th Ann. Meet. MSA (eds. by G.W. Bailey and A.J. Garrett-Reed). pp. 134-135. San Francisco Press, San Francisco.
Chen, Y., Centonze, V.E., Verkhovsky, A., Nibert, M.L. and Borisy, G.G. (1994) Macromolecular imaging of cytoskeletal elements and reovirus by ultra-high resolution cryoscanning electron microscopy. In: Electron Microscopy 1994--XIIIth International Congress on Electron Microscopy (eds. by B. Jouffrey and C. Colliex). pp. 25-26. Les Editiond de Physique, Les Ulis.
Ya Chen Integrated Microscopy Resource University of Wisconsin 608-263-8481
I'm weeding my way through the equations used in x-ray analysis. In particular I was reading Reed's "Electron Microprobe Analysis, Second Ed." and I have run into a problem. Chapter 13, X-ray generation and stopping power, discusses ionisation cross-section with refence to an article by Powell, C.J. (1990) Microbeam Analysis-1990 p.13.
Unfortunately I have no access to this reference except through "Inter-Library Loans" (possibly taking 2 months). I was wondering if someone could spare the few minutes it would take to fax me this article. Please contact me directly at henne-at-sfu.ca so I can give you my fax number.
Thanks for your time. Dan Henne Simon Fraser University Sunny Vancouver Canada henne-at-sfu.ca
I am looking for advice on the purchase of a critical point dryer. We are interested in preparing platelets and Staph. Epi. for high res. EM imaging. Any comments you might have on generally salient issues or particular machines and their strengths and weaknesses would be much appreciated.
Thanks Steven J. Eppell Facility Coordinator Center for Cardiovascular Biomaterials Case Western Reserve University sje-at-po.cwru.edu
Dysktra in his book on EM tech advocated a plain 4:1 formaldehyde:glut fixative for TEM, using commercial formaldehyde. His TEMs of kidney (?) looked fine, even the specimens that were months old before embedding. Anyone else tried his recipe? Phil Oshel poshel-at-luc.edu
Thanks for the many responses to my initial posting Re: Fixative Quality Control. I've done some more reading, called a few suppliers and incorporated the responses from the microscopy listserver to create a memo for the labs I work with. A copy follows for those who are interested: ************************************************************************** We have been doing some research about the shelf life of 37% Formaldehyde. There is no definitive age after which 37% Formaldehyde is no longer useful as a stock solution. Formaldehyde chemistry is moderately complex, but after discussions with other microscopists, manufacturers and reviewing pertinent texts, the following observations are applicable. Formaldehyde should be stored at room temperature, cold temperatures encourage the formation of trioxymethylene with a resulting white precipitate. Formaldehyde should be stored tightly sealed, since exposure to air encourages the oxidation of formaldehyde to formic acid (37% formaldehyde is usually shipped with 10-15% methanol to inhibit this change). Our recommendation is, if the 37% formaldehyde solution is clear, colorless and has no precipitate, and has been stored at room temperature in a tightly sealed bottle that has not been exposed to sunlight, it should be good, however, we still do not recommend using a stock bottle that is older than 1 year, bottles that are already opened should not be used more than 6 months. Consequently, we recommend that labs purchase their formaldehyde more frequently and in smaller quantities than perhaps they have done in the past. ***************************************************************************** There's more, but this should suffice. The only additional comment is that 37% formaldehyde is not recommended for EM work and that a higher grade "methanol-free" formaldehyde or a solution made from paraformaldehyde should be used instead.
Thanks for your help:-{)
Douglas W. Cromey, M.S. Cell Biology and Anatomy Arizona Health Sciences Center 1501 N. Campbell Ave. Tucson, AZ 85724 (602)626-2824 dcromey-at-ccit.arizona.edu
I would suggest a used Reichert Ultracut E, this machine is proven (bullet proof).it cuts great sections, does well with cryo attachments, and can be cleaned and maintained easily without messing up any internal microciroelectronics. No I don't have one to give or sell I wish I still had one. PS- stay away from the RMC 6000. (you've been warned) the RMC 7000 is a far better machine, and is worth consideration, test it against a REichert. -Mike Rock
On Thu, 1 Dec 1994, Todd Voiles wrote:
} } We're looking for both advice and information on the purchase } (new or used) of an ultra-microtome for use in our EM facility } } Does anybody have a used one they want to sell/give us? } } Any suggestions on the type that is the best or of reputable companies } and/or sources? } } Thanks } } Center for Materials Research and Analysis } Central Facility for Electron Microscopy } University of Nebraska at Lincoln } } Todd Voiles } tvoiles-at-unlinfo.unl.edu } }
If, in your inquiries, you find that you have any use for a microtome (not ultra-microtome), we have an unused Reichert-Jung Histocut 820-II which we would part with at a very reasonable price.
Arthur Gillman Princeton, NJ
Todd Voiles wrote:
} We're looking for both advice and information on the purchase } (new or used) of an ultra-microtome for use in our EM facility } } Does anybody have a used one they want to sell/give us? } } Any suggestions on the type that is the best or of reputable companies } and/or sources? } } Thanks } } Center for Materials Research and Analysis } Central Facility for Electron Microscopy } University of Nebraska at Lincoln } } Todd Voiles
One fixative that really works much better than it should is McDowall- Trumps, which is a modified Karnovsky-type fix. It is prepared from bulk (reagent grade) glut and commercial formalin in a phosphate buffer. Its popular in clinical laboratories where much of the tissue received is pathological or from necropsy. People around here like it because its cheap (a couple of dollars per gallon) and is stable for months.
I personally like fixatives that incorporate picric acid, for its ability to retain proteins. Picric acid is not a fixative per se, but acts by precipitating proteins, rendering them insoluble. We routinely use 2% GTA- 2% formaldehyde-0.5% picric acid in cacodylate buffer. It produces good contrast in just about everything vertebrate. This "yellow" fix is stable for at least 2 months.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
When we were looking at ultramicrotomes about 5 years ago, we evaluated the RMC MT-7 and the Reichert Ultracut E. Both of these machines were very impressive. Since neither of them are the most recent models, you may be able to find a used machine, but it won't be easy.
Early in 1995 we will have one or two positions available for postdocs with TEM and/or STEM experience on semiconductors. Most of the work is on III-V systems (GaAs and InGaAs). For more details contact me direct.
Peter Goodhew goodhew-at-liv.ac.uk ---------------------------------------------------------------------------------------------------------- Professor Peter J Goodhew, Department of Materials Science & Engineering University of Liverpool LIVERPOOL Fax (44) (0)51 794 4675 L69 3BX, UK Tel (44) (0)51 794 4665 (secretary Debra) ---------------------------------------------------------------------------------------------------------- inter alia: Director of the MATTER project for educational software ----------------------------------------------------------------------------------------------------------
Does anyone have a FC4S cryomicrotomy unit for use with an Ultracut E microtome that they would like to sell? If so, please call (314-577-8480) or e-mail me back and I will contact you. Thanks Jan Ryerse, St. Louis University Health Science Center.
Isn't picric acid dangerous? What precautions do you take??On Fri, 2 Dec 1994, W.L. Steffens wrote:
} One fixative that really works much better than it should is McDowall- } Trumps, which is a modified Karnovsky-type fix. It is prepared from bulk } (reagent grade) glut and commercial formalin in a phosphate buffer. Its } popular in clinical laboratories where much of the tissue received is } pathological or from necropsy. People around here like it because its } cheap (a couple of dollars per gallon) and is stable for months. } } I personally like fixatives that incorporate picric acid, for its ability } to retain proteins. Picric acid is not a fixative per se, but acts by } precipitating proteins, rendering them insoluble. We routinely use 2% GTA- } 2% formaldehyde-0.5% picric acid in cacodylate buffer. It produces good } contrast in just about everything vertebrate. This "yellow" fix is stable } for at least 2 months. } } -=W.L. Steffens=- } College of Veterinary Medicine } University of Georgia }
} Date sent: Sat, 3 Dec 1994 15:14:59 -0600 (CST) } From: Joyce Craig {bafpjec-at-uxa.ecn.bgu.edu} } Send reply to: Joyce Craig {bafpjec-at-uxa.ecn.bgu.edu} } Subject: Re: fixatives } To: "W.L. Steffens" {STEFFENS.B-at-calc.vet.uga.edu} } Copies to: Microscopy-at-aaem.amc.anl.gov
} Isn't picric acid dangerous? What precautions do you take??On Fri, 2 Dec } 1994, W.L. Steffens wrote:
Picric acid (trinitrophenol) has similar chemical properties to its cousin, trinitrotoluene (TNT). It is only dangerous when dry. In labs that use it in fixatives, it is made up as a saturated aqueous solution stock solution...there is no reason to keep the dry powder around. If you do have stocks of the powder, keep a cm or so of water in the bottle, and avoid getting it on the threads.
Picric acid is commonly used in histology laboratories, as it is a key ingredient in the popular Bouins fixative. I've never known anyone who has had problems with it, other than trying to ship it in the mail.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
I'm of the opinion that most critical point dryers are overloaded with bells and whistles, and have too little sample capacity (and cost too much). The one I usually tell people to look at is the Polaron. It's a good sized cylinder with inlets & outlets for water & CO2, and a couple of gauges (and a safety valve). Very simple, sturdy, usuable by anyone and reliable. The problem is finding the supplier--the way companies have been going out of business, being bought up, etc.. I *think* that it's now carried by Energy Beam Sciences, but.... maybe Ted Pella? (I forget the model name/#, but there was only one like it. No electroincs or electric anything--heating & cooling are done by water from the hot & cold taps.) Phil Oshel poshel -at-luc.edu
In message {see2d46a.052-at-wpo.it.luc.edu} Philip Oshel writes: } Steven Eppell, } } I'm of the opinion that most critical point dryers are overloaded with bells } and whistles, and have too little sample capacity (and cost too much)......
} ........No electronics or electric anything--heating & cooling are done by } water from the hot & cold taps.)
I've already replied to Steven Eppell's query on CPD's privately, but I wanted to respond on the net to Philip Oshels recent comments:
While I generally agree with Philip that the fewer bells 'n whistles on a CPD the better, I do like the electric heating feature of my Ladd CPD (Ladd Reasearch Industries, Burlington Vermont, 802-658-4961); no hot water lines to hook up, no drain needed. Twelve years ago I had a Bomar brand CPD (no longer in business) and it was heated and cooled by tap water. In the summer months, I could not get cold enough water for cooling so I had to rig up circulation through a mixture of ice and water; I built an accessory plumbing unit with 4 valves for mixing, switching hot water, cool and ice water etc, etc. It was a lot of fun to operate and even more fun to teach to others to operate.
The Ladd unit cools by expanding CO2 gas into and quickly out of the chamber, the ol' Joule-Thompson effect in action, so its clean, no extra plumbing, and makes a pleasant gurgling sound which sooths a troubled mind.
Its sample chamber is 1-3/8 inches diameter, 3-3/8 inches deep which is quite large enough for our needs here and it will hold a lot of sample baskets of various sizes.
The January 1993 list price was $3995.00.
Another lab nearby has a totally automatic CPD and they have had occasional problems with the auto-controls from time to time. In 12 years of running my manual Ladd unit, no reapairs of any kind needed to date. However, if your lab needs a CPD running very frequently, perhaps the extra expense and occasional repair is worth it.
Disclaimer: I'm not associated with Ladd in any way, other thatn being a satisfied user of their CPD.
--
Gib Ahlstrand, MMS Newsletter Editor Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.crl.umn.edu
Message-Id: {MAILQUEUE-101.941205093220.352-at-vanlab.paprican.ca} To: bafpjec-at-uxa.ecn.bgu.edu
I would just like to throw in a little more information regarding picric acid. It can be used safely so long as you are aware of the hazards. My (quite old) copy of "Manual of hazardous chemical reactions" from the NFPA has entered under Picric Acid:
Picric acid and bases form explosive salts. The salts with heavy metals are very sensitive to primary explosives.
Contact between picric acid and concrete floors leads to the formation of more explosion-sensitive salts, such as calcium picrate.
Cheers, Laurie
} Isn't picric acid dangerous? What precautions do you take??On Fri, 2 Dec } 1994, W.L. Steffens wrote: }
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
} I've been using that mixture (McDowall-Trumps) and as you sais it is } an excellent fixative. Could you send me the original reference, please? } Thanks in advance
I consider it a good general purpose fix and a starting point. I believe that optimal results can only be attained by experimentation. Its supposed to be a hyper-osmotic fix (about 1100 mOsmol) but much of this is contributed by the formaldehyde, which does not excert an effective osmotic pressure. The effects of it suggest that it is hypoosmotic, evidenced by the tendency for mitochondria and lysosomes to swell.
Original reference:
McDowell, E.M., and B.F. Trump. 1976. Histologic fixation suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med. 100: 404 - 414.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
Aloha microscopists, We are trying to fix dissociated Xenopus muscle cells, growing on glass coverslips, for antibody labelling and high res SEM. Unfortunately, the cell membranes are blebbing a few minutes after fixative is applied. We are trying a light pre-fix in paraformaldehyde or para/glutaraldehyde, followed by ab labelling, then we can postfix in a higher concentration of glut and osmium. Except for the first time (beginner's luck) everything has caused blebbing. Does anyone have any suggestions? Thanks in advance!
