We have been experimentally measuring electron gun brightness of LaB6 and CeB6 sources in our TEM's here and find that values are factors of 4-5 times lower than TEM manufacturers claims.
For example, at 400 kV experimentally we determine values of ~ 2-3 x 10**6 A/cm**2/sr while values quoted are more typically } 1 x 10**7 A/cm**2/sr. Small variances are found when one adjusts filament temp, bias, wehneldt gap & filament but not enough to make up the difference.
The brightness (b) is determined by experimental measurements in the electron microscope using the following equation:
b = 4I/(pi**2d**2a**2)
where I = probe current pi=3.14159, d = beam diameter (FWTM), a = incident beam divergence half angle. All parameters are quantitatively measured in the instrument.
We have found no reports of similiar measurements in the literature, except for the initial studies of source brightness done years ago which of course everone quotes. None of which were done in TEM's, but most were rather "bench" measurements. Has anyone else actually measured the performance of their guns in a TEM (or SEM)?
} Since I decided to bit the bullet and try to get this } system working let me post the first real message to } the Microscopy Listserver. } } =============================================================== } } We have been experimentally measuring electron gun brightness } of LaB6 and CeB6 sources in our TEM's here and find that } values are factors of 4-5 times lower than TEM manufacturers claims.
** specific stuff deleted **
I don't have any hard data on operating conditions, but we have been wondering why LaB6 cathodes don't seem significantly brighter than tungsten in our TEM. We had expected LaB6 to be like looking at the sun, but that's not the case. Could it be that the setup needs to be tweaked more critically?
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
} Hello everybody, } } I am looking for dichroic mirrors for epifluorescence, which are able to } reflect several ranges of wavelengths and allow transmission of light in } the ranges outside. Does anybody know of companies which are designing such } mirrors? It would be a great help to me if I know whom to contact. } Thanks a lot } Franz Keller } *--------------------------------------------------------------------* } | Dr.Franz Keller Tel.:089-8578-3688 | } | MPI f. Psychiatry Fax 089-8578-3939 | } | Dept. Neuromorphology | } | Am Klopferspitz 18a EMail | } | W-8033 Martinsried keller-at-vms.biochem.mpg.de | } *--------------------------------------------------------------------*
The most respected source right now is a company called Chroma Technologies Corp. They are a bunch of techies that left Omega Optical to pursue the realm of multiple pass dichroic mirrors for biomedical research. They can be reached at:
} I don't have any hard data on operating conditions, but we have been } wondering why LaB6 cathodes don't seem significantly brighter than tungsten } in our TEM. We had expected LaB6 to be like looking at the sun, but that's } not the case. Could it be that the setup needs to be tweaked more } critically?
Minor clarification. The LaB6 values we measured are low relative to calculations and claims however, they are better than W values! I'll dig up the relative comparisons and post them later.
Are you running your LaB6 at 1st or 2nd x-ver of the source?
} John C. replies } } } I don't have any hard data on operating conditions, but we have been } } wondering why LaB6 cathodes don't seem significantly brighter than tungsten } } in our TEM. We had expected LaB6 to be like looking at the sun, but that's } } not the case. Could it be that the setup needs to be tweaked more } } critically? } } Minor clarification. The LaB6 values we measured are low relative } to calculations and claims however, they are better than } W values! I'll dig up the relative comparisons and post } them later. } } Are you running your LaB6 at 1st or 2nd x-ver of the source? } } Nestor Zaluzec } ANL EM Center
At the moment, we're not running LaB6. But the reasons why are for another post. :-) I have always run LaB6 at the first maximum brightness I get to, (1st X-over?) when illumination from the sides of the crystal merge with that from the tip.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Does anyone with a LaB6 in a JEOL TEM ever see a second cross over? I see them in Phillips microscopes but not in JEOL microscopes. What is your experience?
Roseann Csencsits ANL 708-252-4977 or 708-252-7902
} Regarding first and second x-ver of the LaB6--Do you have a JEOL or } Phillips TEM? I have never seen the second cross over in a JEOL TEM. } } Roseann Csencsits } Argonne National Lab } 708-252-7902
I used a Philips 400 with LaB6 years ago when I was a graduate student. I don't remember seeing a second brightness peak. When I got to CSU last summer, our JEOL 2000 had LaB6, but we couldn't check the vacuum with the original set of guages. We had to add a Penning guage that would give an accurate readout to the 10\-7 range. Before we went back to tungsten, I don't remember seeing that second peak.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Seems like we have a fair number of people who are not reading the subscription "destructions". In it I ask new subscribers to reply to the listserver-at-anlemc.msd.anl.gov to verify their address before I add them to the list. Most of you did this correctly. :-)
I did this to minimize the possiblity of "bad addresses" causing Email bounce where everyone on the list would receive copies of "undeliverable" mail messages. for days on end.
However a number new subscribers appear to be replying to Microscopy-at-anlemc.msd.anl.gov. I'll try to make the message clearer or sort out another way to check out/confirm the addresses.
Please bear with it for a few more days.
Thanks - Nestor
P.S. I've have gotten 2 bad Email bounces both from Germany, where the message repeated ~ 10 times/day for a few days, so the extra work did stop that problem, only to create the other. :-(
University of Southern California Los Angeles CA 90089 Ph: (213) 740-1992 FAX: (213) 740-7797 X-Mailer: ELM [version 2.4 PL21] Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit Content-Length: 1352
I am seeking a challenging position as a research Electron Microscopist, so I thought this group would be appropriate to post this article.
I graduated from the University of Southern California in May 1993 with a Ph.D. in Materials Science. As a graduate research associate, I was involved extensively with conventional and high-resolution transmission electron microscopy in research projects relating to deformation twinning, martensitic transformations, and studies of ordered and premartensitic phases. I have considerable experience in working with shape memory alloys, intermetallic compounds, titanium alloys, superalloys and ceramics. Further, I also have considerable research experience with SEM, EDS, x-ray diffraction, AES and metallography. With my present experience, I am confident I will be able to undertake projects relating to research and development of a wide variety of materials. I would certainly appreciate an opportunity to discuss with you how I could contribute to you/your organization. Please do not hesitate to call, or send email to me.
Sincerely,
ANANDA S. MURTHY
Center for Electron Microscopy EMail: ANANDAMU-at-USC.EDU & Microanalysis Phone: (213) 740-1992 University of Southern California Fax: (213) 740-7797 Los Angeles, CA 90089-0101
This mailing list was not intended as a place to post resume's to distribute to the world. Please refrain from such usage. If you will recall in the Welcome message it was pointed out that this was to be a discussion/question/answer/information forum.
If you wish you may post this type of information (ie. resume's) instead on the MSA BBS system (accessible by Internet & modem) which (if you are a member of MSA) has a positon/job placement service available to members at no charge.
Posting of Position/Job openings is stretching the limits of what the intention of this list is supposed to address, however, I am willing to consider this as potentially within the bounds. What is the opinion of the readers on this last issue (i.e. positon/job openings in the Microscopy community)
G'day, Yes your message got through and my address is correct. Regards, Gerald Little. Dr Gerry Little | Ph (049) 215618 Discipline of Anatomy | Fax (049) 216903 Faculty of Medicine | Email ANGJL-at-medicine.newcastle.edu.au University of Newcastle
} Posting of Position/Job openings is stretching the } limits of what the intention of this list is } supposed to address, however, I am willing to consider } this as potentially within the bounds. What is the } opinion of the readers on this last issue (i.e. } positon/job openings in the Microscopy community) } } } Nestor J. Zaluzec } ANL EM Center
Just for saving bandwidth and not inconveniencing uninterested people, I'd say posting a message that states you are looking for a position, or have a position open, is fine. This can be done in a few short sentences. Make the details available privately.
I think distribution of an announcement to this very specialized group of readers is completely appropriate, and even an important function of this group.
Just my opinion.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
We are in the same process. The machine you buy is very tightly related to the type of work you need to do. Do you need vacuum? Are you going to be using STM alone, or also AFM, MFM, LFM, M.O.U.S.E., ....? Do you want to crack it open and make your own code? The Nanoscope III seems to be the better for versatility and routine use, while Topometrix gives you the source code of the software so you can do modifications.
} To all: } } I counted no less than fifteen mail messages on the subject of resumes, in } the last day. I think this is far more messages than the resumes you'll see } posted in the next six months. } } We all have control over what we read. If you don't want to read another } resume, just delete the message. This, of course, assumes that the } responsible resume poster will use the subject line and state that the } message is a resume. } } I vote that this is a valid subject (and a much needed service) on this } list. With one ground rule, and that is that the writer must state in the } subject line that the message is a resume. } } Let's face it, looking for a job is tough, especially in our tough econonmic } times. } } John Crum } San Diego State University }
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: microscopy-at-anlemc.msd.anl.gov
G'day Microscopy Subscriber's
The system has been down for most of Thurs. while I did network upgrades, hope this hasn't caused too much trouble.
I've corrected the welcome notice about replies to the list and tried to make the directions VERY CLEAR so most of you should not be receiving copies of confirmation notices any longer.
For those that have asked we currently have ~ 250 subscribers, not all of whom have reconfirmed yet.
Secondly, when replying to a real comment/question posted on the list please make sure that the message you send is addressed to:
MICROSCOPY-at-ANLEMC.MSD.ANL.GOV
and not just a reply to the sender. There are many different type of Email systems out there and not all of them sort out the To;, From: CC: of the Email header. in the same way. Thus you may have the intention of replying publically (i.e. to the list) but your EMail system may direct that message instead to the indivdual, who may have posted the original question and then the remainder of readers will not see the information you are providing in response. (Assuming of course that you wish public viewing which is the purpose of this list).
Third, and lastly, on the question of resume posting.
I think we now have a concensus and let's call the subject closed and get back to microscopy. The replies most of which came to me directly (instead of the list) are definitely in favor of posting announcements (~} 95%) of position's/jobs and overwhealming opposed to resume's (} 90%). I've also updated the welcome notice to make this clear too.
----------------------------------------------------- Nestor J. Zaluzec Electron Microscopy Center For Materials Research Argonne National Laboratory, Argonne, Ill. USA
Further to HJOAN's query on stm purchase, our Park Autoprobe is great (i.e., fast and relatively inexpensive) for changing between techniques, iff you want to do things other than stm.
X-Warning: Original message contained 8-bit characters, however during the SMTP transport session the receiving system was unable to announce capability of receiving 8-bit SMTP (RFC 1425-1428), and as this message does not have MIME headers (RFC 1341) to enable encoding change, only option was to STRIP sent characters to 7-bit :-(
} On Thu, 7 Oct 1993 15:57:00 +0200, { HJOAN%CLEMSON.CLEMSON.EDU-at-ANLVM.CTD.ANL.GOV} wrote: } } } } } resume's - Maybe in the future, this is just starting we need to give it a chanc } } e. Question. We are in the process of purchasing STM. What are the important fea } } tures to look for? Any information would be very much appreciated. Thank you for } } your time, JoAn } Hello JoAn,
Based on experience of one year using Nanoscope II, I would say that one of the most important features is the easy of localisation of the sample area. It can never be very easy but many things can help you. There should be a proper light microscope throug which you should be able to observe both the specimen and the tip simultaneously perpendicular to the sample. This means, you should have a reverse microscope and a stereomicroscope fitted to the STM/AFM. Furthermore, the stereomicroscope should easily be turned so, that you can observe the sample and the tip from the side.
These were on the top of my memory. In case there is more in my FILO- memory, I will let you know.
Best regards, Jouko
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Mki Navigare necesse est... Laboratory Manager Laboratory of Electron Microscopy University of Turku Kiinamyllynkatu 10 FIN 20520 TURKU INTERNET: jouko.maki-at-utu.fi Tel.: + 358 21 633 7318
X-Sender: Zaluzec-at-anlemc.msd.anl.gov (Unverified) Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: microscopy-at-anlemc.msd.anl.gov
On Oct. 6th AG wrote:
} Anybody have access to power spectral density calculation programs for } atomic force microscopy data? My AFM vendor is slow to provide this } function, and I need it to help understand the "wavelength" of roughness } on silicon surfaces. Any help appreciated!
} Andy Gilicinski
Can someone briefly explain "power spectral density calculations" in this context and how it relates to surface roughness? I'm sure I do not know the answer, but am interested in the question, what does the calculation do? Is it a fourier transform? If so there are plenty out in the public domain.
----------------------------------------------------- Nestor J. Zaluzec Electron Microscopy Center For Materials Research Argonne National Laboratory, Argonne, Ill. USA
For a 400kV microscope what is the amorphous material of choice for point to point resolution tests? Carbon or Germanium? I am using an optical bench to determine resolution.
S. Bunasawa asks: Is there a public domain or shareware EDS out there for the PC? I'm looking for books and some reference so I can write my own EDS. Can someone recommend a good source?
Reply, Send an Email message to EMMPDL-at-ANLEMC.MSD.ANL.GOV in the message say "SEND HELP" you will get instructions on how to access the EMMPDL by Email. FTP will not be up for few weeks (minimum).
There are EDS programs in the XEDS directory in that library.
Some time ago the NIST published DTSA (Desk Top Spectrum Analyser) for Mac systems. Does anyone know if there is a PC version available (or something similar) which can accept either EMSA format files or Noran Voyager format files. Is there something at the EMMPDL address (given yesterday) which may be of use? If so, is that address an open one?
NIST/DTSA is available through: Office of Standard Reference Data 221/A323 National Institute of Standards and Technology Gaithersburg, MD, 20899 USA Tel (301) 975 2208 Fax (301) 975 0416
It was written by Chuck Fiori, Carol Swyt and Robert Myklebust.
This information is as printed on the manual and may have changed in the past year or so. I have no idea regarding the price! 8-)
If you want more info. give me a call.
Colin Veitch
##################################################################### ******************************* * Intuition is reason in a * 0------* hurry. * } ---|--- { * H. Jackson * | ******************************* / \ _/ \_
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
We have taken some HREM micrograph of a composite ceramic material, made with a crystalline phase dispersed into an amorphous glassy phase. In the glassy phase there are small crystallites whose diameter is of the order of few nanometers. We would like to identify these nano-crystals. By measuring their lattice spacing no conclusive identity can be assigned. It is also almost impossible to do an EDS on such a small area.
We would appreciate very much if somebody can give us some idea regarding how to identify these crystallites with certain degree of confidence. Thank you.
At the presents DTSA only runs on a Mac platform. I do not know if any plans are being made to expand it to PC.
The cost of DTSA is ~ $900-1000 US (as of a few months ago). and it can be purchased directly from NIST. Demo copies are available for the Mac platform. I do not have the address handy here at home I will post the information tomorrow from the lab.
One of the priniciple authors of the program Chuck Fiori died suddenly earlier this year. The program is still being supported and presumably updated by colleagues at NIST, however, I'm not sure if they wish their Email address to be given out. I will contact them and ask them. If they concur I will post it on the listserver.
The EMMPDL has PC based programs for EDS data reduction, however, they are not as comprehensive as DTSA. The EMMPDL is an open library, you may receive information & source code by EMail to EMMPDL-at-ANLEMC.MSD.ANL.GOV in your message include the single line "SEND HELP". Internet & FTP access will be operating later this year (when I get all the bugs fixed!). You may also login by telephone (however I see that the questions come from colleagues in Australia), or you can make copies of the library at you next local/national (?) EM society meeting.
Complete copies of the EMMPDL will be available at the next Australian meeting in Brisbane, ICEM in Paris, and MSA in New Orleans and any other EM Society meeting that makes arrangement with me in advance.
MAIL FROM: {cmac-at-dmp.csiro.au} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 11-OCT-1993 07:46:57
JOAN, I have recently bought a PARK AutoProbe LS AFM/STM with Electrochemical AFM and STM. The list of requirements when considering buying such instruments is quite long. Briefly consider what sample size you wish to scan, what detail do you want to see, what modes of operation, AFM, STM, ECAFM, ECSTM, LFM, MFM etc, what optical resolution do you need to locate your features of interest, are you happy with a windows look a like interface or do you want a true Windows interface.
If you approach PARK they will supply you with a booklet "How to Buy a Scanning Probe Microscope". It will be quite helpful. Try to visit SPM labs to see how samples are prepared and imaged. As a matter of interest the ACEM-13 conference to be held in Queensland will have a one day workshop on SPM. As I am one of the workshop organisers I think it would be quite useful. However, a long way to travel. I'm sure there must be closer conferences.
Good luck
Colin MacRae Electron Microscopy Section
Commonwealth Scientific and Industrial Research Organisation. _--_|\ cmac-at-dmp.CSIRO.AU Division of Mineral Products / \ +61 3 647 0296 PO Box 124, Port Melbourne 3207 \_.--._/ AUSTRALIA v
The Positron Annihilation facility at Argonne National Lab was shutdown about a year ago, when the funding was cut. In a loose interpretation of "microscopy" as those techniques which allow the experimentalist to determine the structure of a material which cannot be analyzed by the naked eye, then PAS fits. Remember we are not prejudice to any specific microscopy in this forum...
Thank you for adding my name to the list server. My correct address is DUNLAP-at-utkvx.utk.edu. Best Wishes John Dunlap The University of Tennessee Knoxville, TN 37996-0810
MAIL FROM: {jmichael-at-vnet.IBM.COM} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 12-OCT-1993 08:44:53
Hallo everybody:
We have taken some HREM micrograph of a composite ceramic material, made with a crystalline phase dispersed into an amorphous glassy phase. In the glassy phase there are small crystallites whose diameter is of the order of few nanometers. We would like to identify these nano-crystals. By measuring their lattice spacing no conclusive identity can be assigned. It is also almost impossible to do an EDS on such a small area.
We would appreciate very much if somebody can give us some idea regarding how to identify these crystallites with certain degree of confidence. Thank you.
The mailserver system had locked up on a series of addresses to Germany, resulting in a huge queue of possible lost/deleted/replicatd mail.
You may be already aware of this as you may be seeing duplicates of mail you already have received. This should only take a day to clear up. There was a queue of about 100 messages here at this end, some fraction of which were destined for the Microscopy server. The rest to users of the EM Center here at Argonne. Sorry for the duplicate traffic, but I was not able to spend the time necessary to read each of these messages and then sorting out to whom it belonged and if it had already been sent and the simplest method was just to restart the system and let it clear itself out.
I'm not sure what the cost of a CCD camera is but I would guess that it is near the cost of a low-light video camera and control box from Custom Camera in the UK. The majority of the excess cost in once commercially available systems was going to the vendor, not the manufacturer of the camera. The advantage of the low-light video camera and control box is that the pattern is instantaneous. My understanding of the CCD camera is that it takes a period of time to accumulate each pattern. This makes simple tasks such as comparing adjacent grains or aligning a specimen more time consuming.
I'm not sure what the cost of a CCD camera is but I would guess that it is near the cost of a low-light video camera and control box fom Custom Camera in the UK. The majority of the excess cost in once commercially available systems was going to the vendor, not the manufacturer of the camera. The advantage of the low-light video camera and control box is that the pattern is instantaneous. My understanding of the CCD camera is that it takes a period of time to accumulate each pattern. This makes simple tasks such as comparing adjacent grains or aligning a specimen more time consuming.
JOAN, I have recently bought a PARK AutoProbe LS AFM/STM with Electrochemical AFM and STM. The list of requirements when considering buying such instruments is quite long. Briefly consider what sample size you wish to scan, what detail do you want to see, what modes of operation, AFM, STM, ECAFM, ECSTM, LFM, MFM etc, what optical resolution do you need to locate your features of interest, are you happy with a windows look a like interface or do you want a true Windows interface.
If you approach PARK they will supply you with a booklet "How to Buy a Scanning Probe Microscope". It will be quite helpful. Try to visit SPM labs to see how samples are prepared and imaged. As a matter of interest the ACEM-13 conference to be held in Queensland will have a one day workshop on SPM. As I am one of the workshop organisers I think it would be quite useful. However, a long way to travel. I'm sure there must be closer conferences.
Good luck
Colin MacRae Electron Microscopy Section
Commonwealth Scientific and Industrial Research Organisation. _--_|\ cmac-at-dmp.CSIRO.AU Division of Mineral Products / \ +61 3 647 0296 PO Box 124, Port Melbourne 3207 \_.--._/ AUSTRALIA v
I forgot to mention that I have captured EBSP patterns on a Mac and imported them into the Image 1.47 program. I think the macro language of the program could be used to index the patterns but have never had time to try. Has anyone else? If not, the code of the program could be readily modified to do the job. Has anyone explored this option?
Thanks llsutter-at-mtu.edu Larry Sutter Scattering Events R' Us
MAIL FROM: {beanland-at-liverpool.ac.uk} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 12-OCT-1993 06:43:27
Microscopy Subscribers
The purge of old message files is done, and the duplicate messages should be gone. I believe the software update to Mutlinet is now complete and things should be back to normal. Thanks for your patience.
Reply_ Re: EBSP Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu A student here has modified NIH-Image 1.45 to do some basic analysis of EBSPs. We capture EBSPs directly into Image with a Scion LG-3 frame grabber and Dage-MTI camera. The camera points at a phosphor screen through the viewport of the SEM (we actually use an ESEM - nice, plenty of room and no worries about light leaks!!). We have a paper explaining the design and construction almost ready for submission. Preprints will be available on request. The program modifications are a currently little cryptic (i.e. no manual) and only work on cubic crystals but they do allow you to do orientation analysis. The output is further analyzed by programs called MisMat and FindCSL which are both in both the EMMPDL and Microbeam Analysis Society Software Library. They are Mac programs. I will ask the student if he will release his Image code early if people are interested. If he does, no complaints please, he's not a programmer, just a Nuc Eng student trying to get a Ph.D! Modofocations are in Pascal not the built in macro language. The other programs I mentioned above are available on freebie.engin.umich.edu in the dir /pub/MSA+MAS/ both the EMMPDL and MASSL are available there for anonymous ftp. If you have difficulty connecting or downloading any software from this site send mail to me.
OK? John Mansfield --------------------------------------
I forgot to mention that I have captured EBSP patterns on a Mac and imported them into the Image 1.47 program. I think the macro language of the program could be used to index the patterns but have never had time to try. Has anyone else? If not, the code of the program could be readily modified to do the job. Has anyone explored this option?
Thanks llsutter-at-mtu.edu Larry Sutter Scattering Events R' Us
------------------ RFC822 Header Follows ------------------ Received: by mse.engin.umich.edu with SMTP;12 Oct 1993 21:56:51 U Received: by totalrecall.rs.itd.umich.edu (5.67/2.2) with X.500 id AA25695; Tue, 12 Oct 93 21:54:33 -0400 with SMTP id AA25690; Tue, 12 Oct 93 21:54:31 -0400
Reply_ CCD for EBSP Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu } } I'm not sure what the cost of a CCD camera is but I would guess } that it is near the cost of a low-light video camera and control box } from Custom Camera in the UK. The majority of the excess cost in once } commercially available systems was going to the vendor, not the } manufacturer of the camera. The advantage of the low-light video camera } and control box is that the pattern is instantaneous. My understanding } of the CCD camera is that it takes a period of time to accumulate each } pattern. This makes simple tasks such as comparing adjacent grains or } aligning a specimen more time consuming. } } Thanks } } } } Larry Sutter } Scattering Events R' Us
Not really true! Both cost wise and time wise. A slow scan camera would be exepnsive but we use a TV rate Camera (A Dage-MTI CCD72) and a cheap frame capture board (Scion LG3) to record our EBSPs. We use the video rate capture of the LG3 to grab several consecutive frames of video and average those. It is obviously not as noise free as a good slow scann camera but our EBSPsystem cost less than $12K including the Macintosh! Works nicely too.
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How does the 6100 compare to the 6300F? The 6300 has a BNC port that is live from the monitor. We wheeled a MacII cx outfitted with a Data Transl ation frame grabber and NIH Image into the scope room. The DT board was connected with a coax cable to the bottom-most BNC connector on circuit board U-34. Get to this board by removing the kick panel below the operator's console. Look for a series of boards to your left. You have to time the capture to avoid getting a scan shift if you have any image drift - simply issue the command to grab as the scan gets to the bottom. You can't use the SR mode with this. If you can access the video on the 610, then you can get near publication quality images with Image. I even got some 4X5 publication quality prints by helping the contrast with Adobe Photoshop and printing it out on our campus print shop's Linotronic press. Of course you might try calling JEOL technical folks. On Wed, 6 Oct 1993, Bob Keller wrote:
} We would like to set up a simple system for recording electron backscattering } patterns in a JEOL 6100, without resorting to some of the (overpriced) } commercial systems currently available. At the moment, we are considering the } use of a phosphor screen and CCD set-up, using an optical viewing port in the } SEM. As the patterns would be used simply for orienting specimens for the time } being, publication-quality images are not necessary. We do have access to } image processing via NIH-Image for contrast enhancement/noise reduction after } acquiring images. Has anyone tried doing this with success? Without success? } } Bob Keller } NIST-Boulder }
As promised here I checked with NIST and here is the information about the DTSA (Desk Top Spectrum Analyzer) Program for EDS data analysis.
ORDERING INFORMATION: contact Joan Sauerwein Email: srdata-at-enh.nist.gov
Technical Questions: contact Bob Myklebust Email: myklebust-at-gapnet.nist.gov
General questions can always be posted here at the Microscopy server since the NIST group is one of our subscribers. By posting some of the questions here you may receive additional comments insight and/or answers from other users on this server. Remember we will all benefit from this type of discussion in the long run!
Has anyone measured the energy spread of a 400kV beam in a JEOL 4000 with a saturated or undersaturated LaB6 filament? Often we assume a 1 ev energy spread but is this reasonable or is it more like 3-4ev energy spread?
Thanks for your help.
Roseann Csencsits Electron Microscopy Center-Argonne Nat. Lab
} How does the JEOL 6100 compare to the 6300F? The 6300 has a BNC port that is live from the monitor. We wheeled a MacII cx outfitted with a Data Translation frame grabber and NIH Image into the scope room. The DT board was connected with a coax cable to the bottom-most BNC connector on circuit board U-34. Get to this board by removing the kick panel below the operator's console. Look for a series of boards to your left. You have to time the capture to avoid getting a scan shift if you have any image drift - simply issue the command to grab as the scan gets to the bottom. You can't use the SR mode with this. If you can access the video on the 6100, then you can get near publication quality images with Image. I even got some 4X5 publication quality prints by helping the contrast with Adobe Photoshop and printing it out on our campus print shop's Linotronic press. I was tipped off about the presence of this port while speacking to our local JEOL rep. about getting my hands on the images stored on the 6300's internal harddrive. The stored images were going to require about $20,000 to get at since many years ago JEOL adopted an obscure and difficult (their words) image storage format.
I've tried to post this several times and our mail server has been having problems. If this got through earlier, sorry for adding to the chatter.
Your dark "contamination" spots are "cracked" hydrocarbons from the a variety of sources. 1.) Diffusion Pump Oils, 2.) Surface contamination (fingerprints etc...), 3.) Electropolishing solutions.... They are attracted electrostatically to the the beam perimeter (small probes are worse than large probes) and essentially get baked on to your specimen. The phenomenon occurs in all microscopes I've used (and I've used a few), however, it can be minimize depending upon it's source. If you have a poor vaccum ( ~ 10-7 Torr) then cool your specimen to ~ -30 C. The residual H20 vapor in your scope will begine to collect near your specimen surface, the action of the electron beam will dissociate the H & O, the O will react with the C to form CO2 gas and the contamination will cease. Of course if your specimen is carbon based then you drill holes (I did a nice job on a diamond specimen once and learned the hard way). You can also heat your specimen but this doesn't always work as well. Spreading the beam to cover a large area and heating the local region (remove all apertures and go to the largest spot size) will temporarily immobilize things but eventually (after about 1/2-1 hour) things will begin to diffuse across your specimen to the beam area again. There are a variety of other tricks which depend on your specimen. For example semiconductors contaminate less than metal (in clean vaccum system { 10-9 T) A starting reference you should look up is Introduction to Analytical Electron Microscopy, (1st edition not the 2nd) Ed. Hren, Goldstein, Joy, 1979, Plenum Press, look at Chapter 18, by John Hren entitled "Barriers to AEM: Contamination and Etching".....
Oh yes it appears dark because it is so thick!. The spots can build up to heights of microns. You can easily see this by tilting your specimen 30-45 degrees after the spots form. The nice round spots become tilted cones......
It is bad policy to believe any manufacturers claim on the probe size. It will be a function of C2 aperture size, lens excitation, specimen height in the lens and a variety of other parameters. You can get a quick and dirty measurement by doing a line scan across a sharp edge and looking at the signal rise profile when going from a "hole" to a specimen, however, you will need a calibrated magnification to do this. Another bad policy of the manufacturers is that they will claim the probe size based upon FWHM measurements, you should of course be using at least FWTM or even better the first zero of the crossing of the interference function.
A better way to measure the probe size is to image the STEM probe in the TEM mode. Can you turn on your post-specimen lenses and configure them to image the probe in the JEOL 2000? This is possible in some microscopes, but I'm not sure about the 2000 series...
Message-Id: {9310151659.AA15024-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb Subj: GRAIN SIZE DISTRIBUTION MEASUREMENT Orig-Author: {QJXNJ21-at-memrqa.sps.mot.com}:ddn:wpafb ----------------------------------------------------------- Hello readers, Thanks to all who responded to my question about contamination spots. This IS a great format to have such questions answered. Now I have another problem which I think many TEMmers must have been up against. How to measure grain size distributions from TEM images of polycrys- talline materials e.g. polySi or Al etc. What kind of image proces- sing algorithms should be used and with what strategy? I realize that a generalized algorithm may not work for every image but there must be some general principles. I've heard that the Laplacian filter shows up edges but I also heard John Russ of NCSU say how he uses repetitive filtering and subtraction procedures to achieve grain boundary delineation. It is not intuitively apparent to me how this multiple filtering is used. Any tips or insights will be highly appreciated. We have Gatan's Digital Micrograph and the NIH Image 1.4 software. I dont want to use the crude straight line method because I want to calculate size distributions. Thanks for your time. Vidya Kaushik TEM Facility, MOTOROLA INC., Austin, TX, 78721 (512)-928-5134; (512)-928-6664 fax QJXNJ21-at-MEMRQA.SPS.MOT.C
I would like to find out how laser scanning confocal microscopy is being used industry (other than semiconductors). Does this microscopy really provide a significant improvement over 'ordinary' light microscopy?
Mark E. Cavaleri 3M CRL/A&PRL Light Microscopy 3M Center 201-1E-15 St. Paul, MN 55144-1000 (612) 733-3247 (612) 733-0648 FAX
Using the signal rise profile technique to get a nominal probe size is valid for both convergent & parallel probes, whether you are in STEM, TEM, SEM or any other mode..... It's major draw back is that it is not sensitive to weak low intensity tails which can cause signal generation in modes other than CBED, for example, as in X-ray analysis.
I would like to find out how laser scanning confocal microscopy is being used industry (other than semiconductors). Does this microscopy really provide a significant improvement over 'ordinary' light microscopy?
Mark E. Cavaleri 3M CRL/A&PRL Light Microscopy 3M Center 201-1E-15 St. Paul, MN 55144-1000 (612) 733-3247 (612) 733-0648 FAX
Is anyone out there using NIH Image and a Macintosh to capture images from a scanning electron microscope? If so, what is the resolution (i.e. image size) that you are getting? What hardware (i.e. image capture board and Mac) are you using? What does it cost?
The reason I ask is that our EM facility is in the market for a new SEM. A nice feature we have now on our SEM is image capture, using a part of a Princeton Gamma Tech EDS system. The core of that system, though, is a Sun 3/60, which doesn't really work well and will need a costly upgrade. We can capture images of 512 pixels and 1024 pixels sqaure. New software (on top of hardware upgrade) will capture 2kX2k images.
So, the ultimate question is: should we adapt the system we have now to a new SEM, or buy a new Mac/Image based system?
Hi, I'm interested in hooking a video camera to my TEM (JEOL 100B): 1)To demonstrate procedures such as filament saturation and 'scope alignment when teaching my EM class, and 2) To capture "quickie" EM prints to a video printer for students in my cell bio and vertebrate histology classes. I'm looking for about 640x480 pixels and 8 bits, and would like something that covers about the same area at each magnification as the film camera. I know about Gatan's systems, and I've been told "look at the Fullam system before you buy it". Is there anything else out there that I should know about? Have any of you used either the Gatan system or the Fullam system, and how satisfied are you with it? Any help/feedback would be appreciated. TIA Julian Smith III (smithj-at-winthrop.edu)
This is in response to Julian Smith's query about video systems on TEM's.
I only have experience with the old Gatan system, so can't really comment on the questions directly. There is an issue, however, with just taking 'Quickie video prints'. A video printer captures only a single frame of the RS170 video - that is, it is equivalent to a 1/30 sec. exposure. During this time, one can, (depending on the microscope operating conditions) get significant shot noise if the video system is reading out the electron image in real time (as does the old Gatan image intensifier system). Such a print can be next to useless. Since our eyes respond much more slowly, and in any case we are viewing a phosphor (either the microscope screen or the screen of the CRT) which adds its own time constant, we don't notice the effect so much (although it is resonsible for the "liveliness" one sees when viewing a high magnification image on the viewing screen).
This is not an issue when reading out a framestore loaded from a slow-scan system, but they are expensive for just demonstrating saturation and taking quickie video prints. The Fullam system is a light camera viewing a phosphor screen - you will have to experiment to see if it has enough time constant for your needs.
The RS170 can be frame-averaged either by a stand-alone system such as is sold by Gatan or Synoptics (not terribly expensive) or by a board from one of many vendors which plugs into a PC. The advantage of the latter, besides being even less expensive, is that it also can transfer the image to the PC for networking, image processing, etc.
I am looking for a stand-alone ZAF program that I can run on a PC or Mac that will allow me to import k-ratios or app. concentration. I specificly want to do carbonate analysis. A stand alone Bence-Albee program would also suffice. Any thoughts...
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I am interested in using a cooled ccd camera for imaging fluorescence immunostaining. I understand that there are advantages to the cooled ccd-fluorescence microscope combination over a confocal microscope. I would appreciate any information about cooled ccd camera-microscope configurations from anyone who is (has) using one. What is the best manufacturer? What microscope configuration is best? What about software?
Denis Baskin, Ph.D. VA Medical Center and University of Washington Seattle, WA (206) 764-2138 FAX (206) 764-2164
Subject: Time:8:24 AM OFFICE MEMO Acid phosphatase Date:10/19/93 Can anyone out there recommend a protocol for acid phosphatase staining of 1 micron, epon embedded kidney sections? I have tried a couple of kits that are suppose to remove the epon and the fixatives, but the results have not really been spectacular.
If there are any biological electron microscopists out there who could give me a reference, I would really appreciate it.
For our JEOL 6300F we found that we could simply string a coax cable from the Data Translation frame grabber board in a Mac to a live video port hidden behind the kick panel below the operator's console. This allowed us to capture the signal to the SEM monitor with NIH Image. The command to grab had to be synched by eye so that the scan raster had reached the screen bottom. The images were decent quality, and with some Photoshop grayscale and brightness adjustments we obtained publication quality prints on the campus Linotronic press. We aren't using this for anything useful yet for lack of a Mac to leave in the 'scope room.
Help! I have been searching for a monoclonal antibody, Neural Cell Adhesion Molecule (N-CAM), which will react with tissue that has been fixed in at least 4% paraformaldehyde, preferably also with glutaraldehyde. We are using cryo-ultramicrotomy on rat neural tissue for TEM localisation of specific epitopes, N-CAM being one of them. So far, none of the companies I have approached (Sigma, Amersham, Serotec, Boehringer-Mannheim, Dako, Sera-lab) have an N-CAM antibody which will work with formaldehyde fixed tissue. Would any one out there know of a company which would have this particular antibody. Thanks in anticipation. Gerry Little. Dr Gerald Little | Ph (049) 215618 The Neuroscience Group | Discipline of Anatomy | Fax (049) 216903 Faculty of Medicine | The University of Newcastle | Email ANGJL-at-medicine.newcastle.edu.au Australia, 2308 |
To date, there has been much activity on this list, mostly related to electron microscopy in one form or another. It seems to me this much traffic warrants a list of its own, and so I'd like to suggest that just such a "splinter" list is created.
My personal reason for suggesting this is that, not being involved in electron microscopy myself, I simply don't have the time to look through all the EM messages.
Of course, those interested in all topics could subscribe to both lists and be no worse off than now.
-David. - - David Abbott | Email: dfa-at-physiol.unimelb.edu.au School of Physics & Dept. of Physiology | Phone: +61-3-344-5465 The University of Melbourne, Australia. | Fax: Available on request.
For those Microscopy Listserver user that were trying. The Internet link to the MSA BBS system is back up and running. There was a software bug which took the line down for about a week. It appears that everything is back to some degree of normality......
I recognize David's concern about lots of electron vs light microscopy, but my personal feeling is that a single microscopy list is still the best way to go. In our lab, most of the EM folks end up doing a bit of LM and vice versa. Watching everything go by keeps the mind open and active. Message deletion is a command-D away and the need can usually be determined within the first sentence. If it gets to the point where LM is as busy as EM, then perhaps we should consider a split, but until then my vote is a single list.
