Good morning, I'm looking for a water chiller for a TEM and SEM. The unit looking for is a Haskris R075 or equivalent. Options: water cooled 1 supply, 2 returns(minimum) Hot gas bypass preferable, but not necessary 115VAC
Thanks in advance for any info, advice, recommendations. Vaughn Fierke SNBL USA, Ltd
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Hi, We are part of a clinical pathology lab, so we do a lot of muscle, nerve & kidney biopsies. The nerves have been a continual problem. They rarely want to lay flat, once dried on the hot plate. This causes a lot of frustration for us and the doctors. The nerve cross-sections are cut at 1000nm, and then dried on the hot plate (95-100 degrees C). After staining with Toluidine Blue, the creases and bubbles are very clear. We have not experienced this problem with any of the other tissues.
Does anyone have any suggestions to get the sections to lay flat on the glass slide?
Thank You, Lauren Simmerman NHS EM Lab Omaha, NE 402-559-7729
Just a quick note to mention that the session "Museum Applications of SEM" will take place again at Scanning 2003 (San Diego, May 3-5th; http://www.scanning-fams.org).
The deadline for abstract submission is Dec. 2nd. The museum session debuted at Scanning 2001 and was very successful. Looking forward to an interesting program again this year.
Best regards,
Angela
----------------------------------------- Angela V. Klaus, Ph.D. Director, Microscopy and Imaging Facility American Museum of Natural History Central Park West at 79th Street New York, NY 10024 USA Email: avklaus-at-amnh.org Tel: 212-769-5977 Fax: 212-496-3480 -----------------------------------------
I am looking for an ESEM that will resolve 100nm-wide structures under minimal evacuation, which can also be modified (reversibly, of course) for manipulation experiments. Does anyone know of a facility in the San Francisco Bay Area with these capabilities? I am a graduate student at UC Berkeley with considerable SEM experience. I'd be happy to provide a more detailed explanation of my research if necessary.
Thank you, Anne Peattie -- ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ PolyPEDAL Lab University of California, Berkeley Department of Integrative Biology 3060 VLSB, #3140 Berkeley, CA 94720-3140 (510) 643-5183 http://polypedal.berkeley.edu/anne
Nerves can be a real challenge. Here's my mantra for nerves: Leave in half and half overnight (Spurr). Use only great glass knives. Be picky and do not use any knife with only an OK edge. We cut at 0.3 to 0.5 microns (if my math is correct you are cutting at 1 micron); I pick up specimens with the beveled tip of a TB or equiv needle. Make sure that the tip is smooth and not jagged and scrupulously clean (clean with 100% EtOH and wipe with lens tissue). Slides are dried on a hot plate at a lower temp than you: probably around 65-70 degrees for 15 min. Block face 0.5 to 1.5 mm. When staining: take slide with heated Tol Blue off of hot plate and immerse in HOT tap water, not COLD. Rinse in running hot tap water. Dry completely on hot plate. (Walk away and do something else). Let slide come to room Temp, then dip in xylene, coverslip with standard histo mounting media (Cytoseal, etc,).
Good luck and let me know if any of this helps. Becky Garrison Pathology Supervisor 904-244-6237; 6067
-----Original Message----- } From: Lauren [mailto:simmerman_2000-at-netzero.com] Sent: Friday, November 01, 2002 1:45 PM To: Microscopy-at-sparc5.microscopy.com
Hi, We are part of a clinical pathology lab, so we do a lot of muscle, nerve & kidney biopsies. The nerves have been a continual problem. They rarely want to lay flat, once dried on the hot plate. This causes a lot of frustration for us and the doctors. The nerve cross-sections are cut at 1000nm, and then dried on the hot plate (95-100 degrees C). After staining with Toluidine Blue, the creases and bubbles are very clear. We have not experienced this problem with any of the other tissues.
Does anyone have any suggestions to get the sections to lay flat on the glass slide?
Thank You, Lauren Simmerman NHS EM Lab Omaha, NE 402-559-7729
I have a Reichert binocular microscope, ivory in color, that is probably from the 1960's or so. How can I look up the serial numbers to gain information about this scope. I also have about 5 wooden boxes of assorted objectives, oculars, condensers, and a camera. Thanks for any help you can provide.
I have been asked to investigate the upgrade of several Nikon microscopes to digital cameras. I welcome any recomendations based upon experience or the sharing of any difficulties experienced in your labs since the transition.
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Dear all, Has anyone ever prepared TEM specimens from small samples of the size 10-100 microns. We have a student who is studying phase transitions in a diamond anvil cell and wants to put the samples in the TEM. The resulting samples are usually of the order of 50 microns in diameter, and rather thinner in cross section. This falls in an inconvenient size range: I normally work with either bulk materials which can be thinned, or with nanoscale particles which can be dispersed in something and dropped onto a holey carbon film. I was wondering if there was perhaps some way of embedding the samples and then thinning the resulting composite. The material is a hard glassy or crystalline ceramic, depending on the exact treatment in the diamond anvil cell.
Thanks for any leads you can give.
Best wishes -- Ian MacLaren Strukturforschung, Materialwissenschaft, TU-Darmstadt, Germany ian.maclaren-at-physics.org / http://homepages.tu-darmstadt.de/~maclaren/
Please ban this address richard-at-51translation.com or even everything from 51translation.com. It is a SPAM written in Chinese. I know most of the listers can not read it, although I can :-).
Shuyou.
On Mon, 4 Nov 2002 17:07:19 +0200 "Coetzee, Mr S. H Physics Science" {COETZEES-at-mopipi.ub.bw} wrote:
I am looking for some information on the next meeting listed below, but could not find a link at http://www.msa.microscopy.com/MSAMeetings/MSAMeetings.html
Hello, A really good book (not only the atlas) with lot of images and valuable comments is: BIOMEDICAL ELECTRON MICROSCOPY by A.V. Maunsbach and B.A. Afzelius, Academic Press 1999. Best regards Oldrich Benada
On 4 Nov 2002 at 15:05, Hong Yi wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I am looking for a good EM atlas to buy. Any recommendations? Thank } you. } } Hong } } }
+-----------------------------------+ Oldrich Benada Acad. Sci. CR Institute of Microbiology Laboratory of electron microscopy Videnska 1083 CZ - 142 20 Prague 4 - Krc Czech Republic +------------------------------------+ Phone: +420-241062399 Fax: +420-241062347 WEB: http://www.biomed.cas.cz/mbu/lem113/lem.htm
Try Cross and Mercer, Cell and TIssue Ultrastructure, A functional perspective, Freeman Press.
DL
On Mon, 4 Nov 2002, Hong Yi wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I am looking for a good EM atlas to buy. Any recommendations? Thank } you. } } Hong } } } }
______________________________________________________________________ Donald L. Lovett e-mail: lovett-at-tcnj.edu Assoc. Professor, Dept. of Biology voice: (609) 771-2876 P.O. Box 7718 fax: (609) 637-5118 The College of New Jersey Ewing, NJ 08628-0718
I use a textbook in my ultrastructure class called, "Cell and Tissue Ultrastructure: A Functional Perspective," by Patricia Cross and K. Lynne Mercer. Although it is getting a bit old (1993), it still has a good assortment of electron micrographs. The small amount of text is at times dated.
Martin A. Levin Director of the Center for Educational Excellence (CEE) and Professor of Biology Eastern Connecticut State University J. Eugene Smith Library, Room 431 Willimantic, Connecticut 06226 (860)465-5589/4324 FAX (860)465-5522/5213 Email: levin-at-easternct.edu
} ---------- } From: Hong Yi } Sent: Monday, November 4, 2002 3:05 PM } To: Microscopy-at-sparc5.microscopy.com } Subject: EM Atlas } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I am looking for a good EM atlas to buy. Any recommendations? Thank } you. } } Hong } } }
Hi group - not owning a critical point dryer, etc., what would be the best solution in preparing a fresh core sample from fresh water so that the bacteria will not become desiccated? I do have opportunity to run SEM on low vacuum which will help. Thanks Barb
Hi, I'm trying to immuno gold stain adeno-associated virus adsorbed to parlodion coated copper grids. I UV-irradiate the grids for 30 min. to help the virus stick. If I just uranyl acetate stain the sample, I see plenty of viral particles. However, after the immuno staining procedure (1: Wash, 2: primary AB, 3: Wash, 4: secondary AB, 5: wash, 6: uranyl acetate staining) I can't detect any virus particles anymore. All I see is low background staining with immuno gold.
I would appreciate any suggestions. Is there any way to crosslink the virus to the grid? Or would fixing with Glutaraldehyde before or after adsorption help?
I'm not a regular visitor to this list, I would appreciate if you could e-mail me a copy of the answer directly to: thomas.weber-at-mssm.edu.
Thanks for any help.
Thomas
P.S.: I don't have really access to a glow discharge unit.
I have some information posted at my website regarding the Call for Papers etc. for the San Antonio meeting which might be helpful although it's from an International Metallographic Society (one of the participating organizations) perspective. Take a look at http://www.metallography.com/ims/2003call.htm and http://www.metallography.com/ims/2003.htm.
Jeff Stewart Metallographic Laboratory Manager Stern-Leach Company 49 Pearl Street Attleboro, MA 02703
"Dwight Arrieche (IIBCA)" wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Dear Microscopist } } I am looking for some information on the next meeting listed below, but } could not find a link at } http://www.msa.microscopy.com/MSAMeetings/MSAMeetings.html } } Microscopy & Microanalysis 2003 } } Sponsored by MSA, MAS, and CIASEM } } August 3-7 in San Antonio Texas. } } Greatly appreciate your help
I am trying to do pre-embedding ultrasmall-gold immunolabeling of mouse optic nerve. I am trying to do it on 10 um-thick frozen sections attached to gelatin-coated superfrost/plus glass microscope slides. I am in the middle of the first run on these samples, and I am finding that my first challenge is to get the tissue to stick to the slides. For fluorescence immuno-labeling, one can allow the frozen sections to dry onto the slides. For TEM, though, the tissue can?t dry out. I froze the slides immediately after the sections melted onto them, and now some of the tissue is hanging by a thread onto the slide, some has dislodged entirely, and some remains adherent.
Also see http://www.msa.microscopy.com/MSAMeetings/MM03/MMHomePage.html
Jeff Stewart Metallographic Laboratory Manager Stern-Leach Company 49 Pearl Street Attleboro, MA 02703
"Dwight Arrieche (IIBCA)" wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Dear Microscopist } } I am looking for some information on the next meeting listed below, but } could not find a link at } http://www.msa.microscopy.com/MSAMeetings/MSAMeetings.html } } Microscopy & Microanalysis 2003 } } Sponsored by MSA, MAS, and CIASEM } } August 3-7 in San Antonio Texas. } } Greatly appreciate your help
There is the "Particle Atlas" produced by the McCrone Institute. I've never seen it, and I hear it's quite expensive, but as I understand it, it contains many SEM images of common particles and materials. I'm not sure of the exact name, but if you go to the McCrone Institute website, it should be described there.
F.C. Thomas Geological Survey of Canada (Atlantic) Dartmouth, Nova Scotia
Hi Thomas, One suggestion would be to cut down on the washes. You could try adding the primary and then after the incubation simply adding the secondary right on top of that. Then you just need one wash, not two. This might increase your background but perhaps not too bad. Another thing you can try is checking out the washing solution. Are you using PBS to wash? try diluting it or even increasing the ionic strength. Some funny things happen with stickiness and salt concentration.
Hope this helps, Tobias
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Ian MacLaren wrote: ======================================================== Has anyone ever prepared TEM specimens from small samples of the size 10-100 microns. We have a student who is studying phase transitions in a diamond anvil cell and wants to put the samples in the TEM. The resulting samples are usually of the order of 50 microns in diameter, and rather thinner in cross section. This falls in an inconvenient size range: I normally work with either bulk materials which can be thinned, or with nanoscale particles which can be dispersed in something and dropped onto a holey carbon film. I was wondering if there was perhaps some way of embedding the samples and then thinning the resulting composite. The material is a hard glassy or crystalline ceramic, depending on the exact treatment in the diamond anvil cell. =========================================================== We have been embedding samples in this size range, on and off for more than thirty years.
Actually they are quite easy to handle, at least with practice. I think that some of the catalyst samples we have thin sectioned, such as zeolites, would fall into the size range you mentioned. We have (of course) a preference for our own SPI-Pon™ 812 Epoxy Resin system, but at least some of the "Epon substitutes" offered by our major competitors should work just as well. The catalyst samples have some internal porosity so vacuum impregnation is standard. But that might not be necessary if the smaples were diamond anvil pressed and dense.
We use our own SPI Materials Science Diamond Knives, 45 deg., and generally have not had any particular problems. The smaller the particles, the better the sections. They might not be perfect, but they should show you the structure and morphology of the particles. We have found that the 55 deg. knives tend to pull particles out of the block because of compression effects.
Disclaimer: SPI Suppplies is a source for embedding resins and mateials science diamond knives so we have a vested interest in the promotion of these products.
Chuck
PS: Remember that we are striving to be 100% paperless, therefore there are no paper copies kept of this correspondence. Please be sure to always reply by way of "reply" on your software so that the entire string of correspondence can be kept in one place. ============================================
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Ian; A dual beam FIB equipped with an Omniprobe micromanipulator should do the job.
John Mardinly Intel
-----Original Message----- } From: Ian MacLaren [mailto:maclaren-at-tu-darmstadt.de] Sent: Monday, November 04, 2002 7:36 AM To: Microscopy
Dear all, Has anyone ever prepared TEM specimens from small samples of the size 10-100 microns. We have a student who is studying phase transitions in a diamond anvil cell and wants to put the samples in the TEM. The resulting samples are usually of the order of 50 microns in diameter, and rather thinner in cross section. This falls in an inconvenient size range: I normally work with either bulk materials which can be thinned, or with nanoscale particles which can be dispersed in something and dropped onto a holey carbon film. I was wondering if there was perhaps some way of embedding the samples and then thinning the resulting composite. The material is a hard glassy or crystalline ceramic, depending on the exact treatment in the diamond anvil cell.
Thanks for any leads you can give.
Best wishes -- Ian MacLaren Strukturforschung, Materialwissenschaft, TU-Darmstadt, Germany ian.maclaren-at-physics.org / http://homepages.tu-darmstadt.de/~maclaren/
Ron Anderson and company presented work a few years ago where they put small sand-sized pieces of material into holes in Si that they put there by using a special rounded-solid tool for an ultrasonic drill that scalloped the round hole. They then covered the sample with epoxy and another piece of Si and used their tripod polishing to prepare the sample normally. A modification of this theme would probably work for you. Of course there is always FIB if you can get access to one.
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: Ian MacLaren [mailto:maclaren-at-tu-darmstadt.de] Sent: Monday, November 04, 2002 10:36 AM To: Microscopy
Dear all, Has anyone ever prepared TEM specimens from small samples of the size 10-100 microns. We have a student who is studying phase transitions in a diamond anvil cell and wants to put the samples in the TEM. The resulting samples are usually of the order of 50 microns in diameter, and rather thinner in cross section. This falls in an inconvenient size range: I normally work with either bulk materials which can be thinned, or with nanoscale particles which can be dispersed in something and dropped onto a holey carbon film. I was wondering if there was perhaps some way of embedding the samples and then thinning the resulting composite. The material is a hard glassy or crystalline ceramic, depending on the exact treatment in the diamond anvil cell.
Thanks for any leads you can give.
Best wishes -- Ian MacLaren Strukturforschung, Materialwissenschaft, TU-Darmstadt, Germany ian.maclaren-at-physics.org / http://homepages.tu-darmstadt.de/~maclaren/
The M&M-2003 Call for Papers is printed and will be mailed along with the November issue of Microscopy Today and the M&M-2003 poster in about two to three weeks. Nestor, the MSA Listserver friendly SysOp. Has the Call for Papers files and will post them as soon as his schedule permits, probably in the next week.
If anyone URGENTLY needs any information from the Call for Papers, they can contact me. Or, you can wait a little bit. :-)
Ron Anderson Microscopy Today and Call for Papers Editor
-----Original Message----- } From: Jeff Stewart [mailto:jeff-at-metallography.com] Sent: Tuesday, November 05, 2002 11:33 AM To: Dwight Arrieche (IIBCA) Cc: microscopy-at-sparc5.microscopy.com
Dwight,
I have some information posted at my website regarding the Call for Papers etc. for the San Antonio meeting which might be helpful although it's from an International Metallographic Society (one of the participating organizations) perspective. Take a look at http://www.metallography.com/ims/2003call.htm and http://www.metallography.com/ims/2003.htm.
Jeff Stewart Metallographic Laboratory Manager Stern-Leach Company 49 Pearl Street Attleboro, MA 02703
"Dwight Arrieche (IIBCA)" wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Dear Microscopist } } I am looking for some information on the next meeting listed below, but } could not find a link at } http://www.msa.microscopy.com/MSAMeetings/MSAMeetings.html } } Microscopy & Microanalysis 2003 } } Sponsored by MSA, MAS, and CIASEM } } August 3-7 in San Antonio Texas. } } Greatly appreciate your help
Why are you using gelatin on Superfrost plus slides - I thought the point of S'frost was that they 'did the business' - or have I missed something? Why not just use the S'fost on their own?
I would be interested in comments.
Gareth
At 12:06 2002-11-05 -0500, Dorothy Sorenson wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Obs/NB New postal/visiting address from July 2002!
Gareth Morgan MPhil MSc FIBMS, Institute for Microbiology, Pathology and Immunology (IMPI), H5, Karolinska Institutet, Huddinge Universitetssjukhus, F46 SE 141 86 Stockholm Sweden
OBS! Besöksadress: F-Huset, Forskningsgatan 2 F52, Rum 2.10. Laboratoriet för klinisk patologi och cytologi.
NB! Visiting address: Building F, Research Corridor 2 F52, Room 2.10. Clinical Histo- and Cytopathology Laboratory.
Cell Structure; an Introduction to Biomedical Electron Microscopy
Churchill Livingstone ISBN 0 443 02324 7
My 3rd edition dates from 1982 but is still nevertheless a very good book and even has sections on techniques and applications as well as being an atlas with good descriptions,
check it out,
Cameron Hind Analytical Resources group Baxter R&D Europe Belgium
Histology: A text and atlas takes a lot of beating - it goes from LM through to EM. It is out of print now but Amazon.com still has some copies available.
Gareth
At 15:17 2002-11-05 -0400, Frank Thomas wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Obs/NB New postal/visiting address from July 2002!
Gareth Morgan MPhil MSc FIBMS, Institute for Microbiology, Pathology and Immunology (IMPI), H5, Karolinska Institutet, Huddinge Universitetssjukhus, F46 SE 141 86 Stockholm Sweden
OBS! Besöksadress: F-Huset, Forskningsgatan 2 F52, Rum 2.10. Laboratoriet för klinisk patologi och cytologi.
NB! Visiting address: Building F, Research Corridor 2 F52, Room 2.10. Clinical Histo- and Cytopathology Laboratory.
If you mean Ross, Romrell, and Kaye, it is just leaving its third edition, with a new edition due out this December (which is why, I guess, the third is now "out of print"). Great book for a histology course. Wouldn't necessarily be my first choice for an EM atlas, though. JSIII
} Histology: A text and atlas takes a lot of beating - it goes from LM } through to EM. It is out of print now but Amazon.com still has some } copies available. } } Gareth } } -- Julian P.S. Smith III Dept. of Biology Winthrop University Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax)
} Hi group - not owning a critical point dryer, etc., what would be the } best solution in preparing a fresh core sample from fresh } water so that } the bacteria will not become desiccated? I do have opportunity to run } SEM on low vacuum which will help. } Thanks } Barb
Do you have a cooling Peltier stage? Low vacuum by far is not enough to keep specimens wet. If you do not have real wet mode (ESEM), then better to fix specimens in gluteraldehyde (2%) for 0.5 - 1 hr, dehydrate in graded alcohol (for example, 30,50,75,96 and 100% for 10 min each), and then place in HMDS (hexamethyldesilazane) for 20 min and air dry for 1 hr. With HMDS you do not need to use critical point dryer and usually it works well for bacteria. In many cases you even do not need absolute (100%) alcohol, 96% often works fine as a final step in dehydration.
} Dear Listers: } } I have recently had several requests to process and embedd plant } tissue. All of my previous EM experience has involved only human and } animal tissue. } } Those of you who routinely do botanical specimens can you recommend } some EM books or atlases which cover processing methods? } } Are there any atlases that provide ultrastructural images so I can } learn the morphology/terminology of different types of plant specimens. } Right now I will be looking at potato leaves and tubers. Later I am } supposed to get a tomato for EM processing. } } Thanks for your help! } } Karen L. Bentley, M.S.(previously Jensen) } Associate Scientist & Project Manager } Electron Microscope Research Core } University of Rochester Medical Center } Rochester, NY 14642 } 585-275-1954 }
Hi, I have a quick question. Does anything besides water cause small pinholes throughout tissue? Specifically, we had a fairly large piece of brain tissue 3cmx1cmx.5cm. We cut out small cubes from this. The large piece was originally formalin fixed and the cubes were post fixed in MPG. The sections on the grid were full of tiny holes. If my memory serves me right, we have had this problem before with brain tissue? Any connection? A fresh pint of absolute ETOH was used, and we are always very careful to keep all water out of processing.
Thank You, Lauren Simmerman Nebraska Health System EM Lab
Here is a good method from Hsiung's Diagnostic Virology Book, page 102 (4th ed.).
1)Mix Virus suspension with homologous antiserum at predetermined dilution.
2)Incubate mixture -at-37C for 1 hour or overnight -at-4C
3)Centrifuge virus-antibody mixture at 15,000xg for 30 min.
4)Resuspend pellet with small amount of dh20
5)Apply suspension of virus-antibody complexes to a formvar coated nickel grid and remove the excess by blotting with a piece of filter paper. Immediately stain with 2% PTA for 1 minute (avoid drying during preparation).
6)Examine grid under EM.
Good Luck!
Karen Bentley
Karen L. Bentley, M.S.(previously Jensen) Associate Scientist & Project Manager Electron Microscope Research Core University of Rochester Medical Center Rochester, NY 14642 585-275-1954
-----Original Message----- } From: Thomas Weber [mailto:thomas.weber-at-mssm.edu] Sent: Tuesday, November 05, 2002 10:44 AM To: Microscopy-at-sparc5.microscopy.com
Hi, I'm trying to immuno gold stain adeno-associated virus adsorbed to parlodion coated copper grids. I UV-irradiate the grids for 30 min. to help the virus stick. If I just uranyl acetate stain the sample, I see plenty of viral particles. However, after the immuno staining procedure (1: Wash, 2: primary AB, 3: Wash, 4: secondary AB, 5: wash, 6: uranyl acetate staining) I can't detect any virus particles anymore. All I see is low background staining with immuno gold.
I would appreciate any suggestions. Is there any way to crosslink the virus to the grid? Or would fixing with Glutaraldehyde before or after adsorption help?
I'm not a regular visitor to this list, I would appreciate if you could e-mail me a copy of the answer directly to: thomas.weber-at-mssm.edu.
Thanks for any help.
Thomas
P.S.: I don't have really access to a glow discharge unit.
You can try cutting a thicker frozen section, say around 40-50 microns and place them into PBS filled wells for labeling as floating sections. We use a brush to transfer sections during the immunolabeling procedure. If you want the procedure just let me know and I'll send it to you.
I assume you have cryoprotected the tissue prior to freezing? Perhaps the the sucrose is causing them to not adhere to the gelatin.
We never use gelatin or anything else on top of the superfrost plus slides. Those types of slides have a charge on them which is supposed to hold the tissue without the need of other types of coatings. So I would try the sections on the naked superfrost plus slides.
Karen
Karen L. Bentley, M.S.(previously Jensen) Associate Scientist & Project Manager Electron Microscope Research Core University of Rochester Medical Center Rochester, NY 14642 585-275-1954
-----Original Message----- } From: Dorothy Sorenson [mailto:dsoren-at-umich.edu] Sent: Tuesday, November 05, 2002 12:06 PM To: microscopy-at-sparc5.microscopy.com
Dear Listers,
I am trying to do pre-embedding ultrasmall-gold immunolabeling of mouse optic nerve. I am trying to do it on 10 um-thick frozen sections attached to gelatin-coated superfrost/plus glass microscope slides. I am in the middle of the first run on these samples, and I am finding that my first challenge is to get the tissue to stick to the slides. For fluorescence immuno-labeling, one can allow the frozen sections to dry onto the slides. For TEM, though, the tissue can?t dry out. I froze the slides immediately after the sections melted onto them, and now some of the tissue is hanging by a thread onto the slide, some has dislodged entirely, and some remains adherent.
Dear List, I am posting this message for Robert in England, since his Hotmail account does not seem to be able to access the Listserver. ----- Original Message ----- } From: "Robert H. Olley" {hinmeigeng-at-hotmail.com} To: {mager-at-interchange.ubc.ca} Cc: {R.H.Olley-at-reading.ac.uk} ; {hinmeigeng-at-hotmail.com} Sent: Wednesday, November 06, 2002 12:59 AM
Yes, you can. You do not need any holder for the TEM scanner (but you do need a negative adapter, not just a regular scanner). Just put it down on the surface. good luck.
Fang
} } } } } Does anyone have experience of Epson scanners for TEM negatives? I have } } enquired of the company whether the Epson Perfection 2450 Photo can take } our } } } } 4 x 3-and-a quarter inch TEM plates, } } } } but all I can get is the standard answer "we have film holders", which } } include 120 roll film and 5 x 4 inch, but not the size we want. Can one } } simply place the negative straight down on the scanner surface? } } } } Any help would be much appreciated, } } } } (Sorry if you've received this message before - I've had one or two failed } } attempts at sending). } } } } ----------------------------------- } } Robert H. Olley, Physics Dept, } } Univ. Reading, RG6 6AF, England. } } E-mail: R.H.Olley-at-reading.ac.uk } } URL: http://www.rdg.ac.uk/~spsolley } } ----------------------------------- } } } } } } _________________________________________________________________ } } STOP MORE SPAM with the new MSN 8 and get 2 months FREE* } } http://join.msn.com/?page=features/junkmail } } } Mary Mager } Electron Microscopist } Metals and Materials Engineering } University of British Columbia } 6350 Stores Road } Vancouver, B.C. V6T 1Z4 } CANADA } tel: 604-822-5648 } e-mail: mager-at-interchange.ubc.ca
-- ------------------ Department of MSE Cornell University Ithaca, NY14853 (607)-2559461 ------------------
Placing the negatives directly on the scanner plate will often result in Newton rings in the resulting image. keeping it just off the glass prevents this. We put our 4 x 3.25 negatives in the 5 x 4 holders where they are supported on 3 sides and this works well. good luck. tom
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-- Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility
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In Nestor's absence, let me stick my nose into the matter.
I ran into a similar situation a few weeks back. I replied to a poster's message someone-at-yahoo.com and copied the list. The message never came through and I think I got a notification back from the list that the message was blocked as potential SPAM.
What had happened was that the filter triggered on yahoo.com in the address headers. Apparently the rules are set so that yahoo or hotmail appearing in the headers is taken as a sign of SPAM. Many spammers will have multiple addresses in the headers and will likely include plenty of Yahoo and Hotmail.
Strictly speaking, mail really intended for the list should not have to be copied to multiple addresses, including ones at yahoo or hotmail. You might say "well, why can't I?" I suppose it could be allowed, but I expect that the rule does stop a number of SPAM messages. I would allow Nestor the prerogative to set the rules as he sees fit. It seems he is doing a pretty good job of catching SPAM. A little gets through, but I am certain many more messages do not.
If you still want to copy yourself or any other address at Yahoo or Hotmail, there is a simple solution. Simply put that address in the BCC:, blind carbon copy, field. That way the list server would not even see the address to choke on it. This practice is also good for other applications where messages are going out to many recipients and it is not necessary for the recipients to see the pages and pages of other recipient addresses. When I receive a message intended for the engineering faculty and staff, it really isn't necessary for me to see hundreds of addresses before I get down to a three-line message. {g}
Try it and see if that helps.
Warren
At 09:29 AM 11/6/02 -0800, you wrote:
} Dear List, } I am posting this message for Robert in England, since his Hotmail account } does not seem to be able to access the Listserver. } } ----- Original Message ----- } } From: "Robert H. Olley" {hinmeigeng-at-hotmail.com} } To: {mager-at-interchange.ubc.ca} } Cc: {R.H.Olley-at-reading.ac.uk} ; {hinmeigeng-at-hotmail.com} } Sent: Wednesday, November 06, 2002 12:59 AM } Subject: TEM neg scanners: Can't get through } } } } Dear Mary, } } } } I've recently rejoined the Micro Listserver, using the hotmail acount in } the } } cc: line in order to keep the correspondence from getting mixed up with } the } } rest of my university email. While I receive all (?) of the other items } } posted to the Micro group, none of my own messages get through: also } } attempts to contact Nestor seem to fail. I don't know if for some reason } } I'm getting "spammed out", but I would appreciate it if you could post } this } } enquiry to the listserver and see what happens; please also cc: to myself } at } } both addresses above. } } } } Best regards, } } } } Robert H. Olley.