Aloha kakou,
Tina Weatherby Carvalho Biological EM Facility University of Hawaii tina-at-ahi.pbrc.hawaii.edu
Tina Carvalho in Hawaii asks about alternative fixatives to avoid membrane blebbing. Try the recipe in J. Histochem. Cytochem. 37:75-82, 1989, by Luther and Bloch. It might help.
**************************************************** * MICROSCOPY OF SEMICONDUCTING MATERIALS * * * * 20-23 MARCH 1995 * * * * University of Oxford * **************************************************** This conference will focus on the latest developments in the study of the structural and electronic properties of semiconducting materials. Main topic areas are: (1) Characterization of as-grown semiconductors (2) Investigation of lattice defect and impurity behavior (3) Study of the effects of semiconductor processing treatments (4) Assessment of finished electronic devices.
Special conference session on use of HRTEM, nature of epitaxial layers, metal-semiconductor contacts, exploitation of advanced scanning techniques.
The meeting is sponsored by the Institute of Physics, London (e-mail IOPCONF-at-ulcc.ac.uk.) and organized by Dr. A G Cullis and Dr.Anne Staton-Bevan.
DEADLINE FOR THE SUBMISSION OF ABSTRACTS IS DECEMBER 1 BUT LATE PAPERS WILL BE ACCEPTED UP TO DEC.20TH. IF YOU WISH TO SEND AN ABSTRACT NOW PLEASE ALSO FAX A COPY TO Dr.A G Cullis at FAX +44 684-894311
We have a project, looking at morphology of red cells in patients with sepsis. The cells need to be studied with SEM (normal and ultrahigh resolution) as well as TEM. We do not need to fix the samples to retain immunocytochemical characteristics. Some of the published hypo- and hyper- osmotic fixatives that we have tried do not really preserve the shape of the red cells very well. Someone with experience of this type of preparation - please help. We need a fixative that will cause the least possible amount of morphological changes such as swelling or crenation. Due to transport requirements, samples need to be left in the fixative for a few hours at least. At present we are using 2.5% phospate buffered glut with added NaCl (0.075M phosphate, pH 7.4 and 0.075M NaCl) and this seems to be the best we have tried to date. This fixative is close to being iso-osmolar to normal serum if you disregard the contribution of the Glutaraldehyde.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Has anyone heard of a new MSDS coming out for Toludine Blue that says it is now for hood use only because it has been found to be cancer causing? A friend of mine who is an EM Tech at University of Chicago informed me that he recieved the new MSDS, but it has not found its way to my lab yet.
Any information is appreciated.
Thanks,
Dennis Shubitowski Michigan Diabetes Research and Training Center
I am interested in what protocol people are using to en bloc stain with UrAc. Specifically, what buffer, pH, concentration, duration and stage. Do Uranyl acetate solutions go bad? John Johnson started a thread touching on this a while back but the responses were mostly how to avoid or remove pepper staining artifacts. TIA.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
We use a saturated solution in dH2O, pH'ed to 3.3. Tissue pieces are keeped in solution for 1 hour after the osmimium, then start with ethanol dehydration after UA.
I just returned from the Dominican Republic (Nov. 12-20, 1994) where I lectured at the Pontificia Univ. Catolica M & M. There I met Dr. Andres Peralta professor and Director of the International Programs at the Univ. Dr. Peralta founded and directs the Oncologic Regional Cibao Center. On the list of wanted equipment is an EM and assorted lab. equipment. The center is the only non-profit hospital in the Dom. Rep. offering free consultation and cancer therapy. The center is partially furnished by donations from the USA and CANADA: Beds and X-rays donated by the Santiago Cancer Foundation's branch of MIAMI; Operating table and lights donated by Hospital of Saint George Quebec; Radiotherapy Cobalt 60 unit donated by Misericordia Hospital USA, and Puerto Rico. Please contact me if you know of any surpplus hopital equipment they can use. I was very impressed by Dr. Peralta, and must say that besides the nuns I mentioned before, he is a Dominican I trust. You can either deal with the foundation directly or through me. The foundation will pay for packing, transportation and custums the Dominican Republic. The irony is that I know of their needs after giving away a Philips 301 for parts just months ago.
************************************************************ *Cesar D. Fermin, Ph.D \|*|/ Fax (504) 587-7389 * *Tulane Medical School /|*|\ Answ. Mach.(504) 584-261 * *Pathology/SL79 \|*|/ Secretary (504) 584-2436 * *New Orleans, La 70 112 /|*|\ Lab (504) 5841 * *Fermin-at-TMC.Tulane.edu -} Director of Morphological Services* ************************************************************
I have been using 0.5% UAc in dH2O following the rinse step after the OsO4 for 10- 18 hours (i.e. overnight - provides a nice break for day one), followed by 4x rinse with dH2O -at- 10-15 min.. Advantages of this are: you don't have to worry about UAc crystals (Saturation is approx. 3.5-4%), I also use this as a post sectioning stain, and it provides a good break step for things like dinner and sleep!
I have heard that UAc is light sensitive, but I'm unsure of this and I've never had any problems with storing it in clear glass.
I have discovered that UAc in acetone does breakdown - I have no idea to what but after 3 months there where some very beautiful dark golden brown crystals which had grown...if I could only figure out how to get them out of the vial and into the SEM.....
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
The December 1994 issue of the Journal of Microscopy is a special issue featuring papers presented at the meeting on Confocal and Near-Field Microscopy and Three- Dimensional Image Processing in Microscopy, held in Munich, Germany, on the 25 - 28 April 1994.
Contents
In situ analysis of microbial consortia in activated sludge using fluorescently-labelled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy by M. Wagner, B. Assmus, A. Hartmann, P. Hutzler & R. Amann.
In vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy by F. A. Merchant, S. J. Aggarwal, K. R. Diller & A. C. Bovik.
Scanning interference and confocal microscopy by R. Juskaitis & T. Wilson.
Time- and wavelength-resolved spectroscopy in two-photon excited fluorescence microscopy by S. Andersson-Engels, I. Rokahr & J. Carlsson.
Simultaneous confocal recording of multiple fluorescent labels with improved channel separation by K. Carlsson, N. Aslund, K. Mossberg & J. Philip.
Imaging in the far-red with electronic light microscopy: requirements and limitations by C. Cullander.
Optoelectronic detector probes for scanning near-field optical microscopy by H. U. Danzebrink.
Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study by S. Meschini, A. Molinari, A. Calcabrini, G. Citro & G. Arancia.
Scanning force microscopy on live cultured cells: imaging and force-versus-distance investigations by D. Ricci & M. Grattarola.
Modelling of inclined and curved surfaces in the reflection scanning acoustic microscope by W. Weise, P. Zinin & S. Boseck.
Studies of porphyrin containing specimens using an optical spectrometer connected to a confocal scanning laser microscope by O. Trepte, I. Rokahr, S. Andersson-Engels & K. Carlsson.
The tetrahedral tip as a probe for scanning near-field optical microscopy at 30nm resolution by U. C. Fischer, J. Koglin & H. Fuchs.
A versatile tilting device for fluorescence microscopes by J. Bradl, M. Hausmann, B. Schneider, B. Rinke & C. Cremer.
Continuous wave excitation two-photon fluorescence microscopy by P. E. Hanninen, E. Soini & S. W. Hell.
Refractive index induced aberrations in two-photon confocal fluorescence microscopy by H. Jacobsen, P. E. Hanninen, E. Soini & S. W. Hell.
If you do not subscribe to the Journal, Copies can be ordered from Blackwell Science Ltd at a cost of œ14.9 pounds sterling, including postage & packing for UK delivery (orders outside the UK please add œ1.50 pounds sterling for postage).
Please send your order to Anna Rivers, Blackwell Science Ltd, Osney Mead, Oxford OX2 0EL, United Kingdom. Telephone +44 865 206206, fax +44 865 206096.
Materials Science and Engineering Laboratory National Institute of Standards and Technology Gaithersburg, MD
The MSEL anticipates an opening within the next 6-9 months for a person with expertise in transmission electron microscopy of metallic and ceramic materials. Extensive experience in HREM, image simulation, PEELS required. Experience with Gatan imaging filter and running a user facility highly desired. This position is initially a term appointment with an excellent chance of conversion to permenant within 2 years. Because we are a government laboratory, strong hiring preference will be given to US citizens. Also, because of the critical need for the aforementioned expertise, persons requiring training in these areas are not likely to be considered.
The MSEL microscope facility consists of a new JEOL 3010 TEM with PEELS, Gatan IF, etc, Philips 430 EM and 400 STEM, sample prep and image processing/simulation facilities. The position is expected to consist of 50% collaboration with researchers, 25% administration/training/vendor stuff, and 25% independent research.
A formal search announcement will be published in the usual journals in the near future.
For details or to submit your name for consideration, please contact:
Tim Foecke NIST Building 223, Rm B254 Gaithersburg, MD 20899 fax: 301-926-7975 email: tfoecke-at-nist.gov
The US government is an EEO. Women and minorities are encouraged to apply.
I worked with TEM of blood many years ago, primarily granulocytes. Millonig's phosphate buffered 3% glutaraldehyde was what worked best. Can't remember right now if that included sucrose or not, files are at home.
Conventional metallographic preparation involves frequent rinsing of the mounted sample between grinding and polishing stages. The final rinse is typically followed by rinsing in alcohol then acetone.
We find that using filtered and dessicated compressed air through a small diameter blow gun is usually superior in removing residues. The sample is rinsed in water alone, though sometimes a dilute soap solution is required prior to drying. This technique reduces our alcohol/acetone consumption to a mere fraction of what is was. Solvent fumes in the lab are minimal.
The multi-stage filter we use is manufactured for spray painting and is readily available from numerous sources.
Please direct any questions or feedback to: Alan Stone ASTON Chicago, IL compuserve 73004,1733
in order to compare some results I'm looking for up-to-date binding energies (/and or work funktions) measured by XPS and/or XAS from XTiO3 (X=Ba,Ca,Sr,Pb). The values should be refferenced to the Au 4f level or the fermi energy of the material.
By the way, is there a standart data base for such measured or calculated numbers on the net?
The president of the PNW Microscopy Society is Bob Kayton, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portalnd, OR 97201; phone (503) 494-2504.
On Tue, 6 Dec 1994, Daniel Possin wrote:
} Hello everyone - } } How does one contact the Pacific Northwest Microscopy Society? } } Answers to question are greatly appreciated. } } Dan }
In article tphillips-at-biosci.mbp.missouri.edu (Tom Phillips) writes: } } I am interested in what protocol people are using to en bloc stain with } UrAc.
Our protocol refined over the years is to Fix in osmium tetroxide in cacodylate (or, rarely a phosphate buffer), then wash in 1% sodium acetate to remove the buffer which can precipitate the uranium, then use 2% uranyl acetate unbuffered in dh2O for one hour. we follow that with the alcohol dehydration. Use longer uranyl acetate for darker stain. The sodium acetate is a really important step as uranyl phosphate is insoluble in water and cacodylate also can precipitate it. Refer to
Terzakis J.A. (1968) Uranyl acetate, a stain and a fixative. J Ultrastructure Res. 22- 168. Positive Staining for Electron Microscopy. M. A. Hayat 1975 Van Nostrand Reinhold NY. Page 33 onward
Research Project: Atomic Resolution TEM Analysis of Intergranular Fracture ___________________________________________________________________________
A post-doctoral research fellow is required to carry out experimental and theoretical analyses in the study of interfacial segregation. The project will involve extensive use of high-resolution imaging coupled with detailed analyses using valence and core-loss EELS. Furthermore, the project will require careful modelling of EELS spectra using various theoretical approaches. Accordingly, the candidate must be a good experimentalist and have sufficient experience with electron scattering theory.
The research project is sponsored by The Ontario Centre for Materials Research and is linked with industrial partners interested in a more fundamental understanding of intergranular embrittlement in various metallic alloys.
The project will begin April 1, 1995 for up to as many as 3 years.
The research fellow will spend the majority of his/her time at nearby McMaster University where a new JEOL JEM 2010F Field-emission TEM with EDX and PEELS has recently been installed.
Those who are interested and qualified should contact me for consideration.
D.D. Perovic Department of Metallurgy and Materials Science, University of Toronto 184 College Street Toronto, Canada Tel: (416) 978-5635 Fax: (416) 978-4155 Internet: perovic-at-ecf.utoronto.ca
} I am interested in what protocol people are using to en bloc stain with } UrAc. Specifically, what buffer, pH, concentration, duration and stage. } Do Uranyl acetate solutions go bad? John Johnson started a thread touching } on this a while back but the responses were mostly how to avoid or remove } pepper staining artifacts. TIA. } }
Tom:
When using UA as an en bloc stain, do not use phosphate buffers in your fixation or you will most likely have a ppt problem. I use caco-buffer fixes and then generally stain in aqueous UA after the Osmium has been washed out (I dont buffer the stain) - an alternative is to incorporate the staining into the dehydration schedule, using an alcoholic UA
I millipore filter the stain immediately before use and incubate for 0.5 to 1 hr
Microscopical Society of Canada 22nd Annual Meeting
The MSC Executive and the Local Organising Committee cordially invite you to attend and participate in the 22nd Annual Meeting of the Microscopical Society of Canada This meeting will be held in the University Centre Building, University of Ottawa, Ottawa , Ontario, Canada, June 4-7, 1995. A varied and interesting scientific program has been planned and will consist of a combination of interdisciplinary symposia presented by speakers from around the world - separate physical and biological symposia, oral and poster presentations, a workshop on TEM specimen preparation of materials, and commercial exhibits.