The mailing list for NIH Image (subscribe NIH-Image yourname-at-your.address to listserv-at-soils.umn.edu) includes a fair amount of general digital image acquisition and analysis discussion, often dealing with LM issues.
Lastly, if you plan to be away from your electronic in-box for a few days and don't want to wade through buckets-o-bits when you return, unsubscribe from your lists for the time you're gone.
Regards,
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 ==========================
In reply to Gerald Little's request for sources of N-CAM monoclonals, I am consulting the MSRS catalog (it's in front of me now!), and have come up with this list of potentials:
Chemicon (definitely have polyclonals, call about monoclonals) BioDesign, clone NKINBL1, cat # M6127oM Accurate, clone NCAM-OB11, cat # BYA-6075-1 Development Studies Hybridoma Bank, 5B8 Development Studies Hybridoma Bank, 4d Development Studies Hybridoma Bank, 5e Development Studies Hybridoma Bank, 5A5 (sialylated form) Santa Cruz Biotechnology, clone ERIC1, cat # sc-106 American Qualex, cat # M1180 Bioprobe/Thamer (?), 123C3 (I am not familiar with this company) British Biotechnology, clone ERIC-1, cat # BCA8 Becton-Dickinson, clone MY31, cat# 550009 CRB/ICI, clone ERIC-1, cat # AM-19-500
I hope this helps!
David Morilak Dept Psychiatry Stanford Univ morilak-at-cmgm.stanford.edu
On Tue, 19 Oct 1993, Nestor J. Zaluzec (708)-252-5075, -4964 wrote:
} } For those Microscopy Listserver user that were trying. } The Internet link to the MSA BBS system is back up and } running. There was a software bug which took the line } down for about a week. It appears that everything is back } to some degree of normality...... } } Nestor Z. } ANL EMCenter
Just a quick question - what is the MSA BBS and how does one gain access to it ?? Darcy Clark Uni. of QLD Brisbane.AUSTRALIA
Do the subscribers of the list get that amount of mail every day ? I didn't receive any messages between Oct 6 and today. Did I miss something important or does the list allow hidden options (USA anly)
I would like to initiate a discussion on the subject of cross-sectional TEM of multilayers (esp. metal/a-silicon). I am interested in the alternate techniques to HREM that have appeared in the literature such as the High-Angle Annular Dark Field (HAADF) technique practiced by Stearn's group at ASU (ref. Cheng et al., 1992, Proc. 50th Ann. Meet. EMSA, p.122) and the Fresnel Method employed by Stobb's group at Cambridge (ref. Shih and Stobbs, 1990, Ultramicroscopy, 32, p. 219).
The existence of these techniques arose from the concern of the ability of HREM to characterize the roughness of the interface between the alternating material layers which comprise the multilayer. Since many multilayers are composed of two materials with vastly different Z (e.g. X-ray mirrors) the strong scattering at the interface may complicate the interpretation of HREM images.
What I would like to know is why these two techniques are not more widely used. Although all of the HAADF work I have seen reported has been done on VG-STEMs, I have seen a report from the Philips Electron Optics Lab that the technique can be done on CM12,20,&30 TEM/STEMs with annular dark field detectors (Otten, 1991, J. Electron Microscopy Tech., 17, p.221)
As for the Fresnel Method (fitting exp. through-focal fresnel-fringe line-profile intensities to those based on mixed scattering potentials at the interface) I have counted over 20 papers from the Cambridge group but have not come across one from anywhere else. One impediment is the need to digitize the fresnel-fringe profiles with fairly high resolution, which can be a problem for those of us without access to a microdensitometer or the ability to collect the images digitally in the first place. But is the method acceptable on a theoretical basis?
If anyone has had experience with or has critically evaluated either technique I would like to hear from you.
Thank You,
David A. Howell | Ph (517)337-2943 Dept. of Materials Science & Mech. | Fax (517)353-9842 Michigan State University | E. Lansing, MI 48824-1226 | Email howelld-at-egr.msu.edu
} Just a quick question - what is the MSA BBS and how does one gain access } to it ??
In addition to this Listserver the ANL EMC and MSA have several other "on-line" so to speak services. One is the EMMPDL (the Microscopy and Microanalysis Public Domain Library of Software) and the Electronic Bulletin Board System. Access to information about the EMMPDL and the BBS as well as their contents are available via conventional telecommunications (modem lines) or over Internet (via Telnet, not yet FTP , but it is coming).
The BBS is a more selective forum than this list having society information, announcement, job placement, topical discussion session, public and private Email..... that is the type of information which (for the most part) doesnot belong on this Email list.
To get internet information about the EMMPDL send an Email message to "EMMPDL-at-ANLEMC.MSD.ANL.GOV" with the message Send Help EMMPDL
To get internet information about the BBS send an Email message to "EMCBBS-at-ANLEMC.MSD.ANL.GOV" with the message Send Help EMCBBS
I have received several questions regarding the MSRS catalog of Primary Antibodies that I mentioned in a previous transmission, so I am sending this to both the microscopy and NIH image distribution lists. The MSRS catalog is indeed a catalog of primary antisera, both those available commercially and those available from individual labs who choose to have their antisera listed. I have found it to be an extremely useful first reference, though it is, as might be expected, not 100% comprehensive. I often will call the sources listed to find out what else they have available that is not in the catalog (e.g. polyclonals vs monoclonals, different host species, different epitopes etc.). I ordered it at last years Neuroscience meeting, and an update (I am told) will be available approximately annually. The cost was somewhere around $75 (US). (MSRS stands for "Manufacturer's Specifications and Reference Synopsis"). I don't have a phone number, but here is the address off the order form:
MSRS catalog of Primary Antibodies AERIE Corporation Box 1356 Birmingham, MI 48012-1356 -------
} I have received several questions regarding the MSRS catalog of Primary } Antibodies that I mentioned in a previous transmission, so I am sending this } to both the microscopy and NIH image distribution lists. The MSRS catalog is } indeed a catalog of primary antisera, both those available commercially and } those available from individual labs who choose to have their antisera listed.
How does the MSRS compare to Linscott's Directory?
Glen MacDonald Hearing Development Laboratories UW
Does anyone know whether there is a Email or NEWS group specifically relating to cytogenetics (methodology etc.)? If so, please drop me aline where to find them.
Lauran Oomen, Netherlands Cancer Institute, Amsterdam, The Netherlands
We consider buying a microscope-CCD set up, which will be used in a multi-user environment. Applications will comprise a.o. detection of (low levels of) immunofluorescent signals in tissues and cells, in situ hybridization signals and very low levels of NADH fluorescence. Precise quantification and a large dynamic range are also a main issue. Any suggestions relating to brands and types for the several components of the total system (microscope, CCD camera, computer hardware and software) will be highly appreciated.
Lauran Oomen, Netherlands Cancer Institute, Amsterdam, The Netherlands
I'm looking for a technique to measure the thickness of surface contamination/oxidation on metal surfaces. I need an image of the surface for mapping purposes, as well as thickness measurements of the contamination. These specimens range in size from 1-5 cm and less than 1 cm thick.
Any suggestions would be welcome.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
There are two articles in the most recent J. Comp. Neurol. (336, no. 4), both of which describe using N-CAM monoclonals in tissue (tongue) that has been perfusion-fixed with 4% para-formaldehyde:
Smith, Akeson and Shipley, JCN 336(4): 493-506 Nelson & Finger, p.p. 507-516
Hope this helps!
David Morilak Dept of Psychiatry Stanford Univ morilak-at-cmgm.stanford.edu -------
The obvious answer is making a cross-section of your specimen and then looking in the appropriate type of microscope (optical, SEM, TEM.....). What material are you looking at? What thickness is the surface layer. Can you x-section the samples or are they unique? Can you use a SIMS system to image the surface (low res) and then sputter through.....?
We are currently looking at the purchase of an infrared microscope to aid us in our failure analysis of integrated circuits. I was wondering if any one had any experience with these beasts and had any comments to make about any of the currently available models? Also, what manufacturers currently make these microscopes, as we are a little in the back waters here this type of information is a little hard to come by.
Our interest is in microscopy in the wavelength region of 800-1800nm and as high a resolution as possible.
Reply_ RE} Electron microscopy list Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu Hi everyone? Why not identify your messages to the list with OM, SEM, TEM, STM, AFM, etc. in the title? That way we can delete the messages tat are of little or no interest to us.
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My personal reason for suggesting this is that, not being involved in electron microscopy myself, I simply don't have the time to look through all the EM messages.
Of course, those interested in all topics could subscribe to both lists and be no worse off than now.
-David. - - David Abbott | Email: dfa-at-physiol.unimelb.edu.au School of Physics & Dept. of Physiology | Phone: +61-3-344-5465 The University of Melbourne, Australia. | Fax: Available on request.
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} RE: Thickness of Surface Layers } } John } } The obvious answer is making a cross-section of your } specimen and then looking in the appropriate type of microscope } (optical, SEM, TEM.....). What material are you looking at? } What thickness is the surface layer. Can you x-section the samples } or are they unique? Can you use a SIMS system to image the surface } (low res) and then sputter through.....? } } Nestor Z. } ANL EMcenter
Yes, one solution would be to section the material, but that wouldn't allow mapping of thicknesses over the surface. This work would be for a client who wants a non-destructive technique. They just want to know the thickness, and not remove it.
One technique suggested by another lab here at CSU is ellipsometry. As I understand it, this is a technique that uses polarized light to determine the thickness of a thin film over a substrate. I don't know what the mapping or resolution limits are. Unfortunately, I don't know the approximate thickness, although I think it's 1-100 nm.
It's becoming apparent to me that I need to find out more about these specimens before I'll be able to help them very much and direct them to the right technique.
Thanks for all the suggestions.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
At the present we only operate one list called Microscopy and have had one vote/request to split off a portion to an optical microscopy list with another vote/comment not to do this. One reason for the split off this is that some users are only interested in optical or electron or whatever based microscopy and have no interest in the other fields. Until I get more feedback we will keep the single list, however, John Mansfield has recently made a good suggestion relative to titling your messages to the Microscopy Listserver, which may improve the operation. Basically he suggested that we add a label indicating the type of microscopy to the message title.
I like John's Idea. So please from now on when entering your Subject/Title of your EMAIL Message, please try to begin your title with an obvious abbreviation for the type of microscopy you will be addressing.
Here's some suggestions (which of course are not binding and only reflect my ignorance if the abbreviations are not current).
LM: Light Microscopy CFM: Confocal Microscopy SEM: Scanning Electron Microscopy TEM: Transmission Electron Microscopy AEM: Analytical Electron Microscopy AFM: Atomic Force Microscopy STM: Scanning Tunneling Microscopy uAnaly: Microanalysis Gen: General Questions Comments etc..
Other suggestions? Basically use something obvious that makes sense.
A typical message title might look something like this
To: microscopy-at-anlemc.msd.anl.gov /\ /\ || || Abbreviation Subject Matter
Again I have no problems in creating additional lists which are directed toward a specific microsocpy/microanalysis methodology. It only requires a sufficient audience.
======================= Nestor Z. ANL EMCenter Email:Zaluzec-at-ANLEMC.MSD.ANL.GOV
Try short times in diluted hematoxylin or thionin. Thionin will pick up more of the cytoplasmic RNS, but we use it routinely, at 4-fold dilution of the Wisconsin thionin recipe for DAB counterstain and for autoradiography. Just keep the staining time short. We have also used methyl green for nickel enhanced DAB and fast green FCF. If working in B&W, try a green filter or light blue filter in the light path to enhance the DAB contrast.
Glen MacDonald Hearing Development Laboratories University of Washington
In relation to the previous messages concerning the possible splitting of the group I would strongly suggest that we stay as one group and definitely adopt the use of appropriate titles to identify the message as suggested by Nestor Zaluzec. I use TEM, LM and CLSM and occasionally SEM for my research and having access to the group allows me to keep up to date with what is going in all these fields.
Regards, Gerry Little.
Dr Gerald Little | Ph (049) 215618 The Neuroscience Group | Discipline of Anatomy | Fax (049) 216903 Faculty of Medicine | The University of Newcastle | Email ANGJL-at-medicine.newcastle.edu.au Australia, 2308 |
I'm interested in hearing from anyone who is involved in TEM of polymers, particularly high resolution. I'm also interested in hearing about any methods of detecting small crystalline regions in amorphous matrices, either during or after imaging. Darcy Clark 03249587-at-minmet.uq.oz.au Dept.Mining & Metallurgical Eng. Uni.of QLD. BRISBANE AUSTRALIA.
We're not doing anything in the way of HREM of polymers here at ANL, however, I vaguely remember some work done by John Hunt of Lehigh University on a VG HB501 STEM. It was published I believe in Ultramicroscopy or the Microbeam Analysis Society Journal (possibly with Dave Williams) within the last three years. He was doing combined HR Analytical Microscopy and imaging.. If I run across the reference I'll post it
Ned Thomas and Weiping Zhang did some very nice HREM imaging of polymers here at MIT (Ned was also involved with a postdoc at Amherst before he came to MIT). The people at Akashi (now TOPCON) took some excellent images of the lattice of Ned's crystalline polymers when I was at their lab trying out the 002B microscope. I don't know where Weiping is now, but if you want to write to Ned, his address is:
Prof E. L. Thomas MIT Room 13-5094 Cambridge MA 02139
If you want to send an E-mail message to him, address it to me and I will forward it. (I haven't asked his consent to put his E-mail address on this forum, although I think it is probably public knowledge).
Reply_ RE} "Polymer Microscopy," Sawyer & Grubb Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu There is apparently a new edition of this book in production and so those of you having difficulty getting copies should perhaps wait until the new ediition hits the bookshelves.
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We have had the same problem here with cardiac muscle from several species. The background fluorescence is seemingly impossible to get rid of in cryo or paraffin sections fixed in 4% paraform. in 0.1 M PB. Any suggestions?
Denis Baskin VA Medical Center, Seattle
On Tue, 26 Oct 1993, Steve Barlow wrote:
} Tissue cryosections and immunofluorescence. } Can anyone suggest some treatments for reducing background } fluorescence in rabbit muscle tissue? We are using either a 3% } formaldehyde/PBS fix (3 hours) followed by storage in 0.5% formaldehyde in } PBS or McClean's sodium periodate/ formaldehyde/ lysine fixative. We are } using EM grade methanol free formaldehyde from vials. After } cryosectioning sucrose embedded frozen tissue, we have tried treating with } 0.2M glycine or 50 mM ammonium chloride for 30-60 min. . After these } treatments, we still have pronounced tissue fluorescence. I've seen in } the literature that sodium borohydride is used, but its explosive } characteristics and its possible alteration of tissue antigenicity seem to } make it a less desireable treatment. } } Any suggestions would be appreciated. } ---------------------------------------------------------- } 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 }
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: 9:44
} I've seen in } the literature that sodium borohydride is used, but its explosive } characteristics and its possible alteration of tissue antigenicity seem to } make it a less desireable treatment. } I have used sodium borohydride frequently for this purpose and it has never yet blown up on me. We have also used immunocytochem on anumber of antigens successfully. So, I don't think its terrrible for antigenicity. If you are pushing things, there may be a small detrimental effect. I use a 1 mg/ml solution in PBS. You have to make up the solution just before use. We keep the powder in a dessicant at room temp.
In one lab I was in, they used to cut the PBS 1:1 with methanol. This slowed down the reaction. But, methanol itself can do some nasty things to your tissue. A ten to 30 min. incubation is typical.Some folks use a few changes. Ie, incubate for 10 min and then change to fresh solution for another 10.
However, my understanding was that NaBH4 works for reducing autofluor from unreacted aldehydes in gluteraldehyde fixation. That is, glut is bi functional, so there can be dangling aldehydes out there. These the borohydride attacks and prevents from forming highly conjugated somethings or other, which are autofluor. In my work, on plant tissue, no help from NABH on autofluor when only pfa is used. Of course, this is no guarantee your tissue's autofluor might not be eased with the NABH4.
Hope this helps, Tobias Baskin ******************************** *************** Tobias I. Baskin /~~~\ Biol. Sci's * Univ. of Missouri c|o o\ Columbia, MO 65211 USA \ = / Tel:314-882-0173 """ FAX 314 - 882 - 0123 baskin-at-biosci.mbp.missouri.edu
} Steve Barlow asked } Tissue cryosections and immunofluorescence. } Can anyone suggest some treatments for reducing background } fluorescence in rabbit muscle tissue?.........(text deleted)
I have absolutely no experience in LM Fluorescence problems but an obvious solution to me (which probably means I'm wrong) is to change the wavelength of the light source. If the illumination system is causing the staining(?) chemical to fluoresce then might not it be possible to change the excitation source? Presumably the staining chemical is sensitive to the radiation you are using thus rather than move to a potential dangerous chemical try changing the source.....
Now somebody please cure my ignornace and tell me why this is probably wrong....
Regarding the use of sodium borohydride, I have used it regularly as a pretreatment for immunoperoxidase when using glutaraldehyde in my fix - it's great at reducing background (presumably by reducing double bonds - potential sites of non-specific DAB oxidation), and also seems to unmask antigenicity. I have been able to increase my primary antibody dilutions several fold in some cases. I have not noticed any difference though when using only paraformaldehyde, so I'm not sure it would help with your background fluorescence problem.
After initial PBS washes
Incubate 30 min in 0.5% NaBH4 in 50 mM Tris, pH 7.4
Then 3 x PBS washes before blocking serum
The borohydride solution should be weighed out and put into solution immediately before use (it goes in fast). It does generate a lot of bubbles, so use plenty of volume on your tissue.
David Morilak Dept of Psychiatry Stanford University morilak-at-cmgm.stanford.edu -------
} } I've seen in } } the literature that sodium borohydride is used, but its explosive } } characteristics and its possible alteration of tissue antigenicity seem to } } make it a less desireable treatment. } I have used sodium borohydride frequently for this purpose and it } has never yet blown up on me. We have also used immunocytochem on anumber
Just a warning: green Bodipy does not work after this treatment.
I have absolutely no experience in LM Fluorescence problems but an obvious solution to me (which probably means I'm wrong) is to change the wavelength of the light source. If the illumination system is causing the staining(?) chemical to fluoresce then might not it be possible to change the excitation source? Presumably the staining chemical is sensitive to the radiation you are using thus rather than move to a potential dangerous chemical try changing the source.....
Nestor:
I'm not sure if this is the answer you're looking for, but here's my shot anyway: The application, if I understand Steve correctly, is in using a fluorescent-conjugate secondary antibody to detect a primary antibody in immunohistochemistry. The conjugates are typically fluorescein (FITC) and rhodamine (TRITC, a fluorescein derivative). The light source is a high energy mercury lamp, and the light passes through a filter cube that is matched to the fluor that is being examined. These cubes are commercially available - the best are probably from Nikon in my experience. The excitation, emission and notch filter combinations result in a fairly tight wavelength window, which is necessary to be able to discriminate the different tags with different filters (for co-localization studies) without bleed-through, since their spectra do overlap considerably. The problem is that, using the excitation/emission settings necessary for viewing FITC and TRITC, there may be some tissue elements that are also excited, or that emit autofluorescence. (A note - never use Kim-Wipes on your slides - they are treated with something, presumably to make them nice and white, that demonstrates a most impressive "autofluorescence"!). So, the options are to switch fluorescent labels to get away from the wavelengths that cause fluorescence in tissue (but the alternate choices and reagents are extremely few), or reduce the tissue fluorescence by a variety of pretreatments.
Here's another suggestion: I have used a mounting medium made from "antifadent" tablets from CitiFluor (City University, London). The mountant is essentially 90% glycerol/PBS with 1 dissolved tablet/10 ml. The tablet is presumably some proprietary detergent concoction, but it does help reduce background fluorescence. It's fairly expensive though - as I remember about $100 (US) for 10 tablets, so I only use it for "critical" work (but isn't it all?).
I hope this is informative!
David Morilak Department of Psychiatry Stanford University morilak-at-cmgm.stanford.edu -------
Steve: The rule of thumb that we use for polyclonal abys when preimmune serum from the same rabbit is not available is to use normal rabbit serum at the same diluton as the primary aby. However, you may have to screen several batches of normal rabbit serum to find one that has low nonspecific binding. Monoclonal aby concentratons are usually given in mg/ml or something like that, and you usually dilute them for use. If, for example, you use a mca at 0.01 mg/ml, then the control would be mouse IgG at the same concentration, although you can also dilute normal mouse serum to the same concentration. The problem we frequently encounter is that normal mouse serum tend to be "sticky" and you may have to screen a dozen samples to find one without a lot of nospecific staining. Other labs have found similar problems with NMS. Just replacing the primary aby with PBS is definitely not adequate!. If you have to use the primary abys at c.a. 1:100 or 1:50 or less, you may be introuble since the NSB can be as high as the SB. All the more reason to serach for a good normal serum with low NSB. Good luck. Denis Baskin Seattle VA Medical Center 206-764-2138
On Thu, 28 Oct 1993, Steve Barlow wrote:
} 1. what does one use for a control when using a monoclonal Ab--there is, } in this case, no 'pre-immune'? Is a monoclonal Ab known not to be expresed } in the sample the best alternative? } } 2. what does one use for a control when using a polyclonal rabbit Ab } (commercial or otherwise) for which no pre-immune is available? } My user is not satisfied using PBS alone in place of a primary. If I use } rabbit normal serum or rabbit IgG--how does one come up with a meaningful } concentration? Presumably this control is to show how 'sticky' the sample }
On Thu, 28 Oct 1993, Nestor J. Zaluzec (708)-252-5075, -4964 wrote:
} } } Steve Barlow asked } } Tissue cryosections and immunofluorescence. } } Can anyone suggest some treatments for reducing background } } fluorescence in rabbit muscle tissue?.........(text deleted) } } } I have absolutely no experience in LM Fluorescence problems } but an obvious solution to me (which probably means I'm wrong) } is to change the wavelength of the light source. If the illumination
I tried to avoid autofluorescence by changing the wavelength. I suppose that might work in some situations where a single specific component with a narrow band of excitation or emission is present. But some things, like the lipofuscin granules in gut will fluoresce at any wavelength. my hypothesis is that most biological autofluoresce is due to a number of molecules (and processing distortions) that will glow over a range of frequencies.
For what its worth, my recent experience with whole vestibular organs showed that FITC, AMCA and Cascade Blue wash out quickly, leaving little backgound. But Texas Red required an overnight wash.
} Some general questions for discussion: } } 1. what does one use for a control when using a monoclonal Ab--there is, } in this case, no 'pre-immune'? Is a monoclonal Ab known not to be expresed } in the sample the best alternative? } } 2. what does one use for a control when using a polyclonal rabbit Ab } (commercial or otherwise) for which no pre-immune is available? My 2 cents: I had gotten by with finding the IgG concentration of the antibody preparation. Then I dilute a normal (non-immune) serum, or purified IgG, from the appropriate species to a similar concentration. IgG and IgM estimates for the normal sera of several species may be found in R. Lindmark, et al, J. Immunmol. Meth., 62:1-13, 1983. Of course, not all vendors know that much about their products, and most homemade antibodies are relatively uncharacterized. Sometimes the IgG content of an immune serum or monoclonal prep. can be estimated by comparing the total protein to that of non-immune serum for that species, or control culture/ascites fluids. I have encountered commercial "non-immune" sera that gave a better anti-cytokeratin label than the monoclonals being evaluated.
I've seen alot of discussion about staining lately which is interesting, but now I have a simple question. We deal mainly with metals/ceramics/semiconductoring materials hence, we do no staining etc.. So for the "novice in me" can someone tell me what IgG and IgM are? I'm not able to deduce the meaning from the context of the discussion. It would help me (and probably a few others) to follow the discussion a little better.
Remember this list has a whole slew of different readers and not all of us know each others "standard" abbreviations from the different fields of microscopy (or even the same field but in different applications). Try to keep this in mind when posting things.
IgG and IgM stand for immunoglubulin G and immunoglobulin M, and in this context refer to different types of antibodies used for immunohistochemistry. For most applications, primary antisera are of the IgG type, but for some applications, and for antibodies derived from certain sources and by different procedures, they may be IgM. This is important to know, since the secondary antibody, which is directed against the primary antibody and usually carries the tag which will allow visualization, must be directed against the appropriate immunoglobulin and the right host species.
There is an excellent review, both for its information content and its historical value, on the basics of indirect immunohistochemiostry by Ludwig Sternberger, 1974. I don't have the reference handy, but I will track it down and report back.
Hope this has been useful...
David Morilak Dept of Psychiatry Stanford university morilak-at-cmgm.stanford.edu -------
IgG and IgM stand for immunoglubulin G and immunoglobulin M, and in this context refer to different types of antibodies used for immunohistochemistry. For most applications, primary antisera are of the IgG type, but for some applications, and for antibodies derived from certain sources and by different procedures, they may be IgM. This is important to know, since the secondary antibody, which is directed against the primary antibody and usually carries the tag which will allow visualization, must be directed against the appropriate immunoglobulin and the right host species.
There is an excellent review, both for its information content and its historical value, on the basics of indirect immunohistochemiostry by Ludwig Sternberger, 1974. I don't have the reference handy, but I will track it down and report back.
Hope this has been useful...
David Morilak Dept of Psychiatry Stanford university morilak-at-cmgm.stanford.edu -------
I've had some real problems trying to send this message to the list, so I hope it gets through this time, and please forgive me if it gets sent a few times:
Nestor:
IgG and IgM stand for immunoglubulin G and immunoglobulin M, and in this context refer to different types of antibodies used for immunohistochemistry. For most applications, primary antisera are of the IgG type, but for some applications, and for antibodies derived from certain sources and by different procedures, they may be IgM. This is important to know, since the secondary antibody, which is directed against the primary antibody and usually carries the tag which will allow visualization, must be directed against the appropriate immunoglobulin and the right host species.
There is an excellent review, both for its information content and its historical value, on the basics of indirect immunohistochemiostry by Ludwig Sternberger, 1974. I don't have the reference handy, but I will track it down and report back.
Hope this has been useful...
David Morilak Dept of Psychiatry Stanford university morilak-at-cmgm.stanford.edu -------
Message-Id: {m0ouTk2-000dBZC-at-unpsun1.cc.unp.ac.za} To: microscopy-at-ANLEMC.MSD.ANL.GOV
Following up my earlier reply, here is the citation for Sternbergers classic reference on immunocytochemical techniques:
Sternberger, Ludwig A. IMMUNOCYTOCHEMISTRY (Englewood Cliffs, N.J. : Prentice-Hall, 1974)
Sternberger, Ludwig A. IMMUNOCYTOCHEMISTRY. 2nd ed. (New York : Wiley, 1979)
Sternberger, Ludwig A. IMMUNOCYTOCHEMISTRY. 3rd ed. (New York : Wiley, 1986)
If anyone is interested, it's a great introduction to immunocytochemistry, and for anyone who is doing it, it is a great look into how the technique has been developed.
Thanks to all for the general info on IgG & IgM. Just goes to show you that us materials types are interested in lots of things too!
BTW I smiled at Michael Herron's closing comment:
} Hey, it ain't no cruise missle but it what we do :^)
Remind me in a less busy time to describe how the ANL EM Center proposed and developed a protocol for using an SEM on Cruise and other Missles (US & Russian) as a nondestructive means for nuclear arms verification. It was actually a small ($20K) but funded program for a year by the START program! The funny thing is the procedure worked. It got killed in the end because anything that inexpensive couldn't work (at least one of the explaination I got)
Message-Id: {m0ouTLM-000dBTC-at-unpsun1.cc.unp.ac.za} To: microscopy-at-ANLEMC.MSD.ANL.GOV
On Wed, 3 Nov 1993, Michael Cammer wrote:
} } We are looking for a system for gross storage and easy retrieval of a } large number of images for both teaching and research. Does anybody have } any suggestions? } } Basically, we have a large (microscope) slide library that we would like } to scan in color. We need the images available for playback on a video } monitor and for photography. Should we use a computer and, if so, with
} what software (e.g. database) and what storage device (for speed, quanity, } compatibility w/ other systems, and an eye towards support in the future)? } The initial application is for a slide library for teaching, but, perhaps, } this could be turned into an interactive system available to students. } Also, I have been thinking that such a system might be useful for storing } our images for research; even without this specific query, we need more } and more compatible storage. } } Any suggestions for specific parts or for whole integrated systems (e.g. } is there something smilar for other fields?) would be welcome. } } Thanks- } } Michael cammer-at-aecom.yu.edu } } }
Cost aside, one alternative is HDTV for capturing the images. I'm not sure what the current state of the art is, but several years ago, Sony offered complete HDTV systems (cameras, analog and digital storage, post-processing hardware, optical fiber closed-circuit transmission, etc.) specifically aimed at the pathology market. I saw the system demonstrated in the Sony showroom in Tokyo; the image quality was extraordinary. As I recall, there are several sites in the US that offer HDTV-based remote consulting.
Apart from cost, one reason to hold off on the Sony system is that their HDTV standard is different than the one that three US developers have decided (at long last) to develop jointly: You could end up with a pricey white elephant.
Storage of massive amounts of data will depend on a number of factors that include required speed of retrieval, number of users who need simultaneous access, available hardware, money. Running all the images onto CD-ROM is not a bad alternative; costs are not terribly bad (if you want to make your own, you'll have too pay around $4K) although speed of retrieval may be frustrating.
I'm quite interested in hearing of ways people have developed archives of microscopy data. Having representative images on-line in conjunction with other data can make a very powerful tool.
Dave Coder Dept. of Immunology Internet: dcoder-at-u.washington.edu
Related to the current discussion thread on imarge archiving - what systems and software do people use for cataloging and retrieving images? We are accumulating stacks of images from calcium ratioing, confocal, serial images from convential microscopy (lm and SEM) and stuff that has been converted to projection slides and Linotronic files.
There are both commercial and shareware programs offered.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206) 543-8360
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: 17:27
X-Warning: Original message contained 8-bit characters, however during the SMTP transport session the receiving system was unable to announce capability of receiving 8-bit SMTP (RFC 1425-1428), and as this message does not have MIME headers (RFC 1341) to enable encoding change, only option was to STRIP sent characters to 7-bit :-(
On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote:
} Date:11/3/93 } NC EMAL } Subject: Critical Point Dryer } I have a colleague who is in the market for a critical point drying system. } Not knowing anything about such things or exactly how they work, I want to ask } if there is a manufacturer or model that is preferred by the majority of people } and if it is possible to dry material that is wet with a liquid other than } water. } Any help or info would be appreciated. } Thanks. } John Mansfield
The idea of critical point drying is to change the liquid to a suitable liquified gas, which has a low enough critical point to change directly fron liquid phase to gas phase. It does not matter wether the change is made directly or via a series of liquids. The only thing which matters is to maintain the sample as intact as possible while changing the liquids. So all you need to know is the solubility of different liquids in each other and finally in the liquified gas.
Hopefully this helps, others can give you more exact advice.
Best regards,
Jouko Mki
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Mki 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
On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote:
} Date:11/3/93 } NC EMAL } Subject: Critical Point Dryer } I have a colleague who is in the market for a critical point drying system. } Not knowing anything about such things or exactly how they work, I want to ask } if there is a manufacturer or model that is preferred by the majority of people } and if it is possible to dry material that is wet with a liquid other than } water. } Any help or info would be appreciated. } Thanks. } John Mansfield
The idea of critical point drying is to change the liquid to a suitable liquified gas, which has a low enough critical point to change directly fron liquid phase to gas phase. It does not matter wether the change is made directly or via a series of liquids. The only thing which matters is to maintain the sample as intact as possible while changing the liquids. So all you need to know is the solubility of different liquids in each other and finally in the liquified gas.
Hopefully this helps, others can give you more exact advice.
Best regards,
Jouko Maki
P.S. Sorry for repeating this message. The first sending contained 8-bit characters causing errors. I hope this goes through properly. 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
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} We are looking for a system for gross storage and easy retrieval of a } large number of images for both teaching and research. Does anybody have } any suggestions? } } Basically, we have a large (microscope) slide library that we would like } to scan in color. We need the images available for playback on a video } monitor and for photography. Should we use a computer and, if so, with } what software (e.g. database) and what storage device (for speed, quanity, } compatibility w/ other systems, and an eye towards support in the future)? } The initial application is for a slide library for teaching, but, perhaps, } this could be turned into an interactive system available to students. } Also, I have been thinking that such a system might be useful for storing } our images for research; even without this specific query, we need more } and more compatible storage. } } Any suggestions for specific parts or for whole integrated systems (e.g. } is there something smilar for other fields?) would be welcome. } } Thanks- } } Michael cammer-at-aecom.yu.edu Michael, We are also looking at alternatives for the long term archiving and easy retrieval of digital images. Many of our images are directly aquired using Perceptics HR 24 RGB frame grabber with a SONY DXC930P (P for Pal) which gives about 1Mb per image of real 24 bit colour with low noise if the lighting is good. Or we use a Videk (Kodak) MegaVision with a Perceptics MegaGrabber for a 1.4Mb Monochrome image, or we use the Data Translation DT 2555 Quik Capture for a 400Kmonochrome image. These images are used for illustration, measurement etc. We have a Richoh read-write optical disc which is useful for storage, and works well. We have now started to worry about back-ups of our now extensive files of images. We are going to make a Photo CD, which will require the production of films or slides from digital images. although next year Kodak will offer a service called portfolio which will allow the production of a CD containing digital only material. The idea of a photo cd is attractive because it is becoming a well accepted plateform for images, and is likely to be durable in the physical and temporal sense. The other advantages are the availability of "juke boxes" for CD's allowing the use of six or so in one machine. This access might not be as quick as from a hard disc, but it will be better than scanning in the original again. Because photo-cd has the "thumbnails", rapid data base access becomes a reality, software is already available, eg shoebox. So we will report tto the list as we learn. Noel T Goldsmith DSTO Aeronautical Research Laboratory 506 Lorimer Street Port Melbourne Vic 3207 Australia
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} } } } } On Wed, 3 Nov 1993 18:25:54 -0600, kartenh-at-Sdsc.Edu said:
} } Cost aside, one alternative is HDTV for capturing the images.
kartenh} Another alternative is the Kodak digital camera back on a kartenh} Nikon 8008. This directly dumps data into a Mac through kartenh} the SCSI port. It provides a color (24 bit?) image with kartenh} about 1500 pixel horizontal resolution X about 1000 kartenh} pixel. It grabs the image in about the same time that kartenh} film does. But it is slow in dumping it into the kartenh} computer. (Each image is about 4.5 MB). They provide a kartenh} plug-in driver for Adobe Photoshop, so you can directly kartenh} manipulate the image. H. Karten
I believe this is the very same DCS2000 I used earlier this year. It is most emphatically *not* a 24 bit scanner, although all the product lit seems to imply it very strongly. Relevant fragments from old sci.image-processing articles follow. As I wrote before, the PhotoCD images I just got were *much* preferable to the DCS2000 images.
|} From: sasrer-at-unx.sas.com (Rodney Radford) |} Subject: Re: Digital Cameras - Suggestions? |} Date: Wed, 9 Jun 1993 23:27:46 GMT
|} John Peterson {jp-at-taligent.com} writes: |} } Kodak DCS. This is a Nikon 35MM with a new back containing a |} } 1024x1024x24 CCD array. Old models required you to lug around a |} } suitcase, newer ones have a box about the size of the camera body |} } attached to the bottom of the Nikon. Suitcase stores about 1-200 |} } photos (depends on compression), smaller one can store photos in |} } small strap on SCSI disk. Mondo expensive, $8-20K. Smaller |} } cameras only take 20-30 photos on a battery charge; suitcase |} } models go longer. Suitcase has an LCD screen to review images. |} |} Most of your description was very accurate, and very |} informative. However, I don't know of any 24bit DCS cameras |} available (someone correct me if I am wrong, and give me the model |} number). We currently have a DCS200 camera, which is a Nikon F3 |} camera with a strap on hard drive capable of storing 50 images. |} However, it is only an 8bit CCD array with an RGB color mask in |} front so that each pixel can be EITHER red, green, or blue, such |} as: |} |} R G R G R G R G .... |} G B G B G B G B .... |} R G R G R G R G .... |} G B G B G B G B .... |} |} The resulting image on the DCS200 is a 1024x1536 image and takes |} 1.5MB (data is NOT compressed in the camera/disk). After this image |} is downloaded into the computer, it is interpolated into a full |} 24bit image by calculating 'suitable' values of red, green, and |} blue for each pixel. The result is pretty good, but not as good as |} a real 24bit image!
|} From: sasrer-at-unx.sas.com (Rodney Radford) |} Subject: Re: Digital Cameras - Suggestions? |} Date: Thu, 10 Jun 1993 13:05:40 GMT |} |} ledwards-at-leland.Stanford.EDU (Laurence James Edwards) writes: |} |} } Not to quibble, but in standard usage (at least on the output |} } side) a 24bit image means 8bits in rgb ... each "pixel" is |} } composed of an rgb triad. So I would say John had the depth right |} } but the resolution wrong ... that is, the unprocessed image is |} } really 512x768x24. Do they use linear interpolation, or something |} } a little more sophisticated?
|} Except that the interpolation algorithm yields a 1024x1536x24 bit |} image, not the 512x768x24 image you mention above. So they 'fake' |} the other 16bits in EVERY pixel location. So, I still stand by my |} original 1024x1536x8 bit description of the CCD array. Below is a |} paragraph from the DCS200 Programmer's Specifications:
|} "The CCD sensor for the DCS200 Digital Camera does not capture all |} three RGB colors for each pixel. Instead, it captures one color for |} each pixel, so that interpolation is required to construct three |} full RGB planes for the image. The grid below represents the pixel |} grid before interpolation. Green pixels predominate the grid because |} Green captures the luminance levels which can translate across to the |} Red and Blue planes."
|} The interpolation algorithm consists of three seperate passes over |} the image, interpolating the green, red and blue values. The Green |} interpolation algorithm basically looks at the 4 nearest neighbors |} as two pairs (up/down, left/right) and calculates the two delta |} values. It then uses the average of the pair with the largest delta |} value. The red/blue algorithms are very similar, except that there |} is no test for largest delta values - instead some pixels always |} use the up/down pair, some use the left/right pair, and others use |} a 4way average all all neighbors.
|} There is also a black-level color correction pass (enhances |} contrast), and a gamma correction pass. There is also a list of |} 'defective' CCD rows in the camera, and how to adjust for them |} (replace with a neighbor row, or average with two other rows).
|} We have received 3 seperate copies of the KCS200 manual, and in |} EVERY one, the algorithms have changed, so I guess they are not |} fixed in stone, and Kodak is still experimenting. Note that it is |} also possible to just use the interpolated Green plane as a B&W |} image.