------------------------------------------- No files should be attached to this message ------------------------------------------- Warren E. Straszheim, Ph.D. Materials Analysis and Research Lab Iowa State University 23 Town Engineering Ames IA, 50011-3232
I have two SEM micrographs on the web: http://www.magma.ca/~scimat/Defect.htm The micrographs show zagged edges taken at 20 kX. Such phenomenon does not present all the time. Can anyone suggest what causes the problem and how to solve it? Thanking in advance.
AnnFook Yang EM Unit, Eastern Cereal and Oilseed Research Centre, Room 2091, Bldg. 20, Central Experimental Farm, Ottawa, Ontario Canada K1A 0C6
I'm looking for a black and white reversal film. I have some old LPD4 but I can't kind listed anywhere (fuji or kodak) if they still make this film. And if they do make it, does it come in little rolls or for a bulk loader?
I'm lazy and I like to make B&W text slides with B&W reversal film. Or can I use a color slide film and still have it turn out OK?
Any information is gladly accepted and I thank you in advance.
Paula :-)
p.s. some of us are old fashioned and like to use film instead of all the fancy-schmancy computer thingies ;)
Paula Sicurello George Washington Univ. Medical Center Dept. of Pathology, Ross Hall rm 505 Electron Microscope Lab 2300 Eye St. Washington, DC 20037 202-994-2930 phone 202-994-2518 fax
As Kodak slowly discontinues the graded Kodabrome II RC papers (no 1, 4 or 5), we are looking for alternatives for our printing.
We have a Durst point source enlarger, and I was wondering if we can switch to Polycontrast paper and filters. If there are any labs that are using this method, I would appreciate any information on how well it works.
We have found that it is still more economical and time efficient to develop and print 8 x 10 study prints for our users, rather than scan in all our negatives, so any old-fashioned wet darkroom suggestions would be appreciated.
Thanks in advance Leslie
Leslie Gunther Cummins Analytical Imaging Facility Albert Einstein College of Medicine 1300 Morris Park Ave. Bronx, NY 10461 718-430-3547
There is an excellent LM/EM atlas available from Jones and Bartlett Publishers (Subdury, MA, 800-832-0034):
"Plant cell biology: structure and function", by B.E.S. Gunning and M.W. Steer paperback ISBN 0-86720-504-0, the edition I have is from 1996
And there is a recent plant-oriented EM technique book:
"Methods in plant electron microscopy and cytochemistry", edited by W.V. Dashek (Humana Press 973-256-1699), ISBN 0-89603-589-1 (2000)
Hope this helps.
Howard
} Dear Listers: } } I have recently had several requests to process and embedd plant } tissue. All of my previous EM experience has involved only human and } animal tissue. } } Those of you who routinely do botanical specimens can you recommend } some EM books or atlases which cover processing methods? } } Are there any atlases that provide ultrastructural images so I can } learn the morphology/terminology of different types of plant specimens. } Right now I will be looking at potato leaves and tubers. Later I am } supposed to get a tomato for EM processing. } } Thanks for your help! } } Karen L. Bentley, M.S.(previously Jensen) } Associate Scientist & Project Manager } Electron Microscope Research Core } University of Rochester Medical Center } Rochester, NY 14642 } 585-275-1954 } } } R. Howard Berg, Ph.D. Director, Integrated Microscopy Facility Danforth Plant Science Center 975 N. Warson Rd. St. Louis, MO 63132
ph 314-587-1261 fx 314-587-1361 cell 314-378-2409 rhberg-at-danforthcenter.org www.danforthcenter.org
We've been struggling trying to cut cryo-sections of mouse hearts. The samples have ben fixed with PFA (4-8%) by perfusion, then sliced into thin ( { 1mm) sections, chopped and infiltrated in 2.3 M sucrose ON before freezing in LN2. Sections are cut at -108°C, with either 35 or 45° cryo-knife, and picked with methyl cellulose - sucrose mix (50::50 or 60::40). The problem is we get too much compression of the sample and too many folds. These folds occur mostly over the sarcolemma, which is unfortunately the area where we try to see labeling! I suspect one of the problems might be the infiltration medium we use (2.3 M sucrose), or the fixation. We have tried to add some glutaraldehyde (0.1%) without success. I would greatly appreciate any suggestion on how to improve our preservation and sectioning of these samples. Thanks
Marc -- Marc Pypaert Department of Cell Biology, Center for Cell and Molecular Imaging and Ludwig Institute for Cancer Research, Yale University School of Medicine 333 Cedar Street New Haven CT 06520 Tel : (203) 785 3681 Fax : (203) 785 7446
Hi! I run a diagnostic TEM laboratory in SC. One tech. 6 to 5 samples every other day at least. What is the turn around time asked for your labs out there that are also doing diagnostic/clinical work and where does that number come from? Thank you for any help. My supervisor is saying 2 days but I think that is close to impossible when only one tech doing everything including transport of tissue from another lab and scoping all case that come in. Sharon Drew Diagnostic Pathology EM Charleston, SC
----- Original Message ----- } From: {mailto:jsmit51-at-tampabay.rr.com} jerry smith To: {mailto:MICROSCOPY-at-MSA.MICROSCOPY.COM} MICROSCOPY-at-MSA.MICROSCOPY.COM Sent: Wednesday, November 06, 2002 11:45 AM
Yup, that's vibrations. As you've found EMs are extremely sensitive seismometers.
Ridding an SEM of such problems can be as much art as science. It's always nice to know the source but they are often created by many activities or sources in a building. I've seen them caused by high speed tanks (a location next to United Technologies M1A1 Abrams tank test track), ocean waves (Pensacola Naval Air Station) and often by nearby heavy vehicle traffic. Most likely, you'll primarily have a combination of vibrations caused by building air handling equipment.
Primarily, the instrument has to be properly setup. There are two or three 'zones' of isolation that have to be paid attention to. The inner most zone, and the one that should have the least vibrations, is the column and sample chamber. This is actually isolated from the electron optics table, usually by elastomeric shock mounts. You'll want to make sure those are in good shape. Ideally, nothing should make any mechanical contact between this inner table and the outside world. However, that's obviously not possible - there are many wires and tubes that have to connect to the components on the inner table, both above and below the sample chamber. And never overlook the little things, like that clipboard or instrument log that's always left on the optics table, sometimes touching the chamber.
The main idea is to make sure that anything that does have to go to the inner table is first secured to the outer table. In the case of wires and tubing, they should be secured in such a way that there is a loose loop formed between the attachment to the inner and outer tables that touches nothing else. The loop (really a U shape) helps to re-direct vibrations to a swing in the wire or cable. The outer table, itself, has to also be considered an isolated zone, albeit with less isolation than the inner table. The mass of the entire electron optics table and its rigidity while only being supported by four thin feet, gives it some isolation even though it is just sitting on the floor. So all wires and tubing that go from the outer table to other things should also be similarly routed. It's usually not possible to loop these to the electronics console or water and electrical outlets, so sometimes sand or lead bags can be used to dampen vibration in them before they loop to the optics table. Bags of lead shot are cheap and easy to get at many sporting stores (used for loading your own shotgun shells).
Adding an EDS detector adds to the problem. The long moment arm formed by the length of the mounting means that any vibrations coming through the electrical connections to the end of the detector will be easily coupled into the column. If possible, secure them to the inner table and then the outer table. Another vexing problem with EDS detectors is their ability to couple acoustic noise from their large cryogenic dewars. Many have this problem - try clapping your hands loudly when you're looking at an image, if your EDS is susceptible to acoustics, you'll see a decaying vibration from the clap. Best I've come up with if you have this problem is to wrap the dewar with some sound deadening foam, this will help stop the vibrations from reaching the dewar and dampen any that do.
Good Luck!
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174
On Wednesday, November 06, 2002 11:46 AM, Ann-Fook Yang [SMTP:yanga-at-agr.gc.ca] wrote: } Hi everyone, } } I have two SEM micrographs on the web: } http://www.magma.ca/~scimat/Defect.htm } The micrographs show zagged edges taken at 20 kX. Such phenomenon does not present all the time. Can anyone suggest what causes the problem and how to solve it? } Thanking in advance. } } } } } } } } } } AnnFook Yang } EM Unit, } Eastern Cereal and Oilseed Research Centre, } Room 2091, Bldg. 20, } Central Experimental Farm, } Ottawa, Ontario } Canada K1A 0C6 } } Tel: 1-613-759-1638 } Fax: 1-613-759-1701 } } e-mail: yanga-at-em.agr.ca } } } }
Ann, I agree with Allen Sampson's comments, with the addition that you could also get the same effect from AC magnetic fields. These can pop up from a number of sources (computer equipment, electroc motors, overhead lights, the list goes on...). A way of checking if it's vibration as opposed to fields is to check the magnitude of the 'sawteeth' at long and short working distances; if it's fields, then the sawteeth will be bigger at longer working distances (because the action of the field will be operating over a longer distance at the longer working distance). This also serves as a crude short-term fix for the problem sometimes, although I'd really recommend finding the piece of equipment that's causing the problem, and fix that instead. Another possible source is a ground loop, where your 'scope is hooked up to another piece of equipment with a differant ground potential. This causes currents to end up flowing through your equipment-'scope connection, generating the AC fields internally. A solution for this is to decide on a single ground potential to use (usually the 'scope's dedicated ground), and then hardwire all the other equipment to this one ground. (The best case, of course, is to sink your own ground, but many people/labs don't have this option available.) If you do have field problems, you can usually track them down using an AC magnetic field hand meter, which you can get for around $35 from an online dealer (I forget where we got ours now, sorry).
I hope this helps,
Ben Simkin (simkin-at-egr.msu.edu) Michigan State University Department of Chemical Engineering and Materials Science
Our turn around time is about 2 days, but we have 2 FTE's doing ~500 Surg Path EM specimens/yr, (that's about one full case/day). Also, my virology EM techs and I can fill in when the SPEM service gets bombed. The TAT will vary more when there's only one person to do everything. When there's a pile up with several samples at once and no back-up tech, obviously, the TAT has to increase. I would think 2-4 days is reasonable, depending on the work flow.
What do you do if you're out sick or on vacation? This supervisor needs to lighten up a little and realize that one person can't have a set TAT unless the cases arrive in a regular and uniform flow--which isn't going to happen.
You might set up some sort of system where the pathologist lets you know that a case is really urgent and put that one in front of others. You can do a better job if you communicate clearly with the folks needing the service. You can give them an approximate, estimated, or usual TAT to expect and then notify them if it must be longer because of whatever (workload, illness, equipment malfunction, vacation, etc). Learn to work with those needing the service and build a good report; then have them back you up wrt the supervisor.
..my $0.02 worth.
Best regards and greetings to "Whit" Sara Miller
On Wed, 6 Nov 2002, Sharon Drew wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hi! } I run a diagnostic TEM laboratory in SC. } One tech. } 6 to 5 samples every other day at least. } What is the turn around time asked for your labs out there that are } also doing diagnostic/clinical work and where does that number come } from? } Thank you for any help. } My supervisor is saying 2 days but I think that is close to impossible } when only one tech doing everything including transport of tissue from } another lab and scoping all case that come in. } Sharon Drew } Diagnostic Pathology EM } Charleston, SC } } }
Sara E. Miller, Ph. D. P. O. Box 3712 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-3265
We have a recently acquired Epson Perfection 2450 Photo scanner and it works very well with TEM negs mounted in the 5 x 4 film holder. You want to avoid placing the negs directly on the glass surface because of Moire fringes. (Curiously we had little trouble with fringes on our old HP transparency scanner.)
The Epson scanner can acquire 16-bit grayscale TIFF images which can be advantageous for negatives with very low contrast. However few software packages seem to handle this format. DigitalMicrograph and analySIS do, while Adobe Photoshop provides some limited support. Are there other programs out there that can work with this format?
With thanks, Thor
} = = = = = = = = = = = = = = = = = = = } } Does anyone have experience of Epson scanners for TEM negatives? I have } enquired of the company whether the Epson Perfection 2450 Photo can take } our 4 x 3-and-a quarter inch TEM plates, } but all I can get is the standard answer "we have film holders", which } include 120 roll film and 5 x 4 inch, but not the size we want. Can one } simply place the negative straight down on the scanner surface? } } Any help would be much appreciated, } } (Sorry if you've received this message before - I've had one or two failed } attempts at sending). } ----------------------------------- } Robert H. Olley, Physics Dept, } Univ. Reading, RG6 6AF, England. } E-mail: R.H.Olley-at-reading.ac.uk } URL: http://www.rdg.ac.uk/~spsolley } ----------------------------------- } =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Dr Thor Bostrom Acting Director, Analytical EM Facility, Faculty of Science, Queensland University of Technology (QUT) GPO Box 2434, Brisbane, QLD 4001, Australia Ph: +61 7 3864-2351 FAX: +61 7 3864-5100 http://www.sci.qut.edu.au/aemf/ =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=
Unless there's something special an Epson scanner does better than other scanners, you really don't need an Epson scanner to manipulate both SEM and TEM negatives. I may be wrong but all you need are a good scanner and a suite of Adobe Photoshop or PaintShop Pro.
All we do is to scan our negatives into either Paintshop Pro or Photoshop. In Paint Shop Pro, for ordinary BF/DF negtaives, I just go to the "Color" menu and select "Negative Image". And my scanned image turns to a "print image". No wasting of developers and fixers. You won't be disappointed with what you would get. Just get a good scanner. We have also got good quality diff images. Of course, if you know how to use the Fourier Transform filter in your graphic suite, you can still do a lot using your Photoshop or Paint Shop Pro. Never have to worry about film holders. We just lay our negatives flat on our scanner and off we drive.
Well, I don't know if this comes close to what you are looking for.
Goodluck.
Ike Oguocha
} Does anyone have experience of Epson scanners for TEM negatives? I } have enquired of the company whether the Epson Perfection 2450 Photo } can take our } } } } 4 x 3-and-a quarter inch TEM plates, } } } } but all I can get is the standard answer "we have film holders", } which } } include 120 roll film and 5 x 4 inch, but not the size we want. } Can one } } simply place the negative straight down on the scanner surface? } } } } Any help would be much appreciated, } } } } (Sorry if you've received this message before - I've had one or two } failed } } attempts at sending).
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I am using a rather old (almost antique) EDS system for my X ray mapping. It is so old that it will not do quantitative analysis. With the economy like it is I will have to make this equipment last a few more years.
Does anyone know of a program that I can use that contains the reference data tables and allows you to compute the ZAF number (or K ratios ) for different elements? Doing this by hand is quite tedious. Thank you.
Could be vibration. Could also be magnetic field. And, could be signal interference, such as ground loop. What type of SEM are you using?
I will omit remedies for the vibration problem, since Allen Sampson did pretty good job addressing that.
Stray AC line frequency magnetic field: try acquiring image at different accelerating voltages. It is very important that all other geometry parameters will remain the same: particular area of a particular specimen, WD, tilt, magnification, spot size. Contrast, brightness, focus, and stigmator settings can be changed. If the problem is much worse at, say, 2 kV, than at, say, 15kV, you are dealing with magnetic field. You may also try to keep 3 axes AC milliGauss meter at the SEM, and notice what it measures, but catching stray magnetic interference this way may be a little tricky. These meters, though, are very inexpensive, and available from many test instruments suppliers.
Now we are getting down to statistically most likely problem- signal interference and ground loops. It is difficult to go any further without knowing the SEM type, and acquisition system (if separate) type. Too many possibilities exist, but the very first- does your acquisition software have an option somewhere in the settings which reads something like "60 Hertz sync", or "AC line synchronization", or "Line sync", etc., etc.? If yes, check (or uncheck) that option and see what happens. Further remedies of this problem will include eliminating ground loops. Example- shielded cable(s) has shield connected on both sides, and shield is used as one of the signal wires. This is frequently done, and does jeopardize instrument interference tolerance. Another example- one of the accessories is susceptible to some kind of interference, and must be powered through the isolation transformer. The latter remedy will only work with decent (better if dedicated) ground. The fact that the problem is not present all the time does not mean that everything is perfect inside the SEM. Besides, going after the EM interference source could be inefficient and frustrating, if not impossible. Improving grounding/shielding/power connection might be easier.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax (678)467-0012 mobile
This message is made of 100% recycled electrons.
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----- Original Message ----- } From: Ann-Fook Yang {yanga-at-agr.gc.ca} To: {microscopy-at-sparc5.microscopy.com} Sent: Wednesday, November 06, 2002 2:45 PM
Could be vibration. Could also be magnetic field. And, could be signal interference, such as ground loop. What type of SEM are you using?
I will omit remedies for the vibration problem, since Allen Sampson did pretty good job addressing that.
Stray AC line frequency magnetic field: try acquiring image at different accelerating voltages. It is very important that all other geometry parameters will remain the same: particular area of a particular specimen, WD, tilt, magnification, spot size. Contrast, brightness, focus, and stigmator settings can be changed. If the problem is much worse at, say, 2 kV, than at, say, 15kV, you are dealing with magnetic field. You may also try to keep 3 axes AC milliGauss meter at the SEM, and notice what it measures, but catching stray magnetic interference this way may be a little tricky. These meters, though, are very inexpensive, and available from many test instruments suppliers.
Now we are getting down to statistically most likely problem- signal interference and ground loops. It is difficult to go any further without knowing the SEM type, and acquisition system (if separate) type. Too many possibilities exist, but the very first- does your acquisition software have an option somewhere in the settings which reads something like "60 Hertz sync", or "AC line synchronization", or "Line sync", etc., etc.? If yes, check (or uncheck) that option and see what happens. Further remedies of this problem will include eliminating ground loops. Example- shielded cable(s) has shield connected on both sides, and shield is used as one of the signal wires. This is frequently done, and does jeopardize instrument interference tolerance. Another example- one of the accessories is susceptible to some kind of interference, and must be powered through the isolation transformer. The latter remedy will only work with decent (better if dedicated) ground. The fact that the problem is not present all the time does not mean that everything is perfect inside the SEM. Besides, going after the EM interference source could be inefficient and frustrating, if not impossible. Improving grounding/shielding/power connection might be easier.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax (678)467-0012 mobile
This message is made of 100% recycled electrons.
This address can not receive messages larger than 15 kb without prior notification.
----- Original Message ----- } From: Ann-Fook Yang {yanga-at-agr.gc.ca} To: {microscopy-at-sparc5.microscopy.com} } Sent: Wednesday, November 06, 2002 2:45 PM } Subject: vibration? } } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } Hi everyone, } } } } I have two SEM micrographs on the web: } } http://www.magma.ca/~scimat/Defect.htm } } The micrographs show zagged edges taken at 20 kX. Such phenomenon does } not present all the time. Can anyone suggest what causes the problem and how } to solve it? } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } EM Unit, } } Eastern Cereal and Oilseed Research Centre, } } Room 2091, Bldg. 20, } } Central Experimental Farm, } } Ottawa, Ontario } } Canada K1A 0C6 } } } } Tel: 1-613-759-1638 } } Fax: 1-613-759-1701 } } } } e-mail: yanga-at-em.agr.ca } } } } } } } }
try kodalith. you get white on black, very fine resolution. and in the dark ages i used to hand colour them with photographic tints and a 00000 red fox brush.
i am still using an old ektamatic processor with polycontrast III professional grade paper. our department of anatomy is also using the same system. the major problem is now getting parts for the processor, the rollers tend to go after about 20 years and i have been ordering replacements on a gradual basis directly from kodak. also, when i ordered chemicals yesterday our systems contract photographic supplier suggested that the two labs using this system are out of touch with reality. polycontrast papers work well. see what kodak now has for a processor. the Durst enlarger should be fine.
as a hint, one benefit of the polycontrast paper comes if you use a two setting digital timer. i can set it to expose for two different times, determine filtration conditions for different areas on the negative, and do double filter exposures, ensuring good results for publications. takes a bit of work, but once you have the conditions right you can run off 10 prints in about 2 minutes. i've personally never seen the ability to get the same quality from digitized images. but then i'm a dinosaur.
Dear Robert and all, We have an Epson 1640SU scanner with film scanning attachment. It came with 2 holders for negatives, but not the 3 1/4 x 4 inch. However, if you canabalise the 35 mm holder by taking out the central bar, then it takes 3 1/4 x 4 perfectly. This is far prefarable to laying the negative on the glass, since (1) we get no Newton's rings and (2) the negative is always in the same place, and you can therefore keep all the scanned area the same for each neg, when scanning a series in, which saves time on always having to preview, select the area and so on.
I find the Monochrome negative film setting in the software also very good, and the automatic adjustment of contrast usually gets it right for the majority of negatives. Only in cases where the neg is very bright, dark, low contrast, or very high contrast, does it have problems with auto adjustment.
I'm just a happy user. No connection with Epson. Perhaps I should have!
I think the only thing that would be really cool would be an automatic negative changer for when I am scanning 30 negs of industrial contract work, where the contrast is almost the same for each one! Anyway, I can dream.
Best wishes
Ian
} } Does anyone have experience of Epson scanners for TEM negatives? I have } } enquired of the company whether the Epson Perfection 2450 Photo can take } } our } } } 4 x 3-and-a quarter inch TEM plates, } } } } but all I can get is the standard answer "we have film holders", which } } include 120 roll film and 5 x 4 inch, but not the size we want. Can one } } simply place the negative straight down on the scanner surface? } } } } Any help would be much appreciated, } } } } (Sorry if you've received this message before - I've had one or two failed } } attempts at sending). } } } } ----------------------------------- } } Robert H. Olley, Physics Dept, } } Univ. Reading, RG6 6AF, England. } } E-mail: R.H.Olley-at-reading.ac.uk } } URL: http://www.rdg.ac.uk/~spsolley } } -----------------------------------
-- Ian MacLaren Strukturforschung, Materialwissenschaft, TU-Darmstadt, Germany ian.maclaren-at-physics.org / http://homepages.tu-darmstadt.de/~maclaren/
A typical turn -around time is 2-3 days, with four days being the latest that samples should be done by. These numbers come from many years of on the job numbers. Does your boss not help out at all in the lab? How many chucks for the microtome do you have? You should have at least 5, so you are not wasting time changing blocks all the time. Tissue processor? Film and paper processors?
On average I was able to complete 2 (possibly 3) cases/day. Of course these are averages. Some times the do-do does hit the fan.
Ed Calomeni currently unemployed EM tech ----- Original Message ----- } From: "Sharon Drew (by way of MicroscopyListserver)" {drewsh-at-musc.edu} To: {Microscopy-at-sparc5.microscopy.com} Sent: Wednesday, November 06, 2002 5:26 PM
Hi Leslie! We have been using polycontrast paper for a long time (more likely always). I find it much easier to handle then graded paper. We change filters to adjust contrats and play with exposure time. I think it is more economical way of printing then using graded paper. Dorota
I think LPD can still be purchased from Freestyle Sales in southern California.
Geoff
Paula Sicurello wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } ** Reply Requested When Convenient ** } } Hi listers, } } I'm looking for a black and white reversal film. I have some old LPD4 } but I can't kind listed anywhere (fuji or kodak) if they still make this } film. And if they do make it, does it come in little rolls or for a } bulk loader? } } I'm lazy and I like to make B&W text slides with B&W reversal film. Or } can I use a color slide film and still have it turn out OK? } } Any information is gladly accepted and I thank you in advance. } } Paula :-) } } p.s. some of us are old fashioned and like to use film instead of all } the fancy-schmancy computer thingies ;) } } Paula Sicurello } George Washington Univ. Medical Center } Dept. of Pathology, Ross Hall rm 505 } Electron Microscope Lab } 2300 Eye St. } Washington, DC 20037 } 202-994-2930 phone } 202-994-2518 fax
-- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
The suggestion of discriminating between AC fields and vibration via working distance is a good one. Another useful test is to change the beam voltage -- the susceptibility to transverse magnetic fields increases as the beam voltage is lowered. The (approximate) equation goes like this: Deflection = (0.5 microns)* B * Z^2 / sqrt( V ) Where B is the transverse field in gauss, Z is the working distance in mm, and V is the beam voltage in kilovolts. So the variation of deflection (sawtooth height) with working distance is stronger, but it isn't always practical to make a large change in WD, whereas it shouldn't be hard to get a factor of two variation by the beam voltage method (e.g., 20 kev and 5 kev). I tend to prefer the beam voltage method because the stage is a complex mechanical device whose suspectibility to vibration may itself be a function of height, thereby sometimes confusing the results.
Fred Schamber ASPEX
Benjamin - Simkin wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Ann, I agree with Allen Sampson's comments, with the addition that you could } also get the same effect from AC magnetic fields. These can pop up from a } number of sources (computer equipment, electroc motors, overhead lights, the } list goes on...). } A way of checking if it's vibration as opposed to fields is to check the } magnitude of the 'sawteeth' at long and short working distances; if it's fields, } then the sawteeth will be bigger at longer working distances (because the action } of the field will be operating over a longer distance at the longer working } distance). This also serves as a crude short-term fix for the problem sometimes, } although I'd really recommend finding the piece of equipment that's causing } the problem, and fix that instead. } Another possible source is a ground loop, where your 'scope is hooked up to } another piece of equipment with a differant ground potential. This causes } currents to end up flowing through your equipment-'scope connection, generating } the AC fields internally. A solution for this is to decide on a single } ground potential to use (usually the 'scope's dedicated ground), and then } hardwire all the other equipment to this one ground. (The best case, of course, } is to sink your own ground, but many people/labs don't have this option } available.) } If you do have field problems, you can usually track them down using an } AC magnetic field hand meter, which you can get for around $35 from an } online dealer (I forget where we got ours now, sorry). } } I hope this helps, } } Ben Simkin (simkin-at-egr.msu.edu) } Michigan State University } Department of Chemical Engineering and Materials Science
As far as I knew, the 16 bit support is in newer versions of Photoshop, but I haven't used it much. It's certainly in version 6, which I use. Perhaps it still has limited functionality. Worth testing, perhaps.
Ian
Thor Bostrom schrieb: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear Robert and listers, } } We have a recently acquired Epson Perfection 2450 Photo scanner and it } works very well with TEM negs mounted in the 5 x 4 film holder. You want } to avoid placing the negs directly on the glass surface because of Moire } fringes. (Curiously we had little trouble with fringes on our old HP } transparency scanner.) } } The Epson scanner can acquire 16-bit grayscale TIFF images which can be } advantageous for negatives with very low contrast. However few software } packages seem to handle this format. DigitalMicrograph and analySIS do, } while Adobe Photoshop provides some limited support. Are there other } programs out there that can work with this format? } } With thanks, } Thor } } } = = = = = = = = = = = = = = = = = = = } } } } Does anyone have experience of Epson scanners for TEM negatives? I have } } enquired of the company whether the Epson Perfection 2450 Photo can take } } our 4 x 3-and-a quarter inch TEM plates, } } but all I can get is the standard answer "we have film holders", which } } include 120 roll film and 5 x 4 inch, but not the size we want. Can one } } simply place the negative straight down on the scanner surface? } } } } Any help would be much appreciated, } } } } (Sorry if you've received this message before - I've had one or two } failed } } attempts at sending). } } ----------------------------------- } } Robert H. Olley, Physics Dept, } } Univ. Reading, RG6 6AF, England. } } E-mail: R.H.Olley-at-reading.ac.uk } } URL: http://www.rdg.ac.uk/~spsolley } } ----------------------------------- } } } =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= } Dr Thor Bostrom } Acting Director, } Analytical EM Facility, } Faculty of Science, } Queensland University of Technology (QUT) } GPO Box 2434, Brisbane, QLD 4001, Australia } Ph: +61 7 3864-2351 FAX: +61 7 3864-5100 } http://www.sci.qut.edu.au/aemf/ } =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= } } }
-- Ian MacLaren Strukturforschung, Materialwissenschaft, TU-Darmstadt, Germany ian.maclaren-at-physics.org / http://homepages.tu-darmstadt.de/~maclaren/
NIST (as I recall) developed the Desk Top Spectrum Analyzer (DTSA) that will import spectra and allow one to run quantitative analysis routines. An e-mail to Dale Newbury (see cc: list) at NIST may help.
Good luck,
Gary M. Brown ExxonMobil Chemical Company Baytown Polymers Center 5200 Bayway Drive Baytown, Texas 77520-2101 phone: (281) 834-2387 fax: (281) 834-2395 e-mail: Gary.M.Brown-at-ExxonMobil.com
"Anthony Rinaldi" {Anthony_Rinaldi-at-J To: "'Microscopy-at-MSA.Microscopy.Com'" abil.com} {Microscopy-at-sparc5.microscopy.com} cc: Subject: ZAF numbers and K ratios 11/06/02 09:59 PM
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Hello,
I am using a rather old (almost antique) EDS system for my X ray mapping. It is so old that it will not do quantitative analysis. With the economy like it is I will have to make this equipment last a few more years.
Does anyone know of a program that I can use that contains the reference data tables and allows you to compute the ZAF number (or K ratios ) for different elements? Doing this by hand is quite tedious. Thank you.
Thank you for the response. I did find that program. It looks like it imports the data file and then analyzes it. I do not believe that my EDS system has the ability to export the data. I have to take a picture of the screen. It has no disk option to transfer a file.
Since I can't transfer the file, I was hoping for something that would help me with the math part. I did get a program from Mr. Kaker which shows promise. However, I will need to learn about what each factor is in order to use it. If you have any recommendations for course or books please let me know.
Again thank you for your response.