Local Organising Committee Jim Corbett (Chairman, Secretary, University of Ottawa Liaison) John McCaffrey (Treasurer, Space Management, Accommodation) Jeff Fraser (Commercial Exhibits, Social Program) Graham Carpenter (Scientific Program Chair, Materials) Larry Arsenault (Scientific Program Chair, Biology) Peter Sewell (Corporate Liaison, Commercial Exhibits) Kamal Botros (Scientific Program) Louise Weaver (Scientific Program) Paula Allan-Wojtas (Registration) Shea Miller (Registration)
DEADLINE FOR RECEIPT OF ABSTRACTS: March 15, 1995 DEADLINE FOR PRE-REGISTRATION: May 1, 1995
For further information contact:
Program:
Jim Corbett Department of Physics University of Waterloo Waterloo, Ontario Canada, N2L 3G1 Tel: (519) 885-1211 Fax: (519) 746-8115 e-mail: corbett-at-physics.watstar.uwaterloo.ca
Registration:
Shea Miller or Paula Allan-Wojtas Centre for Food and Animal Research Agriculture and Agri-Food Canada Room 2016, K.W. Neatby Bldg. Central Experimental Farm Ottawa, Ontario, Canada, K1A 0C6 Tel: (613) 957-4347, ext. 7709 (Shea), 7970 (Paula) Fax: (613) 943-2353 e-mail: millers-at-ncccot.agr.ca allanwojtasp-at-ncccot.agr.ca
We use alcoholic UA for our enbloc staining, particularly for thick section and whole mount samples for our 300 KeV instrument. We found better staining and less problems with precipitate using the alcoholic UA. We use 20% EtOH and incorporate the staining as part of our dehydration schedule. As with any UA enbloc procedures (as discussed in previous parts of this string) good washing to remove buffer is important before staining.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I am looking for a supplier of electron-sensitive film (similar to type 4489 or S0163) in 35mm roll form. Kodak no longer makes such a product. Please include a phone number and address of the distributor if available. TIA
Cheers,
David A. Howell Materials Science Dept. Michigan State University E. Lansing, MI 48824-1226 howelld-at-egr.msu.edu
Mahalo to all of you who posted suggestions for fixing our cultured cells for SEM. We have, of course, played with several factors, primarily osmolarity, with little change. Now we are considering the effects of different ions, temperature, whatever. I will let the group know what happens if/when we come to some conclusions! One of the problems is that we want to do some very high resolution SEM and/or replicas for TEM of the surface of the cells, so we have to be careful not to fix (by chemicals or freezing) any proteins, salts, whatever, from media or buffers onto the surface, since we are not going to fracture. I knew that would be a problem; I just didn't expect to have to fight blebbing, too! Suggestions still gratefully accepted!
It's 76 degrees F, sunny and windy today. Rainbows in the valleys. Happy holidays!
Aloha, Tina Weatherby Carvalho Biological EM Facility University of Hawaii
A few months ago Damon Herr, from FEI company, offered Field Emission reference articles to anyone interested. I have not been able to contact Damon at the email address he gave. Does anoyone else out there have this info. If so could you please forward it to me. I can be contacted via:
I am truly grateful to all of those who responded to my question regarding vibration isolation platforms. If I do install a platform, I shall post a message about its performance. Season's Greetings to all the Micronetters!
************************************************************************* M.V. Parthasarathy Professor of Plant Biology Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
Damon Heer's email address is DLH-at-FEICO.MHS.CompuServe.COM and phone # is(503)640-7500 FYI merock
On Fri, 9 Dec 1994, Leo D Frawley 03 5667464 wrote:
} A few months ago Damon Herr, from FEI company, offered Field Emission reference articles to anyone } interested. I have not been able to contact Damon at the email address he gave. Does anoyone else out there } have this info. If so could you please forward it to me. I can be contacted via: } } email frawley-at-resmel.bhp.com.au } phone 613 560 7066 } fax 613 561 6709 } } BHP Research_Melbourne Laboratories } 245 Wellington Rd, Mulgrave, Victoria 3170, Australia. } } Thanks in advance. } } Leo D Frawley } } }
Jan Coetzee, Please post a summary to the list of the responses that you get rearding fixing RBCs. This is an interesting problem. Also: for SEM, you have more problems than fixation--the drying process shrinks cells, and the shrinkage continues *after* the drying is finished. This shrinkage in not necessarily isotropic, nor consitent. Yet another thing to watch out for. Phil Oshel poshel-at-luc.edu
A comment on Gib Ahlstrand's note re: the Polaron CPD: We've had the same problem with getting cold enough tap water in the summer, but this was cured by coiling the input hose in a bucket of water and ice--no valves, extra plumbing, etc. The input water only has to get to 10 C to cool and 40 C to heat. Otherwise, I can agree that there are advantages to electrical heating, such as more precise control. But... Phil Oshel poshel-at-luc.edu
A hint for picric acid people: if you've got a jar that you suspect has dried partially, turn it upside down in a container of water for an hour or two or overnight. If the picric acid has dried anywhere, it will be on the threads, and this will dissolve it. Silly and abvious, maybe, but there are people who didn't think of it... Phil Oshel poshel-at-luc.edu
Hey Folks: We typically use UA for en bloc staining at 0.5% in a 28mM acetate/veronal buffer (pH 6.0, 1 hr, 20C) after thorough rinsing in A/V buffer to remove OsO4; then proceed with EtOH dehydration.
Good Luck, Greg Martin Cell Biology and Anatomy Johns Hopkins School of Medicine
Subject:Re: 35mm e-sensitive film supplier? } } I am looking for a supplier of electron-sensitive film (similar } to type 4489 or S0163) in 35mm roll form. Kodak no longer makes such } a product. Please include a phone number and address of the distributor if } available. TIA } } Cheers, } David A. Howell } Materials Science Dept. } Michigan State University } E. Lansing, MI 48824-1226 } howelld-at-egr.msu.edu
David, We use Kodak 5302 fine grain release positve film in our 35mm TEM cameras. It is manufactured as a motion picture film but is good for EM too, and very cheap. We use the same safelights, developer and fix as the SO163/4489 EM films ( you may have to tweak the emulsion sensitivity knob on the EM though).
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
I am new to AFM and am trying to establsh protocols for handling, processing, evaluating, and selecting Si AFM cantalevers for phase imaging of magnetiic media. Random sampling of unused cantalevers using FEG/SEM has revealed damage to the very tip of the cantalevers that might be caused by ESD. For phase imaging, the cantalevers are sputtered with CoCr thin films and then the cantalevers are magnetized. I would like to determine the mode of failure as well as at what stage of processing the damage is occurring. Any suggestions will be greatly appreciated.
The aim of the Symposium is to provide a forum where participants may discuss the use of scanning electron microscopy and related methods in their work and research. The emphasis is on applications and techniques. Contributions are sought on the subjects of SEM imaging of biological and materials specimens, field emission SEM, low voltage SEM, X-ray analysis, cryo-SEM, image processing and analysis, AFM, STM, forensic analysis, laboratory management and training methods.
Pre symposium workshops(February 13-14) will be held. Half day Workshops are being given in 1. Principles and Practice of SEM, 2. Principles and Practice of X-ray analysis, 3. FESEM, 4. Advanced X-ray Analysis, 5. Cryo Micrscopy, 6. Image Processing and Analysis.
A one and one half day workshop is being given in 7.Wavelength Dispersive Analysis.
The meeting is being organised by The Australian Microbeam Analysis Society (AMAS) and is being coordinated by John Ward.
Deadline for the submission of abstracts is December 31 1994. If you wish to send an abstract please Fax a copy to Dr. Peter Miller at 613 544 1128 or email miller-at-rivett.mst.csiro.au
I recently did a staining run using a protocal I have used secessfully before. This time there was not much staining. In fact, there was hardly any gold on the formvar portion of the grid. The other times I have done this exp. there was gold stuck to formvar. Could this indecate a problem with the gold I used?
If you get gold particles on the formvar film, then there is probably a problem with you gold even before you do experiments. You should not get that sort of background when you do immunolabeling. As for the lack of labeling, if you are repeating an experiment that has worked in the past and everything is the same then it could either be your gold probe or the primary antibody. Primary antibodies and gold probes can deteriorate upon improper storage. The antibodies can aggregate, fall apart or stick to the sides of the tube. With the gold, the most common cause of signal loss is the dissociation of ligand from the gold particles. The free protein (usually protein A of IgG) occupies binding sites but cannot be seen in the microscope (no gold). Protein A-gold is more stable than IgG-gold and is the better all round marker for immunocytochemistry. Another reason for loss of signal is the use of inappropriate bocking agents which either react with the primary atibody or the gold marker (eg. rabbit serum to dilute protein A-gold; fetal calf serum to dilute antibodies to BSA). To give you a better diagnostic we need more details.
In my experience, gold labeling of formvar is usually more indicative of the primary antibody sticking to the formvar. i.e. with the same batches of goat anti-mouse gold we get different levels of formvar background with different primary antibodies. Background is usually highest with antibodies we know to be particularly "sticky". Assuming you are using the gold in an indirect immunostaining protocol, I would suspect that my primary ab had gone bad before blaming the gold. I look forward to hearing other peoples ideas.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Mon, 12 Dec 1994, Larry Hawkey wrote:
} } Anyone who has done or is doing immuno-gold: } } I recently did a staining run using a protocal I } have used secessfully before. This time there } was not much staining. In fact, there was } hardly any gold on the formvar portion of the } grid. The other times I have done this exp. } there was gold stuck to formvar. Could this } indecate a problem with the gold I used? } } Larry hawkey } hawkey-at-neuro.duke.edu }
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 2:46 PM
Date:12/12/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Just a reminder about the ESEM Symposium at Scanning 95 & The ESEM User Group Meeting that will follow it. Copies of the Call for Papers and User Meeting Info are enclosed but note that info is available on: http://www.engin.umich.edu/~jfmjfm/esem_folder/scanning95.html http://www.engin.umich.edu/~jfmjfm/esem_folder/user_meeting.html. __________________________ Call For Papers
Environmental Scanning Electron Microscopy:
Working in the Micro-Laboratory
A Special Symposium at Scanning 95
March 28-31, 1995
at the Doubletree Hotel
Fisherman's Wharf, Monterey, CA, USA
Dear Environmental SEM User(s):
Whether we call our instruments ESEMs, Wet SEMs or Low Vacuum SEMs, we are all Environmental Scanning Electron Microscopists. The field of Environmental Scanning Electron Microscopy has now become well established, particularly because these instruments have the unique capability of forming secondary or back-scattered electron images, while the sample is in what would typically be considered an extremely poor vacuum. The sample chambers are typically quite large and can accommodate a wide variety of experimental platforms for in-situ observation. Such specialized in-situ systems include high temperature stages, cold stages, uniaxial straining and multi-point bending stages and liquid sample stages. In fact, these novel SEMs are unique micro-laboratories.
This symposium, as part of this year's SCANNING 95 meeting, will focus on ESEMs, Wet SEMs or Low Vacuum SEMs as microcharacterization laboratories. A number of invited speakers will outline application of their particular microscopes in this capacity.
Invited speakers include:
Dale Newbury National Institute for Standards and Technology, MD, USA. Title: "Scanning Electron Microscopy at Elevated Pressure: A Newcomer's Views"
Brendon Griffin Centre for Microscopy and Microanalysis, University of Western Australia. Title: "A review of detection strategies and imaging of hydrated biological specimens in the environmental SEM"
Paul Meredith Polymer & Colloids Group, Physics Department, Cambridge University, UK. Title: "In-situ hydration studies of ordinary Portland cement by environmental SEM"
This is an invitation to you and your colleagues to contribute papers to this symposium. Contributions emphasizing novel in-situ experiments (advances in hot-stage, cold-stage or deformation microscopies), studies on non-conducting materials (ceramics, polymers etc.) and examination of liquids or emulsions are most appropriate. To facilitate the organization of this symposium, please send copies of your abstracts, in the format outlined in the Abstract Preparation Booklet (see below) to the organizers by DECEMBER 15th, 1994.