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} We are looking for a system for gross storage and easy retrieval of a } large number of images for both teaching and research. Does anybody have } any suggestions? ............ } Michael cammer-at-aecom.yu.edu
to which Harvey Karten replied:
} There are several options. Low to medium resolution, use Video. File size } is modest (circa 300KB per image). Resolution is low = despite what your } television salesman tells you, you won't do better than about 350 lines of } resolution. You can then archive them for database using Aldus Fetch or } Multi-User's Search 2.0 image database. } Alternately, use intermediate photos, and send them off to Kodak for } Photo-CD ROMs. They provide a few different levels of resolution (from } video levels up to about 2,000 lines), depending on your display } capability. Increasing amounts of software to access Kodak Photo-CDs. } } For the user demanding higher levels of resolution, you will have to go to } a Scitex or Leaf scanner (costs about $15,000) which will give youj about } 5,000x4,000 pixel resolution. This is slightly better than a sharply } focussed image on Kodachrome 35 mm (also about 20 megapixels, or 4K x 5K.) } } Image database programs for the Mac were reviewed in the Sept. '93 MacUser, p190, a good starting point for comparisons. "Aldus Fetch" (mentioned by Harvey) received a four-mice review. Our graphics group here has set up a very usable multiuser database using Fetch, for drafted figures and scanned images. It is used to organize and access their archive and active files.
But, how to acquire the digital files? Capture by CCD camera directly from the microscope will be a problem regardless of it's CCD array size if you want to capture a large area (ie. low magnification). If you shoot film at low magnification, you can then scan the transparencies with a film scanner (2000-4000dpi,big files !), your own (your time and $$) or Kodak's PhotoCD ($$). You do have control of cropping, resolution, color or exposure control if you do it yourself. If your images have a narrow color range, you can reduce the file size by x3 by transforming it to 8-bit indexed color without much perceptible loss. "Photoshop" or "Fast Eddie" can do this for you. I have had some discussions with people in our photo library (chemical), who want to establish a digital equivalent. They have over 50,000 negatives and slides in the freezers. Let Kodak handle it! (As per Harvey's recommendation). PhotoCD's provide enough resolution (2000x3000 pixels) for direct 300dpi dye-sublimation output of 6.67"x10" prints. Discussion of the adequacy of dye-sub output can be found in the earlier record of this forum. A review of Kodak's image manipulation program, "Kodak PhotoEdge", can also be found in Sept'93 MacUser. Kodak PhotoCD's can even be viewed on the home TV, a nice feature for cramming students, even if not high-res.
I have been playing with a Nikon LS-3510AF film scanner (on loan from my local Nikon representative, S & M Microscopes, Colorado Springs, CO --A "thank you" plug). You know what? You can stick a slide (40mm x40mm scan area) directly in the 35mm slide holder and scan it. No microscope! At the maximum scanner resolution of 3165 dpi that is a resolution of 0.124 micrometers/pixel which is about what I get at x100 on my microscope set-up. It does a nice job with a slide sandwiched between crossed polarizers (we geologists do this a lot). I assume your slides are however of animal or vegetable, not mineral subjects. You wouldn't be able to do fluorescence stains without major, but not impossible, modifications to the scanner ( Is there a market out there?). Will microscopes be obsolete soon? Within the last year there was an article in "Advanced Imaging" about the development of a digital microscope. I can not remember the exact issue off-hand. In that set up the sample is stepped across a high density (4000-6000 pixel) linear array CCD, just like most film scanners do ( but theirs still looked like a microscope).
Good luck with your project. Maybe some of this will help.
============================================================= Robert Zimmermann rzimm-at-usgs.gov (Internet) U.S. Geological Survey 303-236-5626 MS 905 Box 25046 Federal Center Denver CO 80225
} On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote: } } } Date:11/3/93 } } NC EMAL } } Subject: Critical Point Dryer } } I have a colleague who is in the market for a critical point drying system. } } Not knowing anything about such things or exactly how they work, I want to ask } } if there is a manufacturer or model that is preferred by the majority of } } people } } and if it is possible to dry material that is wet with a liquid other than } } water. } } Any help or info would be appreciated. } } Thanks. } } John Mansfield } } The idea of critical point drying is to change the liquid to a suitable } liquified gas, which has a low enough critical point to change directly } fron liquid phase to gas phase. It does not matter wether the change is } made directly or via a series of liquids. The only thing which matters is } to maintain the sample as intact as possible while changing the liquids. So } all you need to know is the solubility of different liquids in each other } and finally in the liquified gas. } } Hopefully this helps, others can give you more exact advice. } } Best regards, } } Jouko Maki
The most common transition fluid used these days is liquid carbon dioxide. Specimens are typically dehydrated through a graded series of organic solvent, ethanol or acetone, then placed in the cooled critical point dryer (CPD). Ethanol and acetone are both miscible with CO2. Once the specimens are in the chamber, liquid CO2 is introduced, and, through several exchanges (filling and draining with CO2 under pressure from the tank), the organic solvent is completely replaced by CO2.
The chamber is kept cool to keep the CO2 in liquid phase. After exchange of liquids, the temperature is increased and the pressure inside the vessel increases also. Once the critical point is passed, 1073 psi and 31.1 deg C for CO2, the vessel is slowly vented. After atmospheric pressure is reached, the chamber is opened and the specimens removed. They are warm and dry, and they tend to rehydrate pretty fast, so having a dessicator handy is a good idea.
The typical CPD device is a high pressure bomb that has valves (3) for introducing liquid CO2 and venting liquid (bottom of chamber) and gas (top of chamber). There is a mechanism for heating and cooling, too. One temperature control system has electronic heating and cooling devices (can't think of the name now) and sensors for temp control. These can be automated. The other kind, which I prefer, has a water jacket around the pressure vessel. You run ice water in to cool and warm/hot water to heat. You have to keep an eye on the chamber to make sure you don't heat too fast. But I watch the chamber, even when it's automated.
I like the water controled units because (1) they are cheaper (2) there are fewer parts to go bad (3) they are smaller. The one I use now is made by Polaron (model 3200) and is sold by Ted Pella. The last price I have is about $3000. Another consideration is the size of the chamber. If you run only a few specimens you might want a smaller one.
Hope this helps, John (and others),
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
If I may add my two bits worth concerning Kodak's Photo CD system: (- and aplogies in advance if this information has already been covered in previous discussions) KODAK offer an Product Information Kit re Photo CD and their image processing covering o Pro Photo CD (6000 by 4000 pixels of resolution) o Lite Box image library system o Photo CD Catalogue (6000 image storage) o Portfolio Photo CD (variable resolution from base allowing 800 images or 1 hour of sound, or any combination in between) and more
How do you unsubscribe from this list? I am going to be away for 6 weeks and want to stop the flow of mail.
Listserv-at--at-anlemc.msd.anl.gov
ignors my requests.
-d
=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= David L. Hirschberg bnhirsch-at-dapsas1.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
} On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote: } } } Date:11/3/93 } } NC EMAL } } Subject: Critical Point Dryer } } I have a colleague who is in the market for a critical point drying system. } } Not knowing anything about such things or exactly how they work, I want to ask } } if there is a manufacturer or model that is preferred by the majority of } } people } } and if it is possible to dry material that is wet with a liquid other than } } water. } } Any help or info would be appreciated. } } Thanks. } } John Mansfield } } The idea of critical point drying is to change the liquid to a suitable } liquified gas, which has a low enough critical point to change directly } fron liquid phase to gas phase. It does not matter wether the change is } made directly or via a series of liquids. The only thing which matters is } to maintain the sample as intact as possible while changing the liquids. So } all you need to know is the solubility of different liquids in each other } and finally in the liquified gas. } } Hopefully this helps, others can give you more exact advice. } } Best regards, } } Jouko Maki
The most common transition fluid used these days is liquid carbon dioxide. Specimens are typically dehydrated through a graded series of organic solvent, ethanol or acetone, then placed in the cooled critical point dryer (CPD). Ethanol and acetone are both miscible with CO2. Once the specimens are in the chamber, liquid CO2 is introduced, and, through several exchanges (filling and draining with CO2 under pressure from the tank), the organic solvent is completely replaced by CO2.
The chamber is kept cool to keep the CO2 in liquid phase. After exchange of liquids, the temperature is increased and the pressure inside the vessel increases also. Once the critical point is passed, 1073 psi and 31.1 deg C for CO2, the vessel is slowly vented. After atmospheric pressure is reached, the chamber is opened and the specimens removed. They are warm and dry, and they tend to rehydrate pretty fast, so having a dessicator handy is a good idea.
The typical CPD device is a high pressure bomb that has valves (3) for introducing liquid CO2 and venting liquid (bottom of chamber) and gas (top of chamber). There is a mechanism for heating and cooling, too. One temperature control system has electronic heating and cooling devices (can't think of the name now) and sensors for temp control. These can be automated. The other kind, which I prefer, has a water jacket around the pressure vessel. You run ice water in to cool and warm/hot water to heat. You have to keep an eye on the chamber to make sure you don't heat too fast. But I watch the chamber, even when it's automated.
I like the water controled units because (1) they are cheaper (2) there are fewer parts to go bad (3) they are smaller. The one I use now is made by Polaron (model 3200) and is sold by Ted Pella. The last price I have is about $3000. Another consideration is the size of the chamber. If you run only a few specimens you might want a smaller one.
Hope this helps, John (and others),
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
I need several options for storage of fixed tissues that might be used later for E.M. Currently I store tissues in the original fixative. Any other options would be appreciated.
I tried posting a message to the list server and got the following reply. Any thoughts?
} Bad address -- {MICROLISTUSA} } Error -- } %MAIL-E-NOSUCHUSR, no such user MICROLISTUSA } } Start of returned message } } Date: Wed, 10 Nov 1993 16:47:40 -0500 } Message-Id: {199311102147.AA09853-at-mtu.edu} } To: microscopy-at-anlemc.msd.anl.gov } From: llsutter-at-mtu.edu (Larry Sutter) } Subject: ESEM } } We are in the initial stages of evaluating an environmental SEM. } I have heard from some people that a field emission SEM with a cryo stage } will provide very comparable performance. This is an important distinction } to make early because we definitely have application for a field emission } SEM for work on photo catalysts, polymers, and materials research. The } primary interest in an environmental SEM is for examining sludges and } soils. If this work can be done on a FE/SEM with a cryo stage, our opinion } is that the FE/SEM would ultimately be more versatile. } } I would greatly appreciate any input on this matter especially from } (but not limited to) people that have used both types of instruments. } } Thanks... } } } End of returned message
In case you haven't noticed there have been some glitches in the system the last few days. The most common problem reported was either NOMAIL or BAD Addresses. I think the system is fixed, however, I'm not 100% sure. The mailing list has grown to ~ 400 users now and I've had to reconfigure the system to handle 2 groups USA vs. the rest of the World. Because of this it was necessary to manually edit system subscription files. I hope no one was erroneously deleted, however, some of you may be receiving 2 copies of messages. In a 2nd mailing I will attempt to identify duplicate addresses. For the time being please excuse the headaches, multiple copies and various test messages.
Please report any errors directly to me at
zaluzec-at-anlemc.msd.anl.gov
Nestor Zaluzec ANL EM Center
======================= BTW here is a message which I believe only reached part of the mailing list. It was originally sent out on Nov. 7th...
========================
ROYAL MICROSCOPICAL SOCIETY MEETINGS CALENDAR
17 March 1994 ANNUAL IMMUNOCYTOCHEMISTRY MEETING
29 March 1994 ANNUAL LIGHT MICROSCOPY MEETING, London
11 - 13 April 1994 MICROSCOPY OF COMPOSITE MATERIALS II, University of Oxford
13 April 1994 ANNUAL CYTOMETRY AND HISTOCHEMISTRY MEETING, Birmingham Organizers: Dr Gillian Lawrence and Dr B. K Shenton
Outline Programme 10.30 am Joint Session, Cytometry and Histochemistry in Pathology 2.00 pm Parallel Sessions A Histochemistry - Viviane Maggi Prize Competition B Cytometry 3.45 pm Pearse Prize Lecture - Professor S. J. Holt 4.30 pm Bingo Meyer Memorial Lecture
Contributed papers are requested for the joint session, the cytometry session and the Viviane Maggi Prize Competition for beginners. The closing date for receipt of abstracts will be 31 January 1994.
18 - 22 April 1994 EM SPRING SCHOOL, University of Sheffield
28 June 1994 FLOW CYTOMETRIC IMMUNOPHENOTYPING OF LEUKAEMIAS AND LYMPHOMAS MEETING, London
27 - 30 June 1994 COMPUTERS IN MICROSCOPY COURSE, University of Cambridge
13 - 14 July 1994 BENCH-TOP FLOW CYTOMETRY WORKSHOP, Newcastle University
17 - 22 July 1994 LM SUMMER SCHOOL, University of Leeds
5 - 9 September 1994 IMMUNOCYTOCHEMISTRY COURSE, Oxford Polytechnic
12 - 15 September 1994 MICRO 94 EXHIBITION AND CONFERENCE, London
12 September 1994 AGM AND PRESIDENTIAL LECTURE, London
19 - 23 September 1994 FLOW CYTOMETRY COURSE, University of Cambridge
October 1994 MICROSCOPY OF CATALYSIS, Joint Royal Microscopical Society/Royal Society of Chemistry Meeting, London
October 1994 FUNCTION TESTING BY FLOW CYTOMETRY WORKSHOP
November 1994 ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY COURSE, Sutton
Details of all meetings are available from 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.
MICRO 94 FIRST CIRCULAR
MICRO 94, International Microscopy and Image Analysis Conference and Exhibition 12-15 September 1994
Earls Court Park Inn International, Lillie Road, London, SW6
Description of Conference
The Conference will consist of tutorial and review lectures, technical lectures organized by the trade, and posters. Experts in the field of instrumentation, analytical methodology in physics and materials sciences, cytometry, cytochemistry, cytology, image analysis and image processing are invited to give the lectures.
Each speaker will provide an overview of the particular topic in question and ample time for discussion will be provided. Seven half-day sessions of tutorial and review lectures and six half-day sessions of technical lectures will be organized.
Full length papers of the invited tutorial lectures will be considered for publication in the Journal of Microscopy.
Contributed papers are welcomed and will appear in poster sessions. Abstracts will appear in the Proceedings of the Royal Microscopical Society.
The Physiological Society and the Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) will organize separate lectures within the scope of the conference.
Outline Scientific Programme
At the MICRO 94 Conference and Exhibition, emphasis will be placed on Image Processing and Analysis. The launch of a new Special Interest Group within the Royal Microscopical Society will also be taking place.
Review and Tutorial Lecture topics are expected to include:-
Image processing and analysis Computer manipulations of images Scanning probe microscopy in materials sciences and in life sciences Near field optical microscopy 3-D microscopy and reconstruction Confocal scanning laser microscopy Materials Microscopy of composite materials Microscopy of electronic materials Nanostructures Cytometry and Cytochemistry Study of cell proliferation with flow cytometry Tumour markers Probes for in situ hybridisation Probes for immunocytochemistry (p53, BrdU, Ki67, PCNA) Molecular probes for analysis of living cells (co-sponsored by The Physiological Society) Proteases in pathological processes Invasion and metastasis of cancer cells Arthritis and rheumatism Infections Environmental SEM
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited.
Furthermore, on the Monday, a special session on how to use the light microscope will take place.
Exhibition
An Exhibition of the latest microscopes, ancillary instrumentation and technology, of material needed for preparation of microscopic specimens (fluorescent probes, antibodies and dyes) and soft and hardware for image processing and image analysis will be held at the Earls Court Park Inn International. Admission to the exhibition is free by conference badge or exhibition only badge which will be obtainable at the registration desk. Exhibition tickets will also be available well before the exhibition takes place.
There will be approximately 50 companies exhibiting, providing an extensive range of equipment for you to view.
If you would like further details of the MICRO 94 Conference and Exhibition, please 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. Conference enquiries should be addressed to Miss Karen Hale, Exhibition enquiries should be addressed to Mrs Allison Winton.
-------------------end of message----------------------
} Details of all meetings are available from 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 believe, the Email address of the RMS is written in the wrong order. To my knowledge, it should read rms-at-vax.ox.ac.uk
Please, correct me if I am wrong.
Jouko Maki
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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
The "small" problem I thought I solved earlier has turned out to be just the tip of an iceberg. The server will keep running however, it will be intermittent while I try to debug the system.
Part of the problem appears to be in "Multinet" the Internet gateway I'm using here at ANL. Some of the messages you will be receiving in the next few days will have double headers while I sort out the works. The first header will likely always show my name, while the second one will be the true sender.
Please keep me (Zaluzec-at-ANLEMC.MSD.ANL.GOV )informed of problems that you see so that I can continue debugging.
MAIL FROM: {ZALUZEC} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 9-NOV-1993 22:37:57
} Kayton asks: I need several options for storage of fixed tissues that might be used later for E.M. Currently I store tissues in the original fixative. Any other options would be appreciated.
====== What is the time scale you need to store your samples? Are they already EM (?TEM/SEM?) specimens?
MAIL FROM: {ZALUZEC} RCPT TO: {MICROSCOPYLISTUSA} ARRIVAL_TIME: 11-NOV-1993 14:44:27
48 hr. post-germination pea root tips and segments through differentiation zone were fixed in 2.5% glutaraldehyde in 0.15 M cacodylate buffer (pH 6.8), followed by 2% OsO4 in same buffer, then dehydrated and embedded in a resin mixture of equal parts epon (substitute=Epox 812): Spurr. 1-2 micron sections do not stain with 1% toulidine blue with varied times and temps. Am interested in good general stain which will also reveal mitotic figures__ANY SUGGESTIONS?
MAIL FROM: {GWERDOS-at-gnv.ifas.ufl.edu} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 11-NOV-1993 08:20:35
Robert Kayton asked about fixed tissue storage. We generally advise the use of Trumps fixative as it has bee repoted good for this purpose. (McDowell & Trump, 1976, Arch. Pathol Lab. Med. 100:405)
Last year there was a paper by Sopsi et al. (Ultrastructural Pathol. 16:351) that reported good results storing fixed tissue frozen at -70 C after cryo- protection with sucrose (10% I think). They report good immunoreactivity after 2 years and it looked pretty good.
Greg Erdos Univ. of Florida gwerdos-at-gnv.ifas.ufl.edu
It's solved. The problem now definitely appears cured.
You can delete the rest of this message now, unless you want to know what happened.
========================
Symptom: Mailserver lockup, the batch server takes } 2 days to deliver mail that previously took 1/2 hour to process.
Cause:
Ultimately the mailserver lockup was caused by a faulty DNS (Domain Name Server) Address. The subscriber provided an address which checked out initially, remember that the instructions ask you to verify your address! However, after being added to the subscription list, something went haywire with one host node. In effect the mailserver would spend ~ 1-2 days searching & eventually finding the location of the subscriber. Unfortunately the way this software runs each subscriber is accessed sequentially and thus the mail would "appear" to hang doing nothing for a day (or more) while this one host is identified using god know how many secondary, teriary, DNS's in effect bouncing it's way around internet.
Solution: I've removed the problem address from the list and will contact the user seperately. Since the mail actually makes it through to the "lost" host I had no direct traceback feature to locate him and it took about a week to find. Some of you may not have noticed a problem since your subscription would have been entered and cleared before the problem one. In effect the only way to find the problem was to manually test the addresses, something I do not recommend that anyone does.
So... sorry that some of you had to put up with: 1.) multiple copies of repeated messages: 2.) excessively long headers with 1/2 the worlds email addresses 3.) multiple test messages
Many of you will not have received all of the messages posted to the listserver during this last week. If I see one that looks very important that in my infinite wisdom should be reposted I'll do so, otherwise it will just sit until the topic becomes posted again by the original author. I have an archive of everything and eventually will setup some way for anyone to search that archive but I've got alot of catching up to do and this is extremely low on my priority list of things to do.
That's all Folks! :-)
Nestor Z. ANL EM Center
Some how I just knew this listserver would become masochistic
In our standard H600 TEM, we have been getting comparable tungsten filament life to that in some of our other machines configured with sputter pumps, for use with LaB6 cathodes. Emboldened by this, we are fitting a Lab6 to this system to see what happens - anybody out there who has already done this, or has any comments? The cathode we have is a Denka, although we are generally moving to Kimball.
---------------------------------------------------------------------- Sally Stowe | Facility Coordinator ANU Electron Microscopy Unit | Australian National University Canberra, AUSTRALIA | Ph 61 6 249 2743 FAX 61 6 249 4891 | Email stowe-at-rsbs-central.anu.edu.au -------------------------------------|-------------------------------- -
John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu
I just sent off a posting suggesting the creation of a micrscopy newsgroup on the UseNet News system.
It's in the following newsgroups: sci.chem,sci.engr.chem,sci.geo.geology,sci.polymers,sci.materials,sci.misc,sci.p hysics,sci.research,sci.techniques.xtallography,sci.optics,sci.archaeology,sci. engr.mech,sci.geo.geology,sci.med
The following text is the full body of the posting, I encourage comments from the microscopy and imaging community. Please feel free to add to the necessary areas of interest, I will post follwo-ups when I have some response. I only posted this about two hours ago and I have had two messages of support already.
This post could equally well be titled: pre-RFD : sci.techniques.microscopy
Following is a proposed RFD (request for discussion) to create a microscopy newsgroup on the internet
Would potential users of this proposed newsgroup like to give their opinions on this (YES or NO) either by following on to this post (which should appear in sci.materials) or as email to (John.F.Mansfield-at-umich.edu).
Would people also consider passing this proposal onto colleagues who may be interested in participating in a microscopy newsgroup. Potential users of this newsgroup might like to consider adding their names to the RFD as co-proposers. It is hoped that at least one-hundred people can give support to this newsgroup before the RFD is officially posted to news.groups so as to show there is decent support for a microscopy newsgroup on the internet. If it looks like this will take a while to do, summaries of responses (who will remain anonymous) will be posted regularly to keep people informed what has been happening.
The reason for the name sci.techniques.microscopy is that the Usenet administrators have created the "techniques" hierarchy for newsgroups such as microscopy. Going with this name has the best hope of avoiding hair splitting arguments on newsgroup "names" that seem to plague the creation of other science newsgroups. The above name does not affect the aims or the charter of what this microscopy newsgroup is trying to achieve.
Please note if a particular topic of microscopy becomes very popular or there is a perceived need to create a subset of this newsgroup such as "SEM", "AFM" or "TEM", etc - it is relatively easy to create a
sci.techniques.microscopy.sem or sci.techniques.microscopy.tem, etc,
Pre - Request For Discussion (RFD) of sci.techniques.microscopy (unmoderated).
Proposers :- John Mansfield (John_Mansfield-at-mse.engin.umich.edu) Lachlan Cranswick (lachlan-at-dmp.csiro.au)
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.
--------------------------------------------
We propose a new unmoderated newsgroup called sci.techniques.microscopy. The main aim of sci.techniques.microscopy is to provide an open forum for the discussion of microscopy and related fields on the internet.
(Could people reading this post mention it to colleagues who might be interested and benefit from a microscopy group but not normally use the internet newsgroups.)
Proposed Name of Group ------------------------- sci.techniques.microscopy (unmoderated)
Purpose of New Newsgroup ---------------------------
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.
Topics for discussion would include :- --------------------------------------- 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 Diffraction Contrast Imaging Phase Contrast Imaging Selected Area Electron Diffraction - SAED or SAD Convergent Beam Electron Diffraction - CBED Image Filtering Specimen Preparation (Electropolishing, Ion Milling, Ultramicrotoming, etc.) 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
Anything else that is relevant to microscopy in general.
What is the Process of Creating a Newsgroup? --------------------------------------------
} (a) RFD: Discussion, i.e., public hearing to take place in the newsgroup news.groups for approximately one month (b) CFV: Call for votes (the voting period will be about 25 days) (c) Counting of votes and public display of votes (d) Announcement of new newsgroup
(a)--} (b) assumes no major disagreements about this newsgroup during discussion. (c)--} (d) assumes that the vote is favourable., i.e., Y } N+100 .and. Y } (2/3)(Y+N) Y being the number of YES votes, N being the number of NO votes for the creation of the proposed newsgroup. =============================================
Please comment/criticise/suggest by followons to this post or email to John.F.Mansfield-at-umich.edu
John Mansfield, North Campus Electron Microbeam Analysis Laboratory, 2455 Hayward, Ann Arbor, Michigan 48109-2143. Ph: (313)-936-3352. __________ YYURYYUBICURYY4ME.
Hitachi HU-11E Parts for free: If you are nursing along an HU-11E TEM, and would like some spares for it, please contact me directly (please don't respond to the list!) --Forepumps and DP are spoken for --Most other parts go to anyone who can use them. I'll ship light stuff inside the continental US for free; you'll have to pay shipping on the heavy stuff.
If I haven't heard from someone within two weeks, it goes in the dumpster. (TBAITW; as in "too big and in the way").
Julian Smith III Winthrop University 803-323-2111 (Vox) 803-323-2246 (Fax)
} Sally Stowe asks about swapping W/LaB6 in a H600.
Sorry but I'm geting old and forget full so I can't remember the details of an H600, but, the appropriate question to ask is why are you attempting to put LaB6 in the H600? not if it will work! The LaB6 filaments when operated properly and under *CORRECT* conditions will give you a greater Brightness and hence more electrons in electron-optically equivalent probes. Thus for small probe/analytical work or for imaging conditions which will improve with a more coherent source (HREM) and in good vacuum systems they work very well. It will improve your uscope work assuming that you are short on electrons in the configuration your are setting up your electron probe (the argument applies to both AEM and/or HREM modes), however, if you poison the filament by using it in a poor vacuum system (i.e form an oxide or carbide on the surface), then the work function will decrease and you will actually get worse performance than a standard W filament and at ten to twenty times the cost of W. So why do you want to do this?
Assuming you have appropriate reasons and a good vacuum (and students who do not dump the vacuum system to air with a hot filament running), then the next question to ask is your uscope equipped with a filament power supply that can be modified to drive the tip?
} Sally STowe asks about using LaB6 under "tungsten" vacuum configuration - ie without sputter pump.
} Nestor Zaluzec asks 1. Why? 2. Can the filament power supply be modified to drive Lab6 tip?
In answer to (1) - we want to get the best resolution we can over the next few months, and at the moment, on smallest condenser aperture, we cant make best use of the "spot size" adjustment because of lack of electrons, although the resolution does seem to be inproving sharply as far as can be seen. This microscope does seem to be particularly prone to charge effects or whatever if the beam is not well spread. Vacuum in the specimen area is 8 x 10-6mbar. The last tungsten filament lasted 330ish hours, so reckon we have some reasonable users at the moment.Touch wood. (2) - yes, filament supply can be switched.
---------------------------------------------------------------------- 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 -------------------------------------|-------------------------------- -
We use a Park Scientific Universal BD2 Scanning Probe Microscope, controlled by a HP 382 workstation. Does anyone know of any software which will easily (and reliably) extract the images into a format which can be read by PC's? It is possible to export a TIFF file but the format appears (according to the operator) to be slighly different to other TIFF formats.
Any help would be appreciated.
Colin V.
##################################################################### ********************** * 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
} Colin V. writes.... } We use a Park Scientific Universal BD2 Scanning Probe Microscope, } controlled by a HP 382 workstation. Does anyone know of any software } which will easily (and reliably) extract the images into a format which } can be read by PC's? It is possible to export a TIFF file but the format
Colin it sounds like Park is at fault. We routine transfer TIFF files from one computer Vax, Mac, PC, Sun... to another an access TIFF files with no difficulty. My guess is that Park took short cuts and did not properly implement TIFF (this happens alot from what I can tell). You should demand that Park rewrite their code to properly implement TIFF which is no at Version 6.0. T
There are lots of programs which can read TIFF files and display them on the PC.
Yes, 1.% Toluidine Blue in 1% sodium borate will stain sodium ethoxide etched Spurr epoxy.
On Fri, 12 Nov 1993, Nestor J. Zaluzec (708)-252-5075, -4964 wrote:
} From: SMTP%"-at-cc1.kuleuven.ac.be:desclinj-at-ulb.ac.be" 12-NOV-1993 02:02:51.52 } To: ZALUZEC } CC: } Subj: Re: LM - Need help on staining epoxy/Spurr embedded plant tissues_ } } From: desclinj-at-ulb.ac.be (Desclin Jean) } Message-Id: {9311120706.AA00745-at-is1e.vub.ac.be} } Subject: Re: LM - Need help on staining epoxy/Spurr embedded plant tissues_ } To: ZALUZEC-at-anlemc.msd.anl.gov (Nestor J. Zaluzec) } Date: Fri, 12 Nov 93 8:06:14 MET } In-Reply-To: {931111144412.202000ba-at-anlemc.msd.anl.gov} ; from "Nestor J. Zaluzec" at Nov 11, 93 2:44 pm } X-Mailer: ELM [version 2.3 PL11] } } Hello! } what is the pH of your toluidine blue solution? It might help } trying out a higher pH than usual (I am afraid I am not familiar } with plant tissues :-(...). } If you are not re-using the semi-thin sections afterwards (i.e. } resectioning for TEM), you might try treating them with NaOH } dissolved in absolute ethanol (to etch the plastic somewhat), such } as used prior to some immunohistochemical procedures. I believe } toluidine blue might work after that. } Good luck! } 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 * } *********************************************************** }
Many thanks to those who offered information regrding my query. All the information has been most illuminating (and useful).
Colin V.
##################################################################### ******************************* * Logic is invincible because * 0------* in order to combat logic it * } ---|--- { * is necessary to use logic. * | * P.Boutroux * / \ ******************************* _/ \_
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
} Colin V. writes.... } We use a Park Scientific Universal BD2 Scanning Probe Microscope, } controlled by a HP 382 workstation. Does anyone know of any software } which will easily (and reliably) extract the images into a format which } can be read by PC's? It is possible to export a TIFF file but the format
Colin it sounds like Park is at fault. We routine transfer TIFF files from one computer Vax, Mac, PC, Sun... to another an access TIFF files with no difficulty. My guess is that Park took short cuts and did not properly implement TIFF (this happens alot from what I can tell). You should demand that Park rewrite their code to properly implement TIFF which is no at Version 6.0. T
There are lots of programs which can read TIFF files and display them on the PC.
I'm starting to do gold labeling of protein in yeast cells and I'm having problems with infiltration. This is my first time in 25 years I've had to work with yeast. Any helpful hints?
Yes, 1.% Toluidine Blue in 1% sodium borate will stain sodium ethoxide etched Spurr epoxy. I've only had toluidine fail in Spurr's if the pH was off (someone didn't make up the correct borate } . For autoradiography and nuclear anntigen immunocytochemistry, it can be diluted to 0.1% or even 0.01% in 1% sodium borate to keep from obscuring the label.
} } If you are not re-using the semi-thin sections afterwards (i.e. } } resectioning for TEM), you might try treating them with NaOH } } dissolved in absolute ethanol (to etch the plastic somewhat), such } } as used prior to some immunohistochemical procedures. I believe } } toluidine blue might work after that. } } Good luck! } } 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 * } } *********************************************************** } } } }
"Nestor J. Zaluzec (708)-252-50" {ZALUZEC-at-anlemc.msd.anl.gov} CC: ZALUZEC-at-anlemc.msd.anl.gov
Reply_ RE} AFM: Imaging 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 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu We have a Nanoscope 3 and Digital Instruments have a TIFF export that NIH-Image doesnt like. When we import the images into image we lose the LUT information. I have found that the MOST useful piece of software for manipulating images, except NIH-Image of course, is Photoshop. If you are at a Univ you can get really good prices on it and it happily reads almost anything I try and read into it.
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Colin it sounds like Park is at fault. We routine transfer TIFF files from one computer Vax, Mac, PC, Sun... to another an access TIFF files with no difficulty. My guess is that Park took short cuts and did not properly implement TIFF (this happens alot from what I can tell). You should demand that Park rewrite their code to properly implement TIFF which is no at Version 6.0. T
There are lots of programs which can read TIFF files and display them on the PC.
Nestor Z. ANL EM Center -------------
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"I need several options for storage of fixed tissues that might be used later for E.M. Currently I store tissues in the original fixative. Any other options would be appreciated."
One alternative is to put the tissue into the buffer originally used with the fixative. The only drawback here is that after a period of time (} 1 year) the buffer usually tends to become acidic, and there goes the tissue preservation. Alternatively, I used this when working years ago in another lab so have no recent knowledge of the pros and cons, but tissue that you expect to use for TEM, process it through osmium and up to 90% alcohol and then store. I know that I did this and then subsequently finished processing the tissue into epon several months later and the ultrastructural preservation was very good. The best option to maintain good quality morphology is of course to process the tissue into resin then you have a fossil which can not deteriorate.
Hope this is of some help,
Gerry Little.
Dr Gerald Little | Ph (049) 215618 The Neuroscience Group | Discipline of Anatomy | Fax (049) 216903 Faculty of Medicine | The University of Newcastle | Email ANGJL-at-medicine.newcastle.edu.au Australia, 2308 |
I passed Daniel Callahan's query to Richard Lee at Argonne. This is his reply:
Our experience with carbon is mostly on diamond using conventional (tungsten) filaments in an SEM. We typically resolve sub-micron filaments and crystallites dowm to 30 to 50 nm with 10 kV operation. Fullerene crystals were difficult for us because of charging--even at 10kV.
Our experience with an high resolution FEG-SEM was also on diamond and was most impressive! They excel at low kV operation with no charging problems. We have taken photos on diamond nanophase materials (coatings) with 3 nm or better resolution.at 5kV using a new Leica-Cambridge 360FE.
You can try conventional SEM's at 5kV, but you will be limited in magnification. If you go much above 10kV you probably will have charging problems and loss of surface detail. The lower the kV, the better the surface detail with a material of low Z.
I passed Daniel Callahan's query to Richard Lee at Argonne. This is his reply:
Our experience with carbon is mostly on diamond using conventional (tungsten) filaments in an SEM. We typically resolve sub-micron filaments and crystallites dowm to 30 to 50 nm with 10 kV operation. Fullerene crystals were difficult for us because of charging--even at 10kV.
Our experience with an high resolution FEG-SEM was also on diamond and was most impressive! They excel at low kV operation with no charging problems. We have taken photos on diamond nanophase materials (coatings) with 3 nm or better resolution.at 5kV using a new Leica-Cambridge 360FE.
You can try conventional SEM's at 5kV, but you will be limited in magnification. If you go much above 10kV you probably will have charging problems and loss of surface detail. The lower the kV, the better the surface detail with a material of low Z.
Reply to: RE} hex. kikuchi maps The program DIFFRACT will do this on a Macintosh computer very nicely. This program is available from Virtual Laboratories, 37 Highland Court., Ukiah, Calif., USA (Attn: Janet Schlinger) FAX: 707-462-5275.
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Does anyone know a programm for calculating hexagonal kikuchi maps on PC and with/without the possibility to vary the c/a-ratio?
Heinrich Kestler Institut fuer Werkstoffwissenscahften Lehrstuhl I Martensstrasse 5 D-91058 Erlangen, Germany
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The are also public domain programs in the EMMPDL for calculating Diffraction Patterns/Kikuchi Maps. They are not all elegant but they work. To access information about the EMMPDL send an Email message to:
EMMPDL-at-anlemc.msd.anl.gov
In the Email message include the text line
SEND HELP EMMPDL
You will get instructions on how to get abstract listings file names and how to get copies of code for the programs.
We are considering the use of a TEM-style cryo stage for some x-ray diffraction experiments on frozen hydrated specimens. Our stability requirements are not at all high (drift is not a problem at the 0.1 micron/hour level), so we could probably do with a older model cryo stage. We would custom-build an airlock around whatever cryo holder, so we are not limited to a stage for e.g., JEOL or Philips. However, we do need a specimen loading and transfer system to go with the stage. We do not need much in the line of tilt control, and copper or other metals are fine (no need for beryllium).