Anthony Rinaldi Lead Failure Analysis Engineer Jabil Circuit, St Petersburg FL 727-803-3695
-----Original Message----- } From: gary.m.brown-at-exxonmobil.com [mailto:gary.m.brown-at-exxonmobil.com] Sent: Thursday, November 07, 2002 10:18 AM To: Anthony_Rinaldi-at-Jabil.com; microscopy-at-sparc5.microscopy.com Cc: newbury-at-enh.nist.gov
Hello,
I am using a rather old (almost antique) EDS system for my X ray mapping. It is so old that it will not do quantitative analysis. With the economy like it is I will have to make this equipment last a few more years.
Does anyone know of a program that I can use that contains the reference data tables and allows you to compute the ZAF number (or K ratios ) for different elements? Doing this by hand is quite tedious. Thank you.
We used to use the polycontrast paper before we switched to the kodabromide II RC paper. Also, since we are in Rochester, NY the home of Eastman Kodak I can tell you that Kodak still produces Kodabromide in grades 2, 3, & 4.
They have also introduced a Polymax II RC paper which has a set of filters which allow for even greater range of contrasts. I would recommend you change to the Polymax II RC paper. For instance using filter 4 gives you equivalent contrast to the Kodabromide grade 4 paper, filter 5 the grade 5 paper. The lower filters give you grades 1-3 equivalents.
Try the Polymax, you'll like it.....
Karen Bentley
Karen L. Bentley, M.S.(previously Jensen) Associate Scientist & Project Manager Electron Microscope Research Core University of Rochester Medical Center Rochester, NY 14642 585-275-1954
-----Original Message----- } From: Leslie Cummins [mailto:gunther-at-aecom.yu.edu] Sent: Wednesday, November 06, 2002 3:06 PM To: Microscopy-at-sparc5.microscopy.com
Hello listers
As Kodak slowly discontinues the graded Kodabrome II RC papers (no 1, 4 or 5), we are looking for alternatives for our printing.
We have a Durst point source enlarger, and I was wondering if we can switch to Polycontrast paper and filters. If there are any labs that are using this method, I would appreciate any information on how well it works.
We have found that it is still more economical and time efficient to develop and print 8 x 10 study prints for our users, rather than scan in all our negatives, so any old-fashioned wet darkroom suggestions would be appreciated.
Thanks in advance Leslie
Leslie Gunther Cummins Analytical Imaging Facility Albert Einstein College of Medicine 1300 Morris Park Ave. Bronx, NY 10461 718-430-3547
I've been trying to using Osteobed resin to embed small calcified bone samples. However I've got problems with the polymerization step, the resin becomes a rubber-like mass. I tried the manufacturer's protocol and few variations for methylmetacrilate found in literature but none of them worked. Any hint?
And regarding Histocryl, has anyone tried any immnuhistochemical or histoenzymology protocol in tissues embedded in it? Does it need the removal of resin prior the reaction?
I would be grateful with any help.
Best regards, Lenaldo
-- Lenaldo Branco Rocha, DDS, MSc Faculty of Medicine of Ribeirão Preto - USP Department of Pathology Av. Bandeirantes, 3900 Monte Alegre Ribeirão Preto - SP - Brazil 14049-900 --------------------------------- Tel: +55-16-602-3132 lenaldo-at-rpa.fmrp.usp.br
(I've tried to post this but it was bounced - and in light of the recent emails about the hotmail I'm trying to send this as plain text)
I sent out an earlier reply to the first post:
I've had zero problems with contrast with Spurr's if the sections are thick enough (gold post heat stretching).
I've used the xylene to stretch them on the boat, but found the heat 'pen' that we got from EMS works just as well without the risk to all the sections in the boat and without the vapors.
Sections are cut so that when they are stretched they are gold.
To pick them up a quick pass through the alcohol flame is good enough to get them to stick, with a trip into the 55 C oven until they reach temp to keep them on the grid.
Staining is simple
20-30 min submerged in a porcelain well filled with a saturated aqueous UA solution (use carpet tape to keep the UA bottle stable and keep a sediment of crystal UA on the bottom - keep the bottle covered and labeled). 2-5 minutes in the calcined lead citrate solution in a CO2 free environment (coverslip with NaOH pellet lining the walls)
quick coat of carbon in the evaporator - and - well I've got new batches of students each spring that don't seem to have any problems staining Spurr's with the above method.
But of Course (car talk) YMMV (your mileage may vary) but to adapt it: YRMV (results)
Geoff Williams Microscopy Facility Supervisor
Checkout the new Biology Department Microscopy Facility web page. Version 1 is now On-Line: www.cst.cmich.edu/users/willi1gl/BDMF/BDMF.htm
I haven't been in a darkroom in a long time, but I did spend many hours in one. I printed with both the Kodabrome II RC and the Polycontrast II RC and very good results with both. You will have to figure out the correlation between the grade for the Kodabrome and the contrast filter to use with the Polycontrast papers, but there are Kodak darkroom guides that you can use. They have images printed on both the Polycontrast and Kodabrome with the different filters and grades. You can use them to give you the equivalent values for what you wanted to do. Once you know what they are, you use the same filters for your images. For example, a soft filter for diffraction patterns and a hard filter for low contrast images such as HRTEM.
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: Leslie Cummins [mailto:gunther-at-aecom.yu.edu] Sent: Wednesday, November 06, 2002 3:06 PM To: Microscopy-at-sparc5.microscopy.com
Hello listers
As Kodak slowly discontinues the graded Kodabrome II RC papers (no 1, 4 or 5), we are looking for alternatives for our printing.
We have a Durst point source enlarger, and I was wondering if we can switch to Polycontrast paper and filters. If there are any labs that are using this method, I would appreciate any information on how well it works.
We have found that it is still more economical and time efficient to develop and print 8 x 10 study prints for our users, rather than scan in all our negatives, so any old-fashioned wet darkroom suggestions would be appreciated.
Thanks in advance Leslie
Leslie Gunther Cummins Analytical Imaging Facility Albert Einstein College of Medicine 1300 Morris Park Ave. Bronx, NY 10461 718-430-3547
Well, for us turnaround is 3 days because we have a digital camera on our microscope. Eliminates darkroom time... There are two of us in the lab. Sometimes I personally can have 3-4 cases a day cut and scoped. On a good day.
Here we get anywhere from 1-12 cases a day in the lab.
} } Hi! } } I run a diagnostic TEM laboratory in SC. } } One tech. } } 6 to 5 samples every other day at least. } } What is the turn around time asked for your labs out there that are } } also doing diagnostic/clinical work and where does that number come } } from? } } Thank you for any help. } } My supervisor is saying 2 days but I think that is close to impossible } } when only one tech doing everything including transport of tissue from } } another lab and scoping all case that come in. } } Sharon Drew } } Diagnostic Pathology EM } } Charleston, SC } } } }
I always used polycontrast paper and filters on a Durst point source enlarger, until everything went digital. I found that I had better control that way, as there is a wider range of filters than papers. It meant a little more work at the beginning, since test strips had to be done one at a time, but the results were worth it.
Lesley Weston.
on 06/11/2002 12:05 PM, Leslie Cummins at gunther-at-aecom.yu.edu wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hello listers } } As Kodak slowly discontinues the graded Kodabrome II RC papers (no 1, 4 or } 5), we are looking for alternatives for our printing. } } We have a Durst point source enlarger, and I was wondering if we can switch } to Polycontrast paper and filters. If there are any labs that are using } this method, I would appreciate any information on how well it works. } } We have found that it is still more economical and time efficient to } develop and print 8 x 10 study prints for our users, rather than scan in } all our negatives, so any old-fashioned wet darkroom suggestions would be } appreciated. } } Thanks in advance } Leslie } } } Leslie Gunther Cummins } Analytical Imaging Facility } Albert Einstein College of Medicine } 1300 Morris Park Ave. } Bronx, NY 10461 } 718-430-3547 } } http://www.aecom.yu.edu/aif/ } }
} From the pictures she posted, the problem is not AC electro-magnetic fields. They occur at 50 or 60 Hertz. Unless she has an SEM capable of incredible secondary electron collection, the images she posted were taken with an exposure time of at least 60 seconds (assuming a 4000 line exposure, 30 sec for 2000 line, 15 seconds for 1000, etc.). At this rate the vibrations shown are much slower than 60 Hz. Just divide the number of saw teeth seen by the exposure time.
For your information, vibrations will also increase with working distance. Within the electron optics column, where the beam is being acted on by the condenser lens and other magnetic lenses, the electron beam is pretty much constrained to the beamline by the action of the lenses - any external motion or magnetic field that attempts to moves the beam will be offset by the lens's symmetrical action to contain the beam. Within the sample chamber, the beam's path has been pre-determined by the actions of the c olumn and is influenced only by the unexpected, and external, influences.
Thanks, however, for mentioning the effect of working distance - I forgot to mention it before. With both vibrations and electro-magnetic interference, the majority of the influence on the beam will be in the portion of time that the beam spends in traveling from the final pole piece to the sample (the working distance). Decreasing the working distance will, in all cases, decrease the affect.
As far as ground loops - this is an area that is generally not very well understood. I have never really seen an instrument installation that suffered from this problem, at least as it is generally understood. The electrical ground is actually not something that generally has an active role in electrical systems. From the wall into your system, you have electrical connections to hot and neutral as well as an electrical ground. The hot and neutral wires are connected to the phase and center tap of the nearest step down transformer respectively. The ground is connected to a local source of ground potential.
Local ground potentials can vary considerably and this connection is primarily used as a safety to ensure that anything contacting a grounded connection will not be at a dangerous voltage level from anything else (such as water lines) that are at a local ground level. The neutral electrical connection will be grounded by the power company, but not necessarily at a point local to the delivery of power.
This can be best explained, perhaps, by the major problem with lightning strikes. In most cases of lightning strikes, the actual power delivery lines are not hit. Instead, the strike hits a tree or the ground nearby, which increases the local ground potential and causes breakdowns between components and ground. The electrical ground is normally only used to protect people by connecting the exterior surfaces of systems to a local potential that won't result in harm to people should a large incursion in local ground potential happen.
Equipment within a building will generally not connect anything to electrical ground other than the external cases. Should anything within the system short to a surface anyone might touch, this assures that the only voltages encountered will be at the local ground potential.
Having said that, I have to point out the poor nomenclature we have chosen in this regard. The ground loops that can cause problems in instruments this sensitive, are actually those on the electrical 'grounds' of the power supplies. The term 'ground', in this case, is normally applied to the common connection when we use multiple power supplies. For example, if we have a system that has both a +5 volt and a +15 volt power supply, we often tie the negative connection from each power supply together into a common 'ground'. If one of those supplies, or the circuits it connects to, tends to pull its negative voltage higher, we have a ground loop as it will also affect the voltage on the other supply. Since both sides of power supplies will often be completely isolated from the incoming power by transformers within the instrument, this 'common' ground may have no relationship whatsoever to any real ground voltage. I know this sounds confusing, but this is the true meaning of ground loop.
In other words, the external connection of individual equipment cases to each other does little good as they are generally already each connected to local ground through the wall connections. The real trick is to connect the power supply common connections in such a way as to provide as low a resistance path as possible to the chosen 'common' power connection.
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174
On Wednesday, November 06, 2002 5:37 PM, Benjamin - Simkin [SMTP:simkin-at-egr.msu.edu] wrote: } } Ann, I agree with Allen Sampson's comments, with the addition that you could } also get the same effect from AC magnetic fields. These can pop up from a } number of sources (computer equipment, electroc motors, overhead lights, the } list goes on...). } A way of checking if it's vibration as opposed to fields is to check the } magnitude of the 'sawteeth' at long and short working distances; if it's fields, } then the sawteeth will be bigger at longer working distances (because the action } of the field will be operating over a longer distance at the longer working } distance). This also serves as a crude short-term fix for the problem sometimes, } although I'd really recommend finding the piece of equipment that's causing } the problem, and fix that instead. } Another possible source is a ground loop, where your 'scope is hooked up to } another piece of equipment with a differant ground potential. This causes } currents to end up flowing through your equipment-'scope connection, generating } the AC fields internally. A solution for this is to decide on a single } ground potential to use (usually the 'scope's dedicated ground), and then } hardwire all the other equipment to this one ground. (The best case, of course, } is to sink your own ground, but many people/labs don't have this option } available.) } If you do have field problems, you can usually track them down using an } AC magnetic field hand meter, which you can get for around $35 from an } online dealer (I forget where we got ours now, sorry). } } I hope this helps, } } Ben Simkin (simkin-at-egr.msu.edu) } Michigan State University } Department of Chemical Engineering and Materials Science } } } }
Most of your concerns I covered in a previous reply. However, I have to take exception to your claim that a large change in the problem's characteristic would result from a change in accelerating voltage only if the problem was electro-magnetic. A reduction in the accelerating voltage will also result in a reduction of the velocity of electrons in the beam. That would cause an increased displacement of the beam as it traverses the sample surface due to vibrations.
In other words, the effects of electro-magnetic interference and vibrations are virtually inseparable except for the frequency involved. Each will affect the beam in similar ways, in regards to accelerating voltage and working distance. Both will have a greater affect with a lower accelerating voltage as well as a greater working distance.
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174
On Wednesday, November 06, 2002 8:58 PM, Vitaly Feingold [SMTP:vitalylazar-at-worldnet.att.net] wrote: } Could be vibration. Could also be magnetic field. And, could be signal } interference, such as ground loop. What type of SEM are you using? } } I will omit remedies for the vibration problem, since Allen Sampson did } pretty good job addressing that. } } Stray AC line frequency magnetic field: try acquiring image at different } accelerating voltages. It is very important that all other geometry } parameters will remain the same: particular area of a particular specimen, } WD, tilt, magnification, spot size. Contrast, brightness, focus, and } stigmator settings can be changed. If the problem is much worse at, say, 2 } kV, than at, say, 15kV, you are dealing with magnetic field. You may also } try to keep 3 axes AC milliGauss meter at the SEM, and notice what it } measures, but catching stray magnetic interference this way may be a little } tricky. These meters, though, are very inexpensive, and available from many } test instruments suppliers. } } Now we are getting down to statistically most likely problem- signal } interference and ground loops. It is difficult to go any further without } knowing the SEM type, and acquisition system (if separate) type. Too many } possibilities exist, but the very first- does your acquisition software } have an option somewhere in the settings which reads something like "60 } Hertz sync", or "AC line synchronization", or "Line sync", etc., etc.? If } yes, check (or uncheck) that option and see what happens. Further remedies } of this problem will include eliminating ground loops. Example- shielded } cable(s) has shield connected on both sides, and shield is used as one of } the signal wires. This is frequently done, and does jeopardize instrument } interference tolerance. Another example- one of the accessories is } susceptible to some kind of interference, and must be powered through the } isolation transformer. The latter remedy will only work with decent (better } if dedicated) ground. The fact that the problem is not present all the time } does not mean that everything is perfect inside the SEM. Besides, going } after the EM interference source could be inefficient and frustrating, if } not impossible. Improving grounding/shielding/power connection might be } easier. } } } Vitaly Feingold } Scientific Instruments and Applications } 2773 Heath Lane, Duluth GA 30096 } (770)232-7785 ph. } (770)232-1791 fax } (678)467-0012 mobile } } This message is made of 100% recycled electrons. } } This address can not receive messages larger than 15 kb without prior } notification. } } ----- Original Message ----- } } From: Ann-Fook Yang {yanga-at-agr.gc.ca} } To: {microscopy-at-sparc5.microscopy.com} } Sent: Wednesday, November 06, 2002 2:45 PM } Subject: vibration? } } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } Hi everyone, } } } } I have two SEM micrographs on the web: } } http://www.magma.ca/~scimat/Defect.htm } } The micrographs show zagged edges taken at 20 kX. Such phenomenon does } not present all the time. Can anyone suggest what causes the problem and how } to solve it? } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } EM Unit, } } Eastern Cereal and Oilseed Research Centre, } } Room 2091, Bldg. 20, } } Central Experimental Farm, } } Ottawa, Ontario } } Canada K1A 0C6 } } } } Tel: 1-613-759-1638 } } Fax: 1-613-759-1701 } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } } } }
Paula, I have an old (1973) Kodak publication entitled, "Small-Batch Reversal Processing of KODAK B/W Films" , #J-1. Not only use of Reversal kits but also make from scratch formulations. It is on its way to you at the FAX number you have listed below. Let me know if it doesn't come thru alright.
Regards,
Fred Monson
Frederick C. Monson, PhD Center for Advanced Scientific Imaging Mail to Geology West Chester University of Pennsylvania Schmucker II Science Center, Room SS024 South Church Street and Rosedale Avenue West Chester, PA, 19383 Phone: 610-738-0437 eMail: fmonson-at-wcupa.edu
-----Original Message----- } From: Paula Sicurello [mailto:patpxs-at-gwumc.edu] Sent: Wednesday, November 06, 2002 2:56 PM To: microscopy-at-sparc5.microscopy.com
** Reply Requested When Convenient **
Hi listers,
I'm looking for a black and white reversal film. I have some old LPD4 but I can't kind listed anywhere (fuji or kodak) if they still make this film. And if they do make it, does it come in little rolls or for a bulk loader?
I'm lazy and I like to make B&W text slides with B&W reversal film. Or can I use a color slide film and still have it turn out OK?
Any information is gladly accepted and I thank you in advance.
Paula :-)
p.s. some of us are old fashioned and like to use film instead of all the fancy-schmancy computer thingies ;)
Paula Sicurello George Washington Univ. Medical Center Dept. of Pathology, Ross Hall rm 505 Electron Microscope Lab 2300 Eye St. Washington, DC 20037 202-994-2930 phone 202-994-2518 fax
This artefact may be due to vibration, but I don't think it is possible to confirm that without additional information. The waves seem suspiciously regular. Acoustic vibration is rarely at a single frequency, and the pitch of the acoustically-induced sawtooth is usually rather variable. Also, acoustically-induced vibration will improve when the stage is locked or clamped (if you have that facility). I would also consider the possibility that there is an electronic problem in the scan generator circuit. On both of my last two sems (different manufacturers) similar faults to those shown were caused by faulty transistors. Other possible causes are electrical interference carried by the electricity supply, or strong electromagnetic fields generated by e.g. nearby electric motors, power transformers, etc. As noted by Benjamin, interference caused by mag field changes with working distance, but so does interference from acoustic vibration, usually appearing worse at long working distance. Chris
} } } Hi everyone, } } } } } } I have two SEM micrographs on the web: } } } http://www.magma.ca/~scimat/Defect.htm } } } The micrographs show zagged edges taken at 20 kX. Such phenomenon } } does not present all the time. Can anyone suggest what causes the } } problem and how to solve it? } } } Thanking in advance. } } } } } } AnnFook Yang } } } EM Unit, } } } Eastern Cereal and Oilseed Research Centre, } } } Room 2091, Bldg. 20, } } } Central Experimental Farm, } } } Ottawa, Ontario } } } Canada K1A 0C6 } } } } } } Tel: 1-613-759-1638 } } } Fax: 1-613-759-1701 } } } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } }
Looks like a failing turbo pump to me. Does this SEM have turbo?
gary g.
At 11:45 AM 11/6/2002, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Hi Thor, ImageJ (the free, multiplatform Java incarnation of NIH Image) will handle up to 32bit grey images. Website http://rsb.info.nih.gov/ij/ cheers
Sally.
} } } Thor Bostrom {t.bostrom-at-qut.edu.au} 11/07/02 01:46PM } } } ------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dear Robert and listers,
We have a recently acquired Epson Perfection 2450 Photo scanner and it works very well with TEM negs mounted in the 5 x 4 film holder. You want to
avoid placing the negs directly on the glass surface because of Moire fringes. (Curiously we had little trouble with fringes on our old HP transparency scanner.)
The Epson scanner can acquire 16-bit grayscale TIFF images which can be advantageous for negatives with very low contrast. However few software packages seem to handle this format. DigitalMicrograph and analySIS do, while Adobe Photoshop provides some limited support. Are there other programs out there that can work with this format?
With thanks, Thor
} = = = = = = = = = = = = = = = = = = = } } Does anyone have experience of Epson scanners for TEM negatives? I have } enquired of the company whether the Epson Perfection 2450 Photo can take } our 4 x 3-and-a quarter inch TEM plates, } but all I can get is the standard answer "we have film holders", which } include 120 roll film and 5 x 4 inch, but not the size we want. Can one } simply place the negative straight down on the scanner surface? } } Any help would be much appreciated, } } (Sorry if you've received this message before - I've had one or two failed } attempts at sending). } ----------------------------------- } Robert H. Olley, Physics Dept, } Univ. Reading, RG6 6AF, England. } E-mail: R.H.Olley-at-reading.ac.uk } URL: http://www.rdg.ac.uk/~spsolley } ----------------------------------- } =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Dr Thor Bostrom Acting Director, Analytical EM Facility, Faculty of Science, Queensland University of Technology (QUT) GPO Box 2434, Brisbane, QLD 4001, Australia Ph: +61 7 3864-2351 FAX: +61 7 3864-5100 http://www.sci.qut.edu.au/aemf/ =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=
Why is it that the directions from Kodak for making a gallon of Rapid Fixer state that I should pour 32oz. of "A" and 104ml of "B" into a half gallon of water and then make it up to 1 gal. but the kit to make 1 Gal. has 33oz of "A" and 106ml of "B"?
Also for people who now seldom need to use fixer - be advised that "A" DOES go bad with age. You will know it when a precipitate forms as "B" is added slowly to the mixture of water and "A". If the "A" is really old there will be the signs of yellow crystals on the sides of the container.
I questioned Kodak representatives in the mid-1980's and again in the 1990's and was told not to worry. I still worry!
Methods in Plant Electron Microscopy and Cytochemistry by William V. Dashek (Editor)
Plant Cell Biology: Structure and Function by Brian E. S. Gunning, Martin W. Steer
I routinely work with plant tissue. if you need any help in the future, just contact me, we can send you protocols.
Good luck.
Soumitra
************************************************************* Soumitra Ghoshroy Ph.D. College Associate Professor Department of Biology Director, Electron Microscopy Lab Box 3EML New Mexico State University Las Cruces, NM 88003 Tel: 505-646-3268 (office), 646-3283 (lab) Fax: 505-646-3282 e-mail:sghoshro-at-nmsu.edu URL:http://confocal.nmsu.edu/eml
On Wed, 6 Nov 2002, Jensen, Karen wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } Dear Listers: } } } } I have recently had several requests to process and embedd plant } } tissue. All of my previous EM experience has involved only human and } } animal tissue. } } } } Those of you who routinely do botanical specimens can you recommend } } some EM books or atlases which cover processing methods? } } } } Are there any atlases that provide ultrastructural images so I can } } learn the morphology/terminology of different types of plant specimens. } } Right now I will be looking at potato leaves and tubers. Later I am } } supposed to get a tomato for EM processing. } } } } Thanks for your help! } } } } Karen L. Bentley, M.S.(previously Jensen) } } Associate Scientist & Project Manager } } Electron Microscope Research Core } } University of Rochester Medical Center } } Rochester, NY 14642 } } 585-275-1954 } } } }
In a separate e-mail, I will be attaching a set of files containing the NIST "CITZAF" stand-alone ZAF analysis package. The files, once extracted contain two PDF files describing the operation of the various programs as well as the CITZAF program package. This program has been around for some time in various forms. The attached files contain the current (beta-test) development version of this program package that we are using at NIST. We are currently modifying the input procedures of this program and "window-fying" it. Once completed, the updated program will be available on the NIST Chemical Science and Technology Laboratory, Surface and Microanalysis Division web page -- www . cstl . nist . gov / div837 / Division . I hope you will find this useful.
Best regards,
John Armstrong
At 10:59 PM 11/6/2002 -0500, Anthony Rinaldi wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
******************************* Dr. John T. Armstrong Nat'l Inst. of Standards & Tech. Chemistry, Bldg. 222, Rm A113 100 Bureau Drive. Stop 8371 Gaithersburg, MD 20879-8371 (301) 975-3929 (301) 417-1321 (FAX) john.armstrong-at-nist.gov
Hello Leslie, I use a Durst Laborator S-45 Special enlarger with a point light source and print all my TEM and 35mm negatives on Ilford Multigrade IV RC DeLuxe paper using Ilford Multigrade filters. It works fine for me and my bosses, but I am sure that other papers like Kodak Polymax II RC, as suggested by Karen Jensen, work just as well. I learned my darkroom work using fiber-based graded papers and drying them on a big mirrored drum, but now must I air dry my prints ever since our dryer broke and couldn't be fixed. This is a bit of a nuisance as the prints take overnight to dry and can't be turned in the same day they're printed. Does anyone have tips on drying resin coated paper quickly??? Dean Abel Biological Sciences 138BB University of Iowa Iowa City IA 52242-1324
SORRY - I forgot to give the author - it is by Rhodin Histology: A text and atlas takes a lot of beating - it goes from LM through to EM. It is out of print now but Amazon.com still has some copies available.
At 09:14 2002-11-06 -0500, Julian Smith III wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Obs/NB New postal/visiting address from July 2002!
Gareth Morgan MPhil MSc FIBMS, Institute for Microbiology, Pathology and Immunology (IMPI), H5, Karolinska Institutet, Huddinge Universitetssjukhus, F46 SE 141 86 Stockholm Sweden
OBS! Besöksadress: F-Huset, Forskningsgatan 2 F52, Rum 2.10. Laboratoriet för klinisk patologi och cytologi.
NB! Visiting address: Building F, Research Corridor 2 F52, Room 2.10. Clinical Histo- and Cytopathology Laboratory.
You may found the following figures entertaining. Natural constants and results of calculations are rounded to the first character after the decimal point, relativity effects are neglected, plain text format is used. I am a bit rusty on my Physics- my apologies if I missed an order or so of magnitude. Makes no difference in this example.
Electron mass ~ 9.1 x 10-31kg Electron charge ~ -1.6 x 10-19 C One electronvolt energy ~ 1.6 x 10-19J Kinetic energy (of electron, or anything having mass) = mV2/2, where m is the mass, V2 is the velocity square.
Simple calculations yield the following velocities in kilometers per second for electrons accelerated by potential differences of: 1 Volt ~ 6 x 100 km/s 200 V ~ 8.4 x 1,000 km/s 2 kV ~ 2,7 x 10,000 km/s 15 kV ~ 7.3 x 10,000 km/s
Amazing, isn't it?
Mechanical vibration velocity must be compatible in magnitude with the above velocities, in order for vibration effects to look different at different beam accelerating voltages. But SEM is made of the materials found on this planet, with limited durability specifications. Thus, in order to vibrate with such velocities, SEM must be either made very tiny, which is not the case, or must disintegrate into dust, but it doesn't.
Velocities of mechanical vibrations affecting SEM are in many orders of magnitude slower than the E-beam velocities.
This is why SEM stage vibration will show up exactly the same at different beam accelerating voltages.
Why then stray magnetic field affects an e-beam? Because electron has huge charge/mass ratio, ~ 10,000,000,000. The interaction is very strong. Thus, effect increases at lower accelerating voltages. Same with working distance- effect increases with the distance increase. But, one needs to change WD substantially, to see the effect for sure. That will reduce the resolution, among other things. I will advice against chamber/beam/sample geometry change during troubleshooting process.
Now down to business with Ann's images. The sawtooth vertical edges appearance is the result of horizontal scan being out of phase with the external source of either vibration, fields, or signal interference. Time delay between the lines at slow scan is in order of tens to hundreds milliseconds, which is within the possible vibration frequency range. Same frequencies will cause wavy image at TV deflection speeds.
Another point is that mechanical vibration in many cases repeats the AC line frequency, as it is caused by electric motor driven devices, so either 60 Hz or it's harmonic(s) are present as mechanical vibrations. The only way to find the problem is to troubleshoot and differentiate.
I agree with other postings- more information needed to develop more accurate idea on how to proceed.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax (678)467-0012 mobile
This message is made of 100% recycled electrons.
This address can not receive messages larger than 15 kb without prior notification.
----- Original Message ----- } From: Allen Sampson {ars-at-sem.com} To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' {Microscopy-at-sparc5.microscopy.com} Sent: Thursday, November 07, 2002 3:50 PM
Dear All,
How much is a reasonable price to pay for a Leica/Leo Stereoscan s440 and/ or what % of the original purchase price should one expect to pay for a ~6 year old SEM in good working order? I would be most grateful for your opinions.
best regards and thanks,
Marion
*********************************************** Dr Marion A. Stevens-Kalceff Deputy Director Electron Microscope Unit University of New South Wales, Sydney NSW 2052 Australia ***********************************************
The exact concentrations of fixers are not very critical, so don't lose sleep over these minor discrepancies. Unlike developers where time, concentration and temperature must be correct to get a reproducible result, you can safely vary fixer concentrations over quite a wide range and fix for the time/temperature required for completion. A good rule of thumb is to fix for twice the time required to clear the emulsion. The yellow crystals are elemental sulphur, indicating that the ammonium thiosulphate is decomposing. The fixer may still work adequately if sufficient thiosulphate remains. Agfa's tip is to compare the film-clearing time with that of a fresh batch of fix. If it takes more than twice as long the fixer is exhausted. Chris
} ----- Original Message ----- } From: "Pat Connelly" {psconnel-at-sas.upenn.edu} } To: "Microscopy Listserver" {Microscopy-at-sparc5.microscopy.com} } Sent: Thursday, November 07, 2002 9:29 PM } Subject: Kodak Rapid Fixer } }
} -------------------------------------------------------------------- ---- } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html
} -------------------------------------------------------------------- ---. } } } Why is it that the directions from Kodak for making a gallon of Rapid } Fixer state that I should pour 32oz. of "A" and 104ml of "B" into a } half gallon of water and then make it up to 1 gal. but the kit to } make 1 Gal. has 33oz of "A" and 106ml of "B"? } } Also for people who now seldom need to use fixer - be advised that } "A" DOES go bad with age. You will know it when a precipitate forms } as "B" is added slowly to the mixture of water and "A". If the "A" is } really old there will be the signs of yellow crystals on the sides of } the container. } } I questioned Kodak representatives in the mid-1980's and again in the } 1990's and was told not to worry. I still worry! } } Pat Connelly } Univ. of Pennsylvania } Biology Dept.