John F. Mansfield Electron Microbeam Analysis Lab. University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313) 936-3352 FAX: (313) 936-3352 email: John.F.Mansfield-at-umich.edu
Stuart McKernan High-Resolution Microscopy Center University of Minnesota 100 Union St. SE Minneapolis MN 55455-0153 Phone: (612) 624-6590 FAX: (612) 626-7530 e-mail: stuartm-at-maroon.tc.umn.edu ------------------------------------------------------------------------
Abstract Preparation Booklets & Program Information:
Mary K. Sullivan FAMS, Inc. Box 832 Mahwah, New Jersey 07430-0832 Phone: (201) 818-1010 FAX: (201) 818-0086 Email: fams-at-holonet.net
The absolute final deadline for abstract submission is February 1st 1995 Please submit TWO PAGE camera-ready manuscripts (as defined in the Abstract Preparation Booklet) & a copy on computer diskette (Mac or PC), if possible, to: ------------------------------------------------------------------------
Dr. Robert P. Becker Editor, Proceedings Issue, SCANNING 95 545 Island Road, Ramsey, NJ 07446 USA Phone: (312) 996-7215 FAX: (312) 413-3034 Email: rpbecker-at-uic.edu
Electroscan ESEM Users please check out the accompanying Web Page which describes the 1995 ElectroScan User's Meeting.
__________________________
ElectroScan User's Meeting
Doubletree Hotel
Fisherman's Wharf, Monterey, CA, USA
Friday March 31st & Saturday April 1st 1995
In recent years this meeting has been held at ElectroScan's headquarters in Massachusetts. The location, and the fact that it is a user forum, rather than a nationally recognized conference, has precluded a number of users from attending. In an attempt to open the User Meeting to a wider audience, we are, therefore, going to hold user meetings in a variety of locations around the United States in conjunction with a national microscopy conference. Therefore, this year's meeting will be held immediately after Scanning 95 at the Doubletree Hotel on Fisherman's Wharf in Monterey, California.
Holding the User Meeting after a conference means that we have an ideal opportunity to hold a symposium at the conference dedicated to environmental scanning electron microscopy. An associated Web Page describes the symposium "Environmental Scanning Electron Microscopy: Working in the Micro-Laboratory" This symposium will, naturally, be open to all who are interested in environmental scanning electron microscopy, while the User Meeting will be limited to ElectroScan users only. Since the conference, and hence the environmental scanning electron microscopy symposium, will precede the User Meeting, the format of the User Meeting will be different to those held in Massachusetts. Users who wish to make presentations describing the novel experiments that have been performed in the ElectroScan ESEM, should submit abstracts for the conference symposium. This will provide a broader audience for the presentations. However, anyone who feels that there are particular details of their work that would be of interest to the ElectroScan users only, should request a time slot at the User Meeting to describe those additional details. As in past User Meetings, representatives from ElectroScan will be present at the meeting to describe the latest developments of the ESEM and the direction in which future developments are heading. They will also discuss the options that current users have for upgrading their current instruments and will be open to suggestions for instrumental improvements. A number of representatives from other vendors may describe how their equipment may enhance the capabilities of the ESEM. There will be an open question and answer period at the User Meeting to allow for general discussion. If you have a topic for discussion you should contact the organizers of the User Meeting as soon as possible so that your topic can be integrated into the schedule.
Tentative Schedule
Friday March 31st
Evening Reception.
Saturday April 1st
Morning Follow-up of "Environmental Scanning Electron Microscopy: Working in the Micro-Laboratory" sessions. Question and answer with the authors. ElectroScan representatives: The Gaseous Secondary Electron Detector (GSED) and comparison with the Environmental Secondary Detector (ESD). Colorview - a new variation on the viewport. Image archiving and networkability. Upgrading E3 instruments. Other discussion announcements.
Lunch
Afternoon ElectroScan representatives continued. Other Vendors Representatives. Open Forum Discussion - Any matters not previously covered.
Questions, comments, suggestions and topics for discussion should be forwarded to the organizers:
Organizers:
John F. Mansfield Electron Microbeam Analysis Lab. University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313) 936-3352 FAX: (313) 936-3352 email: John.F.Mansfield-at-umich.edu
Stuart McKernan High-Resolution Microscopy Center University of Minnesota 100 Union St. SE Minneapolis MN 55455-0153 Phone: (612) 624-6590 FAX: (612) 626-7530 e-mail: stuartm-at-maroon.tc.umn.edu
Ed Griffith ElectroScan 66 Concord Street Wilmington, MA 01887 Phone: (410)643-2494 FAX: (410) 643-237
We want to make sure we have the correct names and addresses of all users, so please fill out the User's Directory Update.
A while ago I corresponded with several people about a SuperCard stack detailing the inner workings of an SEM.......
I never have been able to get ahold of it....does anyone have any information.....I'd really like to get it before the spring semester so I can use it for my class.....
Thanx in advance Center for Materials Research and Analysis Central Facility for Electron Microscopy University of Nebraska at Lincoln
Please send any responses directly to raharris-at-ucdavis.edu. I am no longer on the listserv.
Our facility is considering selling a used JEOL 100SX. It is currently under repair by JEOL and we are wondering if we should continue with the costly repairs and then sell it. Is there any market for a used scope? The scope is like new and has had few users. It was under a service contract but as soon as the contract expired it cooked the objective lens. Our use has gone way down and we wonder if we should continue the repairs and keep the scope or sell the scope. Has anyone had any luck selling used scopes?
Rick A. Harris raharris-at-ucdavis.edu Dept. of Molecular and Cellular Biology University of California, Davis 916 752 2914 fax 916 752 1449
'ordinary' tiff files have 8 bytes at the start encoding the width and height of the file ie how many pixels wide, how many rows long, then the rows of pixels 0=black, 255 = white ie simple numeric grey level directly encoded. after the h rows of w bytes each, there is a variable section that can contain labels etc Look at the file with a utility like LIST by Breug (best $15 I ever spent!!!!!!) in hexadecimal mode If you know width and height eg 512 * 400 for EDAX files then just skip 8 bytes, read binary 512 bytes as first row, etc for 400 times and ignor the rest. There are many tiff formats that are more complex but I believe most conform to this basic structure. I can transfer EDAX files from RT11 (DEC) to pc by a utility rt2pc.exe given me by EDAX, I think it is public domain or close.... This is 12 bit data encoded as integers, 2 bytes/ pixel. converting to 1 byte / pixel unsigned integer ie 0-255 = increasing grey scale gives a tiff file readable by Optimus in the PC and photoshop, etc on the mac alan pooley marine sci sem lab rutgers univ
The aim of the Symposium is to provide a forum where participants may discuss the use of scanning electron microscopy and related methods in their work and research. The emphasis is on applications and techniques. Contributions are sought on the subjects of SEM imaging of biological and materials specimens, field emission SEM, low voltage SEM, X-ray analysis, cryo-SEM, image processing and analysis, AFM, STM, forensic analysis, laboratory management and training methods.
Pre symposium workshops(February 13-14) will be held. Half day Workshops are being given in 1. Principles and Practice of SEM, 2. Principles and Practice of X-ray analysis, 3. FESEM, 4. Advanced X-ray Analysis, 5. Cryo Micrscopy, 6. Image Processing and Analysis.
A one and one half day workshop is being given in 7.Wavelength Dispersive Analysis.
The meeting is being organised by The Australian Microbeam Analysis Society (AMAS) and is being coordinated by John Ward.
Deadline for the submission of abstracts is December 31 1994. If you wish to send an abstract please Fax a copy to Dr. Peter Miller at 613 544 1128 or email miller-at-rivett.mst.csiro.au
We would like to observe the dislocations in Mg-10%Sn alloys and prepared some TEM specimens by Twin-Jet electropolishing method. But I've never got good specimens because of differentiate etching.
Does anyone know good etchants and polishing conditions ?
If I got it right, this is some kind of a mailing list? I would be happy to get any current information on electron microscopy and laboratories working in the field.
Regards, plehtine-at-butler.cc.tut.fi
in real life MSc Pirjo Lehtinen from the Centre for Electron Microscopy, Tampere University of Technology, Finland.
There is regular, full-time position available in the Shared Scientific Services of The Jackson Laboratory, Bar Harbor, Maine. The Jackson Laboratory is a nonprofit, independent laboratory founded in 1929 on the premise that the causes of cancer and other diseases could be discovered through mammalian research. The Laboratory specializes in mammalian genetics using inbred laboratory mice as model systems to study human health problems such as cancer, diabetes, anemias, heart disease, muscular dystrophy, and aging. Located on a large island in the Gulf of Maine and surrounded by Acadia National Park, The Jackson Laboratory is currently undergoing a major expansion of its scientific staff and research facilities.
Duties include operation and training of end users on a PC-based image analysis system, and the performance of customer-directed analysis of biological samples. Also included is the regular maintenance and alignment of upright and inverted research-level microscopes, with the following optics; brightfield, darkfield, phase contrast, differential interference contrast and fluorescence and associated cameras, computers and video equipment.
A successful candidate would have a minimum of 2 years experience in microscopic imaging and/or experience in rudimentary computer programming. The applicant must also be able to work independently in a multi-user facility and to deal with people on a one-to-one basis. Individual will be expected to attend seminars and participate in interest groups disseminating information about current microscopic imaging techniques. The salary range begins at $28,000 plus benefits, and is negotiable depending upon level of experience.
Interested applicants should send CV to:
Joanne C. Bradt Employment Specialist The Jackson Laboratory 600 Main Street Bar Harbor, ME. 04609 (207) 288-3371 ext. 1281 (207) 288-3371 ext 1082 FAX jcb-at-aretha.jax.org
Hey Folks - Everyone's comments are excellent; I would add that gold particles over the formvar may not be the most relevant indication of a problem with your gold probe. The background labeling over the sections in the absence of a specific probe (e.g. primary Ab) may be more important in assessing the "stickyness" of gold probes. I've had batches of IgG-Gold which gave very low (basically nothing) levels of labeling over tissue sections incubated with a mock primary and significantly higher levels over the formvar -- using primary Ab still gave excellent results. Of course this doesn't matter unless you can get away with not taking pictures which include the formvar film! Also -- leaving off the Carbon coating on the grids can reduce binding of gold probes. Since levels of labelling and background can vary so much for probes from different suppliers, different batches from the same supplier, and with regard to different specific probes, it can't be over emphasized to run a lot of negative (and positive) controls to "tease out" what's really going on.
Good Luck! Greg Martin Cell Biology and Anatomy Johns Hopkins School of Medicine
The program Virtual SEM v 1.2 (formerly SuperSEM) written by B. Griffin and A. van Riessen of the Univ. of Western Australia has been donated to the MSA/MAS public domain library for distribution. It can be downloaded from the ANONYMOUS FTP site.
Warning: The program is ~11 Mbytes in size and if you have a slow FTP link be prepared for a long wait! It is a MACINTOSH SuperCard standalone program! So if you don't have a Mac don't bother to download the SuperCard Stack. You do not need Supercard to run the program. You must download this program using BINARY mode. Also note this FTP site has a limited number of accessible lines so if you have trouble getting through try again at a later time.
I am seeking for a software which allows me to build a cell, draw structures, and simulating diffraction patterns and high resolution TEM images. I prefer a software that runs on MacIntosh. Does anyone have any suggestion on the software, and how and where to get or purchase a copy? I was told that CrystalKit and MacTampas will do the job but I don't have any clue how to get or purchase them. Any suggestion on this matter will be appreciated.
Jessica Chang Research Associate School of Electrical Engineering Purdue University W. Lafayette, IN 47907-1285 E-mail: changj-at-ecn.purdue.edu
I am studying cell walls in thick sections using the EM replica technique. Until now I used glass microscope slides as a base, floating the replica's off in HF. For practical reasons I would like to use a degradable base that I can cut to replica size, e.g. with a razor. In the literature acetyl cellulose sheets have been used for this purpose, but I cannot find this product in the catalogs we have here. Does anyone know a (US) supplier or have any other suggestions.
Thanks for any suggestions!
Herman Meekes Biological Sciences ______________ ______________ University of Missouri ---__ \ / __--- 109 Tucker Hall ------__\---/__------ Columbia, MO. 65211 \( )/ Tel: 314-882-0171 V Fax: 314-882-0123 / \ e-mail: hmeekes-at-biosci.mbp.missouri.edu /___\
I am seeking for a software which allows to build cells, draw structures, and simulating diffraction patterns and high resolution TEM images. I prefer a software that runs on Mac. Does anyone have any idea about what software and where to get or purchase a copy?
I was told that CrystalKit and MacTampas will do the job but I don't have any clue how to get or purchase them. Any suggestion on this matter will be appreciated.
Regards,
Jessica Chang Research Associate School of Electrical Engineering Purdue University W. Lafayette, IN 47907-1285 E-mail: changj-at-ecn.purdue.edu
The term TIFF derives from "Tag Image File Format", meaning that the TIFF file header (or trailer) containing information about the data in the image file is made up of fields identified by a unique tag. The tags have been "specified" by software and hardware manufacturers to facilitate the exchange of image data among different operating systems etc. The TIFF format Rev. 4.0 document was published by Aldus and Microsoft and other manufacturers in 1987: newer revisions may have appeared at later dates. In general, the TIFF header is much longer that just a few bytes specifying the width and height of the image: this format is more like the "raw" image format that some commercial instrumentation generates. The TIFF file header is variable in length and can only be deciphered if you know the meaning of the various tags. The length of the header (or trailer) can be easily determined, if you know the size of the image and the pixel data type (8bit, 16bit, or other), by comparing the length of the raw image data with the size of the file. The first eight bytes in a TIFF file header (always at the beginning of the file) have the following meaning: Bytes 0-1 specifies the byte order in the file (essentail for 16- or 32-bit data) II (hex 4949) from least to most significant byte MM (hex 4D4D) from most to least-significant byte
Bytes 2-3 TIFF version number
Bytes 4-7 this long word contains the offset (in bytes) of the first Image File Directory, which may be at any location in the file after the header but must begin on a word boundary. (the first byte in the file has an offset of 0) At this point the discussion gets complicated, because in principle you may have more than one IFD, more than one image per file, and so on. Assuming that you only have one IFD in your file, this IFD consists of a 2-byte count of the number of fields, followed by a sequence of 12-byte field entries. Each 12-byte field entry has the following format: bytes 0-1 Tag for the field bytes 2-3 field type bytes 4-7 length of the field (1=byte, 2=ASCII, 3=SHORT (2-byte unsigned integer), 4=LONG (4-byte unsigned integer), 5=RATIONAL) bytes 8-11 file Offset in bytes of the Value of the field
The entries in an IFD must be sorted in ascending order by tag. If the Value fits within 4 bytes, the Offset is interpreted to contain the Value instead of pointing to the Value (in general,this is the case for width and height values).