If anyone has a cryo specimen holder that they are willing to loan or sell at modest cost, please contact me.
Thanks! Chris Jacobsen Department of Physics SUNY at Stony Brook Stony Brook NY 11794-3800 USA fax 516-632-8101 cjacobsen-at-ccmail.sunysb.edu CJACOBSEN-at-SBCCMAIL.bitnet
Reply_ RE} AFM: NanoIII to NIH Image And more... Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu If I label my Nanoscope III images as "test.tif" or something similar then I can open the images into NIH-Image without losing the LUT. If I use just "test" without the tiff extension then I get gray images. I do not have AccessPC set to automatically open .tif files are NIH-Image files and so I am unsure as to why this is happening. I can drag and drop a filename.tif file onto NIH-Image and it opens just fine. (We have also use the raw data import option but sometimes want to retain the colors of the original image for presenation purposes.) By the way I am getting my images off the Nanoscope by using Farallon's Appletalk PC software, it works OK but it seems a little slow. Has anyone else had experience with this kind of setup? I also cannot get the ethernet card I am using (a 3COM) to work correctly with NCSA Telnet and FTP, which we would like to use to transfer data to workstations and other computers than Macs. --------------------------------------
Regarding John Mansfield's comment, it looks to me as though the Nanoscope III stores raw data with a greyscale LUT. I base this on the fact that changing color table parameters in Top View mode and then selecting another file does not trigger a "Current file has been modified, save it?" query the way changing the
Z scale does.
Libby Shaw MIT CMSE Surface Analysis Facility
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Reply_ RE} TEM: Re: hex. kikuchi maps Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu You do have to be wary of Diffract, it is buggy, even the latest version. There is a new product from those guys called Desktop Microscopist it is suppoedly a complete rewrite. However, after playing with it for 10 minutes at MSA I managed to crash it! Treat all software with care if you havent written and debugged it yourself!!
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Chris Boothroyd
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Dear Colleagues
I have started a new teaching/research position, to find that there is a very nice Jeol 1200, but no ultramicrotome! We have no funds for one in the near future, and such machines in Japan are very expensive, at least twice the price elsewhere.
However, we may be able to find enough funds for a second hand machine. I also need a 'tome like the old JB4 for semi-thin sections, it must have a retracting arm and be able to use glass knives. I don't care how old the machines are as long as they work.
If anyone has an old microtome that they wish to dispose of for YEN, we would be very interested. We will pay all shipping charges etc, of course.
Look forward to many offers!
Robert W. Ridge Biology Department ICU 10-2 Osawa 3-Chome Mitaka-shi Tokyo 181 Japan
Tel: +81-422-33-3484 (0422-33-3484 for callers within Japan) Fax: +81-422-33-1449 Email ridge-at-icu.ac.jp
Phil: there were two errors in the protocol I sent you. 1. You should start out using LR White HARD grade, not medium grade; polymerize this at 50 degrees C for 24 hrs. There may be a small of amount of fluid LR white at the top of the capsule. These block section fine, also. 2. You may need to block free aldehydes to prevent background problems; so you can include a step between the buffer wash and dehydration, adding 1%ammonium chloride to your buffer solution. -Louisa
Reply_ RE} } AFM- NanoIII to NIH Ima Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu Re: the message below that seems to be replyinmg to my gripes about slow data transfer and printing from our Nanoscope connected to the Appletalk net at the U of M. Sorry, I didnt make myself clear about our physical transport layer! We are using the Appletalk PC package on a 3-COM Ethernet card. We have mostly Macs and several color and greyscale capable printers on the network and so it pays us to have Appletalk connectivity. The major problem with the networking and the Nanoscope is that the AFM software walks over the Network software and vice versa. Digital Instruments have fixed the AFM software so that it runs in a reduced memory mode when the Appletalk stuff is loaded. Anyone know if I can fix this by adding more memory? I am a PC neophyte, Macs are my computer of choice. Plus, does anyone know why Digital Instruments used such and slow stodgy 486 for the platform for their instrument (other that it was cheap)?
OK, Opinions are mine and not the U's! Jfm.
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Hi!
Appletalk IS notoriously slow for transferring images, the maximum speed is just over 200 Kb/sec. Compare with ethernets 10Mb/sec. Put ethernet into the MACs... We have a fairly well working connection between PC's and UNIX workstations (Silicon Graphics). We use Walker Richer Quinns reflection software using TCP/IP as communication protocol. There are some very conveniant solutions though. One includes the Lantastic network (Editirs choice in PC-magazin for a number of years). Now there is a TCP/IP module for this network. It supports NFS and other goodies as well. In practice, I think this will provide more of an invisible network than ever before. Lantastic is also available for the MAC. Moreover windows for workgroups has TCP/IP options at very low prices.
Mikael Gustafsson dept medical microbiology Univ. Link0ping MikGu-at-mme.liu.se FAX 046/13/224789 SWEDEN
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Reply_ AFM- NanoIII Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu Apparently I am wrong in assuming that the computer that is used by Digital Instruments for their Nanscope III is a cheap model. Apparently it is all Intel built. For those of you who have an older model with a 33Mhz processor and want a little more speed, call Mark Lean at Digital and get a price for an Overdrive processor to take it up to 66Mhz. (or go third party). Has anyone put a Pentium in a Nanoscope III yet? :-)
Pat Robinson asks...... ....................stuff deleted.............
} this 8500 is quite a different beastie. I'm especially interested in } ways and means of moving images to our network of Digital (DEC) PC's } running desktop publishing software, via TIFF or otherwise.
If you really want to work efficiently. Toss the TN buy yourself a Mac and framegrabber board. Then get a copy of the Public Domain program NIH-Image. It will grab images off your Sony, do some reasonable processing and save files as Tiff which you can then send via your network (use Pathworks, AppleTalk, TCP, just chooze your protocol & hardware) to your DEC machines.
In reference to } From jkelly-at-pruffle.nist.gov Fri Nov 19 15:58:12 1993 } To: microscopy-at-anlemc.msd.anl.gov } Subject: Image Printing } } I am working with image files on PC's - usually TIFF format } on IBM compatibles. I would like to get good quality B & W } plain paper hardcopy of the images. What are the good } combinations of print software and printers for either the PC's } or Mac environment? Thanks in advance.
For computer image printing, two popular solutions are laser printers (dithered black and white) and dye sublimation printers. We use IDL from Research Systems Inc. (Boulder CO) to do many many things, one of which is to generate PostScript files of images. It runs on PC and Mac (and Unix and VMS). We then get pretty good and very cheap B&W prints on any old PostScript laser printer. For the final print, we have a Tektronix Phaser IIsd (the "sd" or "sdx" is important) dye sublimation printer which takes color or B&W PostScript files. Kodak also now sells a similar product. Codonics sells a dye sublimation printer which takes TIFF files directly.
-- ******************* Chris Jacobsen, Asst. Prof., Department of Physics, SUNY at Stony Brook Phone (516) 632-8093, FAX -8101 Bitnet: cjacobsen-at-sbccmail jacobsen-at-xray1.physics.sunysb.edu ALL-IN_ONE: CJACOBSEN *******************
I have some synthetically prepared mineral powders, grain size in the 1-30 micron range, which I would like to get some EDS analyses of. This would mean embedding the microcrystals in epoxy and polishing them to get nice cross sections -- would someone with experience in this kind of sample prep be willing to share some tips/hints?
Thanks,
Bernhardt Saini-Eidukat Dept. of Geosciences North Dakota State University Fargo, ND 58105
We have been transferring files from the Tracor 8500 to PC and Mac formats using the following steps:
1. IBM-compatible computer connected from serial port 7 of 8500 to com2 of IBM. 2. On 8500 convert file to ".tif" format and re-save the file to the EXPORT directory. 3. Files are transferred to an IBM-compatible image handling application, such as Procomm. If using Procomm, open Procomm and input the following file specification: "/u3/export/name.tif". As the file is transferred, a byte count is tallied which should match that read on the 8500. 4. Copy image from Procomm directory to an IBM-formatted diskette.
Cheers, 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: 215-758-4249 FAX: 215-758-4244
Message-Id: {9311221558.AA20274-at-sifon.cc.mcgill.ca} To: Bernhardt Sainieidukat {sainieid-at-badlands.NoDak.edu}
Bernhardt, I've mounted small grains for use in the microprobe on several occasions. Usually the material I work with is a bit larger, but the techniques should still apply if you work carefully. If you have a large sample and are not concerned with individual grains, the simplest way to work is to set them in in epoxy on a standard thin section blank. If you have a reliable thin-section technician he should have no problen grinding it down to a 15 um thickness. Alternatively, if you have a small number of grains or you are interested in specific grains you can mount them in a plexiglass holder. First cut out a piece of plexiglass to fit into your sample holder (the thinner the better, I find 3 mm is about right). Next drill a number of small holes in the holder (I use a 2mm diameter). I mount these holders on a cleaned thin section blank using double sided tape. You should be very careful that there are no bubbles between the tape and the blank. Fill a hole with epoxy (I use Struers Epofix, it's nice and thin). You probably will want to carry out the next steps under a microscope. Using very fine forceps drop a grain into a hole and stir the epxoy with a fine wire to wet the grain and cause it to settle into the epoxy. If necessary you can adjust the grain so that it's resting on its most stable surface. Depending on the grain size I can usually get 10-15 grains in a mount before they start interfering with each other. Once the epoxy has set you can remove the blank slide on the bottom (usually I have to break it off). Polishing these mounts is crucial, if you take to much material off you lose your sample (This can really ruin your day if that was the only sample you had). I generally start with a very light brush with 300 grit SiC paper, just enough to remove any excess epoxy. This is followed by 600 grit paper. At this stage I polish for 30 seconds to a minute and then look at the result in a microscope to see how much exposed material I have. When sufficient material is exposed I do the fine polishing using 3, 1 and then 0.3 um Al2O3 either by hand or on a lap, depending how nervous I am about losing the material. Again I use short polishing periods followed by observation in the microscope. I've generally had good results with this technique and have rarely lost any material. One problem I have with larger grains is that it is sometimes difficult to get a large polished surface when the grain is sitting on it edges. I hope this helps you. Call me back if any of this is not clear
********************************************************************** * 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'm looking to buy a set of ultra long working distance objectives to fit onto an Olympus BHSM microscope. The longest WDs I've found so far belong to Olympus' own objectives (20x with 11mm and 50x with 8mm). Anyone know of any alternatives?
I'm looking to buy a set of ultra long working distance objectives to fit onto an Olympus BHSM microscope. The longest WDs I've found so far belong to Olympus' own objectives (20x with 11mm and 50x with 8mm). Anyone know of any alternatives?
} I'm looking to buy a set of ultra long working distance objectives to } fit onto an Olympus BHSM microscope. The longest WDs I've found so far } belong to Olympus' own objectives (20x with 11mm and 50x with 8mm). } Anyone know of any alternatives?
One trick is to look for U-stage lenses. These are designed for universal-stage mineralogical microscopes where the stage tilts as well as rotates. These lenses have a separate hemispherical glass element that is placed over the thin-section. Used without the hemisphere they have very long working distances.
Another trick is to use reflecting objectives.
I don't know the Olympus BHSM. Assuming it has standard threads (.8in 38tpi) and the equivalent of a 160mm tube length... We use a Leitz UT40 which is a U-stage lens. Without the hemisphere it has a magnification of 25x and a working distance of 14mm. Leitz has a few other U-stage lenses that are parfocal with this lens. Chromatic aberration can be a problem for refracting objectives with these long working distances.
We also use a reflecting objective from Ealing (15x, 21mm or 24mm working distance). Ealing sells several other reflecting objectives with higher magnification. The 15x is identical to the 15x lens made by Beck as far as I can tell. You may be able to purchase the Ealing lens corrected for infinity focus or a 210mm tube length, if that is what the Olympus requires. These reflecting objectives are just the ticket if you can tolerate their large size and the central obscuration.
Jeff Sweeney sweeney-at-dionheinz.uchicago.edu
Some good references are:
%0 Journal Article %A Burch, C. R. %D 1943 %T On aspheric anastigmatic systems %B Proceedings of the Physical Society %V 55 %N 312 %P 433-444 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1943 %T Reflecting microscopes %B Proceedings of the Physical Society %V 59 %P 41-46 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1945 %T Flat-field singlet aplanats %B Proceedings of the Physical Society %V 57 %P 567-576 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1947 %T Semi-aplanat reflecting microscopes %B Proceedings of the Physical Society %V 57 %P 47-49 %K reflecting objective
A couple of things I forgot to mention in my original mailing - the Olympus has infinity corrected optics (I had found that Nikon had a nifty lens, but it turned out not to be infinity corrected) and the microscope is part of a Renishaw imaging Raman spectrometer. I have been told by others that reflecting objectives would be no use since the Raman laser would either be reflected straight back into the spectrometer without reaching my sample or would have to be defocussed, preventing me doing any small area analysis.
Richard Lee of the Energy Technology Division at Argonne National Laboratory answers Daniel Callahan's question of 11 Nov 93:
Our experience with carbon is mostly on diamond with tungsten filaments in a SEM. We typically resolve sub-micron filaments and crystallites down to 30 to 50 nm at 10kV. Fullerene crystal were difficult for us because of charging, even at 10kV.
Our experience with an high resolution FEG-SEM was with diamond and the results were impressive. The FEG-SEM excels at low kV with no charging problems. We have taken photos of diamond nanophase materials (coatings) with 3 nm or better resolution at 5 kV using a new Leica-Cambridge 360FE.
You can try conventional SEM's at 5 kV but you will be limited in magnification. If you go much above 10 kV you probably will have charging problems and loss of surface detail. The lower the kV the better the surface detail with a material of low Z.
} A couple of things I forgot to mention in my original mailing - the } Olympus has infinity corrected optics (I had found that Nikon had a } nifty lens, but it turned out not to be infinity corrected) and the } microscope is part of a Renishaw imaging Raman spectrometer. I have } been told by others that reflecting objectives would be no use since } the Raman laser would either be reflected straight back into the } spectrometer without reaching my sample or would have to be } defocussed, preventing me doing any small area analysis.
Geez, you're making this more difficult :-) Chromatic aberration is not a problem with Ramam since you are working so near the Rayleigh peak, so look for a U-stage lens with infinity focus. You can use a reflecting objective but you must send the probe laser into the lens off-axis. Your imaging system may not accomodate this (probably not, huh). The focus of the laser can be adjusted with the beam expander if your beam expander is adjustable (probable not, huh). Alternatively, the laser can be prefocused with a long focal-length plano-convex lens. You might consider coating the reflecting objective for the laser wavelength. This would help aviod beam damage (not really a problem) and would give better signal in general.
I would like to hear recommendations for text books and review articles on the subject of light microscopy system design. I am interested in both basic information and the most recent technology. I would specifically be interested in hearing about those "desk reference" type books or review articles you always keep near by, and articles that have good bibliographies.
I am working on designing a photoluminescence imaging system (not confocal).
Please send references to me and I will compile a list. Also, if anyone would like a copy of the completed list, just let me know. Thanks.
Erik Reuter, e-reuter-at-uiuc.edu, 217-244-6378 phone, 217-244-6375 fax
And I haven't yet mentioned the high temperatures I want to have my samples at! Also, one of our third year students wants to Raman his diamond coatings at 400 C - What will that do to either ULWD or standard objectives???
Chromatic aberration is not } a problem with Ramam since you are working so near the Rayleigh peak, so } look for a U-stage lens with infinity focus. You can use a reflecting } objective but you must send the probe laser into the lens off-axis. } Your imaging system may not accomodate this (probably not, huh).
I'll go and ask.
The } focus of the laser can be adjusted with the beam expander if your beam } expander is adjustable (probable not, huh).
I already know this is, but you might be able to tell we haven't had the system long and are all still coming to terms with it. Someone here suggested using reflecting objectives with the beam expanded and with a blanking-widget in the lens to prevent direct reflection of the laser back into the spectrometer.
Alternatively, the laser } can be prefocused with a long focal-length plano-convex lens. You might } consider coating the reflecting objective for the laser wavelength. } This would help aviod beam damage (not really a problem) and would give } better signal in general. } } Jeff Sweeney sweeney-at-dionheinz.uchicago.edu } iThankyou,
"Photography Through The Microscope", P-2, cat.# 152-8371 "Kodak Scientific Imaging Products", L-10, cat. # 813-9321
The first has loads of basic information about objectives, condensers, setting-up illumination, flourescence, phase contrast, etc. Both have bibliographies.
The scope is 10 years old but in very good condition. It has been continuously maintained under service contract and is still in daily use. The components and accessories are listed below. All documentation and records are included. We would like to sell this as a complete package.
JEM-200CX TEM - S/N EM132031-86 200CX-SEGZ JEOL 200CX side entry goniometer HM-PP 3.5A, +30o SEG polepiece HA-PP 4.5A, +60o SEG polepiece 200CX-SQH-2 High Mag single tilt holder 200CX-SCSH Std. SEG quick change single tilt holer 200CX-BST double tilt holder (w/ one Gatan Be cup) 200CX-SHH sample heating holder 200CX-SCH single tilt cooling holder 200CX-SHU heating holder control unit 200CX-SEH single tilt straining holder 200CX-SFH faraday cup Haskris recirculating water chiller 200CX-ASID-3D STEM/SEM system 200CX -BEI-3 BSE detector (needs new Si crystals) 200CX-BF/DF STEM BF/DF option 200CX-FLC Free lens control 35-GMC Gamma control HA-EDS high angle EDS interface 200CX-HXS hard x-ray aperture 200CX-IMS-2 Image selector switch 35-MDD Multiple image display 200CX-SRT Scan Rotation & tilt correction 200CX-UHR UHR (photo) CRT w/ Polaroid 545 film back 35-VCA Video control amp. (derivative/filtering) 200CX-WFM-B Tek 501 waveform monitor 200CX-WFM-M lens current readout 35-YMD Y modulation device EM-THG Top entry Ultrahigh resolution goniometer TN-2000 Tracor-Northern TN-2000 EDS system (PDP-11/23 w/ 128kB RAM) SpectraChrome 512 Color monitor Horizontal Be window EDS detector High Takeoff angle (72o) Be window EDS detector EDS preamplifiers (x2) Digital beam control and interface EDS and Image acquisition software DEC LA-120 printer Honeywell Video Graphics recorder JEOL ASD system (projector lens scan coil) with OSU mods for scanning the TEM image) OSU built hollow cone illumination device Manuals and notes misc spare parts and filaments, including pole pieces and defl. coils HP 7221C 8 pen plotter operator's chair Gatan DT cryo low bkg holder Gatan cryo transfer holder
colijn.1-at-osu.edu OSU Campus Electron Optics Facility 292-0674 ------------------------------------------------------------------- Assumption is the mother of all screwups.
Regarding J. Kelly's (jkelly-at-enh.nist.gov) question:
} I am working with image files on PC's - usually TIFF format } on IBM compatibles. I would like to get good quality B & W } plain paper hardcopy of the images. What are the good } combinations of print software and printers for either the PC's } or Mac environment? Thanks in advance.
Have you considered videoprinters? You can videoprint any analog signal: RS-170 (NTSC), PAL (Europe), VGA... at 1200 dots/inch, 64 grey-levels, and it's *fast* (30 sec) compared to digital printing. You get near Polaroid quality for 40 cents a shot (one 8x10 inch, or two different 3x5 inch images can be printed together). I know you specified "plain paper," but the thermal paper is easily handled. Our Seikosha VP-3500 has been so reliable and popular with our customers, we put another on a Sun workstation based SEM. Capital investment is about $7500. One drawback: the images are not as stable. When I put a cup of hot coffee down on a print, it "developed" a black circle. ******************* Craig A. Smith, Honeywell Solid State Electronics Center, MN14-2C25 12001 State Highway 55, Plymouth, MN 55441-4799 USA Phone (612) 954-2895, FAX (612) 954-2040 smithc-at-ccsvax.ssec.honeywell.com *******************
Like J. Kelly, we are interested in printers. Specifically we are interested in purchasing a high-resolution gray scale printer to print TIF files from both PC's and from Silicon Graphic computers. Our files contain light microscopic images of hippocampal dendrites visualized at high magnification using a variety of staining methods (Golgi, biocytin, neurobiotin). I have information from Harris on their PhotoPro2000 and from Alden on their 9315 continuous tone printer. They range in price from $7500-10,000. What has been your experience & what would you recommend? We need a high-res image, and I am a bit concerned about the longevity of the thermal paper images. Many thanks.
Nancy L Desmond Dept. of Neurosurgery Univ. of Virginia Health Sciences Center Charlottesville, VA 22908 804.924.5607 (phone) NLD-at-GALEN.MED.VIRGINIA.EDU
} I have information from Harris on their PhotoPro2000 and from Alden } on their 9315 continuous tone printer. They range in price from } $7500-10,000.
This is the first I've hear of these brands. So I have no opinion, except that they sound expense for thermal printers. Lots of systems were exhibited at the MSA annual meeting for continuous tone printing. You may want to get a copy of the Exhibitors Guide from the MSA meeting and call up a few of the vendors for more information. Try contacting Bill Gunning Dept. of Path. Medical College of Ohio. 3000 Arlington Ave. Toledo, Ohio 43699-0008. He runs the Advertising for the MSA EXPO and can probably tell you how to get a copy. (419) 381-3484
} We need a high-res image, and I am a bit concerned } about the longevity of the thermal paper images.
High res means different things to different people. Do you mean pixel resolution, gray scale resolution, Image size or what? It also depends upon you digitization source (TV, CCD, Scanner....) and it's "pixel/gray scale" resolution. Also what is the purpose of your prints? Publication reports, records, or what.. If you have them on the computer it's certainly archived there resonably well at least as good as the lifetime of data on disks. I've got floppy disks with data on them that are now approaching 15 & 20 years old and to my surprize the data was still readable even though the disks at the time were only supposed to be good for ~ 10 years! Of course that implies you still have the computer to read all that "good" old data :-)
I would not trust thermal paper for archiving but only for a quick look see and maybe a lab notebook record. The best thing to do here is get the manufacturer's your interested in to give you copies of their prints and lay them out in the sunlight and shade for a few days/weeks. See how long they survive and then decide. The different brands will give different results, some are suprizingly good.
Here's a question for those of you that used to use the old Celli tape popular in England about 10+ years ago.
In the past I had some of this tape which I used to use to prepare specimens of graphite for TEM. Here I would use the old trick of just continually touching the surface of the graphite with fresh tape to delaminate the graphite gradually until I made a nice thin & optically transparent section, which stuck to the tape and was usually perfect for TEM. The nice thing about this old type of scotch tape was that the adhesive used would disolve beautifully in Acetone and the backing for that tape would just float away as it was not soluablein the acetone. Unfortuantely, the current type of "SCOTCH BRAND" transparent tape completely dissolves in Acetone and most other solvents I tried.
I'm back to trying to make more graphite samples as my old ones have finally bit the dust so to speak. Since the new scotch tape failed, I tried extraction replica tape which is available from most EM supply vendors, however it does not have the adhearance of either the old Celli tape or the newer scotch tapes. (BTW the new scotch tape very nicely delaminated the graphite and gives beautifully thin sections if I could ever remove them from the adhesive). Does anyone know of a solvent which works on the newer scotch brand tapes or have another/similar idea/procedure for layered structures which are not strongly bound?.
I'd prefer to avoid ion milling the graphite although I may have to resort to this in the end.
Forwarded message: From K.R.Hallam-at-bristol.ac.uk Mon Nov 29 12:21:47 1993 Message-Id: {9815.9311291221-at-irix.bris.ac.uk}
Dear All,
I was asked if I would post a summary of the replies received after my queation over ULWD objectives to attach to an Olympus BHSM / Renishaw imaging Raman system. Here goes....
} From jacobsen-at-xray1.physics.sunysb.edu Tue Nov 23 14:22:40 1993
Nikon makes a toolmaker's lens which can be fitted onto a Nikon Optiphot. It has 10x mag and a working distance of several cm. Very good images...
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 15:26:16 1993
One trick is to look for U-stage lenses. These are designed for universal-stage mineralogical microscopes where the stage tilts as well as rotates. These lenses have a separate hemispherical glass element that is placed over the thin-section. Used without the hemisphere they have very long working distances.
Another trick is to use reflecting objectives.
I don't know the Olympus BHSM. Assuming it has standard threads (.8in 38tpi) and the equivalent of a 160mm tube length... We use a Leitz UT40 which is a U-stage lens. Without the hemisphere it has a magnification of 25x and a working distance of 14mm. Leitz has a few other U-stage lenses that are parfocal with this lens. Chromatic aberration can be a problem for refracting objectives with these long working distances.
We also use a reflecting objective from Ealing (15x, 21mm or 24mm working distance). Ealing sells several other reflecting objectives with higher magnification. The 15x is identical to the 15x lens made by Beck as far as I can tell. You may be able to purchase the Ealing lens corrected for infinity focus or a 210mm tube length, if that is what the Olympus requires. These reflecting objectives are just the ticket if you can tolerate their large size and the central obscuration.
Some good references are:
%0 Journal Article %A Burch, C. R. %D 1943 %T On aspheric anastigmatic systems %B Proceedings of the Physical Society %V 55 %N 312 %P 433-444 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1943 %T Reflecting microscopes %B Proceedings of the Physical Society %V 59 %P 41-46 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1945 %T Flat-field singlet aplanats %B Proceedings of the Physical Society %V 57 %P 567-576 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1947 %T Semi-aplanat reflecting microscopes %B Proceedings of the Physical Society %V 57 %P 47-49 %K reflecting objective
} From treado+-at-pitt.edu Tue Nov 23 21:03:41 1993
In response to your question posted to the Microscopy discussion list November 23, we use Leica ultra long working distance infinity-corrected objectives on our Olympus BHS microscope.
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 21:30:36 1993
Geez, you're making this more difficult :-) Chromatic aberration is not a problem with Ramam since you are working so near the Rayleigh peak, so look for a U-stage lens with infinity focus. You can use a reflecting objective but you must send the probe laser into the lens off-axis. Your imaging system may not accomodate this (probably not, huh). The focus of the laser can be adjusted with the beam expander if your beam expander is adjustable (probable not, huh). Alternatively, the laser can be prefocused with a long focal-length plano-convex lens. You might consider coating the reflecting objective for the laser wavelength. This would help aviod beam damage (not really a problem) and would give better signal in general.
} From e-reuter-at-uiuc.edu Wed Nov 24 00:20:00 1993
I am also interested in learning about ultra long working distance objective. Would you please summarize to the list or to me what you find out in a few days?
} From timonf-at-earth.ruu.nl Wed Nov 24 07:41:09 1993
The problem with U-stage lenses is that the highest magnification I know which is available is a 30 x, and the optical quality is not too good (some distortion of the image and loosing a loss of brightness)
} From muepf-at-iff067.iff.kfa-juelich.de Wed Nov 24 07:52:31 1993
One of the --- expensive --- alternatives is to get a Questar long
[Sorry - I've lost part of the original here because someone picked up the telephone next door while my modem was connected - the message went on to mention a 3000 ECU alternative - Could you please resend the details to me - Thank you]
} From treado+-at-pitt.edu Wed Nov 24 17:14:22 1993
If you are only interested in doing Raman microprobing the reflective objective may be suitable by defocusing and illuminating off-axis. If you want to perform imaging I would use refractive objectives. The imaging quality is far superior with refractive optics.
We use Leica objectives: 5X, 0.10 NA, 50 mm WD and 50X, 0.45 NA, 20.6 mm WD.
We operate at elevated temperatures (} 200 oC) and jacket the objective in a water cooled housing. According to Leica the optical cements fail above 110 degrees.
Forwarded message: From K.R.Hallam-at-bristol.ac.uk Mon Nov 29 12:21:47 1993 Message-Id: {9815.9311291221-at-irix.bris.ac.uk}
Dear All,
I was asked if I would post a summary of the replies received after my queation over ULWD objectives to attach to an Olympus BHSM / Renishaw imaging Raman system. Here goes....
} From jacobsen-at-xray1.physics.sunysb.edu Tue Nov 23 14:22:40 1993
Nikon makes a toolmaker's lens which can be fitted onto a Nikon Optiphot. It has 10x mag and a working distance of several cm. Very good images...
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 15:26:16 1993
One trick is to look for U-stage lenses. These are designed for universal-stage mineralogical microscopes where the stage tilts as well as rotates. These lenses have a separate hemispherical glass element that is placed over the thin-section. Used without the hemisphere they have very long working distances.
Another trick is to use reflecting objectives.
I don't know the Olympus BHSM. Assuming it has standard threads (.8in 38tpi) and the equivalent of a 160mm tube length... We use a Leitz UT40 which is a U-stage lens. Without the hemisphere it has a magnification of 25x and a working distance of 14mm. Leitz has a few other U-stage lenses that are parfocal with this lens. Chromatic aberration can be a problem for refracting objectives with these long working distances.
We also use a reflecting objective from Ealing (15x, 21mm or 24mm working distance). Ealing sells several other reflecting objectives with higher magnification. The 15x is identical to the 15x lens made by Beck as far as I can tell. You may be able to purchase the Ealing lens corrected for infinity focus or a 210mm tube length, if that is what the Olympus requires. These reflecting objectives are just the ticket if you can tolerate their large size and the central obscuration.
Some good references are:
%0 Journal Article %A Burch, C. R. %D 1943 %T On aspheric anastigmatic systems %B Proceedings of the Physical Society %V 55 %N 312 %P 433-444 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1943 %T Reflecting microscopes %B Proceedings of the Physical Society %V 59 %P 41-46 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1945 %T Flat-field singlet aplanats %B Proceedings of the Physical Society %V 57 %P 567-576 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1947 %T Semi-aplanat reflecting microscopes %B Proceedings of the Physical Society %V 57 %P 47-49 %K reflecting objective
} From treado+-at-pitt.edu Tue Nov 23 21:03:41 1993
In response to your question posted to the Microscopy discussion list November 23, we use Leica ultra long working distance infinity-corrected objectives on our Olympus BHS microscope.
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 21:30:36 1993
Geez, you're making this more difficult :-) Chromatic aberration is not a problem with Ramam since you are working so near the Rayleigh peak, so look for a U-stage lens with infinity focus. You can use a reflecting objective but you must send the probe laser into the lens off-axis. Your imaging system may not accomodate this (probably not, huh). The focus of the laser can be adjusted with the beam expander if your beam expander is adjustable (probable not, huh). Alternatively, the laser can be prefocused with a long focal-length plano-convex lens. You might consider coating the reflecting objective for the laser wavelength. This would help aviod beam damage (not really a problem) and would give better signal in general.
} From e-reuter-at-uiuc.edu Wed Nov 24 00:20:00 1993
I am also interested in learning about ultra long working distance objective. Would you please summarize to the list or to me what you find out in a few days?
} From timonf-at-earth.ruu.nl Wed Nov 24 07:41:09 1993
The problem with U-stage lenses is that the highest magnification I know which is available is a 30 x, and the optical quality is not too good (some distortion of the image and loosing a loss of brightness)
} From muepf-at-iff067.iff.kfa-juelich.de Wed Nov 24 07:52:31 1993
One of the --- expensive --- alternatives is to get a Questar long
[Sorry - I've lost part of the original here because someone picked up the telephone next door while my modem was connected - the message went on to mention a 3000 ECU alternative - Could you please resend the details to me - Thank you]
} From treado+-at-pitt.edu Wed Nov 24 17:14:22 1993
If you are only interested in doing Raman microprobing the reflective objective may be suitable by defocusing and illuminating off-axis. If you want to perform imaging I would use refractive objectives. The imaging quality is far superior with refractive optics.
We use Leica objectives: 5X, 0.10 NA, 50 mm WD and 50X, 0.45 NA, 20.6 mm WD.
We operate at elevated temperatures (} 200 oC) and jacket the objective in a water cooled housing. According to Leica the optical cements fail above 110 degrees.
Message-Id: {MAILQUEUE-101.931129104500.288-at-parmly1.parmly.luc.edu} To: Microscopy-at-anlemc.msd.anl.gov
} We are doing microscopic image processing to access physical properties } (and their change) of lipid membranes and polymers in different setups. } In the near future we will face the problem to escape the limits of standard } video frequency (30/25 Hz). Therefore I want to ask whether anybody is } listening, who has experience with higher frame-rate cameras and other } devices and who would be willing to give me some advice in this field. Question deleted... } I am quite unsure whether the answers to this query might be of general } interest or not, so please feel free to email me directly at } mschindl-at-physik.tu-muenchen.de. I think this is of interest...I would certainly like to know the answers to these questions. Phil Oshel
Before I start let me admit that I have not tried this. However, I have heard other people doing it and it works. You can completely dissolve the scotch tape with acetone. I mean you can dissolve both the adhesive and the backing in aceotne leaving no residues. I have heard of acetone evaporaotrs where a little amount of acetone is heated and the vapors condense on the sample. This acetone dissolves any soluble substance and drips down into the bath to be reevaporated. The solubility of both adhesive and backing is higer at near melting point of acetone and hence this process substantially speeds up the removal. Hope this helps.
Naresh Shah University of Kentucky 233 Mining & Mineral Bldg. Lexington, KY 40506-0107 (606)257-4045 naresh-at-funky.mm.uky.edu
International Microscopy and Image Analysis Conference and Exhibition
12 - 15 September 1994 Earls Court Park Inn, Lillie Road, London
Organized by the Royal Microscopical Society in association with Microscopy and Analysis
Second Circular
Dates
Conference: Monday 12 September - 2.00 pm - 4.15 pm Tuesday 13 September - 10.00 am - 4.15 pm Wednesday 14 September - 10.00 am - 4.15 pm Thursday 15 September - 10.00 am - 4.15 pm
Exhibition: Monday 12 September - 2.00 pm - 7.00 pm Tuesday 13 September - 9.30 am - 6.00 pm Wednesday 14 September - 9.30 am - 6.00 pm Thursday 15 September - 9.30 am - 4.30 pm
On the Monday evening there will be a wine reception at 5.30pm, followed by the AGM and Presidental Lecture at 7.00pm.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
CONFERENCE
Scientific Programme The Conference will be run in seven half-day sessions. The Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) and The Physiological Society are each sponsoring separate lectures within the Conference.
The Programme will consist of tutorial lectures and posters and will feature the following topics:-
Monday 12 September (pm) - Materials I þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ EM in the assessment of semiconductor epitaxial growth Reactions to the surface of implanted bioceramics X-Ray microanalysis in biomaterials Optically active nanostructured materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (am) - Materials II (including EMAG) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Auger electron spectroscopy Imaging time of flight SIMS Formation of strained layer superlattices by phase separation Electron microscopy of weakly ordered III-V semiconductor materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (pm) Scanning Probe Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Applications of atomic force microscopy to thin film research and technology Scanning probe microscopy: near field imaging of surfaces using electrons, forces and photons SPM of living biological systems Environmental SEM þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (am) - Image Processing and Analysis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Sampling in 3D for quantitative microscopy Digital image processing techniques Image analysis of multicoloured biological specimens In vivo microscopy by video imaging þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (pm) - 3D Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Supercomputing in confocal microscopy 3D atomic-scale microanalysis of materials Spatial distribution of fibres in composite materials Confocal polarised-light microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (am) - Flow Cytometry and Proliferation Markers þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Cell cycle control Proliferation-related proteins Proliferation in human tumours Apoptosis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (pm) - Living Cell Cytochemistry (including The Physiological Society) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Living cell cytochemistry: Ratio Imaging Living cell cytochemistry: Confocal scanning laser microscopy
Thursday 15 September (pm) - Proteases in (patho) physiological processes
Use of selective protease inhibitors in the study of collagen breakdown Role of proteases in invasion and metastasis of cancer cells, arthritis and rheumatism and infections þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
Experts in the fields listed above have been invited to give lectures. Each speaker will provide a review of the particular topic in question and ample time for discussion will be provided.
In addition to the above, a special session will be held on the afternoon of Monday 12 September on how to use the light microscope.
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
INVITED SPEAKERS
It is hoped that the following people will be presenting papers at the MICRO 94 Conference:-
Dr Paul D. Brown (University of Cambridge) Professor Peter Marquis (University of Birmingham) Dr Henk Koerten (University of Leiden, The Netherlands) Dr Peter Dobson (University of Oxford) Dr R. K. Wild (University of Bristol) Dr Paul Denison (University of Sheffield) Dr Andrew Norman (Imperial College, London) Dr Caroline Baxter (University of Cambridge) Dr Alan Pidduck (RSRE, Malvern) Dr M. Miles (HH Wills Physics Laboratory, Bristol) Dr H Horber(European Molecular Biology Laboratory, Heidelberg, Germany) Mr Chris Gilpin (Manchester Biological EM Centre) Dr Vyvyan Howard (Royal Liverpool Children's Hospital) Dr Dominique Jeulin (Ecole Nationale Superiere des Mines, Fontainebleau, France) Dr Hans Tanke (University of Leiden, The Netherlands) Dr Andreas Kriete (Der Justus Liebig Universitat, Germany) Dr Alfred Cerezo (University of Oxford) Dr A. R. Clarke (University of Leeds) Dr Alan Entwistle (Ludwig Institute for Cancer Research, London) Dr A Bagg (TNO Rijswijk, The Netherlands) Dr Michael Ormerod (Sutton, Surrey) Dr Peter van Mier (Washington University School of Medicine, USA) Professor P. A. McNaughton (King's College London) Dr R. Jacob (King's College London) Dr Vincent Everts (University of Amsterdam, The Netherlands) Dr Ron Van Noorden (University of Amsterdam, The Netherlands)
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
CONTRIBUTED PAPERS In addition to invited papers, contributions are invited on all aspects of microscopy and related techniques.