Reminds me of the very old question of can a scanning electron spot on a CRT exceed the speed of light?
If only it could be as easy to figure as you portray. I'll try to explain the situation, but it is really more complex that I can describe in words here - and being late at night, I really won't spend much time on the matter.
Let's assume that we are working at a working distance of 20mm. For the velocity you give for electrons accelerated at 2KV (27,000 kM/S) or 15KV (73,000 kM/S), that 20mm would be traversed in an exceeding small fraction of a second. However, we aren't looking at the spatial translation of a single spot. In the SEM, the spot formed on the sample is constantly moving - in the case of a record image the movement of the spot is generally determined by the dwell time on each of the individual pixels on each line and the number and resolution of lines in the frame.
In the record mode, most instruments will sync the start of each line to the zero-crossing of the electrical mains. This is an intentional attempt to minimize the effect of 50 or 60 Hz. interference. Digital systems may or may not do this.
If you go vertically down the recorded image, you are actually looking at pixels that are separated by fairly large periods of time, 1/50th or 1/60th of a second in the case of a synched record cycle. Now let's look at your projected travel times using these figures. The difference between the 2KV and 15KV velocities you mention is 46000 kM/S. That gives us a difference in the velocity at these two accelerating voltages of 4.35x10-9 seconds to traverse the 20mm to the sample.
Assuming a 60 Hz system, the difference between two vertically separated pixels is 0.0167 seconds. Let's say that each 1/60th of a second is split into 1500 equal parts - 1000 pixels used per line and 500 portions used for the vertical retrace. That means that each pixel would be 0.000011 seconds apart.
Now let's look at two pixels that are on separate scan lines and separated by one additional pixel. They would be separated by a time period of 0.016678 seconds (adding the pixel dwell time to the line dwell time). Doesn't sound like much, but in EM we have to understand the effects of the orders of magnitude we work with. If you divide the above time difference to traverse the working distance by the pixel time difference just mentioned, you would find that there is a 2.58x10-7 second difference between the pixels mentioned above.
I'm tired and really don't want to extend this too much more, but consider that the vibrations seen on the images at question cover around 20 or more scan lines and figure in the velocities of a nanometer scale RMS vibration. You'll find that there is indeed a considerable, measurable, variation in image vibrations due to accelerating voltage (as well as working distance).
Amazing, isn't it?
By the way, I still claim that the frequency of the problems in the original image is much lower than 50 or 60 Hertz and is the result of vibrations rather than any electro-magnetic or electronic effect. Another poster mentioned the possibility that a turbo pump may be having problems but that is also not a likely cause as the frequencies involved don't match. You might, however, want to check the operation of any water chiller that is in use.
If I have learned anything in working on these instruments for well over twenty years, it's that nothing is simple or straightforward. We're dealing with sub-atomic particle physics here folks, an area that can only be currently described by the statistical methods of quantum theories.
As far as I am concerned, there is no further determination to be made - the problem is one of mechanical vibrations and I would be glad to stake my reputation on it, as I do on any posting I make here.
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174
On Thursday, November 07, 2002 10:54 PM, Vitaly Feingold [SMTP:vitalylazar-at-worldnet.att.net] wrote: } Hey Allen, } } You may found the following figures entertaining. Natural } constants and results of calculations are rounded to the first character } after the decimal point, relativity effects are neglected, plain text format } is used. I am a bit rusty on my Physics- my apologies if I missed an order } or so of magnitude. Makes no difference in this example. } } Electron mass ~ 9.1 x 10-31kg } Electron charge ~ -1.6 x 10-19 C } One electronvolt energy ~ 1.6 x 10-19J } Kinetic energy (of electron, or anything having mass) = mV2/2, where m is } the mass, } V2 is the velocity square. } } Simple calculations yield the following velocities in kilometers per second } for electrons accelerated by potential differences of: } 1 Volt ~ 6 x 100 km/s } 200 V ~ 8.4 x 1,000 km/s } 2 kV ~ 2,7 x 10,000 km/s } 15 kV ~ 7.3 x 10,000 km/s } } Amazing, isn't it? } } Mechanical vibration velocity must be compatible in magnitude with the above } velocities, in order for vibration effects to look different at different } beam accelerating voltages. But SEM is made of the materials found on this } planet, with limited durability specifications. Thus, in order to vibrate } with such velocities, SEM must be either made very tiny, which is not the } case, or must disintegrate into dust, but it doesn't. } } Velocities of mechanical vibrations affecting SEM are in many orders of } magnitude slower } than the E-beam velocities. } } This is why SEM stage vibration will show up exactly the same at different } beam accelerating voltages. } } Why then stray magnetic field affects an e-beam? Because electron has huge } charge/mass ratio, ~ 10,000,000,000. The interaction is very strong. Thus, } effect increases at lower accelerating voltages. Same with working distance- } effect increases with the distance increase. But, one needs to change WD } substantially, to see the effect for sure. That will reduce the resolution, } among other things. I will advice against chamber/beam/sample geometry } change during troubleshooting process. } } Now down to business with Ann's images. The sawtooth vertical edges } appearance is the result of horizontal scan being out of phase with the } external source of either vibration, fields, or signal interference. Time } delay between the lines at slow scan is in order of tens to hundreds } milliseconds, which is within the possible vibration frequency range. Same } frequencies will cause wavy image at TV deflection speeds. } } Another point is that mechanical vibration in many cases repeats the AC line } frequency, as it is caused by electric motor driven devices, so either 60 Hz } or it's harmonic(s) are present as mechanical vibrations. The only way to } find the problem is to troubleshoot and differentiate. } } I agree with other postings- more information needed to develop more } accurate idea on how to proceed. } } } Vitaly Feingold } Scientific Instruments and Applications } 2773 Heath Lane, Duluth GA 30096 } (770)232-7785 ph. } (770)232-1791 fax } (678)467-0012 mobile } } This message is made of 100% recycled electrons. } } This address can not receive messages larger than 15 kb without prior } notification. } } ----- Original Message ----- } From: Allen Sampson {ars-at-sem.com} } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } {Microscopy-at-sparc5.microscopy.com} } Sent: Thursday, November 07, 2002 3:50 PM } Subject: RE: vibration? } } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -------------------------------------------------------------------- ---. } } } } } } Vitaly, } } } } Most of your concerns I covered in a previous reply. However, I have to } } take exception to your claim that a large change in the problem's } } characteristic would result from a change in accelerating voltage only if } } the problem was electro-magnetic. A reduction in the accelerating voltage } } } will also result in a reduction of the velocity of electrons in the beam. } } That would cause an increased displacement of the beam as it traverses } the } } sample surface due to vibrations. } } } } In other words, the effects of electro-magnetic interference and } vibrations } } are virtually inseparable except for the frequency involved. Each will } } affect the beam in similar ways, in regards to accelerating voltage and } } working distance. Both will have a greater affect with a lower } } accelerating voltage as well as a greater working distance. } } } } } } Allen R. Sampson } } Advanced Research Systems } } 317 North 4th. Street } } St. Charles, Illinois 60174 } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } } } } On Wednesday, November 06, 2002 8:58 PM, Vitaly Feingold } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } Could be vibration. Could also be magnetic field. And, could be signal } } } interference, such as ground loop. What type of SEM are you using? } } } } } } I will omit remedies for the vibration problem, since Allen Sampson did } } } pretty good job addressing that. } } } } } } Stray AC line frequency magnetic field: try acquiring image at } different } } } accelerating voltages. It is very important that all other geometry } } } parameters will remain the same: particular area of a particular } } specimen, } } } WD, tilt, magnification, spot size. Contrast, brightness, focus, and } } } stigmator settings can be changed. If the problem is much worse at, say, } } 2 } } } kV, than at, say, 15kV, you are dealing with magnetic field. You may } also } } } try to keep 3 axes AC milliGauss meter at the SEM, and notice what it } } } measures, but catching stray magnetic interference this way may be a } } little } } } tricky. These meters, though, are very inexpensive, and available from } } many } } } test instruments suppliers. } } } } } } Now we are getting down to statistically most likely problem- signal } } } interference and ground loops. It is difficult to go any further without } } } } knowing the SEM type, and acquisition system (if separate) type. Too } many } } } possibilities exist, but the very first- does your acquisition software } } } have an option somewhere in the settings which reads something like "60 } } } Hertz sync", or "AC line synchronization", or "Line sync", etc., etc.? } If } } } yes, check (or uncheck) that option and see what happens. Further } } remedies } } } of this problem will include eliminating ground loops. Example- sh ielded } } } cable(s) has shield connected on both sides, and shield is used as one } of } } } the signal wires. This is frequently done, and does jeopardize } instrument } } } interference tolerance. Another example- one of the accessories is } } } susceptible to some kind of interference, and must be powered through } the } } } isolation transformer. The latter remedy will only work with decent } } (better } } } if dedicated) ground. The fact that the problem is not present all the } } time } } } does not mean that everything is perfect inside the SEM. Besides, going } } } after the EM interference source could be inefficient and frustrating, } if } } } not impossible. Improving grounding/shielding/power connection might be } } } easier. } } } } } } } } } Vitaly Feingold } } } Scientific Instruments and Applications } } } 2773 Heath Lane, Duluth GA 30096 } } } (770)232-7785 ph. } } } (770)232-1791 fax } } } (678)467-0012 mobile } } } } } } This message is made of 100% recycled electrons. } } } } } } This address can not receive messages larger than 15 kb without prior } } } notification. } } } } } } ----- Original Message ----- } } } } From: Ann-Fook Yang {yanga-at-agr.gc.ca} } } } To: {microscopy-at-sparc5.microscopy.com} } } } Sent: Wednesday, November 06, 2002 2:45 PM } } } Subject: vibration? } } } } } } } } } } } } ------------------------------------------------------------------------ } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } America } } } } To Subscribe/Unsubscribe -- Send Email to } } ListServer-at-MSA.Microscopy.Com } } } } On-Line Help } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } } } -----------------------------------------------------------------------. } } } } } } } } } } } } Hi everyone, } } } } } } } } I have two SEM micrographs on the web: } } } } http://www.magma.ca/~scimat/Defect.htm } } } } The micrographs show zagged edges taken at 20 kX. Such phenomenon } does } } } not present all the time. Can anyone suggest what causes the problem and } } how } } } to solve it? } } } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } } } EM Unit, } } } } Eastern Cereal and Oilseed Research Centre, } } } } Room 2091, Bldg. 20, } } } } Central Experimental Farm, } } } } Ottawa, Ontario } } } } Canada K1A 0C6 } } } } } } } } Tel: 1-613-759-1638 } } } } Fax: 1-613-759-1701 } } } } } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } }
A postdoctoral researcher position is available to study the structures of biological macromolecular complexes by electron cryo-microscopy and image analysis. This position requires experience with electron microscopy and image processing.
The Biology Department at Brookheaven National Laboratory (BNL) has excellent facilities for cryo-EM and image processing. Major facilities available include a new JEM 2010F FasTEM, and access to the STEM facility and National Synchrotron Light Source.
Applicants should send in by email a CV, a brief statement of interests, and names and addresses of 2-3 referees.
Huilin Li Brookheaven National Laboratory Biology Department, Bldg. 463 Upton, NY 11973 email: hli-at-bnl.gov phone: (631)344-2931 fax: (631)344-3407
Regal-Arkay and Jobo both offer "roller transport" type RC print dryers. The Regal-Arkay units are high speed professional dryers that will last many years. They are available in 12" and 20" widths. Jobo sells DevAppa dryers in widths from 8" to 20". They are also very good dryers but I do not have as much experience with them. Check them out at http://www.jobo-usa.com/products/dry_prnt.htm For more limited budgets, Premier has the RC-500A, which utilizes filtered air blowing over prints. It will hold up to four 11x14 prints at one time. The RC500A is under $300. Of course, there is always the squeegee!!
George
George Laing National Graphic Supply ph 800.223.7130 x3109 f 800.832.2205 email scisales-at-ngscorp.com
Does anyone have tips on drying resin coated paper quickly???
In doing SEM I occasionally acquire images that contain a gradient. One example would be an x-ray map that I have acquired in an hours time. Typically the gun will drift slightly out of alignment and the end of the image is darker than the beginning. Another example is when I am collecting very low magnification images using the conventional SEM detector (Everhart and Thornley). The top left hand side of the image is much brighter than the bottom right hand side. I would like a routine that averages pixel values, say nearest ten neighbors, to create a new image based on just the averages. I could then perhaps divide the original image by the average image and in effect, normalize the original image.
It would be great if I could do this within Adobe Photoshop only.
Since it is SEM at least we are dealing with just 256 gray levels.
Print dryers for resin coated papers are available, of course right now I can't remember where! Try a google search for print dryers.
Geoff
Dean Abel wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hello Leslie, } I use a Durst Laborator S-45 Special enlarger with a point light source } and print all my TEM and 35mm negatives on Ilford Multigrade IV RC DeLuxe } paper using Ilford Multigrade filters. It works fine for me and my bosses, } but I am sure that other papers like Kodak Polymax II RC, as suggested by } Karen Jensen, work just as well. } I learned my darkroom work using fiber-based graded papers and drying them } on a big mirrored drum, but now must I air dry my prints ever since our } dryer broke and couldn't be fixed. This is a bit of a nuisance as the } prints take overnight to dry and can't be turned in the same day they're } printed. } Does anyone have tips on drying resin coated paper quickly??? } Dean Abel } Biological Sciences 138BB } University of Iowa } Iowa City IA 52242-1324
-- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
DOES ANYONE HAVE ANY GOOD USED LAB-6 TIPS OR A SURPLUS NEW ONE THAT WE COULD OBTAIN, TO COMPLETE A PROJECT, WE HAVE ORDERED ONE BUT IT TAKES 3 WEEKS TO GET. IT IS FOR AN S570 HITACHI SEM. PLEASE REPLY DIRECT TO E-MAIL
----- Original Message ----- } From: Allen Sampson {ars-at-sem.com} To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' {Microscopy-at-sparc5.microscopy.com} Sent: Friday, November 08, 2002 6:43 AM
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (ramkik-at-wuphys.wustl.edu) from http://www.msa.microscopy.com/Ask-A-Microscopist.html#form on Friday, November 8, 2002 at 08:48:27 ---------------------------------------------------------------------------
Question: Is there a way to statistically justify the number of TEM samples that must be prepared to be confident. One answer to this may be on the basis of the feature size beining invesitgated. There are probably two extreme cases to base the answer upon: In one case we have numerous independent prior measurements of the sample properties while in the other we do not. If there are any good journal paper/Book chapter that answers this question, I would appreciate knowing about it. thank you
Hello, Does anyone know where I can buy a small chunk of low resistivity ( {0.1 ohm-cm) Silicon for an experiment that I need to perform? I need a crystal about 10 to 100 cc's in size and it could be a boule top or bottom. Cheers Mike Urbanik Florida
Recently I freeze substitution embedded some skin samples with Lowicryl K4M. The blocks were cured under UV at -35C for 2 days and at room temperature for several days. The blocks looks fine to me after UV polymerization. However, when I section the blocks, blocks surface always get wet and the section swell badly on the water. It seems like sections dissolving into water. I couldn't pick up any intact sections. I used Lowicryl HM 20 before. It worked very well for me. I did read the instruction of Lowicryl resin and notice that the sectioning polar resin is different from nonpolar resin. But I could not get good sections of Lowicyl K4M. I don't know if the blocks are not fully polymerized or I need some special tricks to get sections. Any suggestions are really appreciated!
Shanling
Shanling Shi Advanced Imaging & Measurement Unilever Research & Development -Edgewater 45 River Road Edgewater, NJ 07020 201-840-2340 Shanling.Shi-at-unilever.com
Sharon, Two days seems very fast, type A personalities? Here we have a typical 1 week turnaround time from fixation to sectioning (thick and thin) to viewing and scanning negatives. I think that is a more fair and resonable time frame for an 8 hour day. Mike Delannoy Johns Hopkins School of Medicine Microscope Facility
Ed Calomeni wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Morning Sharon, } } A typical turn -around time is 2-3 days, with four days being the latest } that samples should be done by. These numbers come from many years of on } the job numbers. } Does your boss not help out at all in the lab? } How many chucks for the microtome do you have? You should have at least 5, } so you are not wasting time changing blocks all the time. Tissue processor? } Film and paper processors? } } On average I was able to complete 2 (possibly 3) cases/day. Of course these } are averages. Some times the do-do does hit the fan. } } Ed Calomeni } currently unemployed EM tech } ----- Original Message ----- } } From: "Sharon Drew (by way of MicroscopyListserver)" {drewsh-at-musc.edu} } To: {Microscopy-at-sparc5.microscopy.com} } Sent: Wednesday, November 06, 2002 5:26 PM } Subject: Clinical turn around } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } Hi! } } I run a diagnostic TEM laboratory in SC. } } One tech. } } 6 to 5 samples every other day at least. } } What is the turn around time asked for your labs out there that are } } also doing diagnostic/clinical work and where does that number come } } from? } } Thank you for any help. } } My supervisor is saying 2 days but I think that is close to impossible } } when only one tech doing everything including transport of tissue from } } another lab and scoping all case that come in. } } Sharon Drew } } Diagnostic Pathology EM } } Charleston, SC } }
Shanling: You have discovered the well known downside of K4M but it can be overcome. Half the battle is to get the water level just right. We also find the sectioning characteristics (and I think how easily water jumps to the surface) also improves if we store the blocks in a desiccator overnight before we cut them. Don't use too slow of a cutting speed since that promotes wetting of the surface. If you do get the block face wet, you need to dry both the block face and back of the knife before re-starting or it will wet immediately upon restarting. We prefer LR Gold for this reason but Lowicryl K4M has some features that make it superior for some antigens. Good luck.
At 11:17 AM 11/8/2002 -0500, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Very generally, assuming that column/apertures alignment/centering is correct- it could be a problem with, or an incorrect setting of the SED collector grid voltage (likely), or collector grid could be damaged/dirty (unlikely). Was this problem always there at low mag., or had it started happening recently?
Outside fixing an actual problem- acquire and keep on file a reference image of a perfectly smooth featureless surface (pick proper gain/black level settings). Then run flat field correction routine on your real images, with the use of that image file. Flat field correction can be found in a number of image processing programs. This routine is used to correct vignetting effect of an optical lens, among other things.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax (678)467-0012 mobile
This message is made of 100% recycled electrons.
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----- Original Message ----- } From: Smartech {smartech-at-optonline.net} To: To all on the list {Microscopy-at-sparc5.microscopy.com} Sent: Friday, November 08, 2002 9:03 AM
Remove the long-wavelength intensity fluctuation from your images using a highpass Fourier Transform filter.
This, and other background correction utilities are available (for free) in ImageJ http://rsb.info.nih.gov/ij/ ImageJ is the most versatile 2D ip&a software for those hwo do not require super-high speed.
Cheers, Paul Baggethun Pittsburgh, PA
-----Original Message----- } From: Smartech [mailto:smartech-at-optonline.net] Sent: Friday, November 08, 2002 9:04 AM To: To all on the list
In doing SEM I occasionally acquire images that contain a gradient. One example would be an x-ray map that I have acquired in an hours time. Typically the gun will drift slightly out of alignment and the end of the image is darker than the beginning. Another example is when I am collecting very low magnification images using the conventional SEM detector (Everhart and Thornley). The top left hand side of the image is much brighter than the bottom right hand side. I would like a routine that averages pixel values, say nearest ten neighbors, to create a new image based on just the averages. I could then perhaps divide the original image by the average image and in effect, normalize the original image.
It would be great if I could do this within Adobe Photoshop only.
Since it is SEM at least we are dealing with just 256 gray levels.
I'm looking for a good mercury bulb supplier? Any recommendations? The last few I have gotten have not lasted the 200 hrs they should, so I am looking for a new source.
Thank you in advance! -Holly
Holly L. Aaron Molecular Imaging Center Cancer Research Laboratory University of California, Berkeley 447 Life Sciences Addition Berkeley, CA 94720 http://imaging.berkeley.edu
The reason there is a gradient in your SE image is not due to any problems with the column but is entirely due to the way the ET detector works. The top left portion of your images are brighter because that is where your ET detector is positioned (if it isn't, then there really is something else wrong). The images are brighter at the upper left because that part of the raster is closer to the detector and more secondary electrons reach the detector, then the lower right, which is further away from the detector. All side-mounted ET detectors (I think) show this at low magnifications.
I think the easiest way to correct for this is the flat field method suggested by Vitaly Feingold in the previous message.
************************************************
....amphiboles do violence to history... T. Feininger, 2001. (taken out of context) ****************************
Dr. Scott Kuehner kuehner-at-u.washington.edu Dept. of Geological Sciences ph.206-543-8393 Box 351310 Fax 206-616-6873 The University of Washington Seattle, Washington 98195-1310 ************************************************
On Fri, 8 Nov 2002, Vitaly Feingold wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } What SEM model is it? } } Very generally, assuming that column/apertures alignment/centering is } correct- it could be a problem with, or an incorrect setting of the SED } collector grid voltage (likely), or collector grid could be damaged/dirty } (unlikely). Was this problem always there at low mag., or had it started } happening recently? } } Outside fixing an actual problem- acquire and keep on file a reference image } of a perfectly smooth featureless surface (pick proper gain/black level } settings). Then run flat field correction routine on your real images, with } the use of that image file. Flat field correction can be found in a number } of image processing programs. This routine is used to correct vignetting } effect of an optical lens, among other things. } } Vitaly Feingold } Scientific Instruments and Applications } 2773 Heath Lane, Duluth GA 30096 } (770)232-7785 ph. } (770)232-1791 fax } (678)467-0012 mobile } } This message is made of 100% recycled electrons. } } This address can not receive messages larger than 15 kb without prior } notification. } } ----- Original Message ----- } } From: Smartech {smartech-at-optonline.net} } To: To all on the list {Microscopy-at-sparc5.microscopy.com} } Sent: Friday, November 08, 2002 9:03 AM } Subject: I need help with digital images __ How to subtract a gradaient } fromSEM image } } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } -----------------------------------------------------------------------. } } } } } } In doing SEM I occasionally acquire images that contain a gradient. One } } example would be an x-ray map that I have acquired in an hours time. } } Typically the gun will drift slightly out of alignment and the end of the } } image is darker than the beginning. Another example is when I am } collecting } } very low magnification images using the conventional SEM detector } (Everhart } } and Thornley). The top left hand side of the image is much brighter than } the } } bottom right hand side. I would like a routine that averages pixel } values, } } say nearest ten neighbors, to create a new image based on just the } averages. } } I could then perhaps divide the original image by the average image and in } } effect, normalize the original image. } } } } It would be great if I could do this within Adobe Photoshop only. } } } } Since it is SEM at least we are dealing with just 256 gray levels. } } } } Any suggestions? } } } } Ric Felten } } } } SMARTech } } 860-485-5054 } } www.semguy.com } } 19 Cornwall Drive } } Goshen CT 06756 } } } } } } } } } } }
I am thinking I should be more careful with our liquid nitrogen handling. I would like to monitor oxygen levels in the room we fill dewars.
So far, I have found small, battery operated low oxygen alarms for about $300. But, they only last a year before needing replacement. The permanent installations I have found go for over $1K.
Is this right? Any one with some sage advice? Yes, I know better safe than sorry, but just double checking with the knowledge base.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
One other possibility That we have run into recently is the interference from a computer monitor that produces similar effects as shown in the images. These effects usually are not noticed at low magnifications...usually just when we are working at mags of 20,000x or greater. An easy way to check is just to turn off your computer monitor while you are capturing an image. Wouldn't it be nice if the solution is as easy as this!
Debby
Debby Sherman, Manager Phone: 765-494-6666 Life Science Microscopy Facility FAX: 765-494-5896 Purdue University E-mail: dsherman-at-purdue.edu S-052 Whistler Building West Lafayette, IN 47907
On 11/7/02 3:50 PM, "Allen Sampson" {ars-at-sem.com} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Vitaly, } } Most of your concerns I covered in a previous reply. However, I have to } take exception to your claim that a large change in the problem's } characteristic would result from a change in accelerating voltage only if } the problem was electro-magnetic. A reduction in the accelerating voltage } will also result in a reduction of the velocity of electrons in the beam. } That would cause an increased displacement of the beam as it traverses the } sample surface due to vibrations. } } In other words, the effects of electro-magnetic interference and vibrations } are virtually inseparable except for the frequency involved. Each will } affect the beam in similar ways, in regards to accelerating voltage and } working distance. Both will have a greater affect with a lower } accelerating voltage as well as a greater working distance. } } } Allen R. Sampson } Advanced Research Systems } 317 North 4th. Street } St. Charles, Illinois 60174 } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } On Wednesday, November 06, 2002 8:58 PM, Vitaly Feingold } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } Could be vibration. Could also be magnetic field. And, could be signal } } interference, such as ground loop. What type of SEM are you using? } } } } I will omit remedies for the vibration problem, since Allen Sampson did } } pretty good job addressing that. } } } } Stray AC line frequency magnetic field: try acquiring image at different } } accelerating voltages. It is very important that all other geometry } } parameters will remain the same: particular area of a particular } specimen, } } WD, tilt, magnification, spot size. Contrast, brightness, focus, and } } stigmator settings can be changed. If the problem is much worse at, say, } 2 } } kV, than at, say, 15kV, you are dealing with magnetic field. You may also } } try to keep 3 axes AC milliGauss meter at the SEM, and notice what it } } measures, but catching stray magnetic interference this way may be a } little } } tricky. These meters, though, are very inexpensive, and available from } many } } test instruments suppliers. } } } } Now we are getting down to statistically most likely problem- signal } } interference and ground loops. It is difficult to go any further without } } knowing the SEM type, and acquisition system (if separate) type. Too many } } possibilities exist, but the very first- does your acquisition software } } have an option somewhere in the settings which reads something like "60 } } Hertz sync", or "AC line synchronization", or "Line sync", etc., etc.? If } } yes, check (or uncheck) that option and see what happens. Further } remedies } } of this problem will include eliminating ground loops. Example- shielded } } cable(s) has shield connected on both sides, and shield is used as one of } } the signal wires. This is frequently done, and does jeopardize instrument } } interference tolerance. Another example- one of the accessories is } } susceptible to some kind of interference, and must be powered through the } } isolation transformer. The latter remedy will only work with decent } (better } } if dedicated) ground. The fact that the problem is not present all the } time } } does not mean that everything is perfect inside the SEM. Besides, going } } after the EM interference source could be inefficient and frustrating, if } } not impossible. Improving grounding/shielding/power connection might be } } easier. } } } } } } Vitaly Feingold } } Scientific Instruments and Applications } } 2773 Heath Lane, Duluth GA 30096 } } (770)232-7785 ph. } } (770)232-1791 fax } } (678)467-0012 mobile } } } } This message is made of 100% recycled electrons. } } } } This address can not receive messages larger than 15 kb without prior } } notification. } } } } ----- Original Message ----- } } } From: Ann-Fook Yang {yanga-at-agr.gc.ca} } } To: {microscopy-at-sparc5.microscopy.com} } } Sent: Wednesday, November 06, 2002 2:45 PM } } Subject: vibration? } } } } } } } } ------------------------------------------------------------------------ } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } America } } } To Subscribe/Unsubscribe -- Send Email to } ListServer-at-MSA.Microscopy.Com } } } On-Line Help } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } -----------------------------------------------------------------------. } } } } } } } } } Hi everyone, } } } } } } I have two SEM micrographs on the web: } } } http://www.magma.ca/~scimat/Defect.htm } } } The micrographs show zagged edges taken at 20 kX. Such phenomenon does } } not present all the time. Can anyone suggest what causes the problem and } how } } to solve it? } } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } } EM Unit, } } } Eastern Cereal and Oilseed Research Centre, } } } Room 2091, Bldg. 20, } } } Central Experimental Farm, } } } Ottawa, Ontario } } } Canada K1A 0C6 } } } } } } Tel: 1-613-759-1638 } } } Fax: 1-613-759-1701 } } } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } } } } } } } } } } } } } } }
You will find a good description of how to use a pilot study to determine the number of samples in: * Chapter 10 of Unbiased Stereology by C.V. Howard and M.G. Reed * Stereological Methods Vol 1: Practical Methods for Biological Morphometry by E.R. Weibel. Everett Ramer Cellomics, Inc. Pittsburgh, PA
This may help (may not, though!) - try storing the blocks in a desiccator with dry-rite or some other desiccant when you aren't using them. I've had similar trouble with K4M blocks that were made during humid weather - drying them out helped. Sometimes these resins are a little too hydrophilic!