The Tags and other parameters for image width and height are (N means number of values):
ImageWidth Tag = 256 (hex 100) Type = SHORT N = 1
ImageLength (height) Tag = 257 (hex 101) Type = SHORT N = 1
Many other tags and fields are defined in this document. In general, you should look for these identifiers if you do not know the organization of the TIFF file: of course, if you only deal with one particular file format generated by a unique instrument, you may have to figure out the file organization once and take a few shortcuts in your program thereafter. The contact persons listed in the document are (don't forget, this doc is dated April 31, 1987):
Tim Davenport Aldus Corporation 411 First Ave. South Suite 200 Seattle, WA 98104
Manny Vellon Windows Marketing Group Microsoft Corporation 16011 NE 36th Way Box 97017 Redmond, WA 98073-9717
If you cannot reach anybody at either location, you can contact me and I will send you a copy of the document I have. Best regards, Massimo
_________________________________ Massimo Sassaroli Dept. of Physiology & Biophysics Box 1218 Mount Sinai School of Medicine 1 Gustave L. Levy Pl. New York, NY 10029-6574
You may buy cellulose acetate sheets from Ted Pell Inc., Redding, CA 96099, Tel:916 243 2200, Fax:916 243 3761. Catalog numbers are G255 (150 x 100 mm, thickness 35 microns, pack of 20 sheets), G254 (150 x 150 mm, thickness 100 microns, pack of 20 sheets) and G254A (150 x 150 mm, thickness of 125 microns, pack of 20 sheets).
The full description of the TIFF specification can be obtained from vendors like Microsoft or Aldus. The format is not at all as simple as indicated in a recent mail. However, it is quite possible to create a TIFF header manually without programming. One way to do this is to create a "dummy" file with a package that can save to tiff. Make a TIFF file with the dimensions in pixels and the gray depth (e.g. 8 bit gray) as the one You want. Fill the entire image with either black or white. Then save as TIFF (no compression). Now it is easy to locate and view the TIFF header with a hex dump program.(You can try to repeat with another image resolution and compare the headers for fun). Now, the header can be removed and saved. Then this header can be used as a template and copied (any number of times) to any raw image file that conforms to the data described in the header. I did this for 256x256 and 512x512 grayscale images respectively. Then converting a raw file to TIFF was a matter of using the DOS copy + command. Used properly, this command adds the proper tiff header to a raw image. The best part is that this can be done in batch processing, converting a great number of images in only a few seconds. With some more effort, a modification of the header to compensate for a Biorad header (76 bytes ??) these files can probably be converted in the same way. This way You don4t need a special package to open an image as raw and save it as TIFF, which will take a while to do if You have many files to process. Once the "nasty" part described above is done once (ask Your local computer freek) then conversion is a snap.
This is no method for computer purists but it works very well.
Best luck
============================================= Mikael Gustafsson MD, PhD Dept Med. Microbiology and Dept Internal Medicine, Cardiology section University Hospital of Linkoping S 581 85 LINKOPING SWEDEN
I am a teacher of Electron Microscopy at Seneca College in Toronto, Canada. I would like to subscribe to the forum as I feel it can be a valuable teaching tool. Please advise me as to how to subscribe. Thanks.LAUREL SCHOLLEN SCHOOL OF BIOLOGICAL SCIENCES AND APPLIED CHEMISTRY SENECA COLLEGE OF APPLIED ARTS AND TECHNOLOGY 1750 FINCH AVENUE EAST NORTH YORK, ONTARIO CANADA M2J 2X5 TEL(416)491-5050 EXT 2390 FAX(416)491-0854 EMAIL ADDRESS: SCHOLLEN-at-ASET.SENECAC.ON.CA
I would like to subscribe to this forum. Please advise method. ThanksLAUREL SCHOLLEN SCHOOL OF BIOLOGICAL SCIENCES AND APPLIED CHEMISTRY SENECA COLLEGE OF APPLIED ARTS AND TECHNOLOGY 1750 FINCH AVENUE EAST NORTH YORK, ONTARIO CANADA M2J 2X5 TEL(416)491-5050 EXT 2390 FAX(416)491-0854 EMAIL ADDRESS: SCHOLLEN-at-ASET.SENECAC.ON.CA
We are recently having problems with distortion of single crystal gold (100) foils on copper grids supplied to us. We use these foils as a lattice image resolution check, but are finding the foils are so badly buckled that only very small areas remain at the exact orientation for strong fringe contrast at a given tilt angle. The problem gets worse after heating the foils in vacuum. Does anyone have similar problems and/or can advise on a source of undistorted foils?
I am unclear from your message why you are considering cathodoluminescence? Standard secondary electron detection should work fine for counting cells. The general appearance of each cell type by sem is well known (White et al, 1982, Artery 11:33) and with metal coating any SEM will give you very good low-noise images for rapid counting directly off the CRT screen. If you need to pick out subpopulations of cells by immuno-labeling, colloidal gold (30 nm) works well and can also be visualized by secondary electrons or very rapidly by backscatter. We routinely count blood cell and platelet adherence to foreign material this way. IMHO, analysis of blood cell adherence is generally more rapid and reproducible by SEM than by light microscope coupled image analysis, at least in our hands and for our applications. Is there some special reason you think cathodoluminescence is necessary?
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Tue, 13 Dec 1994 A.Brandwood-at-unsw.EDU.AU wrote:
} We are doing some experiments which involve counting and image analysis of } blood platelets and other cells on different polymer materials. We have } used eosin staining for fluorescence in LM. We would like to be able to } carry out some equivalent technique in SEM, in order to be able to get a } clear image of the cells so that we can later quantitate cell numbers and } shapes without having to attempt too much enhancement of low contrast/noisy } SEM micrographs. } } A colleague has suggested cathodo-luminescence (CL, we have the appropriate } detector on our SEM). Does anyone have any experience with CL? In } particular, what are appropriate stains for cells (something that will bind } to cell membranes would be best in our application. } } I will, of course, post a summary of replies. } } Arthur Brandwood. } - } --------------------------------------------------------------------- } Arthur Brandwood. A.Brandwood-at-unsw.edu.au } Graduate School of Biomedical Engineering } University of New South Wales Phone: +61 2 385 3906 } Sydney, NSW 2052, Australia Fax: +61 2 663 2108 }
From the paper written by Berger, Mayer and Kohl in Ultramicroscopy 55 (1194) 101-112, the focus conditions of energy loss images were different from that of the zero loss image. Because of the large decrease in the intensity, the focus condition of 200 eV loss image was used for Oxygen K-edge images. I had similar situation in acquiring energy loss images, I had to change focus conditions when I shifted from zero loss image to energy loss images.
Now, somebody claimed that the EELS spectrum was shifted by increasing a corresponding increase in high voltage of the microscope (this is true), so the focus condition should not change.
Could you tell me about your experience in acquiring energy loss images in EM912. Did you have to change focus condition when you shifted from zero loss to energy loss images? Thanks.
Have a NICE DAY!!
*************************************************************************** * Jong-Shing Bow ** *** * Center for Solid State Science ** TEL: (602)965-4534 *** * Arizona State University ** FAX: (602)965-9004 *** * Tempe, AZ 85287-1704 ** Email:smtp%"bow-at-csss.la.asu.edu" *** ***************************************************************************
At 8:02 94/12/14 +0200, Jouko K. Maeki wrote: .... Jouko} To awoid telling you things that you have already tried, please, indicate Jouko} which solutions and conditions you have tested. Jouko} Jouko} Jouko M{ki
The solutions and conditions that we've already tried were following as:
Electropolishing solutions: 1. Perchloric Acid 2.5, 5, 10, 25, 100 ml n-Butyl Cellosolve (2-Butoxyethanol) 20 ml Ethanol Bal.
2. Hydrochloric Acid 130 ml, 10 ml n-Butyl Cellosolve 100 ml, 60 ml Methanol 670 ml, 430 ml
3. Nitric Acid 150 ml Ethanol 300 ml
4. Magnesium Perchlorate { Mg(ClO4)2 } 11.2 g Lithium Chloride { LiCl } 5.3 g Methanol 500 ml n-Butyl Cellosolve 100 ml * We've been trying to electropolish using Bernie Kestel's solution.
Electropolishing conditions: Temperature -30 ~ -40 C Voltage 5 ~ 50 V Current ~ 30 mA Cathode SUS
Does anyone know other solutions?
-- Tohoku University Dept. of Materials Processing Hirotoshi Enoki Dept. of Materials Science Mayumi Suzuki
---------------------------------------------------------------- Hirotoshi Enoki E-mail: enoki-at-material.tohoku.ac.jp Dept of Materials Processing, Faculty of Engineering Tohoku University, Sendai, JAPAN ----------------------------------------------------------------
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 9:26 AM
Date:12/15/94 URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html NC EMAL
Hi there, my apologies to people who are not ESEM users but this is the one of the only ways I know to try and get email addresses.
If you are a user of an environmental scanning electron microscope, please reply to this message. I have a regular mailing list of all ElectroScan ESEM users and Hitachi and JEOL low vacuum SEM users, but I am trying to see how many of you I can reach electronically. Thanks. I know Topcon makes a Wet SEM, but I am unable to get the mailing list from their representative, he seemed guard it jealously!
Using image analysis to quantify the shapes of cells, especially platelets, adherent to polymers can be quite difficult since it is very difficult to separate the cell from the background. I have no experience using cathodoluminescence, however I have used SE and BSE SEM, HVEM, and various modes of light microscopy to analyse platelet-material interaction. Since it appears that SEM is required or appropiate my suggestion is to bite the bullet and use your image analyis system to outline the platelets by hand. Getting the right contrast to permit digital recognition of platelets in all morphological forms (from nearly spherical to very flat pancake-like shapes) on less than perfectly smooth surfaces, which also have a variety of electron interaction properties, is very difficult. While the use of markers to specifically label the platelet may work, this too has problems. While we routinely use colloidal gold for many studies of platelet function I would in general not recommend such techniques for this task for the following:
1. There is always some non-specific background adsorption of the probe to the surface. On different polymers this background would be expected to vary since adsorption is a function of the surface chemistry of the polymers. 2. It is difficult to choose an appropiate marker to uniformly label platelets since many platelet structures change their distribution when the platelet morphology or activation level changes. This includes most (by absolute number) of the glycoproteins on the platelet surface, cytoskeletal components, and others. 3. Platelets on surfaces are rarely entirely isolated from other platelets: they aggregate. No matter how good your digital thresholding is, it will be unable to isolate individuals from clusters of platelets.
Since we rarely have a problem in detecting cell borders by eye (brain) I feel that your best bet is to do the isolation using the interactive feature of your digital image analysis sytem. Alternatively, depending on the nature of the platelet-polymer interaction, you may find it appropiate to analyse platelet shapes using non-digital methods (eg: see Goodman et al., J. Biomed. Material Research 23: 105, 1989, and 27:683, 1993). If you have any further questions on the quantification of platelet shapes, I would be happy to discuss them off the Microscopy server.
Steven L. Goodman ----------------------------------------------------------------------------- Steven L. Goodman, Ph.D. Dept. Animal Health and Biomedical Sciences 608-262-0816 (office) University of Wisconsin 608-262-7420 (FAX) 1655 Linden Drive Madison, WI 53706 --------------------------------------------------------------------------
The School of Optometry at the University of New South Wales has for disposal a Siemens I (1960) Elmiskop TEM. Included are complete spare power supplies and 2 spare high voltage cables. Spare parts galore., heaps of filaments.
ALL THIS IS FREE TO SOME LUCKY PERSON WHO WILL TAKE IT AWAY!
To get this fine antique, call Brian Pirie, School of Optometry, Newton Building, University of New South Wales, SYDNEY, NSW 2052 Australia.
I recently received a message from a colleague at CNRS in France. It seems there exists an email file entitled "Good Times" which will reformat your hard drive if downloaded. I wondered whether anyone else has heard about this. The warning is to not download this email file if received.