All contributed papers will appear in the poster sessions of the Conference. Time will be allowed in the Programme for the viewing of posters, and posters will be on display for the maximum time possible. At certain times authors will be in attendance by their posters to discuss their work.
Camera-ready sheets and instructions for the submission of short abstracts can be obtained from the Royal Microscopical Society office. The deadline for submission is 4 May 1994. These abstracts will appear in the Conference Programme, which will be published in a special MICRO 94 issue of the Proceedings of the Royal Microscopical Society.
Authors will be notified regarding acceptance of their papers by the end of June 1994.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
EXHIBITION An Exhibition of the latest microscopes and ancillary instrumentation and equipment will be held at the Earls Court Park Inn, adjacent to the Lecture Theatre. Admission to the Exhibition is free, by conference badge, or by exhibition only badge which will be obtainable at the registration desk.
By 1 November 1993, the following firms had reserved exhibition space:-
Agar Scientific Ltd Alrad Instruments Ltd Bemax (UK) Ltd Bio-Rad Laboratories Ltd British BioCell International Burleigh Instruments (UK) Ltd Cambridge Scanning Co Ltd Confocal Technologies Ltd Cryophysics Ltd Data Cell Ltd Drukker International Edwards High Vacuum International Emitech Ltd Finlay Microvision Co Ltd Fisons Instruments Foster Findlay Associates Ltd Hamamatsu Photonics UK Ltd Hitachi Scientific Instruments ISS Imaging Associates Ltd J K Instruments Ltd JEOL UK Ltd K E Developments Ltd Lasertec Corporation Leica Cambridge Limited Leica UK Limited Microfield Scientific Ltd Microscopy and Analysis Newport Ltd Nikon UK Limited Olympus Optical Co (UK) Ltd Oxford Instruments Microanalysis Group Oxford Instruments Philips Electron Optics Photonic Science Polaroid (UK) Ltd Princeton Gamma-Tech (UK) Ltd Pyser (Holdings) plc Synoptics Ltd Taab Laboratories Equipment Ltd Tracor Europa Carl Zeiss (Oberkochen) Ltd þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
RECEPTION AND ASSOCIATED EVENTS On Monday 12 September there will be a wine reception in the Exhibition between 5.30 pm and 7.00 pm. The Annual General Meeting of the Royal Microscopical Society, and the Presidential Address will also take place during the evening.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ACCOMMODATION Academic Accommodation A limited amount of academic accommodation has been booked for the use of delegates at Imperial College. Rooms have been booked there on a bed and breakfast basis at a cost of 2.00 pounds6 per night. This academic/student accommodation will be filled on a 'first-come first-served' basis. From the nearby Underground Station at South Kensington, Earls Court is two stops along the District or Piccadilly Line.
Hotel Accommodation There are some rooms available in the Earls Court Park Inn at the special MICRO 94 rate of 65.00pounds per night. If you would like to reserve accommodation at these special rates, please contact the Earls Court Park Inn directly, quoting that you are a MICRO 94 visitor. To be sure of booking rooms at these rates, it is advisable that you book well in advance. Telephone: 071 385 1255 Telex: 917728 Fax: 071 381 4450.
Other Hotel Accommodation Delegates who wish to make their own accommodation arrangements may wish to use the services of Expotel Executive Travel - Europe's leading hotel booking agent, who have been appointed the official hotel agency for MICRO 94. The hotel of your choice or a similar alternative can be booked through Expotel often at discounted rates. By making one telephone call to Expotel on 071 735 0060 stating the event code 'MICRO 94', your reservation will be confirmed verbally followed by confirmation in writing. This free booking service is available to anyone attending MICRO 94. Telephone: 071 735 0060 Telex: 8811951 EXPOTL G Fax: 071 735 2839.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
REGISTRATION AND PAYMENT
Registration Registration will take place at the Earls Court Park Inn from 1.00 pm on Monday 12 September 1994, and from 9.00 am on subsequent mornings.
Payment Payment may be made by sterling cheque payable to the Royal Microscopical Society (please add 12.00pounds to cover exchange and bank charges if the cheque is not a UK Bank Cheque) or by credit card (Visa/Barclaycard or Access/Eurocard/Mastercard).
Cancellation and Refunds Cancellations received before 12 July 1994 will be subject to a full refund. No refunds will be made if cancellation is made after this date. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
HOW TO GET THERE
The Conference, Exhibition, Posters, and Refreshments will all take place at the Earls Court Park Inn, Lillie Road, London SW6.
MICRO 94 is organized by the Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, UK, in association with Microscopy and Analysis. Telephone: 0865 248768, Fax: 0865 791237, Email: RMS-at-UK.AC.OX.VAX.
I am now in the process of designing a new EM suite dedicated to biological EM. I would like input from those of you who work in what they feel is an ideal or close to ideal physical arrangement. I would also like to hear from those who have envisioned an ideal EM suite but have never seen it built. The restrictions are that a space of 2200 sq ft should house 2 TEMs and 2 SEMs as well as a prep lab, darkrooms ancillary equipment and 2 offices. The overall design is of more interest than actual use of the stated space. So I would like to receive copies of plans you might have or rough * Director, ICBR EMCL * Any and all contributions would be greatly appreciated. ***************************** * Greg Erdos * * ICBR EM Lab * * 218 Carr Hall * * University of Florida * * Gainesville, FL 32611 * * gwerdos-at-gnv.ifas.ufl.edu * * 904-392-1295 * *****************************
Here's yet another addition to the EMail server. Periodic updates of upcoming Journal articles. Both the Journal of Microscopy and the Microscopy Society of America (MSA) Bulletin have agreed in principle to forward this (abstract) information to me and I will post it to the subscribers. In many cases the information distributed here may preceed the actual publication of the full article by a few weeks.
Hope you find it informative! I certainly did.
Nestor Z. ANL EM Center =============================================================
JOURNAL OF MICROSCOPY - ABSTRACTS OCTOBER 1993 - DECEMBER 1993
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 3--12. Quantitative acoustic microscopy of individual living human cells by G. A. D. BRIGGS,* J. WANG* and R. GUNDLE, *Department of Materials, University of Oxford, Oxford OX1 3PH, U.K. Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford OX3 7LD, U.K.
Summary The elastic properties of cells can be measured with microscopic resolution by acoustic microscopy. By measuring the waveform of very short pulses, the thickness, and the acoustic velocity, impedance and attenuation can be determined from the two separate signals reflected from the top and the bottom of the cell.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 13--21. Random models for morphological analysis of powders by D. JEULIN, Centre de Geostatistique, E.N.S.M.P., 35 rue St-Honore, 77305 Fontainebleau, France
Summary Models to estimate powder size distributions or fractions of components in a mixture of powders were developed and tested for various specimens. The models derived from the Boolean model and from the dead leaves models can be implemented on rough secondary electron scanning electron microscope images, obtained after minimal sample preparation. With the dead leaves tessellation, the size distributions of spherical particles or short fibres can be estimated either from the size distribution of intact grains, or from the area fraction measurement after binary erosions. With the dead leaves random function, there is no need for image segmentation. It provides data on the size and shape of a population of particles from an estimation of the distribution of grey-level images after erosions by convex structuring elements of increasing size. A version of these models for long fibres is developed for estimating their diameter distribution. Allowing for superposition of particles, the proposed methods enable an unbiased estimation of the size distribution and characterization of the shape of complex particles. The approach is illustrated by applications to spherical particles obtained by simulations and from scanning electron microscope micrographs.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 23--29. Spatial distribution of curve length: concept and estimation by N. ROBERTS and L. M. CRUZ-ORIVE*, Magnetic Resonance Research Centre, PO Box 147, University of Liverpool, Liverpool L69 3BX, U.K. *Stereology Unit, Department of Anatomy, University of Berne, Postfach 139, CH-3000 Berne 9, Switzerland
Summary The length of a curvilinear feature, such as a dendrite tree of a neuron, can, in principle, be estimated by the recent, non-invasive method of total vertical projections (TVPs). Curve length is a measure of size, but it reports nothing about curve shape. The shape of a tree-like structure can be described to some extent by the distribution of branch length in properly defined regions of three-dimensional (3-D) space. A definition of curve length distribution in three dimensions is proposed and implemented here on a human neuron. The relevant 3-D regions overlap after projection, and therefore the TVPs method cannot be used directly to estimate the corresponding feature lengths. However, using the ANALYZE software system running on a SUNSPARC workstation, dendrite subsets sitting in predefined regions of space were rendered in different colours and measured separately by the TVPs method using a cycloid test system. In combination with non-invasive image acquisition and processing techniques, the length distribution concept is likely to be useful in the metrical analysis of either microscopic or macroscopic arborizations in a wide variety of contexts, including living cells and organisms.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 31--39. Image sharpness and contrast transfer in coherent confocal microscopy by R.OLDENBOURG,* H. TERADA,~~ R. TIBERIO## and S. INOUE*, *Marine Biological Laboratory, Woods Hole, MA, U.S.A. Martin Fisher School of Physics, Brandeis University, Waltham, MA, U.S.A. ~~Hamamatsu Photonics Kabushiki Kaisha, Hamamatsu City, Japan. ##National Nanofabrication Facility, Cornell University, Ithaca, NY, U.S.A.
Summary Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non-confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non-confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non-confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non-confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 41--48. A light-emitting diode light standard for photo- and videomicroscopy by J. M. BEACH* and B. R. DULING, Departments of *Biomedical Engineering and Physiology, University of Virginia, Charlottesville, VA, U.S.A. Summary A light calibration system consisting of a compact light-emitting diode (LED) source with feedback control of intensity is described. The source is positioned in the focal plane of the microscope objective and produces flat-field illumination of up to 31microW. The source can be easily used to determine the performance of microscope optics and camera response. It can also be used as a standard light source for calibration of experimental systems. Selectable light intensities are produced by controlling the LED input power via a feedback circuit consisting of a photodiode that detects output light intensity. Spectral coverage extends between 550 and 670nm using green, yellow and red LEDs mounted side by side, which are selected individually. The LED chips are encapsulated in plastic diffusers which homogenize the light, and a flat field of illumination is obtained through a thin 1-mm-diameter aperture positioned directly over each chip. Provision is made for insertion of Ronchi rulings over the aperture to enable measurements of contrast modulation in a uniform field. The light may be pulse-modulated to assess camera response times and the device can be synchronized with video frames. Narrow bandpass interference filters can be placed between the objective lens and the LED source to produce monochromatic light without affecting the spacing of controlled light intensities since emission spectra do not shift appreciably over the range of LED powers chosen in this design. Results of tests using controlled light intensity and uniform illumination are presented.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 49--54. Video camera calibration for optical densitometry by R. A. Baldock and I. Poole, MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, U.K.
Summary An efficient technique for calibrating video cameras to record optical density (OD) from microscopic images is described. The method corrects for variation over the field of the brightfield and darkfield intensities, does not assume a linear response of the camera to the incident intensity and requires a single calibration filter.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 55--61. Vitrification depth can be increased more than 10-fold by high-pressure freezing by N. SARTORI, K. RICHTER* and J. DUBOCHET, Laboratoire d'Analyse Ultrastructurale, Batiment de Biologie, Universite de Lausanne, CH-1015 Lausanne, Switzerland
Summary Biological specimens prepared for cryoelectron microscopy seem to suffer less damage when they are frozen under 2kbar pressure rather than under normal conditions. The volume that can be well preserved is larger. This fact has been illustrated in a number of publications on a number of different samples. However, there is a lack of quantitative data concerning the depth of this good specimen preservation. Catalase crystals in various sugar solutions have been used as test objects and vitrification, as determined by electron diffraction, has been used as the criterion for good freezing. Keeping all other conditions equal, the depth of vitrification is approximately 10 times larger with freezing at high, rather than normal, pressure. The high-pressure vitrification depth in a 15--20% sugar solution averages 200micrometre. Fully vitrified specimens up to 700micrometre in thickness are obtained. When crystalline water is observed it is frequently in the form of high-density ice II, III or IX. These results are probably also relevant for typical biological specimens. The advantage of high-pressure freezing must be balanced by the possible consequences of a considerably increased cooling time and by the damage that may be induced by the pressure.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 63--69. A portable cryo-storage system for low-temperature scanning electron microscopy, suitable for international transport of cryo-specimens by C. E. Jeffree* and P. R. van Gardingen, *University of Edinburgh, Science Faculty Electron Microscope Facility, Daniel Rutherford Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JH, U.K.
Summary A cryo-specimen storage system for low-temperature scanning electron microscopy (LTSEM) specimens is described, which: liberates multi-specimen experiments from sampling restrictions imposed by the rate at which LTSEM specimens can be examined in the SEM; provides security against experiment loss resulting from breakdown of the SEM or cryo-system; enables collection of specimens in the field or in laboratories remote from the SEM laboratory; and facilitates international air transport of LTSEM specimens. The com-ponents of the system, which has a capacity of 98 stub-mounted specimens, are readily made in a laboratory workshop. The details of the design may be altered to suit particular specimen types or experimental approaches.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 71--79. Empirically determined freezing time for quick-freezing with a liquid-nitrogen-cooled copper block by P. H. W. W. BAATSEN, Departement de Physiologie generale, FYMU, Universite Catholique de Louvain, Ave. Hippocrate 55, 1200-Brussels, Belgium
Summary A method is presented to determine freezing time empirically. The method is based on determining the amount of stretch of a skinned muscle fibre while it is being frozen. Freezing time, as determined with this method, lies in between 0 and 1ms.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 81--88. An application of scanned focused ion beam milling to studies on the internal morphology of small arthropods by R. J. YOUNG,* T. DINGLE,* K. ROBINSON and P. J. A. PUGH, *FEI Europe Ltd, Brookfield Business Centre, Cottenham, Cambridge CB4 4PS, U.K. British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, U.K.
Summary For the first time a scanned focused ion beam of approximately 50nm diameter has been used to prepare biological material. Small defined areas of the surface were removed by ion etching to allow examination of the underlying structures with a scanning electron microscope. Different milling procedures were carried out on two anatomical features in mites of the genus Halarachne (Halarachnidae: Mesostigmata). In the first, square holes were milled into the surface of the peritrematal plate to reveal the structure of the underlying respiratory peritrematal groove. In the second, transverse cuts were made across the shafts of the sensory sensilli which make up the sensory Haller's organ on tarsus I. This latter procedure revealed detail both within the core and walls of sensilli. Details of specimen preparation and milling procedures, as well as suitability and interpretation of results, are presented.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 89--92. Variable-depth electropolishing of TEM samples by S. W. LEONARD, Department of Physics, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Summary A variable-depth electropolishing technique has been developed for transmission electron microscopy samples using copper as sample material. This was required for an experiment concerning the measurement of the variation of dislocation density with depth for ion-implanted materials. The polishing technique was achieved by a two-step process, involving the measurement and use of the polishing rate to polish to a specific depth and the application of a transparent cover to one side of the sample for back-thinning. With this technique, any sample depth can be made accessible for observation with a transmission electron microscope and the method should be applicable to many different materials and electropolishers.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 97--107. Computer simulation of a mirror STEM by A. V. CREWE, Department of Physics and the Enrico Fermi Institute, The University of Chicago, 5640 S. Ellis, Chicago, IL 60464, U.S.A.
Summary The results of a computer simulation indicate that it is possible to design and build an STEM that is free from spherical aberration and should therefore have a very high resolution. The computer program was written in APL. The calculations include the effects of apertures and, consequently, mimic a realistic situation.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 109--119. The effect of soft X-radiation on myofibrils by P. M. BENNETT,* G. F. FOSTER, C. J. BUCKLEY and R. E. BURGE, *MRC Muscle and Cell Motility Unit, The Randall Institute, King's College London, 26/29 Drury Lane, London WC2B 5RL, U.K. Wheatstone Physics Laboratory, King's College London, The Strand, London WC2R 2LS, U.K.
Summary Myofibrils, the contractile organelles from striated muscles, have been examined in the X-ray microscope to determine the effect of radiation on their function and structure. Using X-rays of energy 350--385eV in the water window we find that after an exposure to 7.5 x (10 to the fifth power) photons per square micrometre (calculated to give an absorbed dose of 20 000 Gy) the myofibrils will no longer contract. The use of the free radical scavenging agent, DMSO, gives some protection to the fibrils. It has also been found that after this much irradiation the fibrils lose up to 20% of their mass. Further substantial mass loss occurs on subsequent irradiation. After 25 times the loss-of-function exposure only 30% of the mass remains. Analysis of a series of images of the same myofibril covering this range of exposures shows that the mass is preferentially lost in some areas of the structure and consequently significant structural changes occur.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 121--129. Scanning luminescence X-ray microscopy: imaging fluorescence dyes at suboptical resolution by C. JACOBSEN, S. LINDAAS, S. WILLIAMS and X. ZHANG, Department of Physics, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A.
Summary Scanning luminescence X-ray microscopy is based on the use of the very small focused probe of a scanning X-ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X-ray source and a Fresnel zone plate to produce a focused X-ray probe, images of P31 phosphor grains with a resolution of 50--75nm have been obtained, and luminescence from polystyrene spheres loaded with 50--100micromol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X-ray probe (the microscope has imaged features at 36-nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye-tagged sites of biochemical activity at the resolution of the X-ray microscope in wet, unsectioned, and unfixed cells, especially with soft X-ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye-tagged features should be 2--22 times lower than it is in transmission X-ray microscopy. A possible extension of the technique for three-dimensional imaging at the transverse resolution of the X-ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 131--136. High-spatial-resolution maps of sulphur from human hair sections: an EELS study by P. HALLEGOT and P. CORCUFF, Laboratoires de Recherche Avancee, L'Oreal, Departement de Biophysique, 1 avenue Eugene-Schueller, 93 600 Aulnay sous Bois, France
Summary High-resolution sulphur maps have been acquired from human hair using a Zeiss CEM 902A transmission electron microscope equipped with an energy filter. Analysis by electron energy-loss spectroscopy (EELS) was performed on ultrathin sections of hair shafts embedded in three different types of resin: Nanoplast (water-soluble), Spurr (epoxy) and Lowicryl (low-temperature resin). Good-quality energy-loss images have been obtained with the three resins, although it was found that Nanoplast gave the best image contrast. For the first time, the results obtained for the detection of sulphur by silver staining of hair sections, which until now has been the only way to map sulphur at the electron microscopic level, have been confirmed. The results are compared with local sulphur concentrations from bulk analysis.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 137--151. Improvements in the technique of vascular perfusion-fixation employing a fluorocarbon-containing perfusate and a peristaltic pump controlled by pressure feedback by J. Rostgaard, K. Qvortrup and S. S. Poulsen, Institute of Medical Anatomy Department B, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, Denmark
Summary A new, improved technique for whole-body perfusion-fixation of rats and other small animals is described. The driving force is a peristaltic pump which is feedback regulated by a pressure transducer that monitors the blood/perfusion pressure in the left ventricle of the heart. The primary perfusate-fixative is composed of a blood substitute---13.3% oxygenated fluorocarbon FC-75---in 0.05 M cacodylate buffer (pH 7.4) with 2% glutaraldehyde. The secondary perfusate-fixative is composed of 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) with 20 mM CaCl2 A double-barrelled, self-holding cannula is used to cannulate the heart; the outer and inner barrels of the cannula are connected to the peristaltic pump and to the pressure transducer, respectively. The tissue oxygen tension in the rat is monitored by a sub-cutaneous oxygen electrode. Measurements showed that tissue hypoxia/anoxia did not develop before or during the perfusion-fixation. Thus, the technique permits study of specimens which do not exhibit fixation gradients and do not contain cells fixed in a state of asphyxia. This is substantiated by electron micrographs of cells from different organs, revealing new fine structural elements. By adding oxygenated fluorocarbon to glutaraldehyde perfusate-fixatives, enough oxygen is made accessible for cellular respiration as well as for the oxygen-consuming chemical reactions of glutaraldehyde with the tissue. Data on anaesthesia, operative manoeuvres, mechanical components of the system, preparation of fixatives and flow of the perfusate-fixatives are furnished and discussed.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 153--156. A device for picking up ultrathin serial sections by E. P. Meyer and V. J. Domanico, Department of Zoology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
Summary Reconstruction from serial sections is often required to understand the architecture of biological tissue. The sampling of serial sections calls for an enormous amount of patience and skill. Sections are cut with a diamond knife, sampled in a trough and must then be collected on grids for examination with the electron microscope (EM). This last step, in which the section ribbons have to be aligned with the EM grid, is especially difficult as both hands have to work in perfect co-ordination. A simple manually operated or motorized mechanical device has been designed which facilitates the collection of EM section ribbons.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 157--162. An unbiased and efficient procedure for 3-D connectivity measurement as applied to porous media by H. Q. ZHAO and I. F. MACDONALD, Porous Media Research Institute, Department of Chemical Engineering, University of Waterloo, Ontario, Canada N2L 3G1
Summary A new stereological technique to measure the mean genus (connectivity) per unit volume of a porous medium is described and applied to a real sandstone sample. The technique is based on the `net volume tangent counts' performed on disector samples, i.e. pairs of consecutive sections of an interconnected structure. It consists of a set of simple counting rules applied to the features on the sections of the structure and can be easily implemented manually. The applicability and efficiency of the procedure is evaluated by applying it to a Berea sandstone sample which has been studied previously using a network analysis approach by interactive three-dimensional computer reconstruction. It is shown that the procedure yields results in good agreement with the network analysis result, but has the advantages that it is much easier to implement, is more flexible in how the data are collected, is more efficient, and is known to provide an unbiased estimate of the mean genus per unit volume of the whole structure.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 163--176. Remapping disparate images for coincidence by W. Galbraith* and D. L. Farkas, *Department of Laboratory Medicine, Allegheny Singer Research Institute, 320 East North Ave., Pittsburgh, PA 15212, U.S.A. Center for Light Microscope Imaging and Biotechnology, Carnegie-Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, U.S.A.
Summary With the development of complex multimode computerized microscope systems, it is possible and necessary to obtain images of the same area of the microscopical preparation by several methods of microscopy, such as differential interference contrast, reflection interference microscopy, several wavelengths of fluorescence microscopy, laser scanning and confocal modes. Thus, varied information may be obtained about a single field, in the form of a set of images, taken at different ports of the microscope, using different digitizing cameras, each appropriate to certain tasks. For comparative purposes, the images should be superimposable, pixel by pixel, but in general they are not --- they differ in image shape and size, magnification, distortion, centration and orientation. This paper shows how the problem may be approached, using an extension of the remapping procedures described in a previous paper, in which images of a separate grid reference slide are used to detect, quantify and correct the image errors. Affine remapping, without the use of grid images, is also described.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 177--180. Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope by O. GUNTINAS-LICHIUS,* J. MOCKENHAUPT,* E.STENNERT and W. F. NEISS*, *Department of Anatomy and Department of Oto-Rhino-Laryngology, University of Cologne, Lindenthal, D-50924 Koln, Germany
Summary A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6micrometre thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 189--194. A compact Schwarzschild soft X-ray microscope with a laser-produced plasma source by Y. HORIKAWA, K. NAGAI, S. MOCHIMARU and Y. IKETAKI, T. Morokuma Research Laboratory, Olympus Optical Co., Ltd, 2--3 Kuboyama-cho, Hachioji-shi, Tokyo 192, Japan
Summary A compact Schwarzschild soft X-ray microscope using a laser-produced plasma soft X-ray source has been developed. The laser-produced plasma source, which is small but of high brilliance, has made it possible to use the soft X-ray microscope in a small laboratory. The microscope is composed of a Schwarzschild objective and a grazing incidence mirror condenser. Image contrast for biological specimens in soft X-ray regions is investigated briefly. It is possible to observe the fine structures of a thin specimen at a wavelength of 15nm; at this wavelength high-contrast images of biological specimens have been obtained with a single laser shot of pulse width of 8ns at a resolution of 0.3micrometre. The resolution of the system is limited by the detector.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 195--203. Liquid substitution: a versatile procedure for SEM specimen preparation of biological materials without drying or coating by H. J. Ensikat and W. Barthlott, Botanisches Institut der Universitat, Meckenheimer Allee 170, 53115 Bonn, Germany
Summary Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo-preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 205--214. Conformational characterization of nucleosomes by principal component analysis of their electron micrographs by M. M. Z. ZABAL*, G. J. CZARNOTA*, D. P. BAZETT-JONES and F. P. OTTENSMEYER*, *Department of Medical Biophysics, The University of Toronto and Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9
Summary Optimized fixation conditions were determined for protein--protein and protein--DNA crosslinking within calf-thymus nucleosomes in low monovalent salt concentrations. Nucleosomes were examined without heavy-atom staining by darkfield electron microscopy. The dimensions of these macromolecular complexes and those of HeLa core particles optimally fixed in divalent salt were analysed using principal component analysis. According to this analysis the structure of the calf-thymus nucleosomes was best presented by a prolate ellipsoid. Particle images had average major and minor axis lengths of 14.1 and 10.5nm, respectively. In contrast, the HeLa nucleosomes were best modelled by an oblate ellipsoid from the analysis of their images, which had average major and minor axes of 13.3 and 11.5nm. The applicability of this multivariate statistical analysis to the interpretation of macromolecular images is illustrated and discussed.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 215--221. An investigation of substrate and sample preparation effects on scanning tunnelling microscopy studies on xanthan gum by M. J. WILKINS, M. C. DAVIES, D. E. JACKSON, C. J. ROBERTS and S. J. B. TENDLER, The Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
Summary Scanning tunnelling microscopy is developing as an important biophysical tool for the molecular imaging of biological material. In this study, the effect of sample deposition technique and substrate employed on the resultant images of a microbial polysaccharide is investigated. Scanning tunnelling microscopy topographs of xanthan gum, pipette deposited and spray deposited onto highly orientated pyrolytic graphite and mica substrates are presented. The use of pipette deposition of the aqueous sample solution is shown to result in a xanthan network. In contrast, images of isolated xanthan molecules are obtained when spray deposition with glycerol is employed. The effect of these deposition techniques on the macroscopic distribution of sample material across substrates is shown using confocal fluorescence microscopy.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 223--232. Fractal characterization by frequency analysis. I. Surfaces by E. ANGUIANO, M. PANCORBO and M. AGUILAR, Instituto de Ciencia de Materiales, Sede B (CSIC), Universidad Autošnoma de Madrid (C-III), 28049-Madrid, Spain
Summary A study of the quality and accuracy of the methods based on frequency analysis for the fractal characterization of surfaces as measured by scanning tunnelling microscopy (or profilometry) is made. The study is based on computer simulation of images of fractal surfaces. A discussion of the mathematical algorithms used for computer generation of fractal surfaces then follows. The main conclusion is that studies of fractal characterization by frequency analysis reported in previous papers in the STM field, as well as conclusions about the performance of the various methods, are doubtful. New methods for frequency analysis that in some cases produce more reliable results are proposed.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 233--238. Fractal characterization by frequency analysis. II. A new method by M. AGUILAR, E. ANGUIANO and M. PANCORBO, Instituto de Ciencia de Materiales, Sede B (CSIC), Universidad AutÆnoma de Madrid (C-III), 28049-Madrid, Spain
Summary A new frequency analysis method, fractal analysis by circular average (FACA), and an image replication procedure are proposed that together produce accurate measurements of the fractal dimension of surfaces and profiles, eliminating Fourier transform artefacts which arise from the lack of periodic continuity in real surfaces and profiles.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 239--248. Effects of noise and anisotropy on the determination of fractal dimensions by J. C. RUSS, Materials Science and Engineering Department, North Carolina State University, Raleigh, NC 27695-7907, U.S.A.
Summary Measurement of the fractal dimension of surfaces imaged by the scanning tunnelling microscope, atomic force microscope or similar instruments can be performed using several different algorithms. Dimensional analysis---plots of log (perimeter) vs. log (area)---are compared to Korcak and slit-island methods, which also employ a horizontal plane section, and to Fourier analysis of the two-dimensional array of elevation values. The effects of surface anisotropy and instrument noise on each of these measurement techniques is investigated.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 249--256. Evaluation of the precision of systematic sampling: nugget effect and covariogram modelling by J. THIOULOUSE,* J. P. ROYET, H. PLOYE* and F. HOULLIER~~, *Laboratoire de Biometrie, Genetique et Biologie des Populations, (U.R.A.CNRS 243), Universite Claude Bernard-Lyon 1, F-69622 Villeurbanne Cedex, France. Laboratoire de Physiologie Neurosensorielle, (U.R.A. CNRS 180), Universite Claude Bernard-Lyon 1, F-69622 Villeurbanne Cedex, France. ~~Ecole Nationale du Genie Rural, des Eaux et Forets, Laboratoire de Recherches en Sciences forestieres, 14, rue Girardet, F-54042 Nancy Cedex, France
Summary Systematic sampling designs are widely used in stereology. When an estimator of the total amount, Q, of the sampled variable is evaluated by such a procedure, the coefficient of error can be predicted by applying Matheron's theory of regionalized variables. To evaluate the accuracy of the estimate of Q, it is necessary to study the behaviour of the regionalized variable and to model its covariogram. Histological data with a low short-range variability and agronomic data with a pronounced nugget effect provided the biological material for extreme case studies. Results show that the short-range variability, if present, cannot be detected when only small samples are available. An underestimation of the coefficient of error is then to be expected. We propose several models of the covariogram, which can be used to test for the presence of a nugget effect. If a nugget effect is present, these models will provide better estimates of the coefficient of error. If there is no nugget effect a simplified method can be used and will provide reliable estimates of the coefficient of error.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 257--261. Directional analysis of planar fibre networks: application to cardboard microstructure by I. MOLCHANOV,* D. STOYAN* and K.FYODOROV, *Bergakademie Freiberg, FB Mathematik, Bernhard-v.-Cotta-Str. 2, D-09596 Freiberg, Germany. Kiev Technological Institute of the Food Industry, Vladimirskaya, 68, 252017 Kiev, Ukraine
Summary This paper discusses the problem of determining suitable roses of intersections for systems of planar thick fibres which cross and overlap. A planar Boolean model with long rectangular (or similar) grains is suggested as an appropriate mathematical model. It leads to a statistical estimator for the rose of intersections of the system of fibre spines. The method is used to analyse the microstructure of the outer layer of two samples of cardboard.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 263--266. Edge detection in petrographic images by J. STARKEY and A. K. SAMANTARAY, Department of Earth Sciences, The University of Western Ontario, London, Ontario, Canada N6A 3B7
Summary The automatic detection of mineral grain boundaries in images obtained from a polarized-light microscope requires special techniques. Observations in both plane- and cross-polarized light may be necessry and the section must be rotated relative to the plane of polarization of the microscope to see all the grain boundaries. In computer-based microscopy this can be accomplished by the sequential accumulation of individual images captured from one microscope field of view with different polarizer orientations. For real-time implementation the sequential images are segmented individually by applying Canny's algorithm. A separable Gaussian mask is used for smoothing and a 3 x 3 convolution mask is used to generate 1-pixel-wide boundaries, which are located at the zero-crossing of the second-order derivative of the intensity gradient. The boundaries are extracted and accumulated in a composite image. The resulting composite image is a synoptic grain-boundary image of the rock.
Message-Id: {9312010147.AA22920-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM:Celli Tape Orig-Author: {"Nestor J. Zaluzec (708)-252-5075, -4964" {ZALUZEC-at-anlemc.msd.anl.gov} }:ddn:wpafb ----------------------------------------------------------- Re: TEM specimen preparation of Graphite:
Here's a question for those of you that used to use the old Celli tape popular in England about 10+ years ago.
In the past I had some of this tape which I used to use to prepare specimens of graphite for TEM. Here I would use the old trick of just continually touching the surface of the graphite with fresh tape to delaminate the graphite gradually until I made a nice thin & optically transparent section, which stuck to the tape and was usually perfect for TEM. The nice thing about this old type of scotch tape was that the adhesive used would disolve beautifully in Acetone and the backing for that tape would just float away as it was not soluablein the acetone. Unfortuantely, the current type of "SCOTCH BRAND" transparent tape completely dissolves in Acetone and most other solvents I tried.
I'm back to trying to make more graphite samples as my old ones have finally bit the dust so to speak. Since the new scotch tape failed, I tried extraction replica tape which is available from most EM supply vendors, however it does not have the adhearance of either the old Celli tape or the newer scotch tapes. (BTW the new scotch tape very nicely delaminated the graphite and gives beautifully thin sections if I could ever remove them from the adhesive). Does anyone know of a solvent which works on the newer scotch brand tapes or have another/similar idea/procedure for layered structures which are not strongly bound?.
I'd prefer to avoid ion milling the graphite although I may have to resort to this in the end.
International Microscopy and Image Analysis Conference and Exhibition
12 - 15 September 1994 Earls Court Park Inn, Lillie Road, London
Organized by the Royal Microscopical Society in association with Microscopy and Analysis
Second Circular
Dates
Conference: Monday 12 September - 2.00 pm - 4.15 pm Tuesday 13 September - 10.00 am - 4.15 pm Wednesday 14 September - 10.00 am - 4.15 pm Thursday 15 September - 10.00 am - 4.15 pm
Exhibition: Monday 12 September - 2.00 pm - 7.00 pm Tuesday 13 September - 9.30 am - 6.00 pm Wednesday 14 September - 9.30 am - 6.00 pm Thursday 15 September - 9.30 am - 4.30 pm
On the Monday evening there will be a wine reception at 5.30pm, followed by the AGM and Presidental Lecture at 7.00pm.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
CONFERENCE
Scientific Programme The Conference will be run in seven half-day sessions. The Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) and The Physiological Society are each sponsoring separate lectures within the Conference.
The Programme will consist of tutorial lectures and posters and will feature the following topics:-
Monday 12 September (pm) - Materials I þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ EM in the assessment of semiconductor epitaxial growth Reactions to the surface of implanted bioceramics X-Ray microanalysis in biomaterials Optically active nanostructured materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (am) - Materials II (including EMAG) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Auger electron spectroscopy Imaging time of flight SIMS Formation of strained layer superlattices by phase separation Electron microscopy of weakly ordered III-V semiconductor materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (pm) Scanning Probe Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Applications of atomic force microscopy to thin film research and technology Scanning probe microscopy: near field imaging of surfaces using electrons, forces and photons SPM of living biological systems Environmental SEM þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (am) - Image Processing and Analysis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Sampling in 3D for quantitative microscopy Digital image processing techniques Image analysis of multicoloured biological specimens In vivo microscopy by video imaging þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (pm) - 3D Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Supercomputing in confocal microscopy 3D atomic-scale microanalysis of materials Spatial distribution of fibres in composite materials Confocal polarised-light microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (am) - Flow Cytometry and Proliferation Markers þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Cell cycle control Proliferation-related proteins Proliferation in human tumours Apoptosis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (pm) - Living Cell Cytochemistry (including The Physiological Society) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Living cell cytochemistry: Ratio Imaging Living cell cytochemistry: Confocal scanning laser microscopy
Thursday 15 September (pm) - Proteases in (patho) physiological processes
Use of selective protease inhibitors in the study of collagen breakdown Role of proteases in invasion and metastasis of cancer cells, arthritis and rheumatism and infections þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
Experts in the fields listed above have been invited to give lectures. Each speaker will provide a review of the particular topic in question and ample time for discussion will be provided.
In addition to the above, a special session will be held on the afternoon of Monday 12 September on how to use the light microscope.
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
INVITED SPEAKERS
It is hoped that the following people will be presenting papers at the MICRO 94 Conference:-
Dr Paul D. Brown (University of Cambridge) Professor Peter Marquis (University of Birmingham) Dr Henk Koerten (University of Leiden, The Netherlands) Dr Peter Dobson (University of Oxford) Dr R. K. Wild (University of Bristol) Dr Paul Denison (University of Sheffield) Dr Andrew Norman (Imperial College, London) Dr Caroline Baxter (University of Cambridge) Dr Alan Pidduck (RSRE, Malvern) Dr M. Miles (HH Wills Physics Laboratory, Bristol) Dr H Horber(European Molecular Biology Laboratory, Heidelberg, Germany) Mr Chris Gilpin (Manchester Biological EM Centre) Dr Vyvyan Howard (Royal Liverpool Children's Hospital) Dr Dominique Jeulin (Ecole Nationale Superiere des Mines, Fontainebleau, France) Dr Hans Tanke (University of Leiden, The Netherlands) Dr Andreas Kriete (Der Justus Liebig Universitat, Germany) Dr Alfred Cerezo (University of Oxford) Dr A. R. Clarke (University of Leeds) Dr Alan Entwistle (Ludwig Institute for Cancer Research, London) Dr A Bagg (TNO Rijswijk, The Netherlands) Dr Michael Ormerod (Sutton, Surrey) Dr Peter van Mier (Washington University School of Medicine, USA) Professor P. A. McNaughton (King's College London) Dr R. Jacob (King's College London) Dr Vincent Everts (University of Amsterdam, The Netherlands) Dr Ron Van Noorden (University of Amsterdam, The Netherlands)
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
CONTRIBUTED PAPERS In addition to invited papers, contributions are invited on all aspects of microscopy and related techniques.