Tamara
On Fri, 8 Nov 2002, Shanling Shi wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Dear listers, } } Recently I freeze substitution embedded some skin samples with Lowicryl K4M. } The blocks were cured under UV at -35C for 2 days and at room temperature for } several days. The blocks looks fine to me after UV polymerization. However, } when I section the blocks, blocks surface always get wet and the section swell } badly on the water. It seems like sections dissolving into water. I couldn't } pick up any intact sections. I used Lowicryl HM 20 before. It worked very well } for me. I did read the instruction of Lowicryl resin and notice that the } sectioning polar resin is different from nonpolar resin. But I could not get } good sections of Lowicyl K4M. I don't know if the blocks are not fully } polymerized or I need some special tricks to get sections. Any suggestions are } really appreciated! } } Shanling } } Shanling Shi } Advanced Imaging & Measurement } Unilever Research & Development -Edgewater } 45 River Road } Edgewater, NJ 07020 } 201-840-2340 } Shanling.Shi-at-unilever.com } } } } } } } }
|--------------------------------------------------| Tamara Howard Department of Cell Biology and Physiology University of New Mexico - Health Sciences Center Albuquerque, NM 87131 thoward-at-unm.edu |--------------------------------------------------|
My apologies to all - I made a couple of postings on this matter. The first laid down the conditions I was using, assumptions that had to be made (I should have just asked the original poster, but what I wrote was intended to be more general in application).
That original reply of mine laid out the assumption that the images were long exposures. All later remarks I made were based on that. If, instead, these were short exposures then the frequencies involved could be 50 or 60 Hz. or higher. My claim that these image problems would be vibrations was based on those conditions.
Sorry if I caused any confusion on that point by not reiterating the conditions in later messages..
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174 ph. (630) 513-7093 fax (630) 513-7092 web http://www.sem.com
On Friday, November 08, 2002 6:57 AM, Vitaly Feingold [SMTP:vitalylazar-at-worldnet.att.net] wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } ----- Original Message ----- } } From: Allen Sampson {ars-at-sem.com} } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } {Microscopy-at-sparc5.microscopy.com} } Sent: Friday, November 08, 2002 6:43 AM } Subject: RE: vibration? } } } } Reminds me of the very old question of can a scanning electron spot on a } } CRT exceed the speed of light? } } } } If only it could be as easy to figure as you portray. I'll try to explain } } the situation, but it is really more complex that I can describe in words } } here - and being late at night, I really won't spend much time on the } } matter. } } } } Let's assume that we are working at a working distance of 20mm. For the } } velocity you give for electrons accelerated at 2KV (27,000 kM/S) or 15KV } } (73,000 kM/S), that 20mm would be traversed in an exceeding small fraction } } of a second. However, we aren't looking at the spatial translation of a } } single spot. In the SEM, the spot formed on the sample is constantly } } moving - in the case of a record image the movement of the spot is } } generally determined by the dwell time on each of the individual pixels on } } each line and the number and resolution of lines in the frame. } } } } In the record mode, most instruments will sync the start of each line to } } the zero-crossing of the electrical mains. This is an intentional attempt } } to minimize the effect of 50 or 60 Hz. interference. Digital systems may } } or may not do this. } } } } If you go vertically down the recorded image, you are actually looking at } } pixels that are separated by fairly large periods of time, 1/50th or } 1/60th } } of a second in the case of a synched record cycle. Now let's look at your } } projected travel times using these figures. The difference between the } 2KV } } and 15KV velocities you mention is 46000 kM/S. That gives us a difference } } in the velocity at these two accelerating voltages of 4.35x10-9 seconds to } } traverse the 20mm to the sample. } } } } Correct. } } } } Assuming a 60 Hz system, the difference between two vertically separated } } pixels is 0.0167 seconds. } } } That is correct for a scan speed of 60 lines per second - frame of 1000 } lines will be scanned in 16 seconds. } } } } Let's say that each 1/60th of a second is split } } into 1500 equal parts - 1000 pixels used per line and 500 portions used } for } } the vertical retrace. That means that each pixel would be 0.000011 } seconds } } apart. } } } Adjacent horizontal pixels will be ~ 0.00001 seconds apart. While adjacent } vertical pixels will be 1/60s ~ 0.016s apart. } } } } Now let's look at two pixels that are on separate scan lines and separated } } by one additional pixel. They would be separated by a time period of } } 0.016678 seconds (adding the pixel dwell time to the line dwell time). } } } Correct. } } } } Doesn't sound like much, but in EM we have to understand the effects of } } the orders of magnitude we work with. If you divide the above time } } difference to traverse the working distance by the pixel time difference } } just mentioned, you would find that there is a 2.58x10-7 second difference } } between the pixels mentioned above. } } } Here is the juice. When you divide a time period by a time period, the } result } can not be time. It can be, however, a number of events, a number of } portions, etc. What portions? The above figure of 2.58x10-7 is the } difference } between the portions of a pixel, which beam will scan across } the specimen surface, equal to the time difference between 2kV and 15kV } electrons crossing of the WD of 20 mm. Beam deflection (scan) times remain } the same for both accelerating voltages, of course. } Can you see such difference on a micrograph? The ~ 3/10,000,000 of a pixel? } This will be the difference attributed to accelerating voltage change from } 2kV to 15kV and vice versa. This is why kV change will not bring any } noticeable change into vibration affected image. } } My whole point was that scan (deflection) times/speeds are compatible with } times/speeds of both mechanical and AC magnetic } events. The electron velocities/timing, in contrary, are not. They are many } orders of } magnitude shorter/faster than mechanical events. } } } } } } I'm tired and really don't want to extend this too much more, but consider } } that the vibrations seen on the images at question cover around 20 or more } } scan lines and figure in the velocities of a nanometer scale RMS } vibration. } } You'll find that there is indeed a considerable, measurable, variation in } } image vibrations due to accelerating voltage (as well as working } distance). } } } } Amazing, isn't it? } } } } By the way, I still claim that the frequency of the problems in the } } original image is much lower than 50 or 60 Hertz and is the result of } } vibrations rather than any electro-magnetic or electronic effect. Another } } poster mentioned the possibility that a turbo pump may be having problems } } but that is also not a likely cause as the frequencies involved don't } } match. You might, however, want to check the operation of any water } } chiller that is in use. } } Perhaps so, perhaps not. It can be chiller, or turbo pump, or anything else. } I can't say without more information. } } } } } } If I have learned anything in working on these instruments for well over } } twenty years, it's that nothing is simple or straightforward. We're } } dealing with sub-atomic particle physics here folks, an area that can only } } be currently described by the statistical methods of quantum theories. } } } } As far as I am concerned, there is no further determination to be made - } } the problem is one of mechanical vibrations and I would be glad to stake } my } } reputation on it, as I do on any posting I make here. } } } } } } Allen R. Sampson } } Advanced Research Systems } } 317 North 4th. Street } } St. Charles, Illinois 60174 } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } Vitaly Feingold } Scientific Instruments and Applications } 2773 Heath Lane, Duluth GA 30096 } (770)232-7785 ph. } (770)232-1791 fax } (678)467-0012 mobile } } This message is made of 100% recycled electrons. } } This address can not receive messages larger than 15 kb without prior } notification. } } } } } } } } On Thursday, November 07, 2002 10:54 PM, Vitaly Feingold } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } Hey Allen, } } } } } } You may found the following figures entertaining. Natural } } } constants and results of calculations are rounded to the first character } } } after the decimal point, relativity effects are neglected, plain text } } format } } } is used. I am a bit rusty on my Physics- my apologies if I missed an } } order } } } or so of magnitude. Makes no difference in this example. } } } } } } Electron mass ~ 9.1 x 10-31kg } } } Electron charge ~ -1.6 x 10-19 C } } } One electronvolt energy ~ 1.6 x 10-19J } } } Kinetic energy (of electron, or anything having mass) = mV2/2, where m } is } } } the mass, } } } V2 is the velocity square. } } } } } } Simple calculations yield the following velocities in kilometers per } } second } } } for electrons accelerated by potential differences of: } } } 1 Volt ~ 6 x 100 km/s } } } 200 V ~ 8.4 x 1,000 km/s } } } 2 kV ~ 2,7 x 10,000 km/s } } } 15 kV ~ 7.3 x 10,000 km/s } } } } } } Amazing, isn't it? } } } } } } Mechanical vibration velocity must be compatible in magnitude with the } } above } } } velocities, in order for vibration effects to look different at } different } } } beam accelerating voltages. But SEM is made of the materials found on } } this } } } planet, with limited durability specifications. Thus, in order to } vibrate } } } with such velocities, SEM must be either made very tiny, which is not } the } } } case, or must disintegrate into dust, but it doesn't. } } } } } } Velocities of mechanical vibrations affecting SEM are in many orders of } } } magnitude slower } } } than the E-beam velocities. } } } } } } This is why SEM stage vibration will show up exactly the same at } } different } } } beam accelerating voltages. } } } } } } Why then stray magnetic field affects an e-beam? Because electron has } } huge } } } charge/mass ratio, ~ 10,000,000,000. The interaction is very strong. } } Thus, } } } effect increases at lower accelerating voltages. Same with working } } distance- } } } effect increases with the distance increase. But, one needs to change WD } } } substantially, to see the effect for sure. That will reduce the } } resolution, } } } among other things. I will advice against chamber/beam/sample geometry } } } change during troubleshooting process. } } } } } } Now down to business with Ann's images. The sawtooth vertical edges } } } appearance is the result of horizontal scan being out of phase with the } } } external source of either vibration, fields, or signal interference. } Time } } } delay between the lines at slow scan is in order of tens to hundreds } } } milliseconds, which is within the possible vibration frequency range. } } Same } } } frequencies will cause wavy image at TV deflection speeds. } } } } } } Another point is that mechanical vibration in many cases repeats the AC } } line } } } frequency, as it is caused by electric motor driven devices, so either } 60 } } Hz } } } or it's harmonic(s) are present as mechanical vibrations. The only way } to } } } find the problem is to troubleshoot and differentiate. } } } } } } I agree with other postings- more information needed to develop more } } } accurate idea on how to proceed. } } } } } } } } } Vitaly Feingold } } } Scientific Instruments and Applications } } } 2773 Heath Lane, Duluth GA 30096 } } } (770)232-7785 ph. } } } (770)232-1791 fax } } } (678)467-0012 mobile } } } } } } This message is made of 100% recycled electrons. } } } } } } This address can not receive messages larger than 15 kb without prior } } } notification. } } } } } } ----- Original Message ----- } } } From: Allen Sampson {ars-at-sem.com} } } } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } } } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } } } {Microscopy-at-sparc5.microscopy.com} } } } Sent: Thursday, November 07, 2002 3:50 PM } } } Subject: RE: vibration? } } } } } } } } } } } } -------------------------------------------------------------------- ---- } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } America } } } } To Subscribe/Unsubscribe -- Send Email to } } ListServer-at-MSA.Microscopy.Com } } } } On-Line Help } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } -------------------------------------------------------------------- } } ---. } } } } } } } } } } } } Vitaly, } } } } } } } } Most of your concerns I covered in a previous reply. However, I have } } to } } } } take exception to your claim that a large change in the problem's } } } } characteristic would result from a change in accelerating voltage only } } if } } } } the problem was electro-magnetic. A reduction in the accelerating } } voltage } } } } } } } will also result in a reduction of the velocity of electrons in the } } beam. } } } } That would cause an increased displacement of the beam as it } traverses } } } the } } } } sample surface due to vibrations. } } } } } } } } In other words, the effects of electro-magnetic interference and } } } vibrations } } } } are virtually inseparable except for the frequency involved. Each } will } } } } affect the beam in similar ways, in regards to accelerating voltage } and } } } } working distance. Both will have a greater affect with a lower } } } } accelerating voltage as well as a greater working distance. } } } } } } } } } } } } Allen R. Sampson } } } } Advanced Research Systems } } } } 317 North 4th. Street } } } } St. Charles, Illinois 60174 } } } } } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } } } } } } } } } } On Wednesday, November 06, 2002 8:58 PM, Vitaly Feingold } } } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } } } Could be vibration. Could also be magnetic field. And, could be } } signal } } } } } interference, such as ground loop. What type of SEM are you using? } } } } } } } } } } I will omit remedies for the vibration problem, since Allen Sampson } } did } } } } } pretty good job addressing that. } } } } } } } } } } Stray AC line frequency magnetic field: try acquiring image at } } } different } } } } } accelerating voltages. It is very important that all other geometry } } } } } parameters will remain the same: particular area of a particular } } } } specimen, } } } } } WD, tilt, magnification, spot size. Contrast, brightness, focus, and } } } } } stigmator settings can be changed. If the problem is much worse at, } } say, } } } } 2 } } } } } kV, than at, say, 15kV, you are dealing with magnetic field. You may } } } also } } } } } try to keep 3 axes AC milliGauss meter at the SEM, and notice what } it } } } } } measures, but catching stray magnetic interference this way may be a } } } } little } } } } } tricky. These meters, though, are very inexpensive, and available } } from } } } } many } } } } } test instruments suppliers. } } } } } } } } } } Now we are getting down to statistically most likely problem- signal } } } } } interference and ground loops. It is difficult to go any further } } without } } } } } } } } knowing the SEM type, and acquisition system (if separate) type. Too } } } many } } } } } possibilities exist, but the very first- does your acquisition } } software } } } } } have an option somewhere in the settings which reads something like } } "60 } } } } } Hertz sync", or "AC line synchronization", or "Line sync", etc., } } etc.? } } } If } } } } } yes, check (or uncheck) that option and see what happens. Further } } } } remedies } } } } } of this problem will include eliminating ground loops. Example- sh } } ielded } } } } } cable(s) has shield connected on both sides, and shield is used as } } one } } } of } } } } } the signal wires. This is frequently done, and does jeopardize } } } instrument } } } } } interference tolerance. Another example- one of the accessories is } } } } } susceptible to some kind of interference, and must be powered } through } } } the } } } } } isolation transformer. The latter remedy will only work with decent } } } } (better } } } } } if dedicated) ground. The fact that the problem is not present all } } the } } } } time } } } } } does not mean that everything is perfect inside the SEM. Besides, } } going } } } } } after the EM interference source could be inefficient and } } frustrating, } } } if } } } } } not impossible. Improving grounding/shielding/power connection might } } be } } } } } easier. } } } } } } } } } } } } } } } Vitaly Feingold } } } } } Scientific Instruments and Applications } } } } } 2773 Heath Lane, Duluth GA 30096 } } } } } (770)232-7785 ph. } } } } } (770)232-1791 fax } } } } } (678)467-0012 mobile } } } } } } } } } } This message is made of 100% recycled electrons. } } } } } } } } } } This address can not receive messages larger than 15 kb without } prior } } } } } notification. } } } } } } } } } } ----- Original Message ----- } } } } } } From: Ann-Fook Yang {yanga-at-agr.gc.ca} } } } } } To: {microscopy-at-sparc5.microscopy.com} } } } } } Sent: Wednesday, November 06, 2002 2:45 PM } } } } } Subject: vibration? } } } } } } } } } } } } } } } } } } } } } } ------------------------------------------------------------------------ } } } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } } } America } } } } } } To Subscribe/Unsubscribe -- Send Email to } } } } ListServer-at-MSA.Microscopy.Com } } } } } } On-Line Help } } } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } } } } } } } } } -----------------------------------------------------------------------. } } } } } } } } } } } } } } } } } } Hi everyone, } } } } } } } } } } } } I have two SEM micrographs on the web: } } } } } } http://www.magma.ca/~scimat/Defect.htm } } } } } } The micrographs show zagged edges taken at 20 kX. Such phenomenon } } } does } } } } } not present all the time. Can anyone suggest what causes the problem } } and } } } } how } } } } } to solve it? } } } } } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } } } } } EM Unit, } } } } } } Eastern Cereal and Oilseed Research Centre, } } } } } } Room 2091, Bldg. 20, } } } } } } Central Experimental Farm, } } } } } } Ottawa, Ontario } } } } } } Canada K1A 0C6 } } } } } } } } } } } } Tel: 1-613-759-1638 } } } } } } Fax: 1-613-759-1701 } } } } } } } } } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } }
You can do what you describe in Photoshop by performing a Gaussian blur with a large radius, to create a background. But you will probably find that you get better results by using the darkest rather than the average of the pixels in the neighborhood (you can do that, too - select Minimum) to create the background. And for even more control, such as fitting a polynomial background and either subtracting or dividing it, the Image Processing Tool Kit plugins for Photoshop (and compatible programs) provides a variety of background leveling options (the tutorial at http://ReindeerGraphics.com has several examples).
John Russ
In a message dated 11/8/02 9:26:12 AM, smartech-at-optonline.net writes:
} In doing SEM I occasionally acquire images that contain a gradient. One } example would be an x-ray map that I have acquired in an hours time. } Typically the gun will drift slightly out of alignment and the end of the } image is darker than the beginning. Another example is when I am collecting } very low magnification images using the conventional SEM detector (Everhart } and Thornley). The top left hand side of the image is much brighter than } the bottom right hand side. I would like a routine that averages pixel values, } say nearest ten neighbors, to create a new image based on just the averages. } I could then perhaps divide the original image by the average image and } in effect, normalize the original image. } } It would be great if I could do this within Adobe Photoshop only. } } Since it is SEM at least we are dealing with just 256 gray levels. } } Any suggestions?
A simple recipe I use for leveling shading of SEM/TEM images and diffraction patterns in Photoshop follows:
1. Open image. 2. Duplicate layer (twice, to keep unaltered original). 3. Apply Gaussian Blur filter to top layer. Start with about a 25 pixel blur radius and adjust to get desired effect. 4. Invert image contrast 5. Select Overlay mode in Layers. 6. Merge Down (once). 7. Repeat process 2 or 3 times as appropriate. (Record as Action).
This method works best with images having no abrupt brightness transitions (such as annotations). Thanks to whoever first showed me this gem.
Larry Thomas Pacific Northwest National Laboratory P.O. Box 999 Mail Stop P8-16 Richland, WA, USA
} ---------- } From: Baggethun, Paul } Sent: Friday, November 8, 2002 9:47 AM } To: Microscopy-at-Sparc5. Microscopy. Com (E-mail) } Subject: RE: I need help with digital images __ How to subtract a } gradaien t from SEM image } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Remove the long-wavelength intensity fluctuation from your images using a } highpass Fourier Transform filter. } } This, and other background correction utilities are available (for free) } in } ImageJ http://rsb.info.nih.gov/ij/ ImageJ is the most versatile 2D ip&a } software for those hwo do not require super-high speed. } } Cheers, } Paul Baggethun } Pittsburgh, PA } } -----Original Message----- } } From: Smartech [mailto:smartech-at-optonline.net] } Sent: Friday, November 08, 2002 9:04 AM } To: To all on the list } Subject: I need help with digital images __ How to subtract a gradaient } from SEM image } } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } In doing SEM I occasionally acquire images that contain a gradient. One } example would be an x-ray map that I have acquired in an hours time. } Typically the gun will drift slightly out of alignment and the end of the } image is darker than the beginning. Another example is when I am } collecting } very low magnification images using the conventional SEM detector } (Everhart } and Thornley). The top left hand side of the image is much brighter than } the } bottom right hand side. I would like a routine that averages pixel } values, } say nearest ten neighbors, to create a new image based on just the } averages. } I could then perhaps divide the original image by the average image and in } effect, normalize the original image. } } It would be great if I could do this within Adobe Photoshop only. } } Since it is SEM at least we are dealing with just 256 gray levels. } } Any suggestions? } } Ric Felten } } SMARTech } 860-485-5054 } www.semguy.com } 19 Cornwall Drive } Goshen CT 06756 } } } }
Jonathan, The least expensive unit I found about 1 1/2 years ago was an AirAware O2 monitor, #OF-67816 from Lab Safety Supply, 800 356-0783. It was twice what I hoped to pay but much less than $1K. AC adaptor included. Jim } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
James S. Romanow The University of Connecticut Physiology and Neurobiology Department Electron Microscopy Facility Unit 2242 354 Mansfield Road Beach Hall, Room 129 Storrs, CT 06269-2242
Dear Friend, I am Mr Onuigbo Baldwin Gozie, Bank Manager of Diamond BANK , Lagos Branch. I have urgent and very confidential business proposition for you Mr. Barry Kelly made a numbered time (Fixed) deposited for twelve calendar months, valued at US$25,000,000.00 (Twenty-five Million Dollars) in my branch. Upon maturity, I sent a routine notification to his forwarding address but got no reply. After a month, we sent a reminder and finally we discovered from his contract employers, Nigerian National Petroleum Corporation that Mr. Barry Kelly died from an automobile accident. On further investigation, I found out that he did not leave a WILL and all attempts to trace his next of kin were fruitless. I therefore made further investigation and discovered that Mr. Barry Kelly did not declare any next of kin in all his official documents, including his Bank Deposit paperwork. This sum of US$25,000,000.00 is still sitting in the Bank and the interest is being rolled over with the principal sum at the end of each year. No one will come forward to claim it. According to the Nigerian Law, at the expiration of 6{Six} years, the money will revert to the ownership of the Nigerian Government if nobody applies to claim the funds. Consequently, my proposal is that I will like you as a foreigner to stand in as the next of kin to Mr. Barry Kelly so that the fruits of this old man's labor will not get into the hands of some corrupt government officials. This is simple; 1) I will like you to provide me immediately with your full names and address so that the attorney will prepare the necessary documents and affidavits, which will put you in place as the next of kin. 2) We shall employ the services of two attorneys for drafting and notarization of the WILL and obtain the necessary documents and letter of probate/administration in your favor for the transfer. 3) A bank account in any part of the world, which you provide, will then facilitate the transfer of this money to you as the beneficiary/next of kin of Mr.Barry Kelly. The money will be paid into your account for us to share in the ratio of 75% for me and 20% for you then 5% will be set aside for any expenses that may occure during the transfer process. There is no risk at all as all the paperwork for this transaction will be done by the attorney and my position as the Branch Manager guarantees the successful execution of this transaction. If you are interested, please reply immediately via the private email address below. Upon your response, I shall then provide you with more details and relevant documents that will help you understand. Please observe utmost confidentiality, and rest assured that this transaction would be most profitable for both of us because I shall require your assistance to invest my share in your country. Awaiting your urgent reply via email or my private telephone 234-1-7763642. Thanks and regards Baldwin Gozie Esq.
Dear Friend, I am Mr Onuigbo Baldwin Gozie, Bank Manager of Diamond BANK , Lagos Branch. I have urgent and very confidential business proposition for you Mr. Barry Kelly made a numbered time (Fixed) deposited for twelve calendar months, valued at US$25,000,000.00 (Twenty-five Million Dollars) in my branch. Upon maturity, I sent a routine notification to his forwarding address but got no reply. After a month, we sent a reminder and finally we discovered from his contract employers, Nigerian National Petroleum Corporation that Mr. Barry Kelly died from an automobile accident. On further investigation, I found out that he did not leave a WILL and all attempts to trace his next of kin were fruitless. I therefore made further investigation and discovered that Mr. Barry Kelly did not declare any next of kin in all his official documents, including his Bank Deposit paperwork. This sum of US$25,000,000.00 is still sitting in the Bank and the interest is being rolled over with the principal sum at the end of each year. No one will come forward to claim it. According to the Nigerian Law, at the expiration of 6{Six} years, the money will revert to the ownership of the Nigerian Government if nobody applies to claim the funds. Consequently, my proposal is that I will like you as a foreigner to stand in as the next of kin to Mr. Barry Kelly so that the fruits of this old man's labor will not get into the hands of some corrupt government officials. This is simple; 1) I will like you to provide me immediately with your full names and address so that the attorney will prepare the necessary documents and affidavits, which will put you in place as the next of kin. 2) We shall employ the services of two attorneys for drafting and notarization of the WILL and obtain the necessary documents and letter of probate/administration in your favor for the transfer. 3) A bank account in any part of the world, which you provide, will then facilitate the transfer of this money to you as the beneficiary/next of kin of Mr.Barry Kelly. The money will be paid into your account for us to share in the ratio of 75% for me and 20% for you then 5% will be set aside for any expenses that may occure during the transfer process. There is no risk at all as all the paperwork for this transaction will be done by the attorney and my position as the Branch Manager guarantees the successful execution of this transaction. If you are interested, please reply immediately via the private email address below. Upon your response, I shall then provide you with more details and relevant documents that will help you understand. Please observe utmost confidentiality, and rest assured that this transaction would be most profitable for both of us because I shall require your assistance to invest my share in your country. Awaiting your urgent reply via email or my private telephone 234-1-7763642. Thanks and regards Baldwin Gozie Esq.
Dear Friend, I am Mr Onuigbo Baldwin Gozie, Bank Manager of Diamond BANK , Lagos Branch. I have urgent and very confidential business proposition for you Mr. Barry Kelly made a numbered time (Fixed) deposited for twelve calendar months, valued at US$25,000,000.00 (Twenty-five Million Dollars) in my branch. Upon maturity, I sent a routine notification to his forwarding address but got no reply. After a month, we sent a reminder and finally we discovered from his contract employers, Nigerian National Petroleum Corporation that Mr. Barry Kelly died from an automobile accident. On further investigation, I found out that he did not leave a WILL and all attempts to trace his next of kin were fruitless. I therefore made further investigation and discovered that Mr. Barry Kelly did not declare any next of kin in all his official documents, including his Bank Deposit paperwork. This sum of US$25,000,000.00 is still sitting in the Bank and the interest is being rolled over with the principal sum at the end of each year. No one will come forward to claim it. According to the Nigerian Law, at the expiration of 6{Six} years, the money will revert to the ownership of the Nigerian Government if nobody applies to claim the funds. Consequently, my proposal is that I will like you as a foreigner to stand in as the next of kin to Mr. Barry Kelly so that the fruits of this old man's labor will not get into the hands of some corrupt government officials. This is simple; 1) I will like you to provide me immediately with your full names and address so that the attorney will prepare the necessary documents and affidavits, which will put you in place as the next of kin. 2) We shall employ the services of two attorneys for drafting and notarization of the WILL and obtain the necessary documents and letter of probate/administration in your favor for the transfer. 3) A bank account in any part of the world, which you provide, will then facilitate the transfer of this money to you as the beneficiary/next of kin of Mr.Barry Kelly. The money will be paid into your account for us to share in the ratio of 75% for me and 20% for you then 5% will be set aside for any expenses that may occure during the transfer process. There is no risk at all as all the paperwork for this transaction will be done by the attorney and my position as the Branch Manager guarantees the successful execution of this transaction. If you are interested, please reply immediately via the private email address below. Upon your response, I shall then provide you with more details and relevant documents that will help you understand. Please observe utmost confidentiality, and rest assured that this transaction would be most profitable for both of us because I shall require your assistance to invest my share in your country. Awaiting your urgent reply via email or my private telephone 234-1-7763642. Thanks and regards Baldwin Gozie Esq.
In a message dated 11/08/2002 9:40:05 AM US Mountain Standard Time, Shanling.Shi-at-unilever.com writes:
{ { Recently I freeze substitution embedded some skin samples with Lowicryl K4M. The blocks were cured under UV at -35C for 2 days and at room temperature for several days. The blocks looks fine to me after UV polymerization. However, when I section the blocks, blocks surface always get wet and the section swell badly on the water. It seems like sections dissolving into water. I couldn't pick up any intact sections. I used Lowicryl HM 20 before. It worked very well for me. I did read the instruction of Lowicryl resin and notice that the sectioning polar resin is different from nonpolar resin. But I could not get good sections of Lowicyl K4M. I don't know if the blocks are not fully polymerized or I need some special tricks to get sections. Any suggestions are really appreciated!
Shanling } }
Shanling,
K4M is fairly hydrophilic, and the sections will swell if they are left on the water for too long. You will probably have to pick up the sections quickly after they are cut. The "dissolving" on the water surface may be a sign of incomplete infiltration, so you might have to use a little vacuum. I assume you did the normal stepwise infiltration in the cold, per the instructions. Did you agitate during dehydration and infiltration?
Regarding the sectioning and wetting of the block face, here are a couple of tips:
1. You will probably have to section the K4M with a very low water level in the boat, much lower than you are used to, to avoid water "jumping" up onto the black face. In fact, you can get fairly good sections when the reflection at the knife edge is dark, rather than the normal reflection you see when the boat is properly filled for normal sectioning.
2. If you are using a diamond knife and the edge is hydrophobic, overfill the boat on the knife to form a positive meniscus and let the knife sit like this for 15-30 minutes, then try to reduce the level in the boat to give a very dark reflection along the knife edge (meaning the water level is lower than normal). In fact, the first 2 or 3 sections may get lost in the dark reflection so that you can barely see them, or not see them at all.
3. In the past, I have also had good luck by doing the following: After facing and trimming the block, approach with your diamond knife with a partially full boat and a *dry* knife edge. On the final approach, with the microtome running and advancing, stop the microtome after the *very first* bit of the block has been cut (usually a partial section is cut). Then gradually bring the water level in the boat up until the water makes contact with the dry section sitting on the knife edge.
4. At this point the water will creep under the section and lift it off the knife. The entire knife edge will probably not be wet, but the area where you are cutting will have water under the section and the knife edge in this area will be wet. Start the microtome again, and gradually add water *dropwise* between cutting cycles to slowly raise the level of water in the boat. This will usually work with K4M, and you should be able to get reasonably good ribbons from the block.