D.D. Perovic University of Toronto perovic-at-ecf.utoronto.ca
------------------- A - T - T - E - N - T - I - O - N ------------------- | CIAC is available 24-hours a day via its two skypage numbers. To use | | this service, dial 1-800-759-7243. The PIN numbers are: 8550070 (for | | the CIAC duty person) and 8550074 (for the CIAC manager). Please keep | | these numbers handy. | -------------------------------------------------------------------------
Welcome to the fourth issue of CIAC Notes! This is a special edition to clear up recent reports of a "good times" virus-hoax. Let us know if you have topics you would like addressed or have feedback on what is useful and what is not. Please contact the editor, Allan L. Van Lehn, CIAC, 510-422-8193 or send E-mail to ciac-at-llnl.gov.
$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$ $ Reference to any specific commercial product does not necessarily $ $ constitute or imply its endorsement, recommendation or favoring by $ $ CIAC, the University of California, or the United States Government.$ $-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$-$
THE "Good Times" VIRUS IS AN URBAN LEGEND
In the early part of December, CIAC started to receive information requests about a supposed "virus" which could be contracted via America OnLine, simply by reading a message. The following is the message that CIAC received:
--------------------------------------------------------------------------- | Here is some important information. Beware of a file called Goodtimes. | | | | Happy Chanukah everyone, and be careful out there. There is a virus on | | America Online being sent by E-Mail. If you get anything called "Good | | Times", DON'T read it or download it. It is a virus that will erase your | | hard drive. Forward this to all your friends. It may help them a lot. | ---------------------------------------------------------------------------
THIS IS A HOAX. Upon investigation, CIAC has determined that this message originated from both a user of America Online and a student at a university at approximately the same time, and it was meant to be a hoax.
CIAC has also seen other variations of this hoax, the main one is that any electronic mail message with the subject line of "xxx-1" will infect your computer.
This rumor has been spreading very widely. This spread is due mainly to the fact that many people have seen a message with "Good Times" in the header. They delete the message without reading it, thus believing that they have saved themselves from being attacked. These first-hand reports give a false sense of credibility to the alert message.
There has been one confirmation of a person who received a message with "xxx-1" in the header, but an empty message body. Then, (in a panic, because he had heard the alert), he checked his PC for viruses (the first time he checked his machine in months) and found a pre-existing virus on his machine. He incorrectly came to the conclusion that the E-mail message gave him the virus (this particular virus could NOT POSSIBLY have spread via an E-mail message). This person then spread his alert.
As of this date, there are no known viruses which can infect merely through reading a mail message. For a virus to spread some program must be executed. Reading a mail message does not execute the mail message. Yes, Trojans have been found as executable attachments to mail messages, the most notorious being the IBM VM Christmas Card Trojan of 1987, also the TERM MODULE Worm (reference CIAC Bulletin B-7) and the GAME2 MODULE Worm (CIAC Bulletin B-12). But this is not the case for this particular "virus" alert.
If you encounter this message being distributed on any mailing lists, simply ignore it or send a follow-up message stating that this is a false rumor.
Karyn Pichnarczyk CIAC Team ciac-at-llnl.gov
- ------------------------------ Contacting CIAC
If you require additional assistance or wish to report a vulnerability, call CIAC at 510-422-8193, fax messages to 510-423-8002 or send E-mail to ciac-at-llnl.gov. For emergencies and off-hour assistance, call 1-800-SKY-PAGE (759-7243) and enter PIN number 8550070 (primary) or 8550074 (secondary). The CIAC Duty Officer, a rotating responsibility, carries the primary skypager. The Project Leader carries the secondary skypager. If you are unable to contact CIAC via phone, please use the skypage system.
- ------------------------------ This document was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation or favoring by the United States Government or the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or the University of California, and shall not be used for advertising or product endorsement purposes.
- ------------------------------ End of CIAC Notes Number 94-04 94_12_06 ****************************************
The so-called "Good Times virus" received a great deal of attention for a time here at the University of Michigan, and warnings were given several times on our network. Some of our computer people eventually decided that it was a hoax. I don't know how they reached this conclusion.
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On Thu, 15 Dec 1994, PEROVIC Doug Dragan wrote:
} } I recently received a message from a colleague at CNRS in France. It seems there } exists an email file entitled "Good Times" which will reformat your hard drive } if downloaded. I wondered whether anyone else has heard about this. The warning } is to not download this email file if received. } } D.D. Perovic } University of Toronto } perovic-at-ecf.utoronto.ca }
Reply... RE} Virus warning Bogus! Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX: (313)936-3352 jfmjfm-at-umich.edu or John_Mansfield-at-mse.engin.umich.edu URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
This message which warns about the so called "Good Times" virus, is a hoax. I have been informed of this from a reliable source (Adam Engst, who publishes the newsletter Tidbits). If you think about it the mere act of reading a text message cannot really hurt your system. Those of you who have automatic downloaders and decoders (zip, binhex etc) like I do could get bitten but the resulting code would have to launch itself and I think that is a little far-fetched. So dont worry! Jfm.
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I recently received a message from a colleague at CNRS in France. It seems there exists an email file entitled "Good Times" which will reformat your hard drive if downloaded. I wondered whether anyone else has heard about this. The warning is to not download this email file if received.
D.D. Perovic University of Toronto perovic-at-ecf.utoronto.ca
------------------ RFC822 Header Follows ------------------ Received: by mse.engin.umich.edu with SMTP;16 Dec 1994 00:27:27 -0400 Received: by judgmentday.rs.itd.umich.edu (8.6.9/2.2) with X.500 id XAA12532; Thu, 15 Dec 1994 23:23:53 -0500 Received: from AAEM.AMC.ANL.GOV by judgmentday.rs.itd.umich.edu (8.6.9/2.2) with SMTP id XAA12527; Thu, 15 Dec 1994 23:23:51 -0500
I would like to request info on Monte Carlo Programs (for broadening of beams w.r.t. to x-ray interaction) available, to be used on a P.C. You can contact me directly if you wish.
Thanks
Fred Pearson McMaster University Phone: (905) 525-9140 ext. 24609 Fax: (905) 521-2773 email: eoptics-at-mcmail.cis.mcmaster.ca
Do NOT use non-polar solvents - such as ethylene dichloride, chloroform, xylene, toluene, propylene oxide, etc. I would stay away from acetone as well although I don't think Formvar is very soluable in it. Use water based solutions or perhaps water/alcohol solutions only. Try to stay away from strong detergents, if possible, to reduce the chance of removing your films accidentally. A relatively weak detergent shouldn't hurt.
I hope this helps.
Dan
On Fri, 16 Dec 1994, Tim Foecke wrote:
} I want to suspend a powder and pick it up on a carbon-coated } Formvar film. What should I NOT use as a suspension medium? } } Tim Foecke, NIST } tfoecke-at-nist.gov } }
We've been discussing computer viruses here and the possibility of infecting a computer via a downloaded file or E-mail. Files other than *.exe, *.sys, and *.com (PC systems) can be infectous if the virus is a FAT table (File Allocation Table) infection. If the virus infects the FAT simply running a DIR of an infected FAT can tigger the virus to become active. Therefore it is possible that an E-mail message could load a virus into your computer but it would have to downloaded to your system as John Mansfield suggested. Simply viewing it on screen using a network mail system would not infect your system. Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
John} This message which warns about the so called "Good Times" John} virus, is a hoax. I have been informed of this from a John} reliable source (Adam Engst, who publishes the newsletter John} Tidbits). If you think about it the mere act of reading a John} text message cannot really hurt your system. Those of you John} who have automatic downloaders and decoders (zip, binhex John} etc) like I do could get bitten but the resulting code would John} have to launch itself and I think that is a little John} far-fetched. So dont worry! Jfm.
Admittedly this "Good Times" virus is a hoax put lest we get over-confident in our security we should remember the "Worm" virus that caused so much damage on the Internet just a few years ago was the result of a security hole in the mail system. Also before mail handlers filtered 8-bit characters you could send escape sequences that would run any command you wanted on a VT-100. Also with some of the new multimedia mail much of the "multimedia" is run through an arbitrary program on you computer. Try running Mosaic on a WWW site that has a postscript file to view. I will generally call ghostscript on your computer to view it. Again, there are hooks in HTML to call an arbitrary program and run it on your computer. That arbitrary program could be "format" for a clever hacker. As Captain Pickard says, "The price we pay is constant vigilance." (The Drumhead).
-- Kevin Burton Noran Instruments voice: (608) 831-6511 x317 2551 West Beltline Highway, Room 532 FAX: (608) 836-7224 Middleton, WI 53562 email: kburton-at-noran.com
In article {3ccd9q$j98-at-lucy.infi.net} , wmdawes-at-infi.net (William Dawes) writes: } } [snip] Is there no place for commercial interests on the internet? Do } they not also serve our needs? Can't they coexist? } } This is important to me, as I am a 'downsized' 30-year scientist who } would like to make a living using the net. What standard is at stake } here? Would you also find it inappropriate to advertise job openings } or oneself through a resume? } Dear William, There are newsgroups for jobs offered and wanted, and perhaps there should also be commercial newsgroups as well. For those with only e-mail access, this would not be useful, so perhaps lists accept- ing commercial announcements--microscopy does this to a limited extent-- should require some indication in the subject heading. Those using a PC for email might have a problem with space, so there are strong arguments for restricting the volume. Time will probably fix this concern to the satisfaction of most internet users, but there is obvious controversy at this time. Yours, Bill Tivol
Hello, I am currently in the position of having to make a scope acquisition decision in only a few days. Under consideration is the Topcon scope SR-50. Has anyone worked with this scope? Please tell me about performance, age or about when this scope came out, and what you thought of it. Is it comparable to a Topcon WB-6 with a LaB6 attachment? Thanks in advance,
Lou Ann Miller University of Illinois Veterinary Medicine 217-244-1566 lmiller-at-ux1.cso.uiuc.edu
There have been a lot of responses regarding my recently posted question about simulation softwares for diffraction patterns and high resultion images. I thank all of you who generously shared your information with me. The following is a summary of the collected information which may be useful to the other microscopists. (Note: Some of the information I got, e.g. phone numbers, are not updated. I have them corrected here. I am not associated with any of the companies.)
1) CrystalKit/MacTempas are available from
TOTAL Resolution 20 Florida Street Berkeley, CA 94707 (510) 527-9100 Fax (510) 527-9151 Attention: Roar Kilaas E-mail: Roar_Kilaas-at-macmail3.lbl.gov
Both programs are available in both 68K and native PowerPC versions. the PowerPC version of MacTempas runs about 7 times faster for large calculations on an 8100/80 than on a quadra 950. CrystalKit likewise runs much faster on the PowerPC platform as well.
2) Simply
This is a freeware developed by Univesity of Lyons - France and runs on PC.
3) EMS, available from P.S. Stadelmann 12M - EPFL Swiss Federal Institute of Technology CH-1015 Lausanne Switzerland
Ref. PA Stadelmann, Ultramicroscopy 21 (1987) 131-146
and Cerius, available from Molecular Simulations 16 New England Executive Park Burlington MA 01803-5297 U.S.A.
These two packages run on Silicon Graphics workstations.
4) Desktop Microscopist. It allows you to build unit cells knowing the atom types and positions. You can then rotate the cell in 3 dimension. It will calculate expected diffraction patterns, stereographics projections, and x-ray patterns.
Best Regards,
Jessica Chang School of Electrical Engineering Purdue University West Lafayette, IN 47907-1285 E-mail: changj-at-ecn.purdue.edu
There have been a lot of responses regarding my recently posted question about simulation softwares for diffraction patterns and high resultion images. I thank all of you who generously shared your information with me. The following is a summary of the collected information which may be useful to the other microscopists. (Note: Some of the information I got, e.g. phone numbers, are not updated. I have them corrected here. I am not associated with any of the companies.)
1) CrystalKit/MacTempas are available from
TOTAL Resolution 20 Florida Street Berkeley, CA 94707 (510) 527-9100 Fax (510) 527-9151 Attention: Roar Kilaas E-mail: Roar_Kilaas-at-macmail3.lbl.gov
Both programs are available in both 68K and native PowerPC versions. the PowerPC version of MacTempas runs about 7 times faster for large calculations on an 8100/80 than on a quadra 950. CrystalKit likewise runs much faster on the PowerPC platform as well.
2) Simply
This is a freeware developed by Univesity of Lyons - France and runs on PC.
3) EMS, available from P.S. Stadelmann 12M - EPFL Swiss Federal Institute of Technology CH-1015 Lausanne Switzerland
Ref. PA Stadelmann, Ultramicroscopy 21 (1987) 131-146
and Cerius, available from Molecular Simulations 16 New England Executive Park Burlington MA 01803-5297 U.S.A.
These two packages run on Silicon Graphics workstations.
4) Desktop Microscopist. It allows you to build unit cells knowing the atom types and positions. You can then rotate the cell in 3 dimension. It will calculate expected diffraction patterns, stereographics projections, and x-ray patterns.