All contributed papers will appear in the poster sessions of the Conference. Time will be allowed in the Programme for the viewing of posters, and posters will be on display for the maximum time possible. At certain times authors will be in attendance by their posters to discuss their work.
Camera-ready sheets and instructions for the submission of short abstracts can be obtained from the Royal Microscopical Society office. The deadline for submission is 4 May 1994. These abstracts will appear in the Conference Programme, which will be published in a special MICRO 94 issue of the Proceedings of the Royal Microscopical Society.
Authors will be notified regarding acceptance of their papers by the end of June 1994.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
EXHIBITION An Exhibition of the latest microscopes and ancillary instrumentation and equipment will be held at the Earls Court Park Inn, adjacent to the Lecture Theatre. Admission to the Exhibition is free, by conference badge, or by exhibition only badge which will be obtainable at the registration desk.
By 1 November 1993, the following firms had reserved exhibition space:-
Agar Scientific Ltd Alrad Instruments Ltd Bemax (UK) Ltd Bio-Rad Laboratories Ltd British BioCell International Burleigh Instruments (UK) Ltd Cambridge Scanning Co Ltd Confocal Technologies Ltd Cryophysics Ltd Data Cell Ltd Drukker International Edwards High Vacuum International Emitech Ltd Finlay Microvision Co Ltd Fisons Instruments Foster Findlay Associates Ltd Hamamatsu Photonics UK Ltd Hitachi Scientific Instruments ISS Imaging Associates Ltd J K Instruments Ltd JEOL UK Ltd K E Developments Ltd Lasertec Corporation Leica Cambridge Limited Leica UK Limited Microfield Scientific Ltd Microscopy and Analysis Newport Ltd Nikon UK Limited Olympus Optical Co (UK) Ltd Oxford Instruments Microanalysis Group Oxford Instruments Philips Electron Optics Photonic Science Polaroid (UK) Ltd Princeton Gamma-Tech (UK) Ltd Pyser (Holdings) plc Synoptics Ltd Taab Laboratories Equipment Ltd Tracor Europa Carl Zeiss (Oberkochen) Ltd þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
RECEPTION AND ASSOCIATED EVENTS On Monday 12 September there will be a wine reception in the Exhibition between 5.30 pm and 7.00 pm. The Annual General Meeting of the Royal Microscopical Society, and the Presidential Address will also take place during the evening.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ACCOMMODATION Academic Accommodation A limited amount of academic accommodation has been booked for the use of delegates at Imperial College. Rooms have been booked there on a bed and breakfast basis at a cost of 2.00 pounds6 per night. This academic/student accommodation will be filled on a 'first-come first-served' basis. From the nearby Underground Station at South Kensington, Earls Court is two stops along the District or Piccadilly Line.
Hotel Accommodation There are some rooms available in the Earls Court Park Inn at the special MICRO 94 rate of 65.00pounds per night. If you would like to reserve accommodation at these special rates, please contact the Earls Court Park Inn directly, quoting that you are a MICRO 94 visitor. To be sure of booking rooms at these rates, it is advisable that you book well in advance. Telephone: 071 385 1255 Telex: 917728 Fax: 071 381 4450.
Other Hotel Accommodation Delegates who wish to make their own accommodation arrangements may wish to use the services of Expotel Executive Travel - Europe's leading hotel booking agent, who have been appointed the official hotel agency for MICRO 94. The hotel of your choice or a similar alternative can be booked through Expotel often at discounted rates. By making one telephone call to Expotel on 071 735 0060 stating the event code 'MICRO 94', your reservation will be confirmed verbally followed by confirmation in writing. This free booking service is available to anyone attending MICRO 94. Telephone: 071 735 0060 Telex: 8811951 EXPOTL G Fax: 071 735 2839.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
REGISTRATION AND PAYMENT
Registration Registration will take place at the Earls Court Park Inn from 1.00 pm on Monday 12 September 1994, and from 9.00 am on subsequent mornings.
Payment Payment may be made by sterling cheque payable to the Royal Microscopical Society (please add 12.00pounds to cover exchange and bank charges if the cheque is not a UK Bank Cheque) or by credit card (Visa/Barclaycard or Access/Eurocard/Mastercard).
Cancellation and Refunds Cancellations received before 12 July 1994 will be subject to a full refund. No refunds will be made if cancellation is made after this date. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
HOW TO GET THERE
The Conference, Exhibition, Posters, and Refreshments will all take place at the Earls Court Park Inn, Lillie Road, London SW6.
MICRO 94 is organized by the Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, UK, in association with Microscopy and Analysis. Telephone: 0865 248768, Fax: 0865 791237, Email: RMS-at-UK.AC.OX.VAX.
Summary of the procedure for cleaving graphite into TEM specimens using 3M brand Scotch (Magic) tape.
Begin using with the Scotch Brand Magic Tape using it to cleave the graphite flakes several times to get a "clean" i.e. fresh surfaces. Then begin with a fresh piece of tape. Continue to delaminate until you see a number (~10+) of optically transparent "flakes" stuck onto the tape (many more will not be optically transparent) . During the delamination /peeling process try not to press the tape pieces together with alot of force this only serves to make complications later. Use only sufficient force to cause the graphite flakes to stick & peel off. Now trim off excess tape where there is no potential graphite specimens attached. Immerse the remaining tape in Toluene at room temp , this starts to seperate/loosen the backing from the glue, after about 5 minutes slowly add Methanol until you get to a ~50/50 mixture Let this sit a few more minutes then gently loosen the glue mass (looks like a wrinkled gelatinous mass and acts like it too) from the backing . To remove the plastic backing, you may need to use a pair of tweezers to entice the seperation where the glue has been forced down into good contact with the plastic backing at the sites of the graphite flakes. Toss the plastic backing in the trash.
Now begin to add acetone which dissolves the glue. As the acetone is added use a pair of tweezers to agitate the glue/graphite mass. This allows the solvent to get inbetween the graphite flakes and glue mass. Remove as much of the glue mass as possible before it completely dissolves in the solvent mixture. This is a real sticky "goo" at this point and it will only serve to contaminate your solution further. Be realistic here you will not get all of the graphite off of the tape. I estimate that there were about 2 dozen specimens on the tape, as the graphite released from the glue it fractured into maybe a hundred smaller flakes which were just floating in the solvent. At some point it nolonger is worth while trying to release the remaining graphite flakes from the glue mass, too much agitation only causes folds which trap the flakes and makes it nearly impossible to release all the graphite. Waiting to long only causes the glue mass to dissolve into your solvent and possibly contaminate your specimens.
Decant off excess solvent and add fresh acetone to continue dissolution of any remaining glue on the flakes. Now just pickup the flakes on grids by floating them over grids and lifting out of the solvent. (i.e. play chase the flake with the grid. As I recall the life science microscopists have a special tool for this but I just went fishing with a pair of tweezers and a grid under a low power steroe microscope). If they (the graphite flakes) are optically transparent (very light gray to clear) you've got a reasonably good specimen to work with at 100 kV.
Via TEM I see the usual basal plane dislocations, and little surface contamination there is some but it is obvious and can be easily avoided. The flakes show the usual bending that I would expect from the peeling procedure, but are reasonably uniform in thickness over wide areas (~sq microns). No thickness measurements yet, I'll report those when the data comes out of the experiments.
Below is the second circular for next year's Micro conference. Please forward to interested parties or post on your bulletin boards (wood or electronic!). Thanks. John Mansfield (This is a little cleaned version c.f.the first copy and is therefore a little more readable for North American readers. There were too many special characters in the first version - pound signs and the like)
-------------------------------------- MICRO 94
International Microscopy and Image Analysis Conference and Exhibition
12 - 15 September 1994 Earls Court Park Inn, Lillie Road, London
Organized by the Royal Microscopical Society in association with Microscopy and Analysis
Second Circular
Dates
Conference: Monday 12 September - 2.00 pm - 4.15 pm Tuesday 13 September - 10.00 am - 4.15 pm Wednesday 14 September - 10.00 am - 4.15 pm Thursday 15 September - 10.00 am - 4.15 pm
Exhibition: Monday 12 September - 2.00 pm - 7.00 pm Tuesday 13 September - 9.30 am - 6.00 pm Wednesday 14 September - 9.30 am - 6.00 pm Thursday 15 September - 9.30 am - 4.30 pm#012#
On the Monday evening there will be a wine reception at 5.30pm, followed by the AGM and Presidental Lecture at 7.00pm.
CONFERENCE
Scientific Programme The Conference will be run in seven half-day sessions. The Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) and The Physiological Society are each sponsoring separate lectures within the Conference.
The Programme will consist of tutorial lectures and posters and will feature the following topics:-
Monday 12 September (pm) - Materials I EM in the assessment of semiconductor epitaxial growth Reactions to the surface of implanted bioceramics X-Ray microanalysis in biomaterials Optically active nanostructured materials
Tuesday 13 September (am) - Materials II (including EMAG) Auger electron spectroscopy Imaging time of flight SIMS Formation of strained layer superlattices by phase separation Electron microscopy of weakly ordered III-V semiconductor materials
Tuesday 13 September (pm) Scanning Probe Microscopy Applications of atomic force microscopy to thin film research and technology Scanning probe microscopy: near field imaging of surfaces using electrons, forces and photons SPM of living biological systems Environmental SEM
Wednesday 14 September (am) - Image Processing and Analysis Sampling in 3D for quantitative microscopy Digital image processing techniques Image analysis of multicoloured biological specimens In vivo microscopy by video imaging
Wednesday 14 September (pm) - 3D Microscopy Supercomputing in confocal microscopy 3D atomic-scale microanalysis of materials Spatial distribution of fibres in composite materials Confocal polarised-light microscopy
Thursday 15 September (am) - Flow Cytometry and Proliferation Markers Cell cycle control Proliferation-related proteins Proliferation in human tumours Apoptosis
Thursday 15 September (pm) - Living Cell Cytochemistry (including The Physiological Society) Living cell cytochemistry: Ratio Imaging Living cell cytochemistry: Confocal scanning laser microscopy
Thursday 15 September (pm) - Proteases in (patho) physiological processes Use of selective protease inhibitors in the study of collagen breakdown Role of proteases in invasion and metastasis of cancer cells, arthritis and rheumatism and infections
Experts in the fields listed above have been invited to give lectures. Each speaker will provide a review of the particular topic in question and ample time for discussion will be provided.
In addition to the above, a special session will be held on the afternoon of Monday 12 September on how to use the light microscope.
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited.
INVITED SPEAKERS
It is hoped that the following people will be presenting papers at the MICRO 94 Conference:-
Dr Paul D. Brown (University of Cambridge) Professor Peter Marquis (University of Birmingham) Dr Henk Koerten (University of Leiden, The Netherlands) Dr Peter Dobson (University of Oxford) Dr R. K. Wild (University of Bristol) Dr Paul Denison (University of Sheffield) Dr Andrew Norman (Imperial College, London) Dr Caroline Baxter (University of Cambridge) Dr Alan Pidduck (RSRE, Malvern) Dr M. Miles (HH Wills Physics Laboratory, Bristol) Dr H Hoirber(European Molecular Biology Laboratory, Heidelberg, Germany) Mr Chris Gilpin (Manchester Biological EM Centre) Dr Vyvyan Howard (Royal Liverpool Children's Hospital) Dr Dominique Jeulin (Ecole Nationale Superiere des Mines, Fontainebleau, France) Dr Hans Tanke (University of Leiden, The Netherlands) Dr Andreas Kriete (Der Justus Liebig Universitat, Germany) Dr Alfred Cerezo (University of Oxford) Dr A. R. Clarke (University of Leeds) Dr Alan Entwistle (Ludwig Institute for Cancer Research, London) Dr A Bagg (TNO Rijswijk, The Netherlands) Dr Michael Ormerod (Sutton, Surrey) Dr Peter van Mier (Washington University School of Medicine, USA) Professor P. A. McNaughton (King's College London) Dr R. Jacob (King's College London) Dr Vincent Everts (University of Amsterdam, The Netherlands) Dr Ron Van Noorden (University of Amsterdam, The Netherlands)
CONTRIBUTED PAPERS In addition to invited papers, contributions are invited on all aspects of microscopy and related techniques.
All contributed papers will appear in the poster sessions of the Conference. Time will be allowed in the Programme for the viewing of posters, and posters will be on display for the maximum time possible. At certain times authors will be in attendance by their posters to discuss their work.
Camera-ready sheets and instructions for the submission of short abstracts can be obtained from the Royal Microscopical Society office. The deadline for submission is 4 May 1994. These abstracts will appear in the Conference Programme, which will be published in a special MICRO 94 issue of the Proceedings of the Royal Microscopical Society.
Authors will be notified regarding acceptance of their papers by the end of June 1994.
EXHIBITION An Exhibition of the latest microscopes and ancillary instrumentation and equipment will be held at the Earls Court Park Inn, adjacent to the Lecture Theatre. Admission to the Exhibition is free, by conference badge, or by exhibition only badge which will be obtainable at the registration desk.
By 1 November 1993, the following firms had reserved exhibition space:-
Agar Scientific Ltd Alrad Instruments Ltd Bemax (UK) Ltd Bio-Rad Laboratories Ltd British BioCell International Burleigh Instruments (UK) Ltd Cambridge Scanning Co Ltd Confocal Technologies Ltd Cryophysics Ltd Data Cell Ltd Drukker International Edwards High Vacuum International Emitech Ltd Finlay Microvision Co Ltd Fisons Instruments Foster Findlay Associates Ltd Hamamatsu Photonics UK Ltd Hitachi Scientific Instruments ISS Imaging Associates Ltd J K Instruments Ltd JEOL UK Ltd K E Developments Ltd Lasertec Corporation Leica Cambridge Limited Leica UK Limited Microfield Scientific Ltd Microscopy and Analysis Newport Ltd Nikon UK Limited Olympus Optical Co (UK) Ltd Oxford Instruments Microanalysis Group Oxford Instruments Philips Electron Optics Photonic Science Polaroid (UK) Ltd Princeton Gamma-Tech (UK) Ltd Pyser (Holdings) plc Synoptics Ltd Taab Laboratories Equipment Ltd Tracor Europa Carl Zeiss (Oberkochen) Ltd
RECEPTION AND ASSOCIATED EVENTS On Monday 12 September there will be a wine reception in the Exhibition between 5.30 pm and 7.00 pm. The Annual General Meeting of the Royal Microscopical Society, and the Presidential Address will also take place during the evening.
ACCOMMODATION Academic Accommodation A limited amount of academic accommodation has been booked for the use of delegates at Imperial College. Rooms have been booked there on a bed and breakfast basis at a cost of 26 pounds sterling per night. This academic /student accommodation will be filled on a 'first-come first-served' basis. From the nearby Underground Station at South Kensington, Earls Court is two stops along the District or Piccadilly Line.
Hotel Accommodation There are some rooms available in the Earls Court Park Inn at the special MICRO 94 rate of 65.00 pounds per night. If you would like to reserve accommodation at these special rates, please contact the Earls Court Park Inn directly, quoting that you are a MICRO 94 visitor. To be sure of booking rooms at these rates, it is advisable that you book well in advance. Telephone: 071 385 1255 Telex: 917728 Fax: 071 381 4450.
Other Hotel Accommodation Delegates who wish to make their own accommodation arrangements may wish to use the services of Expotel Executive Travel - Europe's leading hotel booking agent, who have been appointed the official hotel agency for MICRO 94. The hotel of your choice or a similar alternative can be booked through Expotel often at discounted rates. By making one telephone call to Expotel on 071 735 0060 stating the event code 'MICRO 94', your reservation will be confirmed verbally followed by confirmation in writing. This free booking service is available to anyone attending MICRO 94. Telephone: 071 735 0060 Telex: 8811951 EXPOTL G Fax: 071 735 2839.
REGISTRATION AND PAYMENT
Registration Registration will take place at the Earls Court Park Inn from 1.00 pm on Monday 12 September 1994, and from 9.00 am on subsequent mornings. Cost of registration for the whole conference is 60 pounds for RMS members and 90 pounds for non-members. Daily rates are 20 pounds for RMS members and 30 pounds for non-members.
Payment Payment may be made by sterling cheque payable to the Royal Microscopical Society (please add 12.00 pounds to cover exchange and bank charges if the cheque is not a UK Bank Cheque) or by credit card (Visa/Barclaycard or Access/Eurocard/Mastercard).
Cancellation and Refunds Cancellations received before 12 July 1994 will be subject to a full refund. No refunds will be made if cancellation is made after this date.
HOW TO GET THERE
The Conference, Exhibition, Posters, and Refreshments will all take place at the Earls Court Park Inn, Lillie Road, London SW6.
MICRO 94 is organized by the Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, UK, in association with Microscopy and Analysis. Telephone: 0865 248768, Fax: 0865 791237, Email: RMS-at-UK.AC.OX.VAX.
I've been involved in design of an EM lab, a histology lab and most recently given freehand to design a facility with "ultimate, within the budget" two general histology labs, a microtome room and two microscope/image analysis rooms to be built soon.
Think carefully about work flow and separation of incompatible activities. Traffic patterns can be aggravating if their is a choke point. e.g. our peninsula bench has ahighly used sink on the end and only 3.5 ft to the wall. There is a blackboard and garbage cans along that wall, with lots of traffic through this narrow slot. Don't allow the free edge of open doors to aim toward knees or heads on single doored cupboards. Big, deep double sinks. Double sinks, or many sinks, ensures that processes requiring long washes don't tie up the sink, and minimize splatter. Our PI put in a lot of pocket sinks in our existing lab. they are great if the benches are not cluttered. Otherwise they will fill with pencils, towels and BEEM capsules as well as splatter adjacent equipment. We have 4-12 people woking in 400 sq. ft. We are vey cluttered and the pocket sinks are useless.
Dont put fume hoods in corners. That makes the wall-end foot or so harder to use, especially if you end up storing anything on the floor in that corner. Stuff on the floor also absturcts the cupboard doors below the hood.
It took some doing, but I got approval for an awning hood over one bench in our new histology lab. It is 10 ft. long, wasn't enough room to go longer. All embedding, deparaffinizing, staining and coverslipping will take place under it. An open shelf below will hold vacuum ovens for paraffin and the plastics oven so they can vent upward from the back. Osmium and fix preparation will remain in the standard hood (a 6 ft. hood). Drafts from ventilation have been a major pain for us and have required makeshift deflectors on vents, or finding combinations of closed doors and hood sashes to slow down drafts.
Consider separate 110V circuits for computers and everything else, especially if you have any surge producers like pump motors or uv lamps.
Look out for dust collectors.. They are pricey, but put glass doored cupboard over dust sensitive situations like microtomes and grid staining area. These will not present so many dusty surfaces as will open shelving.
Gotta go, but one last issue, physically touch, test, open and close the furniture before allowing it to go in. some looks great but is worse than useless.
Prior comments about wiring and fields are very true. We are seeking a new home for our SEM because of fields. (Keep an eye on remodelling - I remember a Philips 300 whose beam would deflect everytime the new photocopier next door made a copy)
On Tue, 30 Nov 1993, Greg Erdos ICBR EM Core Lab University of Florida wrote:
} I am now in the process of designing a new EM suite dedicated to biological } EM. I would like input from those of you who work in what they feel is an } ideal or close to ideal physical arrangement. } I would also like to hear from those who have envisioned an ideal } EM suite but have never seen it built. The restrictions are that a space of } 2200 sq ft should house 2 TEMs and 2 SEMs as well as a prep lab, darkrooms } ancillary equipment and 2 offices. The overall design is of more interest } than actual use of the stated space.
Greg, we are well into the final design of our EM facility for the new Cell and Mol. Bio building in the Div. of Bio Sci. I based our design on the existing EM suite which I remodeled about 10 yrs ago. I also used the book by R. H. Alderson, "Design of the Electron Microscope Laboratory", part of Audrey Glauert's series in Practical Methods in Electron Microscopy published by North-Holland/American Elsevier ISBN 0 444 10816 5. I can send you plans although I doubt if the architects would like that. Further, my design was limited to the space available in the new building and the shape of the room modules. Each module is about 10 ft by 30 ft. I used 3 modules for the main lab and office but there is extra space available across the hall where I housed the freeze fracture device, the microtomes, student TEM, and dept. darkrooms (also under my supervision). You can reach me at 916 752 2914, Univ. of Cal. at Davis, Dept. of Evolution and Ecology. Rick A. Harris
Hitachi H-600 STEM: 13 years old, under service contract with Hitachi until this year, in most excellant condition, scope has all service reports and manuals.
Original cost: $175,000 Asking price: Make offer I can't refuse Contact: Phil Rutledge, Director, Center for Electron Microscopy University of Maryland Baltimore County Dept. of Biology Catonsville, MD 21228 USA Phone: (410) 455-3582 FAX: (410) 455-3875 Email: prutle1-at-umbc.edu
I am looking for a supplier of a 2D array detector (for luminescence microscopy measurements) which will operate out to 1.6 microns. Essentially the same thing as a Si CCD except made from a smaller gap material (InGaAs?, Ge?). I would like recommendations of a manufacturer/supplier.
Erik Reuter, e-reuter-at-uiuc.edu, 217-244-6378 phone, 217-244-6375 fax
Here is a list of references I received from my inquiry on light microscopy. Thanks to everyone who contributed!
***
The best set of notes on LM theory I know of are the course notes for the AQLM course run at Woods Hole each year, they are long and complex but in my experience a great resource
Kodak publications: "Photography Through The Microscope", P-2, cat.# 152-8371 "Kodak Scientific Imaging Products", L-10, cat. # 813-9321 The first has loads of basic information about objectives, condensers, setting-up illumination, flourescence, phase contrast, etc. Both have bibliographies.
Title : Optical Microscopy : Emerging Methods and Applications Author : Herman, Brian/Lemasters, John J. (Editors) ISBN : 0123420601 Subject : Non-Fiction Dewey # : 578.00 Publisher: Academic Pr Date Pub : 12/92 Binding : Hardcover+ Price : $ 79.95
Loveland, Roger Platt. Photomicrography; a comprehensive treatise [by] Roger P. Loveland. New York, Wiley [1970] 2 v. (x, 1039, I-xii p.) illus. 23 cm. LC CALL NUMBER: QH251 .L68 SUBJECTS: Photomicrography. SERIES TITLES (Indexed under SERI option): Wiley series on photographic science and technology and the graphic arts DEWEY DEC: 778.3/11 NOTES: Includes bibliographies. ISBN: 0471548308 LCCN: 70-88315 r892
Shillaber, Charles Patten, 1886- Photomicrography in theory and practice, New York, J. Wiley & sons, inc.; London, Chapman & Hall, limited [1944] viii, 773 p. incl. illus., tables, diagrs. 22 cm. LC CALL NUMBER: QH251 .S5 SUBJECTS: Photomicrography. LCCN: 44-6507
Light microscopy in biology : a practical approach / edited by Alan J. Lacey. Oxford, England ; New York : IRL Press, c1989. xviii, 329 p. : ill. ; 23 cm. LC CALL NUMBER: QH207 .L49 1989 SUBJECTS: Microscopy--Technique. ADDED ENTRIES: Lacey, Alan J. SERIES TITLES (Indexed under SERI option): Practical approach series DEWEY DEC: 578/.4 dc19 NOTES: Includes bibliographies and index. ISBN: 0199630364 : $54.00 (U.S. : est.) 0199630372 (soft) : $36.00 (U.S. : est.) LCCN: 88-23505 r92
Pluta, Maksymilian. Advanced light microscopy / Maksymilian Pluta. Warszawa : PWN ; Amsterdam ; New York : Elsevier : Distribution for the USA and Canada, Elsevier Science P ublishing Co., 1988- {1989 } v. 1- {2 } : ill. ; 25 cm. LC CALL NUMBER: QH207 .P54 1988 SUBJECTS: Microscopy--Technique. DEWEY DEC: 502/.8/2 dc19 CONTENTS (Incomplete): v. 1. Principles and basic properties -- v. 2. Speciali zed methods. NOTES: Translated from the Polish manuscript. Includes bibliographical references and index. ISBN: 0444989390 (v. 1) : $175.00 (est.) 0444989188 (v. 2) LCCN: 87-24605 r922
Ploem, J. S. Introduction to fluorescence microscopy / J.S. Ploem and H.J. Tanke. Oxford ; New York : Oxford University Press ; Oxford : Royal Microscopical Society, 1987. vi, 56 p. : ill. ; 24 cm. LC CALL NUMBER: QH212.F55 P57 1987 SUBJECTS: Fluorescence microscopy. ADDED ENTRIES: Tanke, H. J. SERIES TITLES (Indexed under SERI option): Oxford science publications Microscopy handbooks / Royal Microscopical Society ; 10 Microscopy handbooks ; 10. DEWEY DEC: 578/.4 dc19 NOTES: Includes bibliographies and index. ISBN: 0198564082 : $7.50 (U.S.) LCCN: 87-5539
Inoue, Shinya. Video microscopy / Shinya Inoue ; with contributions by Robert J. Walker, Jr. ... [et al.]. New York : Plenum Press, c1986. xxvi, 584 p. : ill. (some col.) ; 26 cm. LC CALL NUMBER: QH222 .I56 1986 SUBJECTS: Video microscopy. DEWEY DEC: 578/.4 dc19 NOTES: Includes index. Bibliography: p. 515-529. ISBN: 0306421208 LCCN: 85-28252
Slayter, Elizabeth M. Light and electron microscopy / Elizabeth M. Slayter, Henry S. Slayter. Cambridge [England] ; New York : Cambridge University Press, 1993. p. cm. LC CALL NUMBER: QH205.2 .S54 1993 SUBJECTS: Microscopy. Compound microscopes. Electron microscopy. ADDED ENTRIES: Slayter, Henry S. DEWEY DEC: 578/.4 dc20 NOTES: Includes indexes. 92-7825 (continued): ISBN: 0521327148 LCCN: 92-7825 r93
Russ, John C. The image processing handbook / John C. Russ. Boca Raton, Fla. : CRC Press, c1992. 445 p. : ill. (some col.) ; 27 cm. LC CALL NUMBER: TA1632 .R88 1992 SUBJECTS: Image processing. ADDED ENTRIES: Image processing. DEWEY DEC: 621.36/7 dc20 NOTES: Includes bibliographical references (p. [435]-442) and index. ISBN: 0849342333 (acid-free) LCCN: 92-4936
Russ, John C. Computer-assisted microscopy : the measurement and analysis of images / John C. Russ. New York : Plenum Press, c1990. xii, 453 p. : ill. ; 26 cm. LC CALL NUMBER: TA1632 .R87 1990 SUBJECTS: Image processing. Microscopy--Data processing. Optical pattern recognition. DEWEY DEC: 502/.8/20285 dc20 NOTES: Includes bibliographical references and index. ISBN: 0306434105 LCCN: 89-70945 r92
Bradbury, Savile. Basic measurement techniques for light microscopy / SavileBradbury. Oxford ; New York : Oxford University Press ; [London] : Royal Microscopial Society, c1991. viii, 97 p. : ill. ; 24 cm. LC CALL NUMBER: QH207 .B74 1991 SUBJECTS: Microscopy--Measurement. SERIES TITLES (Indexed under SERI option): Royal Microscopical Society microscopy handbooks ; 23 Microscopy handbooks ; 23. DEWEY DEC: 502/.8/2 dc20 NOTES: Includes bibliographical references and index. ISBN: 0198564260 : $14.90 LCCN: 90-21567 r92
Bradbury, Savile. An introduction to the optical microscope / Savile Bradbury. Rev. ed. Oxford ; New York : Oxford University Press ; Oxford : Royal Microscopical Society, c1988. 86 p. : ill. ; 24 cm. LC CALL NUMBER: QH205.2 .B67 1988 SUBJECTS: Microscopes. Microscopy. SERIES TITLES (Indexed under SERI option): Microscopy handbooks ; 1 Oxford science publications DEWEY DEC: 535/.332 dc19 NOTES: Includes bibliographical references (p. [82]). ISBN: 0198564198 : $6.00 LCCN: 88-38921 r92
Dictionary of light microscopy / compiled by the Nomenclature Committee of the RMS, S. Bradbury ... [et al.]. Oxford ; New York : Oxford University Press ; Oxford : Royal Microscopical Society, 1989. x, 139 p. : ill. ; 25 cm. LC CALL NUMBER: QH203 .D53 1989 SUBJECTS: Microscopy--Dictionaries. ADDED ENTRIES: Bradbury, Savile. Royal Microscopical Society (Great Britain). Nomenclature Committee. RMS dictionary of light microscopy. SERIES TITLES (Indexed under SERI option): Microscopy handbooks ; 15 Oxford science publications DEWEY DEC: 502/.8/2 dc19 NOTES: Cover title: RMS dictionary of light microscopy. ISBN: 019856421X : $32.00 (U.S.) 0198564139 (pbk.) : $14.95 (U.S.) LCCN: 88-22712 r92
Thomson, D. J. An introduction to photomicrography / D.J. Thomson, Savile Bradbury. Oxford [Oxfordshire] ; New York : Oxford University Press ; Oxford : Royal Microscopical Society, 1987. 74 p. : ill. (some col.) ; 24 cm. LC CALL NUMBER: QH251 .T44 1987 SUBJECTS: Photomicrography. ADDED ENTRIES: Bradbury, Savile. SERIES TITLES (Indexed under SERI option): Microscopy handbooks ; 13 DEWEY DEC: 578/.4 dc19 NOTES: Includes index. Bibliography: p. [69]-70. ISBN: 0198564147 (U.S. : pbk.) : $9.00 LCCN: 86-33220
Bradbury, Savile. The evolution of the microscope, by S. Bradbury. [1st ed.]. Oxford, New York, Pergamon Press [1967] x, 357 p. illus. 22 cm. LC CALL NUMBER: QH204 .B7 SUBJECTS: Microscopes--History. DEWEY DEC: 578/.09 NOTES: Bibliography: p. 347-348. LCCN: 67-18485 r922
Erik Reuter, e-reuter-at-uiuc.edu, 217-244-6378 phone, 217-244-6375 fax
This message was posted on a different forum however it has applicabity here too. I've reflected it for the subscribers:
Nestor Z. ANL EMCenter ====================
To: Multiple recipients of list {nih-image-at-soils.umn.edu}
} I am interested in finding references to algorithms for grain size } measurement. Any help would be welcome. } There are two "standard" methods for measuring the property usually called "grain size." One is based on counting the number of grains visible per unit area on a two-d section through the structure, and the other is based on drawing lines across the same image and coutning the number of intersections of the lines with grain boundaries. Neither actually measues the "size" of the grains, which are 3D entities. From the point of view of stereology, the second method measures the amount of grain boundary area per unit volume in the sample, which is often important to properties (mechanical, electrical, etc.) because many things happen at grain boundaries. The first method measures the total length per unit volume of the "triple line" where three grains meet in the volume. This is particularly important in sintered structures since the pore channels and much diffusion occurs there. But NEITHER is the "grain size." It happens that in many materials (but by no means all), the grain size distribution after standardized heat treatments (e.g., full recrystallization) is sufficiently consistent that there is a more-or-less consistent relationship between the actual size distibution of the grains and these other parameters. This combined with the fact that people (or at least metallurgists) have been measuring these properties for most of this century, and CALLING it grain size, has made these measurements an accepted standard tool for microstructural characterization.
There are a couple of fairly robust computer approaches to measuring each of the two different properties. In the computer case, instead of drawing lines and couting crossing points it is easier to first perform whatever image processing is needed to threshold and skeletonize the grain boundaries and then measure the total length (properly, not just counting pixels but taking into account the geometry of the line). This is simply related to the intercept count and the surface are per unit volume, and hence to the "grain size number." It has the disadvantage that many grain images do not show all of the boundaries well (inadequate etching or polishing, ec.) The algorithm that works best for the other method is to count the triple points in the grain boundary image, which usually do show up well and are easily define as points on the skeleton that have more than 2 neighbors. This gives the total length of triple line, and again a "grain size number."
For details on both algorithms, and the equations to convert the values, see p.147 and p. 225 in Computer Assisted Microscopy, J. C. Russ, 1990, Plenum Press. ISBN 0-306-43410-5
There are other issues to consider as well, including processing operations on grain images that may alter the length of boundaries (watershed segmentation, for instance, is either a boon or a curse), and ways to actually measure the 3D distribution of grain sizes, without resorting to very biased assumptions like treating the grains as spheres (!). The field of stereology has dealt with such problems for decades, and there are actually some pretty good answers to most of them. Good, but not necessarily simple to implement.
__________________________________________________ John Russ (John_Russ-at-ncsu.edu) or (russ-at-mat.mte.ncsu.edu) Materials Science and Engineering Department, North Carolina State Univ., Raleigh, NC 27695-7907 phone: 919-515-3328 fax: 919-515-7724
On Fri, 3 Dec 1993 03:14:37 +0200, Steve Barlow wrote:
} } We are considering the purchase of a new diamond knife and were interested } to know if anyone has had any experience with companies other than } Diatome. In specific, Edgecraft Corp, MicroStar, or DiaTech. } } } From personal experience, I know that Diatome makes an excellent knife. } Dupont knives were good, but I haven't used any of their knives since they } were bought out by DDK. DiaTech has been touting its 'unique' mounting } system that greatly reduces re-sharpening costs. } } If anyone can offer their own experiences, I would appreciate it, } particularly regarding the quality of the resharpening. } } ---------------------------------------------------------- } 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
Greetings from the "old continent" to all microscopy-netters,
We have used Diatome knifes and found them excellent. Now we are going to test-section a Drukker-knife from Drukker International B.V., the Netherlands. I have great expectations for this particular maker, since they claim to have made the diamond surface hydrophilic, thus enabling sectioning with not-filled waterlevel. We will know more about this knife in two months.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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
I have been doing E.M. for 25 years and for the past 10-12years have been using MicroStar knives. The knives are of excellant quality. I have never had a problem with any of their knives. I have used DuPont and Diatome but as long as MicroStar remains in business, I will continue to use their knives. Their people are friendly, informative and very helpful. As a whole, the company is excellant to deal with. I recommend their knives whole heartedly. Questions? Phil Rutledge, Director, Center for E.M. UMBC Dept. of Biology Catonsville, MD 21228 USA Phone:(410)455-3582 FAX: (410)455-3875 Email: prutle1-at-gl.umbc.edu
I have had excellent service from Microstar for many years, Both with new knives and with resharpening knives that originally came from several different vendors. I'd say we have gotten about 20 knives from this company and have always been satisfied. This is not to say that other suppliers couldn't do as well. My recent (10 yrs) experience has only been with Microstar.
From : JOSEPH MCGINN Number : 8 of 9 To : ALL Date : 12/02/93 1:08a Subject : Nicalon-Black Glass Reference : NONE Read : [N/A] Private : NO Conf : 005 - Specimen Preparation
Has anyone had experience with sample preparation of this composite system. There is a real problem retaining the fibers in the matrix during the final stages of mechanical polishing. Has anyone found a trick to get these samples below 20 microns for ion milling?
From : CRAIG LENDING Number : 9 of 9 To : ALL Date : 12/03/93 10:54a Subject : Unicryl embedding Reference : NONE Read : [N/A] Private : NO Conf : 005 - Specimen Preparation
Has anyone had any experience with immunolabelling with the new Unicryl resin that is marketed by SPI? I currently use LR White, but they claim that this resin has better stability under the electron beam and also gives higher levels of labelling than LR White -- at least that's what they say!
I have tried both resins and have had good results with both. The only problem I have had with LR White is getting my students or principle investigators remembering to use 80kv. LR White is best used at voltages above 60kv or you can have resin "droppings" formed on the wall of the projector pole piece. I have 4 E.M.s and since I don't have service contracts I end up cleaning the contaminated scope. If you can use voltages above 60kv, you can get equally good results with either resin. I normally use 80kv on all of my scopes at all times and have never tried Unicryl at 60kv. It may be OK to use at 60kv or you may have to use a higher voltage as I have to do with LR White. The only real difference I have seen is the initial cost of the two resins. Good Luck! Phone: (410)455-3582 FAX: (410)455-3875 Email: prutle1-at-gl.umbc.edu
We are contemplating getting a new ultra-high resolution field emission SEM. This would be for applications in both biological and materials fields. Resolution needs to be better than 1nm at ca 15kV. We are at present operating 2x SEM's and 1 TEM in a service unit, so this new SEM would be dedicated to clean samples for hi-res work. I would appreciate any and all input from users regarding their experience of such equipment (microscopes and necessary prep peripherals). Practical experience of cold versus thermal FEG's (stability, lifetime, running costs, vacuum problems etc) especially welcome. Dr J Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria 0002 Internet:janc-at-ccnet.up.ac.za South Africa
On Fri, 3 Dec 1993, Nestor J. Zaluzec (708)-252-5075, -4964 wrote:
} } } } From : CRAIG LENDING Number : 9 of 9 } To : ALL Date : 12/03/93 10:54a } Subject : Unicryl embedding Reference : NONE } Read : [N/A] Private : NO } Conf : 005 - Specimen Preparation } } Has anyone had any experience with immunolabelling with the new Unicryl } resin that is marketed by SPI? I currently use LR White, but they claim } that this resin has better stability under the electron beam and also } gives higher levels of labelling than LR White -- at least that's what } they say! } } ================ } We use unicryl on a regular basis as our main embedding medium. The immunolabelling characteristics are comparable or better than Lowicryl K4M. The morphology (?) even seems to be improved using the same preparation procedures. The cutting characteristics are very well. We cut it using a diatome knife and also using glassknifes. The person in charge of cutting has a lot of experience with different materials (incl LR White) and she thinks Unicryl cuts at least as good as Epon or Araldite. Hoping to hear from you. We are interested in LR White because of the not complete dehydration. We have tried some LR White but have experienced great difficulties. Blocks tend to break and are difficult to cut. Perhaps you could comment.