5. Remember, always cut with the water level lower than normal. This will prevent the block face from getting wet.
Holly, Try labdepot.com. for bulbs in sealed containers together with reasonable price. Tel 1-800-810-1620 or 763-557-5814
PS: Only for information purpose, no binding or whatsoever with the company. Shiv
Mayandi Sivaguru, Ph.D., Associate Director Molecular Cytology Core Facility Molecular Biology Program 2, Tucker Hall University of Missouri Columbia, MO 65211-7400
Voice: 1-573-882-4895 Fax: 1-573-882-0123
www.biotech.missouri.edu/mcc/
At 09:55 AM 11/8/02 -0800, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
PLEASE TREAT AS URGENT AND CONFIDENTIAL My name is Mr Jim Shobor. I am one of these senior managers in the Bank. I work for (Zenith International Bank Nigeria PLC). And I work under the (Director of Current Account Dept.). I am contacting you presently because I need your urgent assistance in a business transaction that will be of immense benefit to both of us. I have the immediate need to claim money that have long been declared unclaimed by the chairman and some members of the board of directors of our bank. The money is the closing balance of one of our costumers. Late Eng.Temcoson Brown,am american citizen was a contractor with the feeral gov of nigeriaI I was his person account officer in the ADC plane crash of 1996 in Nigeria He supplied and installed equipment and his company creek contractors completed some of the best construction contract in the country. His closing balance in the bank,of the time of his death stood at (US$19.5M) and has been tagged unclaimed because no relative of Eng. Temcoson Brown has COMe forward to make any acclaim. All efforts to contact the relative have not been fruitful in the past four years. We have no knowledge of his next of kin. At this point. I trust you can picture what the situation is like. My colleagues and my self have made several attempts at locating person that could be remotely related to Egn. Temcoson Brown and we have been doing this for about four year (4yrs) now. I am almost alone in this enterprise as the account is forgotten entirely. I have therefore presently decided to solicit your assistance to act as the next of kin of Engr Temcoson Brown and I will be very grateful if you will be willing to help in this regard. My colleagues and I will therefore alter the original forms completed by Eng.Temcoson brown to riflect your name or any name you want us to use for the claim. We will provide you with answer to security question, which you will haveto anwser There will be no need for you to appear here in Nigeria if you follow my instructions closely. Everything should be concluded on telephone and fax between you and the bank of you participate you will be commensurately compensated. We will discuss among other primary details. I make this proposal in trust and good faith, therefore if you are interested and your agree to assist me then contract me immediately you receive this E-mail, there is a lot more to talk about. If you are not interested. Then please, do get rid of this E-mail and please forgive me if this message has upset you in anyway. Thanks you and Best Regards Mr. Jim Shobor -- _______________________________________________ Get your free email from http://mail.usa.com
} I am looking to buy TEM negs of human pathogens, } bacteria, viruses and parasites. } } If you are interested in selling TEM negs in these } categories, please contact me off-list. } } gary g.
as you can see, there are a lot of answers to turn around time. there is an old article on staffing in the Veterans Administration Hospitals for EM labs. i have been trying to find it in my files, but cannot. the article states that the VA standard is (was?) 1 staff member for every 400 samples per year. i could be slightly off on the number, it may be 450. your 5-6 samples every other day suggests that you should have two techs. as far as turn around, if you have sufficient staff and the right equipment, you can give {24 hr for real rush samples, 2 days for standard clinical, and 4-5 days for samples with special requirements. because i need the article for a project i am involved in, i will keep looking and send the precise number later.
now for the bonus - with the current hyper-awareness of bioterrorism, you may get someone who wants a negative stain prep. where it is appropriate for you to handle it you can be expected to give 5-20 minute turn around from time of arrival in the lab. it must be stressed that you should have your local infectious diseases staff decide whether the sample can be looked at locally or requres special treatment in a containment facility or in Atlanta (Winnipeg if you are in Canada). unless you are a virologist, do not assume that you, or the attending physician can make that decision without this input. simply put, there are some things that are simply too dangerous to handle without special training and equipment.
Through a cooperative agreement between the Microscopy Society of America (MSA) and the Royal Microscopical Society (RMS), a travel scholarship is available to a graduate student or post-doctoral research assistant to attend a meeting or course organized by the RMS in 2003. Details of such events can be found under events at http://www.rms.org.uk (remember U.K. date format is day/month/year). MSA will award up to $1000 towards travel and accommodation. RMS will cover the registration costs and partial accommodation costs. The applicant must be a member of MSA in good standing at the time the application is submitted and at the time of the meeting or course. Applications for the award should consist of: 1. Applicants full name, address, email address, and details of academic and/or professional status; 2. A statement of up to 500 words outlining why the applicant wants to attend a named conference or course; 3. A letter from the applicant's faculty advisor/supervisor confirming the status of the applicant and detailing any supplementary financial support; and 4. If required for a conference presentation, an appropriately prepared abstract. This abstract will need to be submitted in sufficient time and to have sufficient scientific content to be accepted into the official program of the conference. Applications should be sent to Dr. J. Bentley, MSA Awards Committee Chair, Oak Ridge National Laboratory, Bethel Valley Road, PO Box 2008, Oak Ridge, TN 37831-6064 (email: bentleyj-at-ornl.gov) to arrive no later than December 10, 2002. The awardee is expected to be notified by December 16, 2002.
-- Jim Bentley MSA Awards Committee Chair Microscopy, Microanalysis, Microstructures Group Metals and Ceramics Division Bldg. 4515, MS-6064 Oak Ridge National Laboratory PO Box 2008 Oak Ridge, TN 37831-6064, USA
Tel: (865)574-5067 Fax: (865)576-5413 bentleyj-at-ornl.gov express mail use "1 Bethel Valley Rd" instead of PO Box. ** Note the new group name, building, mail-stop, fax, and area code **
I'd suggest that if you are not getting the rated life out of your mercury lamps that you check a couple of things. It is usually not the fault of the lamp. If you can give me the specifics of your problem I may be able to suggest what the cause might be. The manufacturer of the lamp will also check and diagnose what the fault may be if you send them the bulb.
1) Be sure that the lamp connections are clean and TIGHT! People are tentitive installing these expensive lamps and frequently under tighten the screws or knobs that clamp them in place. The huge thermal cycle the lamp goes through will tend to loosen the attachments (which are also the electrical connections) and this can shorten the life of the lamp.
2) Make sure that nobody ever turns the mercury lamp off until it has been on a minimum of twenty minutes. Unless it is allowed to reach operating temperture before it is turned off the life will be shortened or the lamp will be ruined. One time can ruin a lamp.
3) Don't use the HBO 103 lamps unless you have a very new power supply that was designed for them. Sales reps will tell you that these new longer life lamps will work in an older system and 98% of the time they are wrong. You will experience shorter lamp life not longer... I'd be happy to explain.
4) The lamps designed for AC power supplies have half the life of the lamps designed for DC power supplies. This is normal.
I have purchased thousands of mercury lamps from both Bulbman and Lamp Technologies. Both have websites. I have never found better prices anywhere. I'd give Bulbman an A++ for customer service, slightly lower for Lamp Technology. Both are excellent sources for all of your microscope lightbulb needs, not just mercury lamps. Both offer discounts for orders of more then a couple of bulbs. Quantities of 6 or 10 of the same type are usually enough to meet the discount point. I have tried several different manufactures of mercury lamps and found that Osram lamps perform the best for our applications.
My only relationship with either of these companies is as a satisfied customer.
David Burton Optical Specialist University of Washington
ImageJ is OK if the file size is small. Otherwise, you get an Out of Memory error, even with 1.5GB of RAM.
gg
At 09:47 AM 11/8/2002, you wrote:
} Remove the long-wavelength intensity fluctuation from your images using a } highpass Fourier Transform filter. } } This, and other background correction utilities are available (for free) in } ImageJ http://rsb.info.nih.gov/ij/ ImageJ is the most versatile 2D ip&a } software for those hwo do not require super-high speed. } } Cheers, } Paul Baggethun } Pittsburgh, PA } } -----Original Message----- } } From: Smartech [mailto:smartech-at-optonline.net] } Sent: Friday, November 08, 2002 9:04 AM } To: To all on the list } Subject: I need help with digital images __ How to subtract a gradaient } from SEM image } } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
The technique you are referring to is sometimes called "unsharp masking" and has been used by astronomers for a long time. It is used to get better contrast from differently illuminated areas, and it will do a background equalization as well.
In general, you get the best results if you can take an image of the background by itself and correct the image with that background. In a TEM that can be done by leaving any settings undisturbed and removing the specimen, then taking a picture of the background. I am not sure that can be done in an SEM, though.
That leaves you with artificial background correction. Unsharp Masking is one way to do this. Other possibilities include the calculation of a background image from areas where the background shows through. You also have to decide whether you want to divide or subtract. In most cases a division is the correct operation (for example, if irregularities in a TEM phosphor need to be taken out).
In most cases, software that deals with microscope images on a regular basis, will have the appropriate functions built-in.
mike
} } } } } } } } } } WE HAVE MOVED { { { { { { { { { please make a note of the new address below
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Thomas, Larry (PNNL) [mailto:Larry.Thomas-at-pnl.gov] Sent: Friday, November 08, 2002 2:20 PM To: Microscopy-at-Sparc5. Microscopy. Com (E-mail); 'Baggethun, Paul'
Hi Tom, and others
You have raised an interesting point that I have never really been able to get a handle on satisfactorily, that is how do you choose the appropriate acrylic resin to use for a new project,
eg,
should I choose LR White or LR Gold for project X, should I chemically polymerise or perhaps use UV ?
or maybe I should try Unicryl, or Lowicryl, but which one HM20, K4M etc.
Are there others I should be considering?
I would be interested in seeing a discussion on how different people go about choosing a particular acrylic resin for a new project they may have to undertake.
Regards
Allan
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You have raised an interesting point that I have never really been able to get a handle on satisfactorily, that is how do you choose the appropriate acrylic resin to use for a new project,
eg,
should I choose LR White or LR Gold for project X, should I chemically polymerise or perhaps use UV ?
or maybe I should try Unicryl, or Lowicryl, but which one HM20, K4M etc.
Are there others I should be considering?
I would be interested in seeing a discussion on how different people go about choosing a particular acrylic resin for a new project they may have to undertake.
Regards
Allan
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If anyone gets an electron to travel faster than the speed of light, please copy me.
Peter
-----Original Message----- } From: Vitaly Feingold [mailto:vitalylazar-at-worldnet.att.net] Sent: Friday, November 08, 2002 9:57 AM To: ars-at-sem.com; 'Ann-Fook Yang'; 'microscopy-at-msa.microscopy.com'
----- Original Message ----- } From: Allen Sampson {ars-at-sem.com} To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' {Microscopy-at-sparc5.microscopy.com} Sent: Friday, November 08, 2002 6:43 AM
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (atgc1-at-comcast.net) from http://www.msa.microscopy.com/Ask-A-Microscopist.html on Sunday, November 10, 2002 at 21:10:51 ---------------------------------------------------------------------------
Email: atgc1-at-comcast.net Name: Adam Georgas
Organization: UMS-Wright
Education: 9-12th Grade High School
Location: Mobile, AL
Question: Hello. I was wondering if you know were I can find a large repository of images of blood smears.
We use a handheld O2 monitor system from Draeger (PAC III). It is good for at least two years and costs under $1K with a charging stand.
Joseph
Joseph M. Oparowski Materials Science Research - Consulting and Failure Analysis Bose Corporation Joseph_Oparowski-at-bose.com The Mountain, M/S 415 Phone: (508) 766-1371 Framingham, MA 01701-9168 Fax: (508) 766-1313
-----Original Message----- } From: jmkrupp-at-cats.ucsc.edu [mailto:jmkrupp-at-cats.ucsc.edu] Sent: Friday, November 08, 2002 2:05 PM To: Microscopy-at-sparc5.microscopy.com
Hi:
I am thinking I should be more careful with our liquid nitrogen handling. I would like to monitor oxygen levels in the room we fill dewars.
So far, I have found small, battery operated low oxygen alarms for about $300. But, they only last a year before needing replacement. The permanent installations I have found go for over $1K.
Is this right? Any one with some sage advice? Yes, I know better safe than sorry, but just double checking with the knowledge base.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
I had a little sideline business for several years, running a black and white darkroom for a couple local firms. My emergency RC paper drier was a cheap Vidal Sasoon hair drier. I could dry an 8x10 print in about 30 sec to a minute. Just make sure there aren't big drops of water on the print by using a squeegee (or just a towel). Of course, this is not the greatest system for large numbers of prints, but for a reasonable quantity it's fast and cheap. And it leaves your prints so soft and manageable....
Randy
Randy Tindall EM Specialist Electron Microscopy Core Facility W122 Veterinary Medicine University of Missouri Columbia, MO 65211 Tel: (573) 882-8304 Fax: (573) 884-5414 Email: tindallr-at-missouri.edu Web: http://www.biotech.missouri.edu/emc/
-----Original Message----- } From: Dean Abel [mailto:dean-abel-at-uiowa.edu] Sent: Thursday, November 07, 2002 6:09 PM To: Leslie Cummins Cc: microscopy-at-msa.microscopy.com
Hello Leslie, I use a Durst Laborator S-45 Special enlarger with a point light source and print all my TEM and 35mm negatives on Ilford Multigrade IV RC DeLuxe paper using Ilford Multigrade filters. It works fine for me and my bosses, but I am sure that other papers like Kodak Polymax II RC, as suggested by Karen Jensen, work just as well. I learned my darkroom work using fiber-based graded papers and drying them on a big mirrored drum, but now must I air dry my prints ever since our dryer broke and couldn't be fixed. This is a bit of a nuisance as the prints take overnight to dry and can't be turned in the same day they're printed. Does anyone have tips on drying resin coated paper quickly??? Dean Abel Biological Sciences 138BB University of Iowa Iowa City IA 52242-1324
On Friday, November 8, 2002, at 09:55 AM, Holly Aaron wrote:
} The last few I have gotten have not lasted the 200 hrs they should, so } I am } looking for a new source. } Dear Holly, I second what David Burton said about turning on the lamp for short periods; in fact I'll go further. When I had an experiment that used a high-pressure Hg lamp, I set things up to run for 24 hours a day and ran the experiment straight through for about three weeks. The lamp was rated at 100 hours lifetime, and it ran for about four times that. After the experiment, I wrapped the lamp in padding and placed it in the back of a hood until disposal. I'm not sure whether such a protocol can work for you, but it is definitely the case that the fewer times one turns a lamp on and off, the longer it will last. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
You cannot generalize that the frequency of the interference on the images is the same as that of the source.I participated in an experiment several years ago in which we intentionally generated an AC field at frequencies a few Hz above 60. The resulting interference on the sem image was the difference between the source and the 60Hz sync. In other words, a 65Hz field would look like 5Hz(65-60). This also works for harmonics of 60Hz. A computer monitor with a refresh rate of 75Hz would look like a 15Hz field on an sem scan synched to 60Hz.
Also, the resonant frequency of the column suspension has to be considered. Microscope columns usually have a resonant frequency between 5-10Hz.
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My apologies to all - I made a couple of postings on this matter. The first laid down the conditions I was using, assumptions that had to be made (I should have just asked the original poster, but what I wrote was intended to be more general in application).
That original reply of mine laid out the assumption that the images were long exposures. All later remarks I made were based on that. If, instead, these were short exposures then the frequencies involved could be 50 or 60 Hz. or higher. My claim that these image problems would be vibrations was based on those conditions.
Sorry if I caused any confusion on that point by not reiterating the conditions in later messages..
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174 ph. (630) 513-7093 fax (630) 513-7092 web http://www.sem.com
On Friday, November 08, 2002 6:57 AM, Vitaly Feingold [SMTP:vitalylazar-at-worldnet.att.net] wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } ----- Original Message ----- } } From: Allen Sampson {ars-at-sem.com} } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } {Microscopy-at-sparc5.microscopy.com} } Sent: Friday, November 08, 2002 6:43 AM } Subject: RE: vibration? } } } } Reminds me of the very old question of can a scanning electron spot on a } } CRT exceed the speed of light? } } } } If only it could be as easy to figure as you portray. I'll try to explain } } the situation, but it is really more complex that I can describe in words } } here - and being late at night, I really won't spend much time on the } } matter. } } } } Let's assume that we are working at a working distance of 20mm. For the } } velocity you give for electrons accelerated at 2KV (27,000 kM/S) or 15KV } } (73,000 kM/S), that 20mm would be traversed in an exceeding small fraction } } of a second. However, we aren't looking at the spatial translation of a } } single spot. In the SEM, the spot formed on the sample is constantly } } moving - in the case of a record image the movement of the spot is } } generally determined by the dwell time on each of the individual pixels on } } each line and the number and resolution of lines in the frame. } } } } In the record mode, most instruments will sync the start of each line to } } the zero-crossing of the electrical mains. This is an intentional attempt } } to minimize the effect of 50 or 60 Hz. interference. Digital systems may } } or may not do this. } } } } If you go vertically down the recorded image, you are actually looking at } } pixels that are separated by fairly large periods of time, 1/50th or } 1/60th } } of a second in the case of a synched record cycle. Now let's look at your } } projected travel times using these figures. The difference between the } 2KV } } and 15KV velocities you mention is 46000 kM/S. That gives us a difference } } in the velocity at these two accelerating voltages of 4.35x10-9 seconds to } } traverse the 20mm to the sample. } } } } Correct. } } } } Assuming a 60 Hz system, the difference between two vertically separated } } pixels is 0.0167 seconds. } } } That is correct for a scan speed of 60 lines per second - frame of 1000 } lines will be scanned in 16 seconds. } } } } Let's say that each 1/60th of a second is split } } into 1500 equal parts - 1000 pixels used per line and 500 portions used } for } } the vertical retrace. That means that each pixel would be 0.000011 } seconds } } apart. } } } Adjacent horizontal pixels will be ~ 0.00001 seconds apart. While adjacent } vertical pixels will be 1/60s ~ 0.016s apart. } } } } Now let's look at two pixels that are on separate scan lines and separated } } by one additional pixel. They would be separated by a time period of } } 0.016678 seconds (adding the pixel dwell time to the line dwell time). } } } Correct. } } } } Doesn't sound like much, but in EM we have to understand the effects of } } the orders of magnitude we work with. If you divide the above time } } difference to traverse the working distance by the pixel time difference } } just mentioned, you would find that there is a 2.58x10-7 second difference } } between the pixels mentioned above. } } } Here is the juice. When you divide a time period by a time period, the } result } can not be time. It can be, however, a number of events, a number of } portions, etc. What portions? The above figure of 2.58x10-7 is the } difference } between the portions of a pixel, which beam will scan across } the specimen surface, equal to the time difference between 2kV and 15kV } electrons crossing of the WD of 20 mm. Beam deflection (scan) times remain } the same for both accelerating voltages, of course. } Can you see such difference on a micrograph? The ~ 3/10,000,000 of a pixel? } This will be the difference attributed to accelerating voltage change from } 2kV to 15kV and vice versa. This is why kV change will not bring any } noticeable change into vibration affected image. } } My whole point was that scan (deflection) times/speeds are compatible with } times/speeds of both mechanical and AC magnetic } events. The electron velocities/timing, in contrary, are not. They are many } orders of } magnitude shorter/faster than mechanical events. } } } } } } I'm tired and really don't want to extend this too much more, but consider } } that the vibrations seen on the images at question cover around 20 or more } } scan lines and figure in the velocities of a nanometer scale RMS } vibration. } } You'll find that there is indeed a considerable, measurable, variation in } } image vibrations due to accelerating voltage (as well as working } distance). } } } } Amazing, isn't it? } } } } By the way, I still claim that the frequency of the problems in the } } original image is much lower than 50 or 60 Hertz and is the result of } } vibrations rather than any electro-magnetic or electronic effect. Another } } poster mentioned the possibility that a turbo pump may be having problems } } but that is also not a likely cause as the frequencies involved don't } } match. You might, however, want to check the operation of any water } } chiller that is in use. } } Perhaps so, perhaps not. It can be chiller, or turbo pump, or anything else. } I can't say without more information. } } } } } } If I have learned anything in working on these instruments for well over } } twenty years, it's that nothing is simple or straightforward. We're } } dealing with sub-atomic particle physics here folks, an area that can only } } be currently described by the statistical methods of quantum theories. } } } } As far as I am concerned, there is no further determination to be made - } } the problem is one of mechanical vibrations and I would be glad to stake } my } } reputation on it, as I do on any posting I make here. } } } } } } Allen R. Sampson } } Advanced Research Systems } } 317 North 4th. Street } } St. Charles, Illinois 60174 } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } Vitaly Feingold } Scientific Instruments and Applications } 2773 Heath Lane, Duluth GA 30096 } (770)232-7785 ph. } (770)232-1791 fax } (678)467-0012 mobile } } This message is made of 100% recycled electrons. } } This address can not receive messages larger than 15 kb without prior } notification. } } } } } } } } On Thursday, November 07, 2002 10:54 PM, Vitaly Feingold } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } Hey Allen, } } } } } } You may found the following figures entertaining. Natural } } } constants and results of calculations are rounded to the first character } } } after the decimal point, relativity effects are neglected, plain text } } format } } } is used. I am a bit rusty on my Physics- my apologies if I missed an } } order } } } or so of magnitude. Makes no difference in this example. } } } } } } Electron mass ~ 9.1 x 10-31kg } } } Electron charge ~ -1.6 x 10-19 C } } } One electronvolt energy ~ 1.6 x 10-19J } } } Kinetic energy (of electron, or anything having mass) = mV2/2, where m } is } } } the mass, } } } V2 is the velocity square. } } } } } } Simple calculations yield the following velocities in kilometers per } } second } } } for electrons accelerated by potential differences of: } } } 1 Volt ~ 6 x 100 km/s } } } 200 V ~ 8.4 x 1,000 km/s } } } 2 kV ~ 2,7 x 10,000 km/s } } } 15 kV ~ 7.3 x 10,000 km/s } } } } } } Amazing, isn't it? } } } } } } Mechanical vibration velocity must be compatible in magnitude with the } } above } } } velocities, in order for vibration effects to look different at } different } } } beam accelerating voltages. But SEM is made of the materials found on } } this } } } planet, with limited durability specifications. Thus, in order to } vibrate } } } with such velocities, SEM must be either made very tiny, which is not } the } } } case, or must disintegrate into dust, but it doesn't. } } } } } } Velocities of mechanical vibrations affecting SEM are in many orders of } } } magnitude slower } } } than the E-beam velocities. } } } } } } This is why SEM stage vibration will show up exactly the same at } } different } } } beam accelerating voltages. } } } } } } Why then stray magnetic field affects an e-beam? Because electron has } } huge } } } charge/mass ratio, ~ 10,000,000,000. The interaction is very strong. } } Thus, } } } effect increases at lower accelerating voltages. Same with working } } distance- } } } effect increases with the distance increase. But, one needs to change WD } } } substantially, to see the effect for sure. That will reduce the } } resolution, } } } among other things. I will advice against chamber/beam/sample geometry } } } change during troubleshooting process. } } } } } } Now down to business with Ann's images. The sawtooth vertical edges } } } appearance is the result of horizontal scan being out of phase with the } } } external source of either vibration, fields, or signal interference. } Time } } } delay between the lines at slow scan is in order of tens to hundreds } } } milliseconds, which is within the possible vibration frequency range. } } Same } } } frequencies will cause wavy image at TV deflection speeds. } } } } } } Another point is that mechanical vibration in many cases repeats the AC } } line } } } frequency, as it is caused by electric motor driven devices, so either } 60 } } Hz } } } or it's harmonic(s) are present as mechanical vibrations. The only way } to } } } find the problem is to troubleshoot and differentiate. } } } } } } I agree with other postings- more information needed to develop more } } } accurate idea on how to proceed. } } } } } } } } } Vitaly Feingold } } } Scientific Instruments and Applications } } } 2773 Heath Lane, Duluth GA 30096 } } } (770)232-7785 ph. } } } (770)232-1791 fax } } } (678)467-0012 mobile } } } } } } This message is made of 100% recycled electrons. } } } } } } This address can not receive messages larger than 15 kb without prior } } } notification. } } } } } } ----- Original Message ----- } } } From: Allen Sampson {ars-at-sem.com} } } } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } } } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } } } {Microscopy-at-sparc5.microscopy.com} } } } Sent: Thursday, November 07, 2002 3:50 PM } } } Subject: RE: vibration? } } } } } } } } } } } } -------------------------------------------------------------------- ---- } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } America } } } } To Subscribe/Unsubscribe -- Send Email to } } ListServer-at-MSA.Microscopy.Com } } } } On-Line Help } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } -------------------------------------------------------------------- } } ---. } } } } } } } } } } } } Vitaly, } } } } } } } } Most of your concerns I covered in a previous reply. However, I have } } to } } } } take exception to your claim that a large change in the problem's } } } } characteristic would result from a change in accelerating voltage only } } if } } } } the problem was electro-magnetic. A reduction in the accelerating } } voltage } } } } } } } will also result in a reduction of the velocity of electrons in the } } beam. } } } } That would cause an increased displacement of the beam as it } traverses } } } the } } } } sample surface due to vibrations. } } } } } } } } In other words, the effects of electro-magnetic interference and } } } vibrations } } } } are virtually inseparable except for the frequency involved. Each } will } } } } affect the beam in similar ways, in regards to accelerating voltage } and } } } } working distance. Both will have a greater affect with a lower } } } } accelerating voltage as well as a greater working distance. } } } } } } } } } } } } Allen R. Sampson } } } } Advanced Research Systems } } } } 317 North 4th. Street } } } } St. Charles, Illinois 60174 } } } } } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } } } } } } } } } } On Wednesday, November 06, 2002 8:58 PM, Vitaly Feingold } } } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } } } Could be vibration. Could also be magnetic field. And, could be } } signal } } } } } interference, such as ground loop. What type of SEM are you using? } } } } } } } } } } I will omit remedies for the vibration problem, since Allen Sampson } } did } } } } } pretty good job addressing that. } } } } } } } } } } Stray AC line frequency magnetic field: try acquiring image at } } } different } } } } } accelerating voltages. It is very important that all other geometry } } } } } parameters will remain the same: particular area of a particular } } } } specimen, } } } } } WD, tilt, magnification, spot size. Contrast, brightness, focus, and } } } } } stigmator settings can be changed. If the problem is much worse at, } } say, } } } } 2 } } } } } kV, than at, say, 15kV, you are dealing with magnetic field. You may } } } also } } } } } try to keep 3 axes AC milliGauss meter at the SEM, and notice what } it } } } } } measures, but catching stray magnetic interference this way may be a } } } } little } } } } } tricky. These meters, though, are very inexpensive, and available } } from } } } } many } } } } } test instruments suppliers. } } } } } } } } } } Now we are getting down to statistically most likely problem- signal } } } } } interference and ground loops. It is difficult to go any further } } without } } } } } } } } knowing the SEM type, and acquisition system (if separate) type. Too } } } many } } } } } possibilities exist, but the very first- does your acquisition } } software } } } } } have an option somewhere in the settings which reads something like } } "60 } } } } } Hertz sync", or "AC line synchronization", or "Line sync", etc., } } etc.? } } } If } } } } } yes, check (or uncheck) that option and see what happens. Further } } } } remedies } } } } } of this problem will include eliminating ground loops. Example- sh } } ielded } } } } } cable(s) has shield connected on both sides, and shield is used as } } one } } } of } } } } } the signal wires. This is frequently done, and does jeopardize } } } instrument } } } } } interference tolerance. Another example- one of the accessories is } } } } } susceptible to some kind of interference, and must be powered } through } } } the } } } } } isolation transformer. The latter remedy will only work with decent } } } } (better } } } } } if dedicated) ground. The fact that the problem is not present all } } the } } } } time } } } } } does not mean that everything is perfect inside the SEM. Besides, } } going } } } } } after the EM interference source could be inefficient and } } frustrating, } } } if } } } } } not impossible. Improving grounding/shielding/power connection might } } be } } } } } easier. } } } } } } } } } } } } } } } Vitaly Feingold } } } } } Scientific Instruments and Applications } } } } } 2773 Heath Lane, Duluth GA 30096 } } } } } (770)232-7785 ph. } } } } } (770)232-1791 fax } } } } } (678)467-0012 mobile } } } } } } } } } } This message is made of 100% recycled electrons. } } } } } } } } } } This address can not receive messages larger than 15 kb without } prior } } } } } notification. } } } } } } } } } } ----- Original Message ----- } } } } } } From: Ann-Fook Yang {yanga-at-agr.gc.ca} } } } } } To: {microscopy-at-sparc5.microscopy.com} } } } } } Sent: Wednesday, November 06, 2002 2:45 PM } } } } } Subject: vibration? } } } } } } } } } } } } } } } } } } } } } } ------------------------------------------------------------------------ } } } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } } } America } } } } } } To Subscribe/Unsubscribe -- Send Email to } } } } ListServer-at-MSA.Microscopy.Com } } } } } } On-Line Help } } } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } } } } } } } } } -----------------------------------------------------------------------. } } } } } } } } } } } } } } } } } } Hi everyone, } } } } } } } } } } } } I have two SEM micrographs on the web: } } } } } } http://www.magma.ca/~scimat/Defect.htm } } } } } } The micrographs show zagged edges taken at 20 kX. Such phenomenon } } } does } } } } } not present all the time. Can anyone suggest what causes the problem } } and } } } } how } } } } } to solve it? } } } } } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } } } } } EM Unit, } } } } } } Eastern Cereal and Oilseed Research Centre, } } } } } } Room 2091, Bldg. 20, } } } } } } Central Experimental Farm, } } } } } } Ottawa, Ontario } } } } } } Canada K1A 0C6 } } } } } } } } } } } } Tel: 1-613-759-1638 } } } } } } Fax: 1-613-759-1701 } } } } } } } } } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } }
I thank everyone who replied to my posting on EM Atlas. For those who requested forwarding of the replies, here is the list;
"Histology: A text and atlas" by Johannes A G Rhodin. 1974, Oxford University Press.