Best Regards,
Jessica Chang School of Electrical Engineering Purdue University West Lafayette, IN 47907-1285 E-mail: changj-at-ecn.purdue.edu
Neuroscience List {neur-sci-at-net.bio.net} , Microscopy List {microscopy-at-aaem.amc.anl.gov} , Info-EI List {info-ei-at-mom.spie.org} , Image Processing List {image-l-at-vm3090.ege.edu.tr} , Functional Neuroimaging List {lat-at-po.cwru.edu} , Confocal Microscopy List {confocal-at-ubvm.bitnet} , Cell Bio List {cellbiol-at-net.bio.net} Message-Id: {Pine.3.05.1.9412171009.A12826-9100000-at-pauline.sdsc.edu} Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII
What are the best meetings worldwide for 3D imaging/cell biology/neuroscience? Please reply to this list for the benefit of all. Thanks for your contribution.
Marc
Marc C. Brande, M.S. SD3D Email List:3D Imaging San Diego 3D Imaging Group To subscribe/unsubscribe:listserv-at-bobcat.etsu.edu 3840 Camino Lindo To post a message:sd3d-at-bobcat.etsu.edu San Diego, CA 92122 Email:BRANDE-at-SDSC.EDU Voice:619-587-4830
We are considering the possibility of purchasing the DEBEN RESEARCH LTD. low vacuum/environmental adapter for SEM. Does anyone have any experience with it? It would be greatly appreciated if someone could advice on its reliability, ease of use, possibility of easy conversion from low to high vacuum operation and back, etc. I would also appreciate any recommendations on other environmental adaptors available.
Thanks a lot, Alex Titkov Electron Microscopy Unit RMIT Applied Physics GPO Box 2476V Melbourne VIC 3001 alex-at-bunyip.ph.rmit.edu.au Fax: (03) 660 3837
Seasons Greetings!! We are trying to use anti-beta-galactosidase as a marker enzyme in some immunohistochemsitry labelling, much like alkaline phosohatase and horse radish peroxidase are used. We have tried it a number of times and get no labelling. We are running regular APAAP controls which do label. Any suggestions?? I have talked to Sigma about this since our components come from them, but they can't figure it out as it works for their ELISA's. Any help greatly appreciated.
Mark Elliott, Pulmonary Research Lab, UBC Vancouver Canada
How was your tissue fixed? Was the antibody made against an antigen that was fixed with the same fixative? Was the antibody selected (if it is a monoclonal) for/by immunocytochemical acitivity? Antibodies made against unfixed antigens often will not recognize that antigen in fixed tissues. It is most often best to match the antigen fixation with the tissue fixation. Check with Sigma about this. Without more information, I can't suggest anything else.
Dan
On Mon, 19 Dec 1994 MELLIOTT-at-prl.pulmonary.ubc.ca wrote:
} Seasons Greetings!! } We are trying to use anti-beta-galactosidase as a marker enzyme in } some immunohistochemsitry labelling, much like alkaline phosohatase } and horse radish peroxidase are used. We have tried it a number of } times and get no labelling. We are running regular APAAP controls } which do label. Any suggestions?? I have talked to Sigma about this } since our components come from them, but they can't figure it out as } it works for their ELISA's. Any help greatly appreciated. } } Mark Elliott, } Pulmonary Research Lab, } UBC } Vancouver Canada }
Hello All, We have a user that is labeling plant tissues with de-colorized Aniline Blue (specific for 1,3-glucans). He is curious about the excitation and emission spectra, and I can't find much info. We are currently using a Nikon UV-1A filter set with a Zenon bulb. His tissue fluorescences from blue to yellow. Any comments or references will be greatly appreciated. TIA,
C. Michael Stanley, Ph.D. Associate Director Molecular Cytology Core Facility Molecular Biology 2 Tucker Hall University of Missouri-Columbia Columbia, MO. 65211 (314) 882-4895 fax= 314-882-0123
A postdoctoral position jointly supported by The West Company, Lionville, PA and Penn State University, University Park, PA is available starting March 1995 for an initial period of two years. Candidates applying for the position should be knowledgeable in the area of cell biology or related field, and competent in designing, implementing and interpreting experiments. Knowledge in Electron Microscopy is essential and familiarity with freeze-fracture is desirable. Successful candidate would be expected to work independently. The main focus of the project is to support the development of novel drug delivery systems.
Competitive salary will be based on experience and include benefits. Applicants should send their curriculum vitae to: Dr. June Rae Merwin, The West Company, 101 Gordon Drive, P.O. Box 645, Lionville, PA 19341-0645, or call Dr. Merwin at 610-594-3187. !
************************************************************************* M.V. Parthasarathy Professor of Plant Biology Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
After receiving a couple of answers to my earlier question, and re-reading my questions I realized I should have made the question a little clearer. We currently use the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system for detecting anitgens in lung tissue. However, the problem we are having is that in guinea pig lung we get a nonspecific binding of the antibodies (primary, secondary and conjugate) within the airway epithelium. This occurs on cryosections and in order to get rid of it we must fix the slides in formalin. This creates a problem when we are staining for antigens which are fixative sensitive. Using the peroxidase-anti-peroxidase (PAP) method is not an option since there is high level of endogenous peroxidase in teh lung. The blocking methods to remove this are quite harsh on the frozen tissue and thus morphology is greatly compromised. Therefore we are looking at using beta-galactosidase as the capture enzyme. The antibody we are using is from Sigma, as is the beta -galactosidase. They use both of these in their ELISA studies at SIGMA. Therefore, the problem of fixation of the tissue etc as suggested by Dan Possin is not relevant. We are using an insoluble substrate so will be appropriate for immunohistochemistry, and indeed in a test tube we get a colour reaction. The problem is we get no reaction on tissue which we know has antigen in it-when we run APAAP on adjacent slides we get good labelling. The problem I think is in trying to conjugate the anti-beta-gal with the beta-gal. Anybody have any methods to verify that this has occured??? Are there any other suggestions??? I have talked with Sigma Tech Services about this a couple of times and they have no idea what might be wrong. Any help would be greatly appreciated.
Inspired by the success of this forum, a group of Canberra EM microscopists thought it would be a good idea to set up something similar within Australia, for the benefit of microscopists within Australia, but also ex-patriates and anyone else who might be interested in messages of less general interest than are posted world-wide.
The computer staff at the Research School of Biological Sciences, Australian National University, have made space available on a server. You can join the list by emailing directly to me, or by emailing to maiser-at-rsbs-central.anu.edu.au , with "subscribe AUSTEM" as the message.
Happy Christmas,
Sally Stowe ---------------------------------------------------------------------- Sally Stowe Australian National Univ. Facility Coordinator Canberra, AUSTRALIA ANU Electron Microscopy Unit Ph 61 6 249 2743 RSBS, Box 475 Email stowe-at-rsbs-central.anu.edu.au FAX 61 6 249 4891
On Sat, 17 Dec 1994 lmiller-at-ux1.cso.uiuc.edu wrote:
} Hello, } I am currently in the position of having to make a scope acquisition } decision in only a few days. } Under consideration is the Topcon scope SR-50. Has anyone worked with this } scope? Please tell me about performance, age or about when this scope came } out, and what you thought of it. Is it comparable to a Topcon WB-6 with a } LaB6 attachment? } Thanks in advance, } } Lou Ann Miller } University of Illinois } Veterinary Medicine } 217-244-1566 } lmiller-at-ux1.cso.uiuc.edu } Dear Lou Ann Without regard to a specific model or manufacturer, if you have the time, establish the features and instrument requirements that you need for your investigations. Some of these are; resolution, sample size, range of sample translation (x,y,z), method of sample exchange, number of operators that will use it, ease of operation, and SERVICE, SERVICE, SERVICE.
Your resolution requirement will tell you what kind of electron gun will be needed (tungsten, LaB6 or FEG), and a large chamber will usually allow increased sample translation, while a vacuum interlock for sample exchange will greatly increase productivity. The types of detectors may also be important to you.
This is by no means a complete list of questions for you to answer, but I hope it will get you started before you sign the check.
Seasons Greetings Has anyone any ideas re the detrimental effects on TEM photographic plates after long term dessication (2-3 months), also long term storage of un-opened packages of same at low temperature (4-10oC)? What are the "symptoms" shown on the exposed micrograph negative? As I purchase in bulk, I would like opinions as to how TEM plates can and should be stored. Thanks in advance John////////
John F. Putterill Electron Microscopy Unit Tel: (Int) 27-12-529-9174 Pathology Section Fax: (Int) 27-12-529-9165 Onderstepoort Veterinary Institute E-mail: john-at-moon.ovi.ac.za Private Bag X05 Onderstepoort 0110 South Africa
} On Tue, 20 Dec 1994 11:27:11 EST1, } SALLY STOWE writes: } } } } } Inspired by the success of this forum, a group of Canberra EM } } microscopists thought it would be a good idea to set up something } } similar within Australia } } but... is there a need? The beauty of Listservs is that national } boundaries don't } matter... } } Arthur Brandwood
I agree with Arthur Brandwood's comments.
Arthur Day, Electron Microscopy Group Ansto Advanced Materials Program Phone: 61-2-717-3457 PMB 1, Menai (Sydney), NSW, 2234 Fax: 61-2-543-7179 Australia Email: ard-at-atom.ansto.gov.au
Dear readers, I think the global list-server is the best forum, rather than a regional group as recently mooted, however I do have a suggestion regarding list structure. I wonder if anyone else finds that about half of the messages posted are ignored (deleted without reading) since the subject is not within their interest range. I know that microscopy is a broad discipline, but lets face it, MSA runs parallel sessions for Biological and Materials Sciences (so do most Microscopy Societies) for the very good reason that the two are very different fields.
Should we have a pair of list-servers, one for Biological Science and one for Materials Science? You could join both just as easily as joining one, you could send information and comments to both almost as easily as to one. I notice quite a lot of people unsubscribing from the list- server recently, why? I did unsubscribe myself once because I found the mail box so full I couldn't afford the time to read it all, happily I find more time now. I don't want to divide the community, I want to get more people involved.
} Seasons Greetings } Has anyone any ideas re the detrimental effects on TEM photographic } plates after long term dessication (2-3 months), also long term } storage of un-opened packages of same at low temperature (4-10oC)? } What are the "symptoms" shown on the exposed micrograph negative?
"Sparking" is the only symptom I have come across personally. This is caused by excessive dryness of the negative prior to loading in the film cassette (metal). A static charge builds up on the film itself which then discharges, sometimes causing visible sparks, when the film is loaded into the plate holder prior to use. SO163 is particularly prone to this if dessicated for more than a couple of days (your local R.H. will affect this). After sparking, negatives often resemble an all-night exposure of the sky during a large thunderstorm - very attractive, but perhaps not quite what is desired!
The leaflet that comes with the film should have details on recommended storage. There may be some detrimental effect on the dynamic response with long-term storage, whilst all film has an expiry date. Hopefully, there's a chemist here who can tell us exactly what happens here.
-------------------------------------------------------------------------- Dr. S.L. King Dept. Electronics and Electrical Engineering, University College London Torrington Place, London WC1E 7JE England
Season Greetings to all of you (including our Australian friends!)
I hope you will NOT create a seperate listserver dedicated to EM in Australia (notwithstanding I only use LM). What purpose might seperation serve. There is already enough separation in the world. Lets stick together and share out knowledge i.s.o. falling apart.
Regards,
Kees.
To my opinion "Knowledge is Power" is wrong and should read "Knowledge is Responsibility" -- Kees van der Wulp TNO - Nutrition & Food Research Institute INTERNET : vanderwulp-at-mbl.tno.nl Division of Toxicology VOICE : +31 15 843101 PO-Box 5815 FAX : +31 15 843989 2280 HV RIJSWIJK (NL) General TNO Info : http://www.tno.nl THE NETHERLANDS
I've recently noticed that some of my older viewgraphs (~2 years old) that were printed on a Tektronix-Phaser dye sublimation printer have started to fade. The degradation is not uniform, but rather occurs in patchy regions. It also appears that the problem is primarily with view-graphs that I have stored in vinyl sheet protectors; viewgraphs stored for a comparable time in polyethylene protectors appear to be ok.
Has anyone had similar problems?
+---------------------------------------------+ ! Douglas L. Medlin ! ! Physical Properties of Materials Department ! ! Organization 8715 ! ! Sandia National Laboratories ! ! Livermore, California 94551 ! ! ! ! (510) 294-2825 ! ! dlmedli-at-california.sandia.gov ! +---------------------------------------------+
We are using a Kodak dye-sublimation engine (Codonics printer) and have had good stability when storing in polyethylene protectors. Pages left out of protectors seem stable except for areas that had fingerprints. Slight fading and coloring of black regions seems to occur for unprotected prints left on the wall. We do not know about long- term stability as our oldest prints are from eight months ago. Any data on long-term stability of dye-sub prints would be interesting.
Most vinyls are not acid-free, hence, the organic dyes will react with the acid residues. Try storing your images, whether they be viewgraphs, slides, negatives, or prints in archival, acid-free, polyethylene sheet protectors.
Russ Cook Argone National Laboratory Argonne, IL 60439
Dear John, We have some old LoDose and MRF-32 film which has been stored in a refrigerator for ~15 years. We get very good performance from it. However, I am aware of problems with over-desiccated film. In particular with LoDose, static discharges can leave some remarkable dendritic patterns. They are quite lovely in themselves, but they do compromise the image one wants. How this translates to less sensitive film (4489, SO163, etc.) I don't know. Our normal EM films get used up fairly quickly (~6 mos. average). Good luck. Yours, Bill Tivol
I took a bit too much for granted in posting the notice about an Australian listserver. To be honest, it never crossed my mind that anyone would assume there was any intention to duplicate the functions of this forum on a smaller scale, as far as questions with any sort of generality are concerned. What on earth would be the point? I would also be sorry to see this server split into Biological and Materials SCience groups - many EM units, including this one, work at least to some extent in both biological and non-biological areas, and the cross-fertilization of ideas is often valuable.