We've had experience with DDK over many years with many knives. Their knife quality is excellent. The resharpening is excellent. An easy company to deal with.
On Thu, 2 Dec 1993, Steve Barlow wrote:
} We are considering the purchase of a new diamond knife and were interested } to know if anyone has had any experience with companies other than } Diatome. In specific, Edgecraft Corp, MicroStar, or DiaTech. } } } From personal experience, I know that Diatome makes an excellent knife. } Dupont knives were good, but I haven't used any of their knives since they } were bought out by DDK. DiaTech has been touting its 'unique' mounting } system that greatly reduces re-sharpening costs. } } If anyone can offer their own experiences, I would appreciate it, } particularly regarding the quality of the resharpening. } } ---------------------------------------------------------- } 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 }
-------------------------------------- Jim Michael
------------------ RFC822 Header Follows ------------------ Received: by quickmail.yale.edu with SMTP;6 Dec 1993 12:13:44 -0500
At Argonne National Laboratory's E. M. Center, we have used a Kodak Royalprint Processor for ten years or so. It will handle any developer- incorpoorated RC paper and give a dry, archival print in about 45 seconds, as Ron Anderson noted. It is not cheaper than using trays, but you'll be able to do more work in less time with less frustration. Any similar processor will give you the same results. If you want to get really efficient, you can get an automatic dodging enlarger from LogEtronics. Then you don't have to spend time or test strips trying to find the correct exposure! I haven't heard of anyone who is using the 3M Dry Silver Processor which Ted Pella, Inc. is marketing. According to it's brochure, the processor is supposed to deliver a dry, archival print in 6 seconds. You'd avoid handling chemicals with that processor. Any experience out there?
} I am in the process of deciding whether or not to purchase a B&W photo paper } processor for our EM Lab darkroom. I have reviewed some product literature and } now would like to hear from anyone who has used such processors. Are they } neater, cleaner, faster and more economical than tray processing? Are they } 'plumber's nightmares'? Are they tied to a specific paper type and chemistry? }
Ted Pella, Inc. has advertised a product called `Pro Automatic Print and Film Processor' in its recent catalog. It is said that this can process negatives (4489 TM, T-MAx etc.) as well as prints using the same developer in one unit. It costs about $4000. There is yet an another product advertised called the Pelco Dry Silver Processor which does not use any chemicals. The product is marked about $1400. We are considering upgrading our darkroom facilities, and so contemplating on purchasing the Print and Film Processor. Does anyone have any experience with it? Thanx in advance for suggestions/advice.
} } Thank you for any information you can provide. } } Gib G. Ahlstrand } Electron Optical Facility } University of Minnesota
Ananda S. Murthy Department of Physics & Astronomy University of Delaware Newark, DE 19716 (302) 831-6811
The mentions of wash by others reminded me: the system I used still required that the photos be fixed as usual for tray developing. (Using Kodak Polycontrast RC paper.) I forget if it was really required, but yellowing etc. wash a problem if photos weren't fixed. Wash was 5-10 min. Phil Oshel
Re} EM practical courses Back by popular demand -even though we never seem to make a profit (or break even).
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 detailson both courses;
Paul Webster, Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510.
We too use Ilford 2150RC units and have no problems with yellowing etc. I should note, however, that we have had a lot of minor problems with one of the two identical units in our building and this has resulted in considerable downtime and frustration. When they work properly, they are wonderful units which increase the work rate by at least a factor of 2-3 over manual developing. If, however, I was based in the states and looking to buy a processor I'd be asking my Ilford representative some very probing questions about service and call-out arrangements in case of problems. Please note that our other 2150RC has given about four years of trouble free service.
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 worked part time for two years processing b&w EMs and had extensive experience w/ b&w photography before the job (including making silk screens, photo lithographs, etc.).
Having a Ektagraphic(?) machine by Kodak greatly sped up making large numbers of notebook prints. The machine developed and stabilized in 20-30 seconds or so. I just piled the prints up and fixed and washed them in a big batch before lunch and before going home. I only had to get my hands wet two or three times each day; the actual printing was almost a dry process.
Granted these prints are not archival, but they last for at least five years without significant fading or discoloring.
Another trick for tray processing is to keep dektol (or your favorite developer) warm ( } 70 degrees F); the paper develops instantly, but the chemicals oxidize fast.
nih-image-request-at-nx1.soils.umn.edu (NIH Image Mailing List Maintainer) Newsgroups: news.announce.newgroups,news.groups,sci.chem,sci.engr.chem,sci.geo.geology,sci.materials,sci.misc,sci.physics,sci.optics,sci.research,sci.physics.accelerators,sci.engr,sci.polymers
FIRST CALL FOR VOTES (of 2)
Unmoderated group sci.techniques.microscopy
Newsgroups line:
sci.techniques.microscopy The field of microscopy.
Votes must be received by 23:59:59 UTC, 2 January 1994.
After this CFV appears on news.announce.newgroups it will be sent to the following mailing 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-nx1.soils.umn.edu {NIH Image Mailing List} maintained by John Ladwig {ladwig-at-soils.umn.edu}
This vote is being conducted by a neutral third party. For voting questions only, contact pschleck-at-unomaha.edu. For questions about the proposed group, contact John F. Mansfield {John.F.Mansfield-at-umich.edu} .
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.
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The Journal of Microscopy has received the following books for review:
Enzyme Histochemistry. A Laboratory Manual of Current Methods By C J F Van Noorden & W M Frederiks. Oxford University Press
X-ray Microanalysis in Biology. Experimental Techniques and Applications Edited By David C Sigee, A John Morgan, Adrian T Sumner & Alice Warley. Cambridge University Press
Immunocytochemistry II Edited by A C Cuello. John Wiley
A Manual of Applied Techniques for Biological Electron Microscopy By Michael J Dykstra. Plenum Publishing
Soil Microscopy and Micromorphology By E A Fitzpatrick. John Wiley
If you are interested in reviewing any of these publications, and can provide a review within two months of receiving the book, please contact Gillian Wilson at RMS-at-uk.ac.ox.vax directly. There is no financial reward, but you can keep the book, of course! I will work on a first-come, first-served basis.
I am also looking for volunteers to review a forthcoming book (which will appear in print in January 1994):
In Situ Hybridization By A R Leitch, T Scwarzacher, D Jackson & I J Leitch. BIOS Publications
Two copies will be available for review. ******************************************************************************
RE} Lowicryl protocol In reply to the request for a Lowicryl protocol that works, from Rajesh Patel. Try this for something a little more exotic.
Fix the lung tissue as well as possible (perfusion fix if possible) in either formaldehyde or glutaraldehyde, 1 -2 hrs is enough. Infiltrate with sucrose (2.3M in PBS) for approx. 1 hr, depending on the size of the pieces. Freeze the cryoprotected pieces on the tips of wire stubs (or cryo ultramicrotome specimen stubs if available) by immersion in liquid nitrogen. Do not let them warm up but transfer them directly to 100% methanol on dry ice (precooled of course). Leave until they fall of the wire (usually overnight is enough for small pieces). Wash with fresh, cold methanol on dry ice then gradually replace the methanol with methanol containing increasing amounts of Lowicryl (we use HM-20 or HM-23 at these depressed temperatures) until you can put the tissue in 100% resin. Leave overnight then embed in fresh resin, still on dry ice, and polymerize, on dry ice, using a UV light as per instructions. Remember that oxygen will interfere with the resin polymerization so use embedding capsules or centrifuge tubes that are filled up with resin and are sealed. Also bubble nitrogen through the resin before use.
This easy freeze substitution protocol can be done in a styrofoam box (which is what we use) or in a Revcco type freezer (the Lowicryl smells bad and is difficult to stop from spilling though!) The cold methanol does not need to be specially dehydrated before use (just open a new bottle) and can contain 1% osmium tetroxide or 1% uranyl acetate to improve contrast. Do not allow the tissues to warm up in the osmium if you are concerned about antigenicity. Surprizingly, the additions to the methanol do not seem to affect antigenicity on most of our systems. For extra polymerization Lowicryl blocks can be put under UV light at room temp (or in the sun - says Heinz Schwarz of Tubingen). A reference for this can be found in van Genderen et al 1991 J. Cell Biol. 115:1009-1019.
If your antibodies do not work on Lowicryl-embedded tissue (and many do not) why not try cryosections. They are not difficult to obtain - even from lung. Good luck.
At Image Analysis we have a Tektronix Phaser IISDX Dye Sublimation printer which is connected simultaneously to a Silicon Graphics Indigo (via a parellel port) and a Macintosh (via appletalk). This printer produces near photographic quality (some people consider it better than photo quality) 8.5 x 11 and legal size 24 bit color prints and transparencies. Used in combination with Adobe Photoshop on the Mac or Showcase on the Silicon Graphics, it produces fantastic results allowing researchers to create hardcopy for posters, publications and portfolios in record time. The printer understands most postscript so that text and object oriented graphics is reproduced with little or no aliasing.
The main drawback with this printer is cost per print which is around $3. The newer printers from tektronix and other vendors are rumored to use regular printer paper rather than requiring the purchase of special paper for the printer. Two other problems with the printer have been inability to read all postscript files (usually solved by reading the files into Photoshop or Showcase first) and matching the intensity of the colors on the computer screen with what is produced by the printer.
boyd
Boyd Knosp 319-335-6715 University of Iowa knosp-at-tessa.iaf.uiowa.edu Image Analysis 77 EMRB Iowa City, IA 52242
On Thu, 9 Dec 1993, L. D. Marks wrote:
} } Any comments on computer image printers ? } } Laurie Marks } Northwestern
I am interested in any problems that other users of the Reichert microtome Ultracut S with FCS cryo attachment have had. I am especially interested in chronic problems with the cryo pump; could you also relay what your solutions have been? I am having the membrane in my pump replaced more often than seems appropriate for a new piece of equipment and wonder if this is typical. Also the poly tube that connects the pump to the liquid nitrogen in the dewar keeps slipping off of its connector - anyone having this problem? Solutions? Thanks.
R} Beef Wenbo Zhang writes that he has pepper on his beef muscle after fixation. The muscle is fixed and postfixed in the presence of phosphate buffer which may be the problem. All the glutaraldehyde must be washed out of the tissue before going into osmium otherwise some sort of reaction occurs to cause a precipitate. This may vary from small particles (pepper) to crystals.
Message-Id: {9312101416.AA12900-at-umail.UMD.EDU} To: microscopy-at-anlemc.msd.anl.gov
Fixation pepper probably results from the interaction of a number of key fixation ingredients including phosphate buffer, glutaraldehyde, uranyl acetate and ethanol. For a nice work on the role of the above participants in the formation of dense deposits see the paper by J. Louw, et al in STAIN TECHNOLOGY 65(5): 243-250, 1990 entitled "Electron dense artefactual deposits in tissue sections: The role of ethanol, uranyl acetate and phosphate buffer."
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
Reply_ RE} Printers Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu OK, we recently (actually Ron Gibala and John Halloran paid for it) bought a Kodak ColorEase 300 PS printer for printing out images. We are talking about a Mac environment here with a few PCs so this discussion will be centered on printing from Macs for the most part. We had a demo of the ColorEase and it during that the printer was not printing as well as the Tektronix Phaser IIsdi. So, we were going to go with the Tektronix (even though it was more expensive). But during the demo, I started using the Apple Laserwriter 8.0 drivers and then the Kodak performed better for grayscale images. We have found for 8bit color and grey images we use the Apple drivers (we have a PPD downloaded from Adobe.com so the applications on the Macs know big the print area is on the Kodak under 8.0. The 8.0 drivers were developed by Apple and Adobe together anyway). There are PPDs at Adobe for most Postscript printers (except maybe the Hewlett Packards). To get them check out ftp.adobe.com. Anyway after that small diversion, we use the ColorEase driver if we want to do 24bit color and try and match what the image looks like on the screen. Since we dont often use true 24bit color we mostly use the Apple drivers. The latest version of NIH-Image when used in conjunction with the Apple drivers will scale the image you are trying to print to fit on the page and so that avoids the clipping warnings you get from Photoshop. Of course, if you want to use 24bit color you need to use Photoshop. It's a nice printer. The paper and transparencies are a lot more heavy weight that the Tek media. Prints cost us about $2 each and transparencies a little more. Considering you can get 3 Polaroid size prints on one sheet that is a considerable cost saving over Polaroid. The Polaroid that we use is in excess of $2.50 per shot and so if you use the Kodak printer, you can pay for the printer after 3500 prints. Given the atrocious Polaroid quality and their vague responses if you try and get credit for a bad batch of film, this printer seems to me like an excellent way to go if you have a good way of electronically acquiring your images. As a disclaimer, my opinions of the corporations that I mention above are my own personal ones and do not in any way reflect the opinions of my employer
We have a Codonics NP-600 dye sub printer. It came to us as part of a confocal package, the price is about $10K. Cost of the standard B&W/color paper is $1.50 a sheet (8" x 8.5"). The 8.5" x 11 is about $3 a print. Microscopy images are as good as one's equipment. Color matching seems good but we may not be as discriminating as others. One major strongpoint is the number of acceptable formats : IFF, GIF, JPEG, PICT, PCX, PPM, RGB, Sun Raster, TIFF, TGA, X-11 Window Dump, CMU WM, IMG, PBM, PGM, and Lisp Machine Bitmap. Codonics is a small outfit out of Ohio and have provided excellent backup. Take a look. ************************************************* * * * * * 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 * * * * * *************************************************
We have a vast amount of experience with the processing of myocardial / muscular tissue. In our experience it is the phosphates found in muscular tissue which play a role in the formation of these dense deposits.
The essential factors in the formation of electron dense deposits appear to be phosphate buffer, ethanol and uranyl acetate. Glutaraldehyde may contribute in a way, while osmium does not appear to play a role. (See our article in STAIN TECHNOLOGY 65: 243-250 (1990). -------
Message-ID: {MAILQUEUE-101.931213111417.384-at-eagle.mrc.ac.za} To: Microscopy-at-anlemc.msd.anl.gov
We have a vast amount of experience with the processing of myocardial / muscular tissue. In our experience it is the phosphates found in muscular tissue which play a role in the formation of these dense deposits.
The essential factors in the formation of electron dense deposits appear to be phosphate buffer, ethanol and uranyl acetate. Glutaraldehyde may contribute in a way, while osmium does not appear to play a role. (See our article in STAIN TECHNOLOGY 65: 243-250 (1990). -------
Message-Id: {MAILQUEUE-101.931213084915.416-at-parmly1.parmly.luc.edu} To: Microscopy-at-anlemc.msd.anl.gov
Greetings, Has anyone had exerience with a diamond knife designed to cut semi-thin sections? That's correct, semi-thin sections, 0.2 - 5 um range. I have just seen an ad for a product from Diatome called the "Histo Knife" which claims to do just that. The main question would seem to be how many sections such a knife can cut before needing to be resharpened. Advice or comments welcome. I will post a summary of any private replies. Thanks in advance, Tobias Baskin
******************************** *************** Tobias I. Baskin /~~~\ Biol. Sci's * Univ. of Missouri c|o o\ Columbia, MO 65211 USA \ = / Tel:314-882-0173 """ FAX 314 - 882 - 0123 baskin-at-biosci.mbp.missouri.edu
I manage a biologically-oriented EM facility at a university. Service fees are supposed to cover all expenses although this has never happened. Actually, we probably could break even if we had 50-75% of full use. However, there are three fully-equipped labs on this campus which make this prospect unlikely. We charge university users $25/room hour for both TEM and SEM, neither of which are equipped with EDAX, etc. We also charge for film. However, sputter-coating, fixation chemicals, resins, grids are all supplied by me. Industrial users are charged $50/room hour. The scope charge is supposed to cover service contract, parts and all prep chemicals, etc. In practice, the SEM costs about half as much as the TEM to run (including sample prep expenses) so the SEM is subsidizing the TEM. This is a friendly and efficient way of running a lab since people aren't being constantly nickel-and-dimed and the function of the lab is kept focused on providing researchers with the most useful services and supplies. Neither is time wasted by billing users for minor items. Furthermore, clients with small projects need not shell out major bucks just to get basic supplies. We used to charge about $1/print to cover paper, chemicals, and rapidprocessor repairs. The darkroom has turned into a general-purpose college facility with the result that the college pays our darkroom expenses. Users buy their paper from me at cost.
Please keep this information absolutely confidential!
Rod Kuehn
On Fri, 10 Dec 1993, Margaret E. Hogan wrote:
} } I am trying to get a feel for the amount to charge for EM services. I recently } took over an EM facility, and was asked to revamp the pricing for service work. } I am interested in any pricing schedule, from private, commercial or university } settings. Any help in this project would be GREATLY appreciated. All info } will be held strictly confidential. } Thanks!! } } Peggy Hogan } The Jackson Laboratory } 600 Main Street } Bar Harbor, Maine 04609 } (207) 288-3371 #1450
I have have been using diamond knives for cutting epoxy embedded tissues for about 20 years. Nearly all of the sections I cut are in the range of 0.5 - 2.0 micrometers in thickness. I started using a diamond knife that was no longer satisfactory for EM work because of small scratches. The sections I have made for LM are superior in quality. I have made 10,000-15,000 (maybe more) sections on a single 3mm knife that originally cost about $300. This knife has saved me many hours of time that would have been devoted to making glass knives. I am now using another knife (4.0 mm) with a larger boat that make makes flawless sections. The older knife is still useful but it makes more scratches than it once did. But, a few scratches are insignificant for some applications.
I assume you have had lots of experience with glass knives and know how to use your microtome skillfully. Be careful to avoid striking the knife edge with the specimen when you set up the microtome. Don't take big bites. Start cutting with specimen block-faces that are no larger than about 0.25 mm on a side (truncated pyramidal shape) and gradually work up to 0.5 mm on a side as you cut. Larger sections can be cut (if essential) after you have gained experience. Clean the edge (front and back) with a small, wetted wedge-shaped piece of polystyrine foam held with forceps. Wipe only parallel to the edge. It is remarkable that even after cutting many semi-thin sections the knives I use for LM will still cut nice looking silver and gold sections although I don't usually use these. I have never used Diatome "Histo Knifes" but I am confident that they would be satisfactory.
I bet there are many diamond knives siting around in drawers in laboratories that need to be resharpened for EM work. Before buying a new "Histo Knife" I suggest that you try to obtain a used diamond knife from someone who has given up EM work or has set aside of knife because of the high cost of resharpening.
I am interested in buying an old diamond knife for use in cutting sections for light microscopy. Anyone who is willing to part with an old, but unused favourite please contact me soon.
Bob
Bob Ridge Biology ICU 10-2 Osawa 3-chome Mitaka-shi Tokyo 181 Japan
Reply_ RE} } Greyscale Printers Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu I just invested in a Laserwriter Pro 630 to replace our aging IIg that had been upgraded from an NTX. At $1740 you cant complain! It is a 600dpi printer using a Canon EX engine. It makes a nice job of greyscale images. I printed a couple of wedges that Mark Disko sent me and It seems to do aboout 60 grey levels that I can count. I plan to use it for student research notebook printing, much cheaper than Polaroid! Faculty like it as it gives good images at a fraction of the cost! We use it for digital images collected from the TEMs, ESEM and AFM on to Apple Macs. We have the DI AFM connected to Appletalk and the software thinks it is printing directly to LPT2. Works well. I have seen output from the Hewlett Packard 600 dpi printers too and they are also excellent, maybe even a little better. I was driven by price considerations here tho' and the Apple was little cheaper and directly replaced our old printer with no additional network connection problems. Just my few cents worth. John Mansfield.
Reply_ RE} AFM to Appletalk question Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu
--------------------------------------
Would you mind providing some information on how did you connect your DI AFM to Appletalk (software and hardware).
Thank you,
Raul Fainchtein fain-at-aplcomm.jhuapl.edu
I am sending this to the list as I think there may be others who are interested in this. Since the AFM is run by a PC and our lab is still mainly Mac based as far as computers are concerned, we have pinters and file servers set up for the Macs, but little support for the PC. Therefore to print and store files on a server we purchased an ethernet card for the PC. We used a 3Com card, a 3C503, a really cheap basic card (ISA). We then bought the Farallon PhonenetPC software. Farallon bought the AppletalkPC software rights from Apple a number of years ago and changed the name. Farallon make a number of networking products, including Timbuktu which allows remote control of Mac and Windows PCs from remote machines of either type (scary seeing a Mac with a full windows interface!! and vice versa!!). The Phonenet software runs on the ethernet card (runs on almost any card these days) and supports multiple protocols, Appletalk, IPX and TCP/IP. You can have these loaded simulataneously, but it eats into the PC memory and the SPM software runs in reduced memory mode when the networking software is loaded on our 8 meg machine. DI have told us we can upgrade to 12meg (max) but we need new ROMs for the SPM hardware. This may help and we are in the process of ordering the upgrade. We hope we could run the full blown version of the SPM software and the networking software too. We could then try running ftp and telnet from the PC to enable wide area file transfer (I plan to try NCSA Telent for the PC). When the Appletalk stack is loaded (we use a number of cute little batch files to reboot the machine with different software configurations loaded --- A digression here, we have a 21M floptical and some of it's software is incompatible with the network software and so beware. Also it is very slow, if youy are thinking of getting one, my advice is don't! Pop the extra cash and buy a 128M mag-opt instead, much better bang for the buck! ---).
So as I say, when the Appletalk stack is loaded you can run a small program called DA which is a little like the Apple Chooser and you can then connect to Appletalk file servers and Appletalk printers and use either postscript or Epson emulation. We use postscript although it is very slow. Part of the reason for this is that the postscript spool file created by the SPM software is huge. It is a bitmap dump of the image screen of the microscope. Instead of using postscript to draw the text,lines and boxes on the screen and then merely bitmapping the image area, the whole screen is bitmapped so a large proportion of the spool file is either black or white space. This is why the text on DI SPM images that are printed on a postscript printer has jagged edges, the text is part of teh bitmap and not postscript fonts. Although I have been chastised in the past for implying that DI do some things on the cheap, I do think that the way the postscript printing has been implemented was a shortcut that was never cleaned up. For presentation purposes it would be nice to have the text of the image be printed in true postscript fonts. This would seriously cut down on the spool file size. I have asked about this in the past and was given the impression that it was a low priority item. Note: THIS IS MY PERSONAL OPINION AND SHOULD NOT BE TAKEN AS THE OPINION OF MY EMPLOYER. Anyway, inspite of this minor criticism, the Appletalk software and ethernet card allows us to share resources effectively. We print to our LaserWriter Pro 630 in our own lab and also to a Kodak Colorease PS300 in another building, saving us the cost of purchasing a dedicated printer for the SPM. We are currently investigating the possibility of using NetWare on the SPM to send print jobs to a NetWare file/print server which then spools the output to a number of printers. This may improve the speed and throughput.
Hope this helps, feel free to ask further questions if any of this is unclear. John Mansfield.
------------------ RFC822 Header Follows ------------------ Received: by mse.engin.umich.edu with SMTP;14 Dec 1993 09:36:36 U Received: from fainchteins_mac (fainchteins_mac.jhuapl.edu) by aplcomm.jhuapl.edu (4.1/SMI-4.1) id AA22735; Tue, 14 Dec 93 09:37:04 EST X-Mailer: InterCon TCP/Connect II 1.1 Message-Id: {9312140937.AA15363-at-fainchteins_mac.aplcomm.jhuapl.edu}
} Greetings, } Has anyone had exerience with a diamond knife designed to cut } semi-thin sections? That's correct, semi-thin sections, 0.2 - 5 um range. I } have just seen an ad for a product from Diatome called the "Histo Knife" } which claims to do just that. The main question would seem to be how many } sections such a knife can cut before needing to be resharpened. Advice or } comments welcome. I will post a summary of any private replies. } Thanks in advance, } Tobias Baskin } We have used the Diatome Histo knife in our lab for several years (where have you been?). They will cut about a gazillion sections then after they are resharpened they will cut a gazillion more.
R. A. Harris EM Facility Evolution and Ecology Univ. of Calif. Davis
I have already posed the following question to the NIH-IMAGE forum, but I would also like to know what members of this forum might suggest. I'm using an annular light ring, attached to my objective lens, to project a direct beam of white light onto my subject, which reflects some of that light back to my objective lens and video camera. This annular light ring isn't perfect in that it causes non-uniform illumination of my subject; in particular, the center of my subject is brighter than average and the edges are darker.
I can, of course, correct my acquired images for shading irregularities by applying any of various digital procedures, but this approach is also not per- fect. For example, to obtain a reference image for the digital shading cor- rection, I use a standard gray card that is well out of focus, which eliminates problems of the heterogeneous surface of the card itself. Unfortunately, the pattern and strength of illumination non-uniformities is dependent on distance from the light source, and the shading pattern on the out-of-focus reference is different enough from that of my in-focus image that the digital correction is not acceptable.
I suppose I can alter the mathematics of my digital shading correction, but that would be an emperical approach that is not satisfying in general. I'm now wondering about inserting a diffusing lens (a ring, ordoughnut, filter) between my light source and subject to convert the direct beam into a diffuse, flat wash of light onto my subject, regardless of its distance to the light source. Has anyone tried this, and if so, will it actually work to solve my problem? Are there any other suggestions?
Thanks in advance,
Paul Sheppard Laboratory of Tree-Ring Research University of Arizona, Tucson GRAD12-at-CCIT.ARIZONA.EDU
The last message I just saw posted on Microscopy Email forum about conducting an EMail campaign directed to the US Government in support of Basic Energy Sciences was not appropriate to this listserver.
Whether I agree or not with the message is irrelevant, the point is that it should NOT have been done without first clearing it with the SysOP, since the posting is not directly associated with Microscopy.
Clearing of messages being sent to a listserver when they DO NOT deal with the general subject matter is a common practice of Email etiquette, which is followed by most responsible users of systems such as this. Since this is an unmoderated listserver, each of us has the duty to police the system appropriately and I would like to remind you each of that obligation.
I will resend the list of rules to the person responsible ldm-at-apollo.numis.nwu.edu (L. D. Marks) for cluttering up the Microscopy Forum with the message.
We are in the process of evaluating LM/Digital Imaging systems for materials research and I would be grateful for any feedback on systems that are in use with regards to ease of use, capabilities, software support, etc. Thanks in advance...
Hi, To add further to the conversation about printers...Has anyone had experience using 1200 dpi laser printers to generate working (i.e. lab notebook-type quality) printouts for use in data acquisition of microscopic images (e.g. determining lengths of dendrites, counting objects). Of course, the images aren't good enough for publication, but the price of these printers is declining (I think I saw one in PC Magazine for about $2500) and the $/page is negligible. Thanks for any comments. Nancy Desmond Neurosurgery Univ. of Virginia ' Charlottesville, VA 22908 804.924.5607 (voice)
I'd like to elaborate on Ron Anderson's comments about how the LogE enlargers work. The newer versions (e.g. EM55) do not have condenser lenses, and the CRT is located directly above the negative, thus eliminating the need to keep any condenser lenses free of dust. The rastered spot on the CRT illuminates only that portion of the negative which is directly below the spot. Also, the photo detector does not measure light reflected from the printing paper, rather the beam splitter (fractionally-silvered mirror), which is below the negative and above the enlarging (objective) lens, reflects a portion of the light which has passed through the negative to a PMT. Thus the PMT and its circuitry measures the relative density of each portion of the negative and adjusts the brightness of the beam spot and its raster speed within a small range in an attempt to print all of the densities within the limited gamma of the printing paper. The electronics also allow the user to adjust or override the exposure decisions made by the enlarger.
Russell E. Cook Electron Microscopy Center for Materials Research Argonne National Laboratory Argonne, IL
} On a number of occasions, I have seen answers to posted notices but } I did not see the original posted notice. As an example, the current } discussion on autofill LN2 systems. The first message I saw was Ron } Anderson's response. I never saw the original message. It has happened on } other occasions. Any thoughts? My main concern is that I may be missing } topics of intrest or replies to messages I've posted.
The problem in many cases is on the recieving end not the posting end. The Mailserver frequently get messages that say something like:
"address not active, "machine is off-line" "address cannot be reached after N-days"
and several other variations. Some can be traced to the fact that some users turn their computers off and the various relay systems will only hold messages so long before they trash them. Others are due to network problems where a NameServer goes off-line etc..
This listserver trys several times to send a message through in case links go down, however, things donot always make it through. Sometimes the backlog of "Requeued Mail gets very large" and I have to clean out the backlog, otherwise the entire system comes to a halt.
You should remember that the mailing list is now OVER 500 subscriptions long and this takes a fair amount of negotiation. I avoid "erasing" names from the subscription list when there are problems as usually the networks address problems clear up in a day or so. If I see messages to the same host bouncing continuously for many days, then I remove that one from the list.
All the messages sent to the list are archived, but I have not gotten around to setting up a review/list etc.. At the moment I just don't have the time. SysOp for this List is only one of the HATS I wear here at ANL, and most of the development work has to be done on the cheap and off-hours at home after my kids get all their homework done and get to bed (Yawn)...
I've also been having abit of hardware problems lately with the main computer which server the ANL EMCenter and this listserver. I'm in the process of replacing it (at least the Purchase Order was filed a few days ago). It will likely take about a month or two (we are a Gov. Lab with all its paperwork) to get it in, installed, and debugged. I plan on trying to make the change over around late Feb, however, Murphy says it won't be that easy and/or simple.
In the interim, the list will have to bear with the glitches.
Sorry-- Nestor Z.
Right now maybe I can get back to my scope and get some work done! I've got some nice angle resolved, energy filtered, dispersion surfaces of graphite measured. Looks really great. Anyone else interested in that sort of thing???
This is in reference to the current topic of Liquid Nitrogen Auto-fill devices. We have never used an LN2 Auto-fill device on our system and from reading all the responses to this topic, I don't think that we will ever get one. I only need to fill our dewar twice a week. First thing Monday morning and on Friday afternoon before I leave for the weekend. We've never had any problems doing it this way. It seems like more trouble than it's worth. I just thought that I would put in my two cents.
We are now up to 4 Diatome Histo Knives. We serial section 4 mm long vestibular and auditory organs, decalcified temporal bones and cochlea, fetal stuff without decal at .5 to 5 microns, 25 micron methacrylate, etc. the oldest is 5-6 years old. Sharpening is largely a function of how often they get dropped, people overlap their dumonts when cutting dry, or cutting bone. They have paid for themselves many times over compared to the section quality and time cost of glass. Cutting cochlea or crunching through the otoconia of vestibular organs raises hell with glass but doesn't faze the histo knives. We would have used old thin sectioning diamonds, but didn't have any. We still use a lot of glass, especially for potentially destructive samples and for teaching. With the newer LKB knifemaker, we have been using thicker glass, which makes glass more attractive.
} } On Mon, 13 Dec 1993, Tobias Baskin wrote: } } } Greetings, } } Has anyone had exerience with a diamond knife designed to cut } } semi-thin sections? That's correct, semi-thin sections, 0.2 - 5 um range. I } } have just seen an ad for a product from Diatome called the "Histo Knife" } } which claims to do just that. The main question would seem to be how many } } sections such a knife can cut before needing to be resharpened. Advice or } } comments welcome. I will post a summary of any private replies. } } Thanks in advance, } } Tobias Baskin } Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Message-Id: {MAILQUEUE-101.931216081620.288-at-parmly1.parmly.luc.edu} To: Microscopy-at-anlemc.msd.anl.gov
} Date: Wed, 15 Dec 1993 12:48:38 -0600 (CST) } From: "Nestor J. Zaluzec (708)-252-5075, -4964" {ZALUZEC-at-anlemc.msd.anl.gov} } To: Microscopy-at-anlemc.msd.anl.gov } Cc: ZALUZEC-at-anlemc.msd.anl.gov } Subject: Missing Messages:
} To All Subscribers on Microscopy Forum: } } On Dec. 15. L.Sutter wrote: } } } On a number of occasions, I have seen answers to posted notices but } } I did not see the original posted notice. As an example, the current } } discussion on autofill LN2 systems. The first message I saw was Ron } } Anderson's response. I never saw the original message. It has happened on } } other occasions. Any thoughts? My main concern is that I may be missing } } topics of intrest or replies to messages I've posted. } } The problem in many cases is on the recieving end not the posting end. } The Mailserver frequently get messages that say something like: } } "address not active, } "machine is off-line" } "address cannot be reached after N-days" } } and several other variations. Some can be traced to the fact that } some users turn their computers off and the various relay systems } will only hold messages so long before they trash them. Others are } due to network problems where a NameServer goes off-line etc.. } } This listserver trys several times to send a message through in case links } go down, however, things donot always make it through. Sometimes the } backlog of "Requeued Mail gets very large" and I have to clean out } the backlog, otherwise the entire system comes to a halt. } } You should remember that the mailing list is now OVER 500 subscriptions long } and this takes a fair amount of negotiation. I avoid "erasing" names from } the subscription list when there are problems as usually the networks } address problems clear up in a day or so. If I see messages to the } same host bouncing continuously for many days, then I remove that one } from the list. } } All the messages sent to the list are archived, but I have not gotten } around to setting up a review/list etc.. At the moment I just don't have } the time. SysOp for this List is only one of the HATS I wear here } at ANL, and most of the development work has to be done on the cheap and } off-hours at home after my kids get all their homework done and get } to bed (Yawn)... } } I've also been having abit of hardware problems lately with the } main computer which server the ANL EMCenter and this listserver. } I'm in the process of replacing it (at least the Purchase Order was } filed a few days ago). It will likely take about a month or two } (we are a Gov. Lab with all its paperwork) to get it in, installed, } and debugged. I plan on trying to make the change over around } late Feb, however, Murphy says it won't be that easy and/or simple. } } In the interim, the list will have to bear with the glitches. } } Sorry-- Nestor Z. } } Right now maybe I can get back to my scope and get some work } done! I've got some nice angle resolved, energy filtered, dispersion } surfaces of graphite measured. Looks really great. Anyone else } interested in that sort of thing???
I've been having lots of trouble lately with undeliverable mail (Nestor: same as last time--then, one of the elves in Computer Headquarters had done a partial recompile of the system. This time...?) So this is a test to see if THIS works... But also...I'm not a materials person, but your nice graphite makes me recall an article from 2 or so years ago where some folks did STM on graphite & demonstrated things like DNA, proteins, etc.. Except, they were using just graphite. (I also haven't seen their work mentioned by anyone else.) The question is, do such structures show up in your preps in the TEM? Phil Oshel
I am new to this forum, and new to microscopy in general. We have recently set up equipment in our lab to study the microstructure of continous fiber reinforced composite materials. In specific we are trying to characterize the "fiber waviness" in composite laminates and cylinders. We are trying to view the fibers in plane (longwise) in hopes of characterizing the observed with some battery of FFT analysis, but this comes with many challenging hurdles, such as intermittent fiber breakages and also 3-D fiber curvature effects which sometimes take the fibers out of view. We are also trying to perform cross-section measurements, sort of a statistical sampling really.
We are grappling with a couple of issues. Optimum specimen preparation for light microscopy, we doing sort of modified metallographic grinding and polishing - but we still have trouble with too much fiber fracturing possibly linked back to the very first cut we did on the material. Anyone who has removed coupons of compostie material from laminates for microscopic evaluation, can you suggest a cost effective technique for this. Our laminates start at 1/8" thich and go up to almost 1/2" thick - presently we use diamond sectioning blades from Metallurgical Supply in Houston mounted on a milling machine at 1400 rpm with thwe table on slowest pass to cut the plate into strips and then we bring the strips to our Buehler Isomet 2000 to make small chips to pot, grind, and polish. This requires a skilled operator in the machine shop, it is very time intensive and still we have fiber fracturing. Is there anyone out there using as tabletop unit witha feed mechainsm like a table saw? Any suggestions?
We are also getting more surface relief thsn we can cope with in the final specimen. Our material is a polysulfone matrix with 7 micron diameter graphite fibers, the matrix is softer than the fibers so we get pitting very easily. We have been able to minimize this by diddling with the polishing sequence. If anyone has tackled this problem and has any insight we would appreciate it.
Thirdly we are using mostly brightfield illumination, but we don't get very good contrast. We are using a Leica MeF3-A metallograph and the contrast just isn't sharp enough or "broad" enough for our subsequent image analysis needs - i.e. can't be thresholded easily. We have tried darkfield which appears to give us sharper contrast but over an even "tighter" range. Has anyone got any experience with this one?
bruce-at-macgate.di.com writes: (regarding dye sublimation printers) } These printers are a sharable resource. Connect your whole department to } it (and get them to help pay for it)
I agree, but do be careful when hooking a dye sublimation printer to a network. Too many times I have had to shut ours down because someone down the hall or outside the building has neglected to change the Chooser setting before submitting a lengthy text file.