“Cell and Tissue Ultrastructure: A Functional Perspective” by Pat Cross and K. Lynn Mercer (1993, ISBN 0-7167-7033-4 )
"The Cell", by Dr. Don Fawcett, 2nd edition, 1981, Saunders, Philadelphia, ISBN: 0-7216-3584-9.
“Cell Structure; an Introduction to Biomedical Electron Microscopy”, 3rd edition, 1982, Churchill Livingstone ISBN 0 443 02324 7,
"Particle Atlas" produced by the McCrone Institute.
“Histology” by Rhodin, (1974, Library of Congress Catalogue No.73-90374)
“BIOMEDICAL ELECTRON MICROSCOPY” by A.V. Maunsbach and B.A. Afzelius, Academic Press 1999.
“The Fine Structure of Nervous System” Peter Davis and Sanford Paley, 3rd edition, 1991, Oxford
"Ultrastructural Pathology of the Cell and Matrix" by Ghadially
On the list, the first two were recommended most frequently. I purchased a used copy of “Cell and Tissue Ultrastructure: A Functional Perspective” from Amazon.com for $18.00.
You've made a great point that would be of considerable interest to many microscopists.
I would love to see this discussion ensue and then a final wrap-up written for publication in Microscopy Today!
Ron Anderson, Editor Microscopy Today
-----Original Message----- } From: Allan Mitchell [mailto:allan.mitchell-at-stonebow.otago.ac.nz] Sent: Sunday, November 10, 2002 2:42 PM To: Microscopy-at-sparc5.microscopy.com Cc: phillipst-at-missouri.edu
Hi Tom, and others
You have raised an interesting point that I have never really been able to get a handle on satisfactorily, that is how do you choose the appropriate acrylic resin to use for a new project,
eg,
should I choose LR White or LR Gold for project X, should I chemically polymerise or perhaps use UV ?
or maybe I should try Unicryl, or Lowicryl, but which one HM20, K4M etc.
Are there others I should be considering?
I would be interested in seeing a discussion on how different people go about choosing a particular acrylic resin for a new project they may have to undertake.
Regards
Allan
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Yep, as long as you can't transmit any information between the spot positions faster than light, you can WARP speed on them.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Dusevich, Vladimir [mailto:dusevichv-at-umkc.edu] Sent: Monday, November 11, 2002 7:29 AM To: microscopy-at-msa.microscopy.com
} } If anyone gets an electron to travel faster than the speed of } light, please } copy me. } } Peter
An electron and a scanning electron spot are two very different things. There are no lows preventing the spot from traveling with any speed.
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
The best I ever used was manufactured by Ilford for their RC multigrade paper. It took up about 40" on a standard bench top, but it processed an 8x10 in about 30 seconds and REALLY created a glossy result. Here's a site that advertises one with a price around $3k (WOW!)
Frederick C. Monson, PhD Center for Advanced Scientific Imaging Mail to Geology West Chester University of Pennsylvania Schmucker II Science Center, Room SS024 South Church Street and Rosedale Avenue West Chester, PA, 19383 Phone: 610-738-0437 eMail: fmonson-at-wcupa.edu
-----Original Message----- } From: Geoff McAuliffe [mailto:mcauliff-at-umdnj.edu] Sent: Friday, November 08, 2002 9:39 AM To: Dean Abel Cc: Leslie Cummins; microscopy-at-msa.microscopy.com
Print dryers for resin coated papers are available, of course right now I can't remember where! Try a google search for print dryers.
Geoff
Dean Abel wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } Hello Leslie, } I use a Durst Laborator S-45 Special enlarger with a point light source } and print all my TEM and 35mm negatives on Ilford Multigrade IV RC DeLuxe } paper using Ilford Multigrade filters. It works fine for me and my bosses, } but I am sure that other papers like Kodak Polymax II RC, as suggested by } Karen Jensen, work just as well. } I learned my darkroom work using fiber-based graded papers and drying them } on a big mirrored drum, but now must I air dry my prints ever since our } dryer broke and couldn't be fixed. This is a bit of a nuisance as the } prints take overnight to dry and can't be turned in the same day they're } printed. } Does anyone have tips on drying resin coated paper quickly??? } Dean Abel } Biological Sciences 138BB } University of Iowa } Iowa City IA 52242-1324
-- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
Very good point. It absolutely could be a beat frequency. Either mechanical, magnetic, or signal interference.
Ann, please do not be discouraged by all seeming complexity. Practical solution could be simpler than bulletproof-correct description covering all possible aspects, causes, and manifestations of the problem.
Vitaly Feingold Scientific Instruments and Applications 2773 Heath Lane, Duluth GA 30096 (770)232-7785 ph. (770)232-1791 fax (678)467-0012 mobile
This message is made of 100% recycled electrons.
This address can not receive messages larger than 15 kb without prior notification.
----- Original Message ----- } From: William J Mushock {wim5-at-Lehigh.EDU} To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' {Microscopy-at-sparc5.microscopy.com} Sent: Monday, November 11, 2002 12:53 PM
Dear Listers:
We are planning to upgrade our TEM digital camera purchased 9 years ago from. We do alot of kidney biopsies as well as various research specimens. Does anyone have any experience with the higher resolution TEM digital cameras? We are looking for something around 2-4 megapixels.
Thanks
Karen
Karen L. Bentley, M.S.(previously Jensen) Associate Scientist & Project Manager Electron Microscope Research Core University of Rochester Medical Center Rochester, NY 14642 585-275-1954
Here is the Table of Contents for the November/December issue of "Microscopy Today" Magazine. A publication of the Microscopy Society of America.
I will close the subscription list for this issue on November 13th. Microscopists in the USA may obtain free subscriptions via the form at www.microscopy-today.com Non-USA members anywhere in the world also receive MT free. Non-USA, non-MSA members are charged a fee to cover postage. Details on the web site.
This issue will mail with the Call for Papers for the 2003 M&M meeting in San Antonio to over 23,000 people, derived from the combined MSA, MAS, IMS, and CIASEM membership as well as the 11,500 regular subscribers to Microscopy Today.
Ron Anderson, Editor Microscopy Today
TABLE OF CONTENTS When Dinosaurs Became Extinct, What Happened To The Insects? Stephen W. Carmichael, Mayo Clinic
A Comparison Of Grain Size Measurements In Al-Cu Thin Films: Imaging Vs. Diffraction Techniques5 L.M. Gignac,* C.E. Murray,* K.P. Rodbell,* M. Gribelyuk+
IBM T.J. Watson Research Center, IBM Microelectronics Division Using a Sony Cyber-Shot Digital Camera for Photomicrography Gregor Overney, Agilent Technologies Inc.
Use Adobe Acrobat to Keep Original Resolutions and to Make TIFF Files From Any Program Jerry Sedgewick, University of Minnesota
Confocal Microscopy System Performance: Laser Power Measurements Robert M. Zucker, PhD, U.S. Environmental Protection Agency
Imaging of Shallow Surface Topography by the Low-Loss Electron (LLE) Method in the Scanning Electron Microscope Oliver C. Wells, IBM Research Division
New and Interesting at Microscopy & Micranalysis-2002
Penetration Rates of Formaldehyde Bryan R. Hewlett, McMaster University Medical Centre
Digital Imaging in K-12 Biology James Ekstrom, Phillips Exeter Academy
Designing A Microscopy/analytical Instrumentation Facility: Step By Step Procedure Judy A. Murphy, San Joaquin Delta College
Preparing Ultra-Smooth SEM Stud Surfaces Dr. Carole Hickman, University of California, Berkeley
Protection from Sulfur Hexafluoride Leaks Mick Thomas, Cornell University
try this one. I used to do this on a regular basis when I was still doing TEM. We looked at anything from heterostructures to dislocations, and had a good success with this technique:
1) buy some 3mm OD steel pipe (or perhaps other strong material)
2) cut rings with a thickness of half a mm to a mm off the pipe. Clean rings thorougly and remove burrs.
3) with the rest of the pipe, drill a ring shaped groove into the material you are interested in around the part you want to prepare.
4) Glue with a strong epoxy one of the rings into the groove.
5) grind sample from backside until you have a freestanding steel ring with the GaAs or other material inside.
6) Dimple
7) Ion mill
8) Enjoy.
the steel ring gives it enough support for handling with tweezers. You probably need to experiment with the thickness of the rings and other parameters, but it works, once you got everything under control.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 3:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
A paper was presented in August at the Ultrapath meeting and will be published in Ultrastructural Pathology where diagnostic EM turnaround time was reduced to 4 hours. It involved digital image processing and storage within patient files and high-speed tissue specimen processing. Such an approach might help you meet your workload. I believe Ron Austin of LSU and Jon Charlesworth of Mayo Clinic, two of the authors on the paper, as well as others might be able to answer questions about this.
Tom Pella
-----Original Message----- } From: Sharon Drew (by way of MicroscopyListserver) [mailto:drewsh-at-musc.edu] Sent: Wednesday, November 06, 2002 2:27 PM To: Microscopy-at-sparc5.microscopy.com
Hi! I run a diagnostic TEM laboratory in SC. One tech. 6 to 5 samples every other day at least. What is the turn around time asked for your labs out there that are also doing diagnostic/clinical work and where does that number come from? Thank you for any help. My supervisor is saying 2 days but I think that is close to impossible when only one tech doing everything including transport of tissue from another lab and scoping all case that come in. Sharon Drew Diagnostic Pathology EM Charleston, SC
Eighth Annual INTERNATIONAL 12-Day Short Course on
3D Microscopy of Living Cells June 15 - 26, 2003 (Pre-course: June 14)
Seventh, Post-course Workshop on
3D Image Processing, June 28 - July 30, 2003
Organized by Prof. James Pawley, (University of Wisconsin-Madison)
in association with
The Departments of Pharmacology and Physiology and the Brain Research Centre, University of British Columbia Vancouver, BC, Canada
DATES
Applications must be received by March 15, 2003 Deposit due April 15, 2003 Registration 5:00 - 7:00 PM Sunday, June 9, 2003 First Lecture 7:30 PM Sunday, June 9, 2003 Live-cell Course ends, noon Thursday, June 20, 2003 3D Image Processing Course, June 22 - 24, 2003
APPLICATIONS DUE BY MARCH 15, 2002
APPLICATIONS Applicants must complete a questionnaire on the web to assess knowledge level, field of interest and proposed personal, live-cell, project. Enrollment will be limited to about 24 participants (exact number depends on number of 3D Systems available). Selection will be made on the basis of background and perceived need. Those without previous LM experience will be provided with access to basic texts to read before the course begins. Application forms requesting information on field of interest and level of experience may be down-loaded from the WWW site at http://www.3dcourse.ubc.ca/application.htm , or obtained from:
Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 JBPAWLEY-at-WISC.EDU
Additional information is available from: http://www.3dcourse.ubc.ca/brochure.htm
We expect to have at least 11, 3D microscope workstations for student use and there will be an international faculty of 20.
Application deadlines:
Application forms must be received for screening by March 1, 2003. Successful applicants will be notified by April 1, and a deposit of 50% must be received by April 15, 2003. In general, refunds of the deposit will only be possible if someone on the waiting list can take the place. The remainder is due before Registration.
Pre-Course Tuition (1/2 day Basic Optical principles) $120 (US) 3D Live-cell Course Tuition (includes lunches and snacks): $2450 (US) Workshop Tuition (includes lunches and snacks): $900 (US)
Room/board about $40/day (US) depending on room type.
I hope that this includes all of the information that you need, but if not, please get back to me. -- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 JBPAWLEY-at-WISC.EDU "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
I highly recommend the small angle cleavage technique for III-V materials. It is a very easy technique to learn, produces superior samples to any other technique, and it is very quick, approx. 9 samples per hour. Out of that 9 samples, about 5-7 will be usable and 2-3 of those will be fantastic. Go to the South Bay Technology web page and check out their microcleave kit. There are some examples of a MQW GaAs-InGaAs structure that I prepared in their document section along with some other examples. Send me your address, and I can send you a disk with a detailed poster on how to do it. You can also go to the #4 TEM sample prep book from MRS proceedings. John McCaffrey and I have a detailed discussion on how to do it.
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 5:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
I would like to estimate the particle size distribution of spherical particles that were embedded in a continuous solid phase and polished to obtain a cross-section. The cross-sectioning could have cut the particles at any point, which means that the size observed is probably not the size of the original particles.Is it possible to deduce the particle size distribution from measurements of the cross-sectional areas ? Can anyone suggest a good reference that may explain how this estimation is accomplished ?
Regards W Erasmus
Willem Erasmus Snr. Scientist, Materials Characterization Group Sasol Technology Tel : +27 +16 960 - 4211/2772 Fax : +27 +16 960 - 2826 E-mail : willem.erasmus-at-sasol.com PO Box 1, Sasolburg, 1947, Republic of South Africa
[All views expressed are my own and not necessarily that of my employer.]
NOTICE: Please note that this eMail, and the contents thereof, is subject to the standard Sasol eMail disclaimer which may be found at: http://www.sasol.com/disclaimer.htm
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We have a small exhaust equipped gas storage room where we fill LN2 dewars. We keep the doors in the area open when we fill. If the flow rate is moderate, we do not have a problem. However, if we get an occasional med pressure dewar, the alarm will sound. The sensor is about 5 feet off the floor. Hope this helps.
Joseph
Joseph M. Oparowski Materials Science Research - Consulting and Failure Analysis Bose Corporation Joseph_Oparowski-at-bose.com The Mountain, M/S 415 Phone: (508) 766-1371 Framingham, MA 01701-9168 Fax: (508) 766-1313
-----Original Message----- } From: psconnel-at-sas.upenn.edu [mailto:psconnel-at-sas.upenn.edu] Sent: Monday, November 11, 2002 7:53 PM To: Oparowski, Joseph Cc: Microscopy-at-sparc5.microscopy.com
In a message dated 11/12/02 5:42:19 AM, willem.erasmus-at-sasol.com writes:
} I would like to estimate the particle size distribution of spherical particles } that were embedded in a continuous solid phase and polished to obtain a } cross-section. The cross-sectioning could have cut the particles at any } point, which means that the size observed is probably not the size of the } original particles.Is it possible to deduce the particle size distribution } from measurements of the cross-sectional areas ? Can anyone suggest a good } reference that may explain how this estimation is accomplished ? } } } } Regards } } W Erasmus
You can find the explanation and math in Practical Stereology, 2nd edition, J. C. Russ & R. T. Dehoff, Plenum Press, 2001. Assuming that you have a way to measure the intersection sizes, the calculations can be done in a spreadsheet. However be warned that the technique, which has been known and used since the 1920's, has two problems. First, it is critically dependent on the assumption that the shape of the features is exactly known and that it remains the same for large and small features. Second, it is mathematically unstable, with the precision of the 3D data being much poorer than that of the 2D intersections. These problems have caused many stereologists to prefer another approach that provides the mean and standard deviation of the distribution without making any shape assumptions. that method is also described in the book. Original references to important papers are provided for all of the stereological techniques.
"Erasmus, Willem (WJ)" To: {Microscopy-at-sparc5.microscopy.com} {willem.erasmus-at-sa cc: sol.com} Subject: SEM particle size measurement
11/12/02 05:07 AM
------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Dear microscopists
I would like to estimate the particle size distribution of spherical particles that were embedded in a continuous solid phase and polished to obtain a cross-section. The cross-sectioning could have cut the particles at any point, which means that the size observed is probably not the size of the original particles.Is it possible to deduce the particle size distribution from measurements of the cross-sectional areas ? Can anyone suggest a good reference that may explain how this estimation is accomplished ?
Regards W Erasmus
Willem Erasmus Snr. Scientist, Materials Characterization Group Sasol Technology Tel : +27 +16 960 - 4211/2772 Fax : +27 +16 960 - 2826 E-mail : willem.erasmus-at-sasol.com PO Box 1, Sasolburg, 1947, Republic of South Africa
[All views expressed are my own and not necessarily that of my employer.]
NOTICE: Please note that this eMail, and the contents thereof, is subject to the standard Sasol eMail disclaimer which may be found at: http://www.sasol.com/disclaimer.htm
If you cannot access the disclaimer through the URL attached and you wish to receive a copy thereof please send an eMail to disclaimer-at-sasol.com. You will receive the disclaimer by return eMail.
Mike Bode's reply reminds me of the method my colleague uses for cross sections. He first sandwiches the sample material between pieces of silicon with G1 epoxy (Gatan) in a vise til the epoxy sets up.Then the sandwich is waxed into a holder for a sonic cutter (again, Gatan). The material cored out is epoxied into a brass tube with a 3mm outside diameter.This tube is placed on a saw with a 3" diameter diamond blade and several disks are cut. Now go to the lap, dimple and ion mill steps.
I have no connection with Gatan; we happily use their products.
--
+++++++++++++++++++++++++++++
R. Ann Bliss Technician, Chemistry and Materials Science Materials Science and Technology Division Lawrence Livermore National Laboratory
I have just read Willingham & Rutherford (1984) J. Histochem Cytochm 32(4):455-460 paper on the use of ferrocyanide reduced osmium with the osmium-thiocarbohydrazide-osmium (OTO) technique of Seligman. I thought the conclusion that R-OTO gave a superior preservation and contrast of membranes in TEM was a good one based on their images. But when I did a Medline search for papers using either the OTO or R-OTO technique, the limited number of papers was essentially limited to SEM work. Perhaps the method is buried in papers and not coming up in a keyword search. Or maybe no one uses it or there are problems with it?? Has any one used the R-OTO (or even OTO) technique with tissue biopsies (Willingham & Rutherford use cell cultures so it is tough to extrapolate the correct osmication times)? Are there hidden pitfalls and disadvantages to this technique? Any comments welcome. Tom
Thomas E. Phillips, PhD Associate Professor of Biological Sciences Director, Molecular Cytology Core 3 Tucker Hall University of Missouri Columbia, MO 65211-7400
Bruce; I don't recommend using steel rings as supports, as these will couple to the filed of the microscope lens and interfere with imaging. You can get brass tube from Gatan (or from a hobby shop for a lot less). Also, get vacuum tweezers if you don't want to fracture your samples. However, you will have a long way to go even after that point. 3-5 compunds containing indium and antimony dissociate during normal ion milling. You will need to consult a series of papers written by Tony Cullis during the late '80s, published in Ultramicroscopy, on the low temperature and iodine techniques necessary to avoid a mess.
John Mardinly Intel
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 2:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
Dear Sharon and Tom: In fact, Prof. Johanessen at Univ. of Bergen and later at Oslo published 4h protocols (from biopsy taking to prints on the table) in the early 80s and summarized in his basic book on ultrastructural pathology. Cheers, Marek.
At 05:58 PM 11/11/02 -0800, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Willem, A great start will be the "Stereology" and "The Image Processing Handbook" by John Russ. Check out Amazon.com or B&N.com. The stereological formula and examples are given there. Probably any other stereology text would also have the examples.
Have Fun,
Jerzy ****************************************************** Jerzy Gazda, Ph.D. Advanced Micro Devices Supervising Engineer 5204 E. Ben White Blvd. - MS 613 PCAL - AIM Section Austin, TX 78741 TEL: 1-800-538-8450, Ext. 51453 FAX: (512) 602-7470 jerzy.gazda-at-amd.com ******************************************************
-----Original Message----- } From: Erasmus, Willem (WJ) [mailto:willem.erasmus-at-sasol.com] Sent: Tuesday, November 12, 2002 4:07 AM To: Microscopy-at-sparc5.microscopy.com
Dear microscopists
I would like to estimate the particle size distribution of spherical particles that were embedded in a continuous solid phase and polished to obtain a cross-section. The cross-sectioning could have cut the particles at any point, which means that the size observed is probably not the size of the original particles.Is it possible to deduce the particle size distribution from measurements of the cross-sectional areas ? Can anyone suggest a good reference that may explain how this estimation is accomplished ?
Regards W Erasmus
Willem Erasmus Snr. Scientist, Materials Characterization Group Sasol Technology Tel : +27 +16 960 - 4211/2772 Fax : +27 +16 960 - 2826 E-mail : willem.erasmus-at-sasol.com PO Box 1, Sasolburg, 1947, Republic of South Africa
[All views expressed are my own and not necessarily that of my employer.]
NOTICE: Please note that this eMail, and the contents thereof, is subject to the standard Sasol eMail disclaimer which may be found at: http://www.sasol.com/disclaimer.htm
If you cannot access the disclaimer through the URL attached and you wish to receive a copy thereof please send an eMail to disclaimer-at-sasol.com. You will receive the disclaimer by return eMail.
You might want to try the high angle polishing technique described in “The Concept of High Angle Wedge Polishing and Thickness Monitoring in TEM Sample Preparation,” Hao Li, and L. Salamanca-Riba, Ultramicroscopy 88, 171-8 (2001). With this technique you can make samples that have electron transparent areas without any ion milling. Because of the high angle polishing the samples are not very thin every where and, therefore, are less likely to break.
I advise you to try the technique first with Si as it is a lot easier to make a TEM sample of Si than InSb. When you try the InSb sample you should use diamond paper with very fine particle size (3 microns) very early in the polishing process because InSb is a very soft material. Then change to finer particle size sooner than you would do with Si so that you can control better how much material you are removing.
With this technique we were able to prepare cross sections of AlSb films grown on GaSb substrates.
Good luck,
Lourdes Salamanca-Riba
----- Original Message ----- } From: "Bruce Brinson" {brinson-at-rice.edu} To: "MSA Listserver" {microscopy-at-sparc5.microscopy.com} Sent: Monday, November 11, 2002 5:06 PM
MURPHY STRIKES AGAIN!
It never fails, I make the announcement and the Course Site goes down (it will be back up soon!). Then several of you point out inconsistencies in the dates in the Announcement. The dates have been fixed below (thanks to Iain Johnson,at Molecular Probes!!) and the Site will soon be repaired.
Thank you for your patience!
Jim P.
Eighth Annual INTERNATIONAL 12-Day Short Course on
3D Microscopy of Living Cells June 15 - 26, 2003 (Pre-course: afternoon, June 14)
Seventh, Post-course Workshop on
3D Image Processing, June 28 - July 30, 2003
Organized by Prof. James Pawley, (University of Wisconsin-Madison)
in association with
The Departments of Pharmacology and Physiology and the Brain Research Centre, University of British Columbia Vancouver, BC, Canada
DATES
Applications must be received by March 15, 2003 Deposit due April 15, 2003 Registration 5:00 - 7:00 PM Saturday, June 14, 2003 First Lecture 7:30 PM Saturday, June 14, 2003 Live-cell Course ends, noon Thursday, June 26, 2003 3D Image Processing Course, June 28 - 30, 2003
APPLICATIONS DUE BY MARCH 15, 2003
APPLICATIONS Applicants must complete a questionnaire on the web to assess knowledge level, field of interest and proposed personal, live-cell, project. Enrollment will be limited to about 24 - 32 participants (exact number depends on number of 3D Systems available). Selection will be made on the basis of background and perceived need. Those without previous LM experience will be provided with access to basic texts to read before the course begins. Application forms requesting information on field of interest and level of experience may be down-loaded from the WWW site at http://www.3dcourse.ubc.ca/application.htm , or obtained from:
Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 JBPAWLEY-at-WISC.EDU
Additional information is available from: http://www.3dcourse.ubc.ca/brochure.htm
We expect to have at least 11, 3D microscope workstations for student use and there will be an international faculty of 20.
Application deadlines:
Application forms must be received for screening by March 15, 2003. Successful applicants will be notified by April 1, and a deposit of 50% must be received by April 15, 2003. In general, refunds of the deposit will only be possible if someone on the waiting list can take the place. The remainder is due before Registration.
Pre-Course Tuition (1/2 day Basic Optical principles) $120 (US) 3D Live-cell Course Tuition (includes lunches and snacks): $2450 (US) Workshop Tuition (includes lunches and snacks): $900 (US)
Room/board about $40/day (US) depending on room type.
I hope that this includes all of the information that you need, but if not, please get back to me. -- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 JBPAWLEY-at-WISC.EDU "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
John is of course correct on both points: the steel could potentially interfere with the magnetic field of the lenses and create unwanted effects. I thought we were using steel (it's been a while), the material was definitely not copper, but perhaps it was some other non-magnetic metal.
For the milling I always used LN2 cooling as John suggests, and I also used Iodine. It helps to keep the milling as short as possible, so a careful dimpling is important.
Thanks, John, for pointing that out.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Mardinly, John [mailto:john.mardinly-at-intel.com] Sent: Tuesday, November 12, 2002 10:15 AM To: Bruce Brinson; MSA Listserver
Bruce; I don't recommend using steel rings as supports, as these will couple to the filed of the microscope lens and interfere with imaging. You can get brass tube from Gatan (or from a hobby shop for a lot less). Also, get vacuum tweezers if you don't want to fracture your samples. However, you will have a long way to go even after that point. 3-5 compunds containing indium and antimony dissociate during normal ion milling. You will need to consult a series of papers written by Tony Cullis during the late '80s, published in Ultramicroscopy, on the low temperature and iodine techniques necessary to avoid a mess.
John Mardinly Intel
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 2:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
While previous replies focused on grid-free technique, I would like to comment on the way you did out there.
As you already realized, the dimpled sample is very hard to handle. This is reasonable because the center of the dimpled area is down to a couple of microns! Accordingly what I normally do is glue the sample onto copper grid BEFORE removing it from the support glass. 1. dimple on a glass post. 2. apply very small amount of glue (M-bond or G1 epoxy). 3. put copper grid on the top of the sample. 4. wait for glue curing. Heat up if necessary. 5. put the post in acetone.
Because M-bond or G1 epoxy can not be removed by acetone, you will have your sample stands together with the copper grid.
if your sample is somewhat thick (more than 70microns), you can also glue copper grid onto the sample before dimpling. Routinely I use both method and the yield is always ~1.
Hope this helps, Shuyou.
On Mon, 11 Nov 2002 14:06:37 -0800 Bruce Brinson {brinson-at-rice.edu} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Hello TEM Microscopist and sample prep masters, } } I've spent a humbling amount of time trying to learn to prepare TEM } cross section samples from GaAs substrates. I should also point out that } I do not have mentor for this project. For the moment I am working with } dummy samples. When I start working with the real material of interest } (InSb AlAs quantum wells), we will typically have enough material for } one attempt... so I need to get the yield (to the TEM) up to or atleast } close to 1. } I am able to block, polish and dimple the material easily enough but } loose many samples transferring from the dimpling block (glass post) to } the Cu support. } } My procedure is .... then } 1. dimple on a glass post. } 2. I then put a piece of filter paper and stack 2 u-scope slides in a } dish. I put the post, sample down, with one edge of the post on the } u-scope slides allowing a space for the dimpled bit to fall off then } immerse in acetone . } To this point my yield is ~1 } } Mounting to the Cu support is where I often loose the samples due to } fracturing. Use fine tipped tweezes typical of those use with 3mm TEM } grids and all the caution I can muster. I lift the sample out and } attempt to lay it on a glue bearing support. Some times I get there in 1 } piece, sometimes I don't. } } Since we need a yield of 1 for 30+ samples this is a real problem. } } Any advice on the sample prep. of this material would be greatly } appreciated. } } } Bruce Brinson } Rice U.
_____________________________ Shu-You Li, Ph.D. Electron Microscopist Electron Probe Instrumentation Center(EPIC) Northwestern University 2220 Campus Drive, 1141 Cook Hall Evanston, IL 60208, USA Ph: (847) 491-7798, Fax: (847) 491-7820 Email: syli-at-northwestern.edu; syli16-at-hotmail.com http://pubweb.northwestern.edu/~sli974
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Dear Sharon and Tom: In fact, Prof. Johanessen at Univ. of Bergen and later at Oslo published 4h protocols (from biopsy taking to prints on the table) in the early 80s and summarized in his basic book on ultrastructural pathology. Cheers, Marek.
At 05:58 PM 11/11/02 -0800, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
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Dear microscopists
I would like to estimate the particle size distribution of spherical particles that were embedded in a continuous solid phase and polished to obtain a cross-section. The cross-sectioning could have cut the particles at any point, which means that the size observed is probably not the size of the original particles.Is it possible to deduce the particle size distribution from measurements of the cross-sectional areas ? Can anyone suggest a good reference that may explain how this estimation is accomplished ?
Regards W Erasmus
Willem Erasmus Snr. Scientist, Materials Characterization Group Sasol Technology Tel : +27 +16 960 - 4211/2772 Fax : +27 +16 960 - 2826 E-mail : willem.erasmus-at-sasol.com PO Box 1, Sasolburg, 1947, Republic of South Africa
[All views expressed are my own and not necessarily that of my employer.]
