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 }
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