Yet another UK microscopy meeting to add to the comprehensive list sent by NJZ on November 9th.
8th September 1994 High Resolution Transmission Electron Microcopy Institute of Physics Headquarters, Belgrave Square, London, UK
This one-day meeting is being organised by the IOP EMAG group in conjunction with the Institute of Materials Metal Science Committee to discuss recent developments in the techniques and applications of HREM. The meeting will consist of overviews by invited speakers including Prof. C.J. Humphries and Dr. J. Hutchison, followed by contributed talks. Prospective speakers are invited to submit abstracts of {250 words before 31st May 1994 to M. Aindow, at the address below, indicating a preference for a 12 or 25 minute talk. Further details will be available from the IOP meetings department shortly.
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.
The mailer appeared to bounce my last attempt to post this - my apologies if you receive two copies of the same message;
Yet another UK microscopy meeting to add to the comprehensive list sent by NJZ on November 9th.
8th September 1994 High Resolution Transmission Electron Microscopy Institute of Physics Headquarters, Belgrave Square, London, UK
This one-day meeting is being organised by the IOP EMAG group in conjunction with the Institute of Materials Metal Science Committee to discuss recent developments in the techniques and applications of HREM. The meeting will consist of overviews by invited speakers including Prof. C.J. Humphreys and Dr. J. Hutchison, followed by contributed talks. Prospective speakers are invited to submit abstracts of {250 words before 31st May 1994 to M. Aindow, at the address below, indicating a preference for a 12 or 25 minute talk. Further details will be available from the IOP meetings department shortly.
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.
This did not go out via the official route as the news.groups official posting has closed down for the holiday. I think we may have enough votes but just in case, if you know someone, with an interest in the newsgroup, who has not voted then please encourage them to do so. Thanks and compliments of the season to you all. John Mansfield. I have forwarded this to the confocal mailing list, the microscopy mailing list and the NIH-Image mailing list.
-------------------------------------- Second CALL FOR VOTES (of 2)
Unmoderated group sci.techniques.microscopy
Newsgroups line:
sci.techniques.microscopy The field of microscopy.
Votes must be received by 23:59:59 UTC, 2 January 1994.
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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.
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Many years ago, at another institution, I used a naphthyl thio acetate esterase histochemical reaction to mark histocytes and noticed that pericytes seemed to react and be labelled. Could never find any refs for esterases in pericytes and we lacked the funds to exhaustively check it out. This might help.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Fri, 17 Dec 1993, Martin B Garment wrote:
} Does any one know of a light microscopy stain that will differentiate between } endothelial nuclei and pericyte nuclei in capillaries, or of an antibody marker } specific for pericytes? } Martin Garment, UW-Madison, Madison, WI } mgarment-at-macc.wisc.edu }
Subject: Time:12:58 PM OFFICE MEMO TEM- JEOL 1010 Date:12/21/93 I'm new to the list and not sure if this inquiry is appropriate for the types of topics discussed on this list. If this type of inquiry is too specific or inappropriate please let me know. We purchased a JEOL 1010 TEM about 1 year ago and have had continual problemns with photography on the scope. We have approx.7 experienced TEM users (} 6 years experience) and several less experienced users which are all experiencing the same problem. Simply, when looking at images on the screen they are in focus, but when we photograph them, it becomes a lottery as to whether the images on the negative are in focus. JEOL has been out several times and has given the scope a clean bill of health each time using standard calibration grids. At their last visit they used our specimans and experienced the same problem. They have concluded that the problem may be speciman related, however, we are skeptical since it is present for a variety of users using different resins, tissue types etc.. Further, when we take these grids to other facilities in the area ( a Philips and a JEOL 1200) we seem not to have this problem. Does anyone else have experience, good or bad with this scope, or have any suggestions as to what the problem might be? We have been told by the service representitives that we are the only 1010 installation in the Eastern United States (we are in New Haven, CT). Does anyone on the list in the have this scope in the Eastern service region for JEOL?
Mike Sect. of Neurobiology Yale Univ. School of Medicine New Haven, CT 06510 Mike_Schwartz-at-qm.yale.edu
Subject: Time:2:44 PM OFFICE MEMO JEM-1010 Reply Date:12/21/93 I have not had direct experience with the model TEM you mention, but have managed electron microscopy labs for a quarter of a century. Users traditionally blame their problems on the instruments, thereby putting the burden of proof back on the Lab Manager. In a case such as this, the best thing to do is to obtain a reliable specimen, and keep it on hand to check the instrument whenever such a problem arises. In that way, you know whether it is the instrument or the users' specimens. I would suggest the 'Image Checker' specimen (No. 10070) and/or the 'Resolution Standard' specimen (No. 10090) supplied by Fullam (although equally suitable specimens are certainly available from other companies that handle EM supplies). Also, be sure that the specimen holder is clean, and that the specimens are being mounted in it so that they are held firmly and make good thermal contact. If this approach doens't clarify the situation to your satisfaction, then you really do have a problem.
} I'm new to the list and not sure if this inquiry is appropriate for the types } of topics discussed on this list. If this type of inquiry is too specific or } inappropriate please let me know. We purchased a JEOL 1010 TEM about 1 year } ago and have had continual problemns with photography on the scope. We have } approx.7 experienced TEM users (} 6 years experience) and several less } experienced users which are all experiencing the same problem. Simply, when } looking at images on the screen they are in focus, but when we photograph them, } it becomes a lottery as to whether the images on the negative are in focus.
This kind of question is very appropriate, Mike.
I manage a lab that has, among other equipment, a JEOL 2000 EX II. I'm not sure what the similarities are with your scope, but I do know that there are things to watch out for with these computer-controlled machines. Many users of our lab don't seem to understand that the scope is similar to a 200 CX. A major difference is that a computer controls the lenses, deflectors, etc, and stores the settings digitally. The guts of the scope, however, is still a series of analog devices, and they do drift. The computer helps maintain better control, but it is not magic.
I think that it is very important that users understand how to do the daily alignment procedures. They are easy to do. We use a brief instruction sheet to help them. I tweak the alignment periodically, to keep it close at all accelerating voltage ranges.
I agree with what Wil said. I have come to trust the machine, until I can absolutely rule out the specimen as the problem. Multi-user facilities are difficult in this regard.
If there is a problem with the scope, I know that JEOL will work with you to fix it. Good luck, and please post again when you find the problem. The solution is just as important as the problem.
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Mike Schwartz's focusing problem Before trying to make suggestions,it would be helpful to have the answers to a few basic questions. 1. Do you use "holey grids" and examine carbon holes to correct for astigmatism? 2. Have you made a recent through focus series of micrographs of carbon holes? 3. Have you made a recent trough-focus series of micrographs of well stained sections of standard histological material? 4. What criteria do you use in evaluating the focus of your negatives? 5. Are you familiar with the practice or art of obtaining "critical underfocus"? RAC
On Tue, 21 Dec 1993 23:26:42 +0200, John Chandler wrote:
} If there is a problem with the scope, I know that JEOL will work with you } to fix it. Good luck, and please post again when you find the problem. } The solution is just as important as the problem. } } John chandler-at-lamar.ColoState.EDU Fort Collins, CO
I agree with John about Jeol to help solving the problem. However, I would add one checkpoint for you. Do you focus using the image wobbler? If so, do you usually have the Optimum Under Focus switch ON? We have in my management a JEM 100C, JEM 100SX and JEM 1200EX, which all have the OUF switch. I have had to switch OFF the OUF because it seemed to bring the images too far underfocus.
Bet regards for Merry Christmas and a happy new year to all microscopy- netters
Jouko Maki
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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
} I agree with John about Jeol to help solving the problem. However, I would } add one checkpoint for you. Do you focus using the image wobbler? If so, do } you usually have the Optimum Under Focus switch ON? We have in my } management a JEM 100C, JEM 100SX and JEM 1200EX, which all have the OUF } switch. I have had to switch OFF the OUF because it seemed to bring the } images too far underfocus.
Yes, I'd forgot about the OUF, because we leave ours turned off. I don't think, however, that this would be the cause of inconsistent focus problems.
As for performance of various OUF's on JEOL's, when I was in a different lab with two 100CX's, we always had the OUF on. We always got beautiful negatives with it up to about 20K mag. I learned to trust it. There was one setting; it was ON or OFF. The 2000EX has OFF and three ON settings, low, medium and high. the medium setting is supposed to be equivalent to the ON setting of the 100CX.
One of the reasons we don't leave ours on now is because our heaviest users are used to the wobbler on the Philips 400. I haven't used one of these for years, but they remind me that it is bang on, even at 100K mag. They still use the wobbler on the JEOL to get close, then fine focus by eye. I have to keep reminding them that the two are different and not to expect them to behave the same.
Cheers,
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
At ANL we sometimes use a combination of crushed dry ice and then methanol in the cooling holder this get you to about -30C. Does your cooling holder have a heater? The Gatan models do, in that case just turn up the heater until you reach a stable temp. It's a bit of guess work but it sometimes is sufficient. The only problem with this method is if the heater oscillates turning on and off, then you get specimen drift.
I need help in determining the best way to prepare a sample for TEM analysis. The specimens are secretory vesicles isolated from yeast. They are in a 0.8M sorbital buffer, to prevent osmotic damage. I was given approximately 50uL of each prep. The goal is to see the ratio of vesicles to sheets of plasma membrane in the prep. The vesicles should be mostly lipid. I wondered about osmium vapor fixation on a formvar grid, or some associated "negative" stain technique. I don't have a lot of specimen to do a routine TEM prep. Any help/suggestions would be greatly appreciated. Thanks in advance. Randy Nessler rnessler-at-emiris.iaf.uiowa.edu
Mike You did not explain "out of focus." When your negative is examined with an 8x lupe, is the grain "stretched" from astigmatism or drift? Or is the grain just very large? Or are the images just lacking in contrast?
Drift problems could be aggravated by excessively long exposures or too powerful an electron beam. Long exposures could be forced by excessively thick sections for your kv. High intensities result from improper "emission" or "bias"/filament heating combinations.
High kv seriously reduce contrast for biologicals.
Contrast can be seriously degraded by underexposure (intensity x exposure time too short) or underdevelopment caused by use of exhausted or cool developer.
Some scopes have low image contrast. Biological samples on such a microscope look crisp only when seriously underfocused so the problem isn't apparent until the negatives are examined. A standard is an inherently more contrasty subject. If it looks good in the scope, it is correctly focused. A good way of checking this would be to examine a section containing a hole. Focus on the membranes, then check the optical fringes in the hole.
Alternatively, your microscopists (being accustomed to a different scope) may be shooting too close to true focus, which delivers a muddy, low-contrast image with biologicals.
Good luck.
Rod Kuehn
} Subject: Time:12:58 PM } OFFICE MEMO TEM- JEOL 1010 Date:12/21/93 } experienced users which are all experiencing the same problem. Simply, when } looking at images on the screen they are in focus, but when we photograph them, } it becomes a lottery as to whether the images on the negative are in focus. } JEOL has been out several times and has given the scope a clean bill of health } each time using standard calibration grids. At their last visit they used our } specimans and experienced the same problem. They have concluded that the } problem may be speciman related, however, we are skeptical since it is present } for a variety of users using different resins, tissue types etc.. Further, } when we take these grids to other facilities in the area ( a Philips and a JEOL } 1200) we seem not to have this problem. Does anyone else have experience, good } or bad with this scope, or have any suggestions as to what the problem might } be? We have been told by the service representitives that we are the only 1010 } installation in the Eastern United States (we are in New Haven, CT). Does } anyone on the list in the have this scope in the Eastern service region for } JEOL? } } Mike } Sect. of Neurobiology } Yale Univ. School of Medicine } New Haven, CT 06510 } Mike_Schwartz-at-qm.yale.edu } }
Subject: Time:1:28 PM OFFICE MEMO JEM-1010 reply2 Date:12/22/93 In describing your problem, you simply stated that the images appeared to be "out of focus". This is a very specific problem that should only result from a variation in the current of one of the image-forming lenses, from variation in the high voltage, or from such effect as that of the OUF device, or perhaps from variation in the current in some device such as a set of deflecting or stigmator coils. All of these are electronic problems, and it should be possible for service engineers to check them out (i.e. to see if all likely circuits performing to specifications. UNSHARP images can be caused by a variety of other factors, however. As already suggested by others, specimen drift is a very common cause. For a discussion of sources and methods of diagnosing such instabilities you might want to read over Ch. 9 (Instabilities) in the book by J.C.H. Spence "Experimental High-Resolution Electron Microscopy", and Ch. 5 (Checking the performance of an EM) in the book "Principles & Practice of EM Operation" by A. W. Agar, et. al. which was published in the series "Practical Methods in Electron Microscopy" A. M. Glauert, Ed.
Grain-Boundary Characterization By Conventional TEM: A Survey Of Current Techniques
STUART MCKERNAN AND C. BARRY CARTER
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN
ABSTRACT
The study of grain boundaries by transmission electron microscopy is now a mature field. Much of the research effort has focused on the analysis of a small set of special grain boundaries by high-resolution electron microscopy. More general boundaries necessarily have to be studied by other techniques. This paper will review the techniques currently available for the characterization of these grain boundaries and consider lines of future research.
National Center for Electron Microscopy, Lawrence Berkeley Laboratory, Berkeley, CA
ABSTRACT
This paper reviews recent progress in high resolution electron microscopy (HREM) of internal interfaces in solids. A brief summary of generic interface features of interest is followed by specific examples of HREM investigations of grain boundaries and heterophase interfaces. Features of interest include interface roughness, rigid body displacements, faceting, steps or ledges, elastic displacement fields, atomic bonding, composition gradients and localized atomic relaxation into structural units. Two types of analysis, direct interpretation and comparison with image simulations, are applicable to different types of interface characteristics. Critical issues of current importance and recent developments in technique are outlined for asymmetrical and symmetrical grain boundaries and for metal-semiconductor and metal-ceramic interfaces.
Synchrotron X-Ray Topographic Studies of Grain Boundaries
FUPING LIU, IAN BAKER
Thayer School of Engineering, Dartmouth College, Hanover, NH
ABSTRACT
Synchrotron white beam X-ray topography (SWBXT) is a powerful tool for studying the evolution of structural changes in bulk crystalline materials. This paper outlines the use of SWBXT in the transmission Laue geometry for studying grain boundaries (GBs). The advantages and disadvantages associated with SWBXT are described with the emphasis on in situ observations of the response of GBs to applied stress, thermal treatment and dislocation impingement.
Compositional Analysis of Interfaces Using X-ray Spectroscopy
ERNEST L. HALL
GE Corporate Research and Development, Schenectady, NY
ABSTRACT
In this paper, we examine the use of x-ray spectroscopy in the AEM to study compositional changes at interfaces in materials. The proper sample and microscope conditions for optimum data collection are discussed. The determination of the spatial resolution of the analysis, and the effect of spatial resolution on the analytical results, are described. Methods for deconvoluting the electron distribution in the sample from the solute distribution are reviewed. The effect of the minimum mass fraction detectable on this type of analysis is shown. Finally, examples of the use of x-ray spectroscopy to measure equilibrium and non-equilibrium composition variations at grain boundaries and interphase interfaces are shown.
EELS at Buried Interfaces: Pushing Towards Atomic Resolution
P.E. BATSON,1 N.D. BROWNING,2 and D.A. MULLER3
1IBM Thomas J. Watson Research Center, Yorktown Heights, NY; 2Oak Ridge National Laboratory, Oak Ridge, TN; 3Department of Applied and Engineering Physics, Cornell University, Ithaca, NY
ABSTRACT
Recently available increases in the sensitivity of electron spectrometry has allowed usable EELS signals to be obtained with the 2 sized probe needed to produce annular dark field (ADF) channeling contrast at 100 KeV. Three applications of this performance are discussed: 1) bonding and electronic structure obtained at a Si/SiO2 interface, 2) elemental Co composition at a CoSi2/Si interface, and 3) imaging using the ã and å carbon transitions at a diamond/Si interface.
Atom-Probe Field-Ion Microscope Studies of the Chemistry of Internal Interfaces on an Atomic Scale
DAVID N. SEIDMAN, BRUCE W. KRAKAUER AND DAVID K. CHAN
Materials Science and Engineering Department and the Materials Research Center, Northwestern University, Evanston, IL
ABSTRACT
We explain the basic physical principles of both the field-ion and atom-probe microscopes with an emphasis on the atomic-scale imaging of atoms, and the concurrent measurement of the chemical identities of individual pre-selected atoms--on the surface of a field-ion microscope specimen--by time-of-flight mass spectroscopy. The advantages and disadvantages of atom-probe microscopy for the study of interfacial chemistry are enumerated. It is shown how an individual internal interface may be prelocated employing transmission electron microscopy, and then the same interface studied via atom-probe microscopy. The atomic-scale spatial-resolution capability of the atom-probe technique for studying interfacial chemistry is illustrated with two specific examples. The first one involves the direct and absolute measurement of the Gibbsian interfacial excess of solute at a grain boundary--a homophase interface in the Fe(Si) system; the depth resolution for the Si solute atom profile associated with the grain boundary is } 0.07 nm. The second application is the study of a metal/ceramic heterophase interface--the cadmium-oxide/silver {222} interface. It is demonstrated, via atom-probe microscopy, that the terminating {222} plane of CdO is the anion plane and not the cation plane; this result corresponds to a depth resolution of 0.136 nm and 0.118 nm in cadmium-oxide and silver, respectively, along a {111} direction. These examples illustrate the unique capabilities of an atom-probe to make standardless and quantitative measurements of interfacial chemistry on an atomic scale.
Three-Dimensional Atom Probe Studies of Solid-Solid Interfaces
THOMAS F. KELLY1,2,3, PATRICK P. CAMUS2, DAVID J. LARSON2,3, AND LOUIS M. HOLZMAN2,3
1Department of Materials Science and Engineering; 2Applied Superconductivity Center; 3Materials Science Program; University of Wisconsin, Madison, WI
ABSTRACT
The origins and underlying concepts behind the three-dimensional atom probe (3DAP) are described. Application of the 3DAP to the study of solid-solid interfaces is discussed in terms of the fundamental limitations of this technique compared with those of other characterization techniques, especially the conventional atom probe. Several examples of actual images from existing 3DAPs are shown with emphasis on their relevance to the study of solid-solid interfaces. Future developments in this form of microscopy which might have beneficial impact on the study of solid-solid interfaces are discussed.
The Tangent Formula in Electron Crystallography--Phase Determination of Copper Perchlorophthalocyanine
DOUGLAS L. DORSET1, MARY P. MCCOURT1, JOHN R. FRYER2, WILLIAM F. TIVOL3, JAMES N. TURNER3
1Electron Diffraction Department, Medical Foundation of Buffalo, Inc. Buffalo, NY; 2Electron Microscope Centre, Chemistry Building, University of Glasgow, Glasgow G12 8QQ, Scotland; 3Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY
ABSTRACT
The tangent formula as an appropriate method for phase determination in electron crystallography was evaluated using 198 experimental intensity data from ca 100 thick, epitaxially-oriented microcrystals of copper perchlorophthalocyanine. If the basis set used for the analysis (here with QTAN) is too small, the phase determination is unsuccessful. This agrees with independent assessments in another laboratory with MULTAN or RANTAN. However, a basis phase set of 27 reflections obtained from evaluation of three- and four-phase invariant sums is sufficient to phase 137 reflections, yielding Eh maps at lowest NQEST values that are readily interpreted in terms of atomic positions. This is an even smaller basis set than the e.g. 47 reflections at 2 resolution obtained from the Fourier transform of electron microscope images, which were shown earlier to be adequate for a successful phase extension.
The History of The Development of The First High-Resolution Electron Microscope
Remembering the Knoll Research Team at The Technical University Berlin, 1927-1934.
MARTIN M. FREUNDLICH
ABSTRACT
The first high-resolution electron microscope was developed by Ernst Ruska working first in cooperation with Max Knoll and later with Bodo V. Borries at the High-Tension Laboratory of the Technical University of Berlin. Though the electron microscope (EM) became one of the greatest and most far reaching achievements of the twentieth century, Ruska had to wait 53 years before being honored with the Nobel Prize. It took this long to sort out the merits of Reinhold Rudenberg's claim to be the sole inventor of the EM. He applied for a patent just 5 days before Knoll reported on the first low-resolution EM.
IBM Research Division, Zurich Research Laboratory, Sumerstr. 4, CH-8803 Rschlikon
ABSTRACT
An introduction to the principles, applications and perspectives of magnetic force microscopy (MFM) is given. Selected examples from magnetic recording as well as from fundamental research in magnetism are presented to demonstrate the type of information presently obtainable by MFM. Factors determining resolution are briefly discussed and some future perspectives for this method are described.
} I am currently working with marine orgasnisms and would appreciate any } suggestions for decalcifing them for em studies. } } Happy Holidays to all! } } Phil Rutledge } Email: prutle1-at-gl.umbc.edu
For EM of auditory receptors we used a decalcifying procedure first published by Baird, Winborn and Bockman (Anat. Rec. 159:281-290, 1967). This was to get rid of otoconia and any remaining otic capsule.
Briefly, we fixed in 4.0% aldehyde in 0.2 M s-collidine buffer with 2.0% sucrose. After buffer washes, specimens were decalcified using 0.1 M tetrasodium ethylenediamine tetraacetic acid (Na4EDTA), pH 7.4 (adjusted using versene acid), with 4.0% glutaraldehyde. Decalcifier was changed every other day, until decalcification was complete, usually 4-6 days. This was followed by post-fixation in OsO4 and normal processing for TEM or SEM. I don't think choice of buffer will affect decalcification.
A friend used a similar, but much higher concentration of EDTA, for dental specimens. EM preservation was still excellent with the higher concentration.
Good luck and Happy Holidays!
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
I've used Warshawsky's EDTA for mammalian cochlea, mammalian and avian temporal bones with good results for EM and LM EDTA-2Na 0.111M 41.3 gm NaOH 0.11 M 44 gm Take to 1 L pH should be 7.4 decal tissues at 4 deg. C and change the solution every couple of days. 10% EDTA works ok, takes more NaOH to get the pH back up, Mori (J. Histochem. Cytochem. has a glycerol/EDTA solution forimmunocytochemisty at the EM level.. All of these work, the Mori is more trouble, I mostly stick with Warshawsky's.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 22 Dec 1993, rutledge phil wrote:
} I am currently working with marine orgasnisms and would appreciate any } suggestions for decalcifing them for em studies. } } Happy Holidays to all! } } Phil Rutledge } Email: prutle1-at-gl.umbc.edu } }
On Wed, 22 Dec 1993 17:34:33 +0200, John Chandler wrote:
} Yes, I'd forgot about the OUF, because we leave ours turned off. } } One of the reasons we don't leave ours on now is because our heaviest users } are used to the wobbler on the Philips 400. I haven't used one of these } for years, but they remind me that it is bang on, even at 100K mag. They } still use the wobbler on the JEOL to get close, then fine focus by eye. I } have to keep reminding them that the two are different and not to expect } them to behave the same. } } Cheers, } } John chandler-at-lamar.ColoState.EDU Fort Collins, CO
Hi again,
In case your problem occurs with a standard specimen, e.g. holey carbon, I strongly suggest you should build up heavy pressure against JEOL service people to find out the reason. It can be even a faulty DAC in the OL control circuit. Before going further you have to explain the exact way you make the exposures - that means the whole procedure. There may be some typical pattern how every user operates and that may help when analysing the reason for the problem. Make a point-to-point list of the procedures and try to find if there is a step which all operators do and which could cause a misfocussing if there exists an electronic fault. I have faced some DAC-troubles in our 1200EX, which is less integrated than 1010. Another source of component trouble is the optocouplers which are used to change the control voltage. I had to change each of them to a more reliable type and that was a lot of work. I hope you can solve the problem with the help of JEOL-people. It only takes some time to get it done.
Best regards,
Jouko
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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
} Date: Wed, 22 Dec 1993 17:06:13 -0500 (EST) } From: rutledge phil {prutle1-at-gl.umbc.edu} } Subject: decalcification } To: microscopy-at-anlemc.msd.anl.gov
} I am currently working with marine orgasnisms and would appreciate any } suggestions for decalcifing them for em studies. } } Happy Holidays to all! } } Phil Rutledge } Email: prutle1-at-gl.umbc.edu } Which marine orgs? Crustaceans? (if so, try the crust- l-at-sivm.si.edu mailserver at the Smithsonian), molluscs? forams? etc. What have you tried? such as EDTA? Phil Oshel po-at-parmly1.parmly.luc.edu
Subject: Time:8:47 AM OFFICE MEMO JEOL 1010 cont. Date:12/23/93 My thanks to the many individuals which have offered suggestions concerning our focusing problem. We are now systematically performing a variety of the solutions/tests that many of you have recommended. Unfortunately, the holidays have intervened and we will not complete this analysis till after the the 1st of the year. I will let you know the results when we are done. Just a little clarification on the nature of the focus problems and our previuos attempts to remedy them. The negatives are not dramatically out of focus look as if there is some drift in the specimans as several of you have suggested. Our initial attempts to address this problem included using 60Kv versus 80Kv as the accelerating voltage, using the wobbler to focus versus focusing "by eye", turning the under focus on or off, using different spot sizes, testing different embedding media (Epon/Arraldite vs. Durcapan) and using slot versus mesh grids. The tissue we use is brain tissue and we have even tested whether the quality of fixation, i.e. lightly fixed for immunohistochemistry or well fixed, makes a difference. While several of these parameters aggravate the problem, as one might expect, none of them rectified the problem. However, it is safe to say that there are certain types of specimans that are less likely to photograph out of focus such as holey grids used for alignment. Although all of these tests suggest that there may be a problem with the specimans, we are disturbed that this problem does not show itself with other scopes. We have taken the "exact" same grids and photographed them on a Phillips and a JEOL 1200 within our building and not experienced these problems. We also had no problem with the JEOL 100S that we traded in for the more sophisticated and versatile 1010. It is also very difficult to assume user error since 4 of us have over 15 years of TEM experience. Although we have tried many solutions, as I said, many of you have provided additional approaches which we have yet to attempt and we are hopeful that one of these may provide a solution. Thanks again for all the advice, and I'll keep you posted on the outcome.
Mike Sect. Neurobiology Yale Univ. Sch. Med. Mike_Schwartz-at-qm.yale.edu
Looking into relative merits of wet etching and plasma etching glass from microelectronic devices. There appears to be a bias towards plasma etching as being cleaner and less destructive to metallization under glass. We have used wet etching (Buffered HF solution) without any noticeable effect on metallization. However, the wet etch is at times incomplete and some areas of the die may have residue remaining. Any comments would be appreciated.
Also I would appreciate any information on the commercial plasma etchers available and their relative merits. Cost considerations are primary.
Thanks.
Richard Sartore at RSARTORE-at-MONMOUTH-ETDL1.ARMY.MIL
US ARMY RESEARCH LABORATORY AMSRL-EP-RA FORT MONMOUTH, NJ 07703-5601
Subject: Time:2:08 PM OFFICE MEMO TEM JEM-1010 reply3 Date:12/23/93 I strongly disagree with Ron Anderson's comments that the on-going discussion of the problem with 'unfocused' images on the JEM-1010 is inappropriate to this forum. I have used JEOL TEMs for many years, and agree that they are splendid instruments. In fact, I have probably been one of JEOL's strongest supporters. HOWEVER, subtle problems can, and unfortunately do, occur on virtually every model instrument, and are likely to be more difficult to diagnose on the more sophisticated models. Furthermore, service engineers are only human, and consequently the attention they provide varies with their individual background, their personal problems at any given moment, and a variety of other factors. I have also encountered a couple of service engineers, admittedly EXCEPTIONS to the general rule, who were outright belligerent and only marginally competent. I have not seen anything in this series of comments that should be taken as detrimental to JEOL instruments in general. The fact that the service engineer obtains what he claims are good micrographs suggests that the problem is in the users specimens or their techniques. (If faulty technique in using the instrument is the problem, then a good service engineer should be able to be helpful in overcoming it.) The fact that good micrographs are obtained from the users' specimens on other instruments (some of which are JEOL) suggests a problem with this particular instrument. Although I do not know them personally, at least some of the users involved here apparently are microscopists with a great deal of experience, and they seem to be taking an OBJECTIVE approach to attempting to obtain help with what is to them a very vexing problem. I see nothing wrong with discussing methods of resolving a situation of this kind. In fact, this is the kind of thing a forum such as this can be most helpful in dealing with.
Subject: Time:3:09 PM OFFICE MEMO JEM-1010 reply2 Date:12/23/93 OFFICE MEMO JEM-1010 reply2 Date:12/22/93 In describing your problem, you simply stated that the images appeared to be "out of focus". This is a very specific problem that should only result from a variation in the current of one of the image-forming lenses, from variation in the high voltage, or from such effect as that of the OUF device, or perhaps from variation in the current in some device such as a set of deflecting or stigmator coils. All of these are electronic problems, and it should be possible for service engineers to check them out (i.e. to see if all likely circuits are performing to specifications. UNSHARP images can be caused by a variety of other factors, however. As already suggested , specimen drift is a very common cause. For a discussion of this and other sources and methods of diagnosing such instabilities you might want to read over Ch. 9 (Instabilities) in the book by J.C.H. Spence "Experimental High-Resolution Electron Microscopy", and Ch. 5 (Checking the performance of an EM) in the book "Principles & Practice of EM Operation" by A. W. Agar, et. al. which was published in the series "Practical Methods in Electron Microscopy" A. M. Glauert, Ed.
Subject: Time:11:02 AM OFFICE MEMO JEOL 1010 Ethics Date:12/24/93 I would like to respond to Ron Anderson's comments that the discussion of our focusing difficulties is unethical and detrimental to JEOL. Firstly, we are long time users of JEOL TEMs and purchased the 1010 on the basis of our satisfaction with our prior JEOL TEM and our feeling the JEOL service has always been exempelary. My queries to this forum were not intended to impune the well deserved reputation for high quality that JEOL has come to enjoy as a result of good service and product. Rather, we have been trying to determine, in anyway possible, how we might improve the performance of the 1010 using both JEOL's resources, as well as those of any other informed sources. This serves to beneifit ourselves, may be of use to service technicians from JEOL which may have had only limited experince with this relatively new machine and may prove useful to future and current users of the JEOL 1010. We would not have even put this issue before the group if we had not obtained excellent results with the same tissue, grids and magnifications on other JEOL and Philips TEMs in our facility. Perhaps the 1010 is more sensitive to speciman parameters, if so, it is important that we and others know this. On the other hand many members of this list have provide several excellent suggestions for things to look at with regard to speciman preparation and possible machine tuning which we and the JEOL people had not yet explored. We are hopeful that these may help us resolve our problems. Perhaps, many members of the list feel that it is unethical to discuss particular products. If so, this should be made a part of the rules governing the use of the list which are sent to new members such as myself. However, I feel that I have benefited from feedback on particular commercial products on other lists. This has often saved me from purchasing inappropriate or substandard equipment or products.
I think the JEOL discussion has run it's course, let's let it cool off!
Part of the percieved problems MAY be due to the fact that messages occassionally get lost/trashed and not all subscribers see EVERY message and hence all readers do not see the entire discussion (I'm working on trying to fix this it appears to be an intermittent problems which randomly affects the listserver, and not the same individuals every time!).
Let me add the following:
* It is appropriate to discuss instrument problems. * It is appropriate to discuss specific manufacturers. * It is not appropriate to abuse a specific manufacturer user, or agency. * I read every message and take most with a large grain of salt, remember the ultimate goal of this forum is to let Microscopists help each other, and no one is perfect not even me :-)
I think there was abit of over-reaction here, but that's okay once and awhile. I did not think the discussion had gone out of bound yet, both points of view had valid points about making sure things were covered and still to remind everyone that a rumor could be ruinous to a company, should it not be true.
So to end on a more cheery note:
--------------------------------------------------------------- The Microscopy Listserver wishes everyone a good holiday season ---------------------------------------------------------------
Nestor Z. ANL EM Center ===============================================================
Electron Beams, Ion Beams (with apologies to the authors of Jingle Bells)
Dashing down the column traveling at hundreds of K. Through the sample they go scattering along the way. Mag and Beam up high making screens glow bright Oh what fun it is to see - atoms- day or night.
Electron Beams, Ion Beams, Photons on the Way! Oh what fun it is to be, in the E-M-C to-day T-E-M's, S-E-M's, Op-ti-cal scopes too! Scru-ti-nizing matter is what we love to do. E-D-S., E-L-S., Auger spectra too! Analyzing data it's all in store for you.
Electron Beams, Ion Beams, Photons on the Way! Oh what fun it is to see - atoms- day or night. A-F-M's, S-T-M's, Confocal scopes abound. We're here to study matter - from the whole world all around. Microtomes, Diamond Wheels, Lectro-polishing too. all this prep equipment and answers to be found..
Electron Beams, Ion Beams, Photons on the Way! Oh what fun it is to see - atoms- day or night. C-C-D's, V-C-R's, Video screens glowing bright displaying lots of data far into the night. Macrographs, - Micrographs, - Computed pictures too! Far too many pixels - to know with, what to do!
Electron Beams, Ion Beams, Photons on the Way! Oh what fun it is to see - atoms- day or night. Happy Holiday's from Us - and a Prosperous New Year too!
-------------------------------------------------------------- All the crew of the ANL EM Center:
Russ, Ed, Stan, Bob, Roseann, Charlie & Nestor --------------------------------------------------------------
On Thu, 23 Dec 1993 17:37:59 +0200, Ronald M. Anderson wrote:
} This is really simple! If anyone can take sharp pictures using standard } specimens then the machine is working fine! JEOLs are great machines } but I don't think they are smart enough to realize whose specimens } have been loaded and produce sharp pictures for one set of specimens } and 'out of focus' pictures for another set. } If the *JEOL* people get sharp pictures on standard specimens and } out of focus pictures on your specimens, why the skepticism? } It's your specimens! Are your specimens well bonded to a small mesh grid? } } In any case, I question the ethics of holding this dialog in this open formum. } Jouko Maki and others are searching for what could be wrong with the } instrument when it is not at all clear that the instrument is involved } and that you have not received satisfactory attention from the JEOL people. } Many readers will forget, or not read, these fine points and will carry } away the notion that the JEOL instrument has focus problems, therby } impacting their sales. That isn't fair!
Excellent,
I was not reading the original text carefully enough. Anyway, it essential to find out the procedure how people are making the exposures. It is also possible to find the reason for unsharp pictures from that starting point. It is quite common even to experienced researchers to learn to "bad habbits" while taking very many pictures. It would be interesting to know what other microscopes they are using.
As a comment to the in-focus standard specimen pictures, I would myself try what has already been told - that is: take a biological sample which normally gives unsharp pictures, find a hole in the support film (which I believe you are using?). Take test pictures by focussing to the hole-edge and to the normal biological structures. Compare the results. If the first is O.K. and the second not, You have solved the case - it is the method of focussing.
I am the last person to say anything negative about JEOL microscopes - if anyone has understood me to misevaluate them, that has not been my purpose. I have been using JEOL-microscopes since 1968 and found them very reliable and easy to use. I have also only positive things to tell about JEOL- service. What I am afraid is, that many microscopists are not "speaking the same language" with the service engineers.
I sincerely hope I have not created any negative pressure against JEOL by analysing the possible sources for un-focussed images.
Hello all, I work in the electron microscopy lab at the University of Iowa. We are in the process of purchaseing a SPM package from one of the commercial vendors (DI, Park, Topometric, etc). I am interested in hearing any information or experiences anyone has had with any of these systems. Please email me or post to microscopy-at-anlemc.msd.anl.gov. Thank you, Randy Nessler rnessler-at-emiris.iaf.uiowa.edu
We have a problem with an extraction voltage power supply on an HB-501 and would like to have copies of schematic # 139C-984 and 139C-974 if you have these available. The main schematic for these drawings is 139C-213-1. If you have the drawings needed would you mind sending a FAX to me at 602-965-9004. We have 139C-213-1 but not the others. Thank you. John C. Wheatley Arizona State University 602-965-3831
My apologies to the person whom I am addressing with this request for having forgotten your name. You requested opinions on automatic print processors. Were you able to draw any conclusions from the responses that you received?
Have you made a decision on a purchase. Did any general consensus materialize concerning one machine over another?
In this same regard it seems that whem using an automatic processor one should also have a means of automatically controlling expossure as well
What are the experiences of those out there using automatic exposure systems? ****************************** Greg Erdos * * Director, ICBR EMCL * * University of Florida *
My apologies to the person whom I am addressing with this request for having forgotten your name. You requested opinions on automatic print processors. Were you able to draw any conclusions from the responses that you received?
Have you made a decision on a purchase. Did any general consensus materialize concerning one machine over another?
In this same regard it seems that whem using an automatic processor one should also have a means of automatically controlling expossure as well
What are the experiences of those out there using automatic exposure systems? ****************************** Greg Erdos * * Director, ICBR EMCL * * University of Florida *