The intention of AUSTEM is to act as a clearing house for local questions of the "borrowing a cup of sugar" sort. Someone in Canberra may need a machine part, some film, particular sized grids or stubs really urgently - it makes sense to call the rest of Australia, but not Wheeling, West Virginia. Somebody may need a lift from Cairns to the next conference in Melbourne...there isn't much point clogging a terminal in Amsterdam.
We actually have a Canberra-wide listserver as well - it isnt used much, but it's there if we need it. Its been running about a year, and takes almost no time to maintain.
I dont see anything wrong with one global/many national/myriad local listservers. All it needs is a modicum of common sense on the part of the person making the posting. The local servers should take much less maintenance than the global one - I really appreciate the job Nestor is doing, but I wasnt proposing to lighten his load!
Sally ---------------------------------------------------------------------- Sally Stowe Australian National Univ. Facility Coordinator Canberra, AUSTRALIA ANU Electron Microscopy Unit Ph 61 6 249 2743 RSBS, Box 475 Email stowe-at-rsbs-central.anu.edu.au FAX 61 6 249 4891
For those of you that remember, we had a round robin test of greyscale printers at last year's MSA meeting in NewOrleans. I have kept all the submission from that test and intend on bringing them back to the next meeting in Kansas City. I will send out a call to each person who submitted a print(s) to the round robin test and ask that they print a new copy (if possible) of the same test image. We will then have a direct comparison of data that is very nearly 1 year old. All prints are being stored together in a plain folder in an office/lab environment, out of direct sunlight in a file cabinet.
Cheers.. Nestor Zaluzec Your Friendly Neighborhood SysOp
} I've recently noticed that some of my older viewgraphs (~2 years old) } that were printed on a Tektronix-Phaser dye sublimation printer } have started to fade. The degradation is not uniform, but rather } occurs in patchy regions. It also appears that the problem is } primarily with view-graphs that I have stored in vinyl sheet protectors; } viewgraphs stored for a comparable time in polyethylene protectors } appear to be ok. } } Has anyone had similar problems? } ! Douglas L. Medlin !
We have had Mitsubishi CP100B video copier prints get stuck to the inside of (presumably pvc) photograph wallet things, with loss of colour when you extract them.
I have recently been doing research on obtaining the tip geometry of atomic force microscope probes using the AFM and VLSI calibration grids. The procedure yeilds three dimensional information of the tip which can be used as an inspection technique or for "deconvoluting" the images.
Thus far, we have used the grids supplied by Digital Instruments. These are circular depressions about 500 nm in diameter and spaced 1000 nm apart. These are limited for our purposes when examining square pyramidal probes (think of fitting a square peg in a round hole).
I would therefore like to ask you if you would know of a manufacturer of other VSLI grids. I would be most interested in the sources of such standards rather than the vendors.
Any comments or assistance would be appreciated.
Peter Markiewicz, pmarkiew-at-alchemy.chem.utoronto.ca
Subject: Time:12:12 PM OFFICE MEMO RE:Files for film Date:12/21/94
I have obtained crystal clear plastic files which are advertised as being made of archival-quality polypropylene, containing no PVC, and being suitable for long-term protection of all photographs, slides, and negatives, from 20th. Century Plastics, 3628 Crenshaw Blvd., Los Angeles, CA 90016 (800-767-0777). Results to date have been good.
} In article {3ccd9q$j98-at-lucy.infi.net} , } wmdawes-at-infi.net (William Dawes) writes: } } } } [snip] Is there no place for commercial interests on the internet? Do } } they not also serve our needs? Can't they coexist? } } } Dear William,
what you hve to remember is that many of the folks on newsgroups acess ther internet through commercial companies so they are paying for each message they recieve - alowing commercial messages in a regular newsgroup is like sending advertising through regular mail with postage due
a separate commercial newsgroup is the only alternative that I see
Subject: Time:12:12 PM OFFICE MEMO RE:Files for film Date:12/21/94
I have obtained crystal clear plastic files which are advertised as being made of archival-quality polypropylene, containing no PVC, and being suitable for long-term protection of all photographs, slides, and negatives, from 20th. Century Plastics, 3628 Crenshaw Blvd., Los Angeles, CA 90016 (800-767-0777). Results to date have been good.
I use this same product from 20th Century Plastics for my personal photos. Over the last 3 yrs at least, kept in a dry, dark cabinet the photos have aged respectably (no detrimental effects).
P. Joyce
Peter J. Joyce Graduate Research Assistant - Materials Science & Engineering University of Texas at Austin (512) 471-5723
Use either sodium methoxide or sodium ethoxide. These can be made by making a saturated solution of sodium hydroxide in either 100% ethanol or methanol. It takes a little time to mature and you end up with a sherry coloured liquid. By sherry colour I mean a fino not an amontillado or an oloroso ! When using, dilute 1:1 in either EtOH or MeOH. It's powerful stuff so use with caution.
Best wishes for the holiday season
Patrick Echlin Director, Multi-Imaging Centre, Cambridge UK
At the University of Delaware we are planning to put all our electron microscopes under one umbrella organization and are looking into the idea of using third party vendors for some/all of our service contract needs. We have JEOL, Philips, Zeiss, Cambridge and Amray TEMs and SEMs along with Kevex and EDAX EDS systems. Questions: a) Does anyone know of independent vendors for service contracts on TEMs and SEMs? b) What sort of experiences have folks had with them? c) Do folks think service contracts are really worthwhile for EDS detectors and are there third party vendors?
Feel free to reply to me personally and I'll post a synopsis of useful replies later. Happy holidays.
Rick Hall Materials Science University of Delaware
Message-Id: {9412221559.AA02912-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Dissolving Araldite Orig-Author: {"Ronald M. Anderson" {ron-anderson-at-VNET.IBM.COM} }:ddn:wpafb ----------------------------------------------------------- We potted some complex shaped parts in Araldite epoxy. Now we want to dissolve the epoxy completely and get the parts back. (It's a long story......).
Does anyone know how to chemically dissolve Araldite? Private response suggested. I'll summarize if there is anyone else who will admit to smearing goop all over valuable specimens.
Thanks to everyone who has provided help to me and my organization throughout this past year. May everyone be blessed with a happy and joyful holiday and the best of wishes for the coming year! MERRY CHRISTMAS AND HAPPY NEW YEAR! Phil
SOLICITATION FOR NRC OR ASEE-ONT POSTDOCS AT NRL TRIBOLOGY SECTION, SURFACE CHEMISTRY BRANCH, NRL.
Dear University Colleague,
We are looking for post-doctoral candidates interested in tribology research from a surface science point of view. The NRL Tribology program offers cooperative research associateships (to US citizens only - sorry) in programs covering surface films, tribochemistry, microscopic aspects of adhesion/friction/wear and new approaches to probing friction at the atomic scale. These programs are described in more detail in the two write-ups below. (The deadlines for the two 1995 NRC competitions are 15 Jan 1995 and 15 April, repectively.) Please pass this message on to potential candidates.
CONTACT: Dr. Irwin L. Singer Naval Research Laboratory-Code 6176 Washington DC 20375 TEL: (202) 767-2327; FAX: (202) 767-3321; E-MAIL: SINGER-at-CHEM.NRL.NAVY.MIL
or, the post-doc coordinator:
Ms. Jessica Hileman NRL Prgram Coordinator Naval Research Laboratory-Code 1005.7 Washington DC 20375 TEL: (202) 767-3865 E-MAIL: HILEMAN-at-UTOPIA.NRL.NAVY.MIL !#1. "MACHO" COATINGS BUILT ATOM-BY-ATOM
Protective coatings for metals and ceramics will enable common engineering materials to be used in extreme environments. Until recently, however, poor coating adhesion and uncontrollable film microstructures prevented their full exploitation. Ion beam processing adds a new measure of control for tailoring the chemistry and structure of coatings. The Tribology Group at NRL is using ion-beam assisted deposition techniques to control the elastic-plastic properties of thin films designed to protect surfaces subjected to stress and oxidation during sliding contact. A dual ion beam deposition system, with a third ion assist gun, makes it possible to grow multilayer composite films with compositions and structures never before achievable by physical vapor deposition techniques. Thin films have been grown with microstructures that vary from amorphous to polycrystalline to single crystal, depending on the bombardment energy of the ion-assist beam. Friction coefficients of solid lubricant films grown by these techniques have 10 times lower friction than the best lubricating oils. Can you figure out the chemical and structural basis for protective coatings? To assist you, we supply extensive analytical facilities, including high resolution SAM, XPS, and Raman for characterizing the film before, during (Raman, SAM) and after wear; we use simple testing devices (computerized friction and wear machines, scratch testers, indentation mechanics techniques,...) to understand sliding behavior as well as film failure; we develop sliding contact models to interpret sliding wear processes; we use thermochemical calculations to account for the tribochemical products formed during sliding; and, most importantly, we look for post-docs who can dream up clever testing and analytical approaches to reveal the structure-property relationships of protective coatings.
REFERENCES I.L. Singer et al., Appl. Phys. Lett., 57, (1990) 995-997. I.L. Singer Surf. Coatings Technol., 49 (1991) 474-481. I.L. Singer in Fundamentals of Friction eds. I.L. Singer and H.M. Pollock (Kluwer Academic Publishers, Dordrecht, 1992) pp. 237-261. L.E. Seitzman, et al., Surf. Coatings Technol. 52 (1992) 93-98. P.D. Ehni and I.L. Singer, Appl. Surf. Sci., 59 (1992) 45-53. I.L. Singer et al., Appl. Phys. Lett., 65 (1994) 3191-3193. !#2. NEW WAYS OF PROBING THE PHYSICS AND CHEMISTRY OF FRICTION AT THE ATOMIC SCALE.
Fundamental studies of sliding contacts can lead to new understanding of friction processes and wear reduction. The processes are extremely complex, but can be linked to chemical reactions or structural instabilities at the velocity accommodating interface. What makes this science doubly difficult is that sliding takes place at a buried interface. New proximal probes, like atomic force microscopy, are being developed to examine these changes locally. The evolving surface compositions and microstructures are analyzed by proximal probes as well. More traditional microscopies (SAM, SIMS, XPS, FTIR, EDX, TEM) help characterize evolving surface compositions and microstructures, providing data to model the tribochemical and tribomechanical events. But how do we follow the chemical and structural changes that take place in the buried interface in real time? Atomic interactions occur in picoseconds and materials transform in microseconds. New and clever experimental and theoretical approaches are needed to unravel friction processes at the time and length scales on which they occur. Have you got any ideas how to probe friction and wear processes in real time: e.g. to quantify energy dissipated during sliding via phonon spectroscopy? Can you devise experiments with the AFM/FFM that can investigate the relationships between static friction and fracture processes, between kinetic friction and surface forces? Can you perform simulations of friction events, develop hybrid models that can extend computational simulations from the nm/femtosec scale to the !m/!sec scale? Do you have ideas for bridging the gap between surface chemistry and deformation structures? This solicitation is for new approaches to identify the microscopic phenomena underlying macroscopic behavior during sliding.
REFERENCES Fundamentals of Friction eds. I.L. Singer and H.M. Pollock (Kluwer Academic Publishers, Dordrecht, 1992). I.L. Singer, "Friction and Energy Dissipation at the Atomic Scale - A Review," J. Vac. Sci. Technol., A 12 (1994) 2605-2616. Irwin ****************************************** Irwin Singer NRL - Code 6170 Washington DC 20375 tele: 202-767-2327; fax: 202-767-3321 *******************************************
I'm currently working on importing some artifically created images (e.g. bitmaps) into the NanoScope III file format used by the software of NanoScope III AFM/STM by Digital Instruments. For that purpose i've to assign height information to the colours in the bitmap. I'm using 256-indexed bitmaps. This would mean that in case of a depth scale of lets say 10 nm, i've to assign 0.039 nm / colour step.
According the NanoScope III manual the height of a data point can be derived from the values in the file using the following formula (height data assumed):
h = (d/65536)*C
with : h ... height [nm] d ... data point (signed int)
and C :
C = Zscale * (Zattenuation/65536) * ((Zmax*2)/65536) * Zsensitivity
This means i can create the signed int value used in the NanoScope file format using the given height for each bitmap colour by:
d = (65536 * h)/C (cutting off decimals from height being a float)
it now turns out that in the simplest case of a bitmap with 2 colours (0 and 255) and an assigned max height difference of lets say 10 nm, the resulting height difference in the NanoScope III software when displaying the image is only 5 nm. This means, the resulting height difference is only half of the value it should be.
My question now is : does anyone here know the formula given by Digital in detail? Where comes the 2 in the term "Zmax*2" in C from? Has anyone already experienced similar effects?
And: no, i'm not calculating this on a Pentium ;)
Thanx for your help in advance ...
-gmf
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