NOTICE: Please note that this eMail, and the contents thereof, is subject to the standard Sasol eMail disclaimer which may be found at: http://www.sasol.com/disclaimer.htm
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Bruce;
Mike Bode's reply reminds me of the method my colleague uses for cross sections. He first sandwiches the sample material between pieces of silicon with G1 epoxy (Gatan) in a vise til the epoxy sets up.Then the sandwich is waxed into a holder for a sonic cutter (again, Gatan). The material cored out is epoxied into a brass tube with a 3mm outside diameter.This tube is placed on a saw with a 3" diameter diamond blade and several disks are cut. Now go to the lap, dimple and ion mill steps.
I have no connection with Gatan; we happily use their products.
--
+++++++++++++++++++++++++++++
R. Ann Bliss Technician, Chemistry and Materials Science Materials Science and Technology Division Lawrence Livermore National Laboratory
"Erasmus, Willem (WJ)" To: {Microscopy-at-sparc5.microscopy.com} {willem.erasmus-at-sa cc: sol.com} Subject: SEM particle size measurement
11/12/02 05:07 AM
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Dear microscopists
I would like to estimate the particle size distribution of spherical particles that were embedded in a continuous solid phase and polished to obtain a cross-section. The cross-sectioning could have cut the particles at any point, which means that the size observed is probably not the size of the original particles.Is it possible to deduce the particle size distribution from measurements of the cross-sectional areas ? Can anyone suggest a good reference that may explain how this estimation is accomplished ?
Regards W Erasmus
Willem Erasmus Snr. Scientist, Materials Characterization Group Sasol Technology Tel : +27 +16 960 - 4211/2772 Fax : +27 +16 960 - 2826 E-mail : willem.erasmus-at-sasol.com PO Box 1, Sasolburg, 1947, Republic of South Africa
[All views expressed are my own and not necessarily that of my employer.]
NOTICE: Please note that this eMail, and the contents thereof, is subject to the standard Sasol eMail disclaimer which may be found at: http://www.sasol.com/disclaimer.htm
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Willem, A great start will be the "Stereology" and "The Image Processing Handbook" by John Russ. Check out Amazon.com or B&N.com. The stereological formula and examples are given there. Probably any other stereology text would also have the examples.
Have Fun,
Jerzy ****************************************************** Jerzy Gazda, Ph.D. Advanced Micro Devices Supervising Engineer 5204 E. Ben White Blvd. - MS 613 PCAL - AIM Section Austin, TX 78741 TEL: 1-800-538-8450, Ext. 51453 FAX: (512) 602-7470 jerzy.gazda-at-amd.com ******************************************************
-----Original Message----- } From: Erasmus, Willem (WJ) [mailto:willem.erasmus-at-sasol.com] Sent: Tuesday, November 12, 2002 4:07 AM To: Microscopy-at-sparc5.microscopy.com
Dear microscopists
I would like to estimate the particle size distribution of spherical particles that were embedded in a continuous solid phase and polished to obtain a cross-section. The cross-sectioning could have cut the particles at any point, which means that the size observed is probably not the size of the original particles.Is it possible to deduce the particle size distribution from measurements of the cross-sectional areas ? Can anyone suggest a good reference that may explain how this estimation is accomplished ?
Regards W Erasmus
Willem Erasmus Snr. Scientist, Materials Characterization Group Sasol Technology Tel : +27 +16 960 - 4211/2772 Fax : +27 +16 960 - 2826 E-mail : willem.erasmus-at-sasol.com PO Box 1, Sasolburg, 1947, Republic of South Africa
[All views expressed are my own and not necessarily that of my employer.]
NOTICE: Please note that this eMail, and the contents thereof, is subject to the standard Sasol eMail disclaimer which may be found at: http://www.sasol.com/disclaimer.htm
If you cannot access the disclaimer through the URL attached and you wish to receive a copy thereof please send an eMail to disclaimer-at-sasol.com. You will receive the disclaimer by return eMail.
I need service for my RMC MT6000-XL ultramicrotome. It's located at UCLA, Los Angeles, CA. If you could recommend real person/company I would really appreciate. Thanks. Sergey
_____________________________________
Sergey Ryazantsev Ph. D. Electron Microscopy UCLA School of Medicine Department of Biological Chemistry Box 951737 Los Angeles, CA 90095-1737
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John is of course correct on both points: the steel could potentially interfere with the magnetic field of the lenses and create unwanted effects. I thought we were using steel (it's been a while), the material was definitely not copper, but perhaps it was some other non-magnetic metal.
For the milling I always used LN2 cooling as John suggests, and I also used Iodine. It helps to keep the milling as short as possible, so a careful dimpling is important.
Thanks, John, for pointing that out.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Mardinly, John [mailto:john.mardinly-at-intel.com] Sent: Tuesday, November 12, 2002 10:15 AM To: Bruce Brinson; MSA Listserver
Bruce; I don't recommend using steel rings as supports, as these will couple to the filed of the microscope lens and interfere with imaging. You can get brass tube from Gatan (or from a hobby shop for a lot less). Also, get vacuum tweezers if you don't want to fracture your samples. However, you will have a long way to go even after that point. 3-5 compunds containing indium and antimony dissociate during normal ion milling. You will need to consult a series of papers written by Tony Cullis during the late '80s, published in Ultramicroscopy, on the low temperature and iodine techniques necessary to avoid a mess.
John Mardinly Intel
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 2:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
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I have just read Willingham & Rutherford (1984) J. Histochem Cytochm 32(4):455-460 paper on the use of ferrocyanide reduced osmium with the osmium-thiocarbohydrazide-osmium (OTO) technique of Seligman. I thought the conclusion that R-OTO gave a superior preservation and contrast of membranes in TEM was a good one based on their images. But when I did a Medline search for papers using either the OTO or R-OTO technique, the limited number of papers was essentially limited to SEM work. Perhaps the method is buried in papers and not coming up in a keyword search. Or maybe no one uses it or there are problems with it?? Has any one used the R-OTO (or even OTO) technique with tissue biopsies (Willingham & Rutherford use cell cultures so it is tough to extrapolate the correct osmication times)? Are there hidden pitfalls and disadvantages to this technique? Any comments welcome. Tom
Thomas E. Phillips, PhD Associate Professor of Biological Sciences Director, Molecular Cytology Core 3 Tucker Hall University of Missouri Columbia, MO 65211-7400
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Bruce; I don't recommend using steel rings as supports, as these will couple to the filed of the microscope lens and interfere with imaging. You can get brass tube from Gatan (or from a hobby shop for a lot less). Also, get vacuum tweezers if you don't want to fracture your samples. However, you will have a long way to go even after that point. 3-5 compunds containing indium and antimony dissociate during normal ion milling. You will need to consult a series of papers written by Tony Cullis during the late '80s, published in Ultramicroscopy, on the low temperature and iodine techniques necessary to avoid a mess.
John Mardinly Intel
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 2:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
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Dear Bruce,
You might want to try the high angle polishing technique described in “The Concept of High Angle Wedge Polishing and Thickness Monitoring in TEM Sample Preparation,” Hao Li, and L. Salamanca-Riba, Ultramicroscopy 88, 171-8 (2001). With this technique you can make samples that have electron transparent areas without any ion milling. Because of the high angle polishing the samples are not very thin every where and, therefore, are less likely to break.
I advise you to try the technique first with Si as it is a lot easier to make a TEM sample of Si than InSb. When you try the InSb sample you should use diamond paper with very fine particle size (3 microns) very early in the polishing process because InSb is a very soft material. Then change to finer particle size sooner than you would do with Si so that you can control better how much material you are removing.
With this technique we were able to prepare cross sections of AlSb films grown on GaSb substrates.
Good luck,
Lourdes Salamanca-Riba
----- Original Message ----- } From: "Bruce Brinson" {brinson-at-rice.edu} To: "MSA Listserver" {microscopy-at-sparc5.microscopy.com} Sent: Monday, November 11, 2002 5:06 PM
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You cannot generalize that the frequency of the interference on the images is the same as that of the source.I participated in an experiment several years ago in which we intentionally generated an AC field at frequencies a few Hz above 60. The resulting interference on the sem image was the difference between the source and the 60Hz sync. In other words, a 65Hz field would look like 5Hz(65-60). This also works for harmonics of 60Hz. A computer monitor with a refresh rate of 75Hz would look like a 15Hz field on an sem scan synched to 60Hz.
Also, the resonant frequency of the column suspension has to be considered. Microscope columns usually have a resonant frequency between 5-10Hz.
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My apologies to all - I made a couple of postings on this matter. The first laid down the conditions I was using, assumptions that had to be made (I should have just asked the original poster, but what I wrote was intended to be more general in application).
That original reply of mine laid out the assumption that the images were long exposures. All later remarks I made were based on that. If, instead, these were short exposures then the frequencies involved could be 50 or 60 Hz. or higher. My claim that these image problems would be vibrations was based on those conditions.
Sorry if I caused any confusion on that point by not reiterating the conditions in later messages..
Allen R. Sampson Advanced Research Systems 317 North 4th. Street St. Charles, Illinois 60174 ph. (630) 513-7093 fax (630) 513-7092 web http://www.sem.com
On Friday, November 08, 2002 6:57 AM, Vitaly Feingold [SMTP:vitalylazar-at-worldnet.att.net] wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } ----- Original Message ----- } } From: Allen Sampson {ars-at-sem.com} } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } {Microscopy-at-sparc5.microscopy.com} } Sent: Friday, November 08, 2002 6:43 AM } Subject: RE: vibration? } } } } Reminds me of the very old question of can a scanning electron spot on a } } CRT exceed the speed of light? } } } } If only it could be as easy to figure as you portray. I'll try to explain } } the situation, but it is really more complex that I can describe in words } } here - and being late at night, I really won't spend much time on the } } matter. } } } } Let's assume that we are working at a working distance of 20mm. For the } } velocity you give for electrons accelerated at 2KV (27,000 kM/S) or 15KV } } (73,000 kM/S), that 20mm would be traversed in an exceeding small fraction } } of a second. However, we aren't looking at the spatial translation of a } } single spot. In the SEM, the spot formed on the sample is constantly } } moving - in the case of a record image the movement of the spot is } } generally determined by the dwell time on each of the individual pixels on } } each line and the number and resolution of lines in the frame. } } } } In the record mode, most instruments will sync the start of each line to } } the zero-crossing of the electrical mains. This is an intentional attempt } } to minimize the effect of 50 or 60 Hz. interference. Digital systems may } } or may not do this. } } } } If you go vertically down the recorded image, you are actually looking at } } pixels that are separated by fairly large periods of time, 1/50th or } 1/60th } } of a second in the case of a synched record cycle. Now let's look at your } } projected travel times using these figures. The difference between the } 2KV } } and 15KV velocities you mention is 46000 kM/S. That gives us a difference } } in the velocity at these two accelerating voltages of 4.35x10-9 seconds to } } traverse the 20mm to the sample. } } } } Correct. } } } } Assuming a 60 Hz system, the difference between two vertically separated } } pixels is 0.0167 seconds. } } } That is correct for a scan speed of 60 lines per second - frame of 1000 } lines will be scanned in 16 seconds. } } } } Let's say that each 1/60th of a second is split } } into 1500 equal parts - 1000 pixels used per line and 500 portions used } for } } the vertical retrace. That means that each pixel would be 0.000011 } seconds } } apart. } } } Adjacent horizontal pixels will be ~ 0.00001 seconds apart. While adjacent } vertical pixels will be 1/60s ~ 0.016s apart. } } } } Now let's look at two pixels that are on separate scan lines and separated } } by one additional pixel. They would be separated by a time period of } } 0.016678 seconds (adding the pixel dwell time to the line dwell time). } } } Correct. } } } } Doesn't sound like much, but in EM we have to understand the effects of } } the orders of magnitude we work with. If you divide the above time } } difference to traverse the working distance by the pixel time difference } } just mentioned, you would find that there is a 2.58x10-7 second difference } } between the pixels mentioned above. } } } Here is the juice. When you divide a time period by a time period, the } result } can not be time. It can be, however, a number of events, a number of } portions, etc. What portions? The above figure of 2.58x10-7 is the } difference } between the portions of a pixel, which beam will scan across } the specimen surface, equal to the time difference between 2kV and 15kV } electrons crossing of the WD of 20 mm. Beam deflection (scan) times remain } the same for both accelerating voltages, of course. } Can you see such difference on a micrograph? The ~ 3/10,000,000 of a pixel? } This will be the difference attributed to accelerating voltage change from } 2kV to 15kV and vice versa. This is why kV change will not bring any } noticeable change into vibration affected image. } } My whole point was that scan (deflection) times/speeds are compatible with } times/speeds of both mechanical and AC magnetic } events. The electron velocities/timing, in contrary, are not. They are many } orders of } magnitude shorter/faster than mechanical events. } } } } } } I'm tired and really don't want to extend this too much more, but consider } } that the vibrations seen on the images at question cover around 20 or more } } scan lines and figure in the velocities of a nanometer scale RMS } vibration. } } You'll find that there is indeed a considerable, measurable, variation in } } image vibrations due to accelerating voltage (as well as working } distance). } } } } Amazing, isn't it? } } } } By the way, I still claim that the frequency of the problems in the } } original image is much lower than 50 or 60 Hertz and is the result of } } vibrations rather than any electro-magnetic or electronic effect. Another } } poster mentioned the possibility that a turbo pump may be having problems } } but that is also not a likely cause as the frequencies involved don't } } match. You might, however, want to check the operation of any water } } chiller that is in use. } } Perhaps so, perhaps not. It can be chiller, or turbo pump, or anything else. } I can't say without more information. } } } } } } If I have learned anything in working on these instruments for well over } } twenty years, it's that nothing is simple or straightforward. We're } } dealing with sub-atomic particle physics here folks, an area that can only } } be currently described by the statistical methods of quantum theories. } } } } As far as I am concerned, there is no further determination to be made - } } the problem is one of mechanical vibrations and I would be glad to stake } my } } reputation on it, as I do on any posting I make here. } } } } } } Allen R. Sampson } } Advanced Research Systems } } 317 North 4th. Street } } St. Charles, Illinois 60174 } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } Vitaly Feingold } Scientific Instruments and Applications } 2773 Heath Lane, Duluth GA 30096 } (770)232-7785 ph. } (770)232-1791 fax } (678)467-0012 mobile } } This message is made of 100% recycled electrons. } } This address can not receive messages larger than 15 kb without prior } notification. } } } } } } } } On Thursday, November 07, 2002 10:54 PM, Vitaly Feingold } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } Hey Allen, } } } } } } You may found the following figures entertaining. Natural } } } constants and results of calculations are rounded to the first character } } } after the decimal point, relativity effects are neglected, plain text } } format } } } is used. I am a bit rusty on my Physics- my apologies if I missed an } } order } } } or so of magnitude. Makes no difference in this example. } } } } } } Electron mass ~ 9.1 x 10-31kg } } } Electron charge ~ -1.6 x 10-19 C } } } One electronvolt energy ~ 1.6 x 10-19J } } } Kinetic energy (of electron, or anything having mass) = mV2/2, where m } is } } } the mass, } } } V2 is the velocity square. } } } } } } Simple calculations yield the following velocities in kilometers per } } second } } } for electrons accelerated by potential differences of: } } } 1 Volt ~ 6 x 100 km/s } } } 200 V ~ 8.4 x 1,000 km/s } } } 2 kV ~ 2,7 x 10,000 km/s } } } 15 kV ~ 7.3 x 10,000 km/s } } } } } } Amazing, isn't it? } } } } } } Mechanical vibration velocity must be compatible in magnitude with the } } above } } } velocities, in order for vibration effects to look different at } different } } } beam accelerating voltages. But SEM is made of the materials found on } } this } } } planet, with limited durability specifications. Thus, in order to } vibrate } } } with such velocities, SEM must be either made very tiny, which is not } the } } } case, or must disintegrate into dust, but it doesn't. } } } } } } Velocities of mechanical vibrations affecting SEM are in many orders of } } } magnitude slower } } } than the E-beam velocities. } } } } } } This is why SEM stage vibration will show up exactly the same at } } different } } } beam accelerating voltages. } } } } } } Why then stray magnetic field affects an e-beam? Because electron has } } huge } } } charge/mass ratio, ~ 10,000,000,000. The interaction is very strong. } } Thus, } } } effect increases at lower accelerating voltages. Same with working } } distance- } } } effect increases with the distance increase. But, one needs to change WD } } } substantially, to see the effect for sure. That will reduce the } } resolution, } } } among other things. I will advice against chamber/beam/sample geometry } } } change during troubleshooting process. } } } } } } Now down to business with Ann's images. The sawtooth vertical edges } } } appearance is the result of horizontal scan being out of phase with the } } } external source of either vibration, fields, or signal interference. } Time } } } delay between the lines at slow scan is in order of tens to hundreds } } } milliseconds, which is within the possible vibration frequency range. } } Same } } } frequencies will cause wavy image at TV deflection speeds. } } } } } } Another point is that mechanical vibration in many cases repeats the AC } } line } } } frequency, as it is caused by electric motor driven devices, so either } 60 } } Hz } } } or it's harmonic(s) are present as mechanical vibrations. The only way } to } } } find the problem is to troubleshoot and differentiate. } } } } } } I agree with other postings- more information needed to develop more } } } accurate idea on how to proceed. } } } } } } } } } Vitaly Feingold } } } Scientific Instruments and Applications } } } 2773 Heath Lane, Duluth GA 30096 } } } (770)232-7785 ph. } } } (770)232-1791 fax } } } (678)467-0012 mobile } } } } } } This message is made of 100% recycled electrons. } } } } } } This address can not receive messages larger than 15 kb without prior } } } notification. } } } } } } ----- Original Message ----- } } } From: Allen Sampson {ars-at-sem.com} } } } To: 'Vitaly Feingold' {vitalylazar-at-worldnet.att.net} ; 'Ann-Fook Yang' } } } {yanga-at-agr.gc.ca} ; 'microscopy-at-msa.microscopy.com' } } } {Microscopy-at-sparc5.microscopy.com} } } } Sent: Thursday, November 07, 2002 3:50 PM } } } Subject: RE: vibration? } } } } } } } } } } } } -------------------------------------------------------------------- ---- } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } America } } } } To Subscribe/Unsubscribe -- Send Email to } } ListServer-at-MSA.Microscopy.Com } } } } On-Line Help } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } -------------------------------------------------------------------- } } ---. } } } } } } } } } } } } Vitaly, } } } } } } } } Most of your concerns I covered in a previous reply. However, I have } } to } } } } take exception to your claim that a large change in the problem's } } } } characteristic would result from a change in accelerating voltage only } } if } } } } the problem was electro-magnetic. A reduction in the accelerating } } voltage } } } } } } } will also result in a reduction of the velocity of electrons in the } } beam. } } } } That would cause an increased displacement of the beam as it } traverses } } } the } } } } sample surface due to vibrations. } } } } } } } } In other words, the effects of electro-magnetic interference and } } } vibrations } } } } are virtually inseparable except for the frequency involved. Each } will } } } } affect the beam in similar ways, in regards to accelerating voltage } and } } } } working distance. Both will have a greater affect with a lower } } } } accelerating voltage as well as a greater working distance. } } } } } } } } } } } } Allen R. Sampson } } } } Advanced Research Systems } } } } 317 North 4th. Street } } } } St. Charles, Illinois 60174 } } } } } } } } phone (630) 513-7093 fax (630) 513-7092 http://www.sem.com } } } } } } } } } } } } On Wednesday, November 06, 2002 8:58 PM, Vitaly Feingold } } } } [SMTP:vitalylazar-at-worldnet.att.net] wrote: } } } } } Could be vibration. Could also be magnetic field. And, could be } } signal } } } } } interference, such as ground loop. What type of SEM are you using? } } } } } } } } } } I will omit remedies for the vibration problem, since Allen Sampson } } did } } } } } pretty good job addressing that. } } } } } } } } } } Stray AC line frequency magnetic field: try acquiring image at } } } different } } } } } accelerating voltages. It is very important that all other geometry } } } } } parameters will remain the same: particular area of a particular } } } } specimen, } } } } } WD, tilt, magnification, spot size. Contrast, brightness, focus, and } } } } } stigmator settings can be changed. If the problem is much worse at, } } say, } } } } 2 } } } } } kV, than at, say, 15kV, you are dealing with magnetic field. You may } } } also } } } } } try to keep 3 axes AC milliGauss meter at the SEM, and notice what } it } } } } } measures, but catching stray magnetic interference this way may be a } } } } little } } } } } tricky. These meters, though, are very inexpensive, and available } } from } } } } many } } } } } test instruments suppliers. } } } } } } } } } } Now we are getting down to statistically most likely problem- signal } } } } } interference and ground loops. It is difficult to go any further } } without } } } } } } } } knowing the SEM type, and acquisition system (if separate) type. Too } } } many } } } } } possibilities exist, but the very first- does your acquisition } } software } } } } } have an option somewhere in the settings which reads something like } } "60 } } } } } Hertz sync", or "AC line synchronization", or "Line sync", etc., } } etc.? } } } If } } } } } yes, check (or uncheck) that option and see what happens. Further } } } } remedies } } } } } of this problem will include eliminating ground loops. Example- sh } } ielded } } } } } cable(s) has shield connected on both sides, and shield is used as } } one } } } of } } } } } the signal wires. This is frequently done, and does jeopardize } } } instrument } } } } } interference tolerance. Another example- one of the accessories is } } } } } susceptible to some kind of interference, and must be powered } through } } } the } } } } } isolation transformer. The latter remedy will only work with decent } } } } (better } } } } } if dedicated) ground. The fact that the problem is not present all } } the } } } } time } } } } } does not mean that everything is perfect inside the SEM. Besides, } } going } } } } } after the EM interference source could be inefficient and } } frustrating, } } } if } } } } } not impossible. Improving grounding/shielding/power connection might } } be } } } } } easier. } } } } } } } } } } } } } } } Vitaly Feingold } } } } } Scientific Instruments and Applications } } } } } 2773 Heath Lane, Duluth GA 30096 } } } } } (770)232-7785 ph. } } } } } (770)232-1791 fax } } } } } (678)467-0012 mobile } } } } } } } } } } This message is made of 100% recycled electrons. } } } } } } } } } } This address can not receive messages larger than 15 kb without } prior } } } } } notification. } } } } } } } } } } ----- Original Message ----- } } } } } } From: Ann-Fook Yang {yanga-at-agr.gc.ca} } } } } } To: {microscopy-at-sparc5.microscopy.com} } } } } } Sent: Wednesday, November 06, 2002 2:45 PM } } } } } Subject: vibration? } } } } } } } } } } } } } } } } } } } } } } ------------------------------------------------------------------------ } } } } } } The Microscopy ListServer -- Sponsor: The Microscopy Society of } } } } America } } } } } } To Subscribe/Unsubscribe -- Send Email to } } } } ListServer-at-MSA.Microscopy.Com } } } } } } On-Line Help } } } } http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } } } } } } } } } } } } -----------------------------------------------------------------------. } } } } } } } } } } } } } } } } } } Hi everyone, } } } } } } } } } } } } I have two SEM micrographs on the web: } } } } } } http://www.magma.ca/~scimat/Defect.htm } } } } } } The micrographs show zagged edges taken at 20 kX. Such phenomenon } } } does } } } } } not present all the time. Can anyone suggest what causes the problem } } and } } } } how } } } } } to solve it? } } } } } } Thanking in advance. } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } AnnFook Yang } } } } } } EM Unit, } } } } } } Eastern Cereal and Oilseed Research Centre, } } } } } } Room 2091, Bldg. 20, } } } } } } Central Experimental Farm, } } } } } } Ottawa, Ontario } } } } } } Canada K1A 0C6 } } } } } } } } } } } } Tel: 1-613-759-1638 } } } } } } Fax: 1-613-759-1701 } } } } } } } } } } } } e-mail: yanga-at-em.agr.ca } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } }
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Yep, as long as you can't transmit any information between the spot positions faster than light, you can WARP speed on them.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Dusevich, Vladimir [mailto:dusevichv-at-umkc.edu] Sent: Monday, November 11, 2002 7:29 AM To: microscopy-at-msa.microscopy.com
} } If anyone gets an electron to travel faster than the speed of } light, please } copy me. } } Peter
An electron and a scanning electron spot are two very different things. There are no lows preventing the spot from traveling with any speed.
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Pat,
We have a small exhaust equipped gas storage room where we fill LN2 dewars. We keep the doors in the area open when we fill. If the flow rate is moderate, we do not have a problem. However, if we get an occasional med pressure dewar, the alarm will sound. The sensor is about 5 feet off the floor. Hope this helps.
Joseph
Joseph M. Oparowski Materials Science Research - Consulting and Failure Analysis Bose Corporation Joseph_Oparowski-at-bose.com The Mountain, M/S 415 Phone: (508) 766-1371 Framingham, MA 01701-9168 Fax: (508) 766-1313
-----Original Message----- } From: psconnel-at-sas.upenn.edu [mailto:psconnel-at-sas.upenn.edu] Sent: Monday, November 11, 2002 7:53 PM To: Oparowski, Joseph Cc: Microscopy-at-sparc5.microscopy.com
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Jonathan,
We use a handheld O2 monitor system from Draeger (PAC III). It is good for at least two years and costs under $1K with a charging stand.
Joseph
Joseph M. Oparowski Materials Science Research - Consulting and Failure Analysis Bose Corporation Joseph_Oparowski-at-bose.com The Mountain, M/S 415 Phone: (508) 766-1371 Framingham, MA 01701-9168 Fax: (508) 766-1313
-----Original Message----- } From: jmkrupp-at-cats.ucsc.edu [mailto:jmkrupp-at-cats.ucsc.edu] Sent: Friday, November 08, 2002 2:05 PM To: Microscopy-at-sparc5.microscopy.com
Hi:
I am thinking I should be more careful with our liquid nitrogen handling. I would like to monitor oxygen levels in the room we fill dewars.
So far, I have found small, battery operated low oxygen alarms for about $300. But, they only last a year before needing replacement. The permanent installations I have found go for over $1K.
Is this right? Any one with some sage advice? Yes, I know better safe than sorry, but just double checking with the knowledge base.
Jonathan Krupp Microscopy & Imaging Lab University of California Santa Cruz, CA 95064 (831) 459-2477 jmkrupp-at-cats.ucsc.edu
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Bruce,
try this one. I used to do this on a regular basis when I was still doing TEM. We looked at anything from heterostructures to dislocations, and had a good success with this technique:
1) buy some 3mm OD steel pipe (or perhaps other strong material)
2) cut rings with a thickness of half a mm to a mm off the pipe. Clean rings thorougly and remove burrs.
3) with the rest of the pipe, drill a ring shaped groove into the material you are interested in around the part you want to prepare.
4) Glue with a strong epoxy one of the rings into the groove.
5) grind sample from backside until you have a freestanding steel ring with the GaAs or other material inside.
6) Dimple
7) Ion mill
8) Enjoy.
the steel ring gives it enough support for handling with tweezers. You probably need to experiment with the thickness of the rings and other parameters, but it works, once you got everything under control.
mike
Michael Bode, Ph.D. Soft Imaging System Corp. 12596 West Bayaud Avenue Suite 300 Lakewood, CO 80228 =================================== phone: (888) FIND SIS (303) 234-9270 fax: (303) 234-9271 email: mailto:info-at-soft-imaging.com web: http://www.soft-imaging.com ===================================
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 3:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
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In a message dated 11/12/02 5:42:19 AM, willem.erasmus-at-sasol.com writes:
} I would like to estimate the particle size distribution of spherical particles } that were embedded in a continuous solid phase and polished to obtain a } cross-section. The cross-sectioning could have cut the particles at any } point, which means that the size observed is probably not the size of the } original particles.Is it possible to deduce the particle size distribution } from measurements of the cross-sectional areas ? Can anyone suggest a good } reference that may explain how this estimation is accomplished ? } } } } Regards } } W Erasmus
You can find the explanation and math in Practical Stereology, 2nd edition, J. C. Russ & R. T. Dehoff, Plenum Press, 2001. Assuming that you have a way to measure the intersection sizes, the calculations can be done in a spreadsheet. However be warned that the technique, which has been known and used since the 1920's, has two problems. First, it is critically dependent on the assumption that the shape of the features is exactly known and that it remains the same for large and small features. Second, it is mathematically unstable, with the precision of the 3D data being much poorer than that of the 2D intersections. These problems have caused many stereologists to prefer another approach that provides the mean and standard deviation of the distribution without making any shape assumptions. that method is also described in the book. Original references to important papers are provided for all of the stereological techniques.
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Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
You will find a description of what you want in the book "Morphometry" by Aherne WA and Dunnill MS, Pub. Arnold, ISBN 0 7131 4538 2. It is out of print but Amazon are selling it second hand at
http://www.amazon.com/
I would be interested in knowing what other suggestions people come up with.
At 12:07 2002-11-12 +0200, Erasmus, Willem (WJ) wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Obs/NB New postal/visiting address from July 2002!
Gareth Morgan MPhil MSc FIBMS, Institute for Microbiology, Pathology and Immunology (IMPI), H5, Karolinska Institutet, Huddinge Universitetssjukhus, F46 SE 141 86 Stockholm Sweden
OBS! Besöksadress: F-Huset, Forskningsgatan 2 F52, Rum 2.10. Laboratoriet för klinisk patologi och cytologi.
NB! Visiting address: Building F, Research Corridor 2 F52, Room 2.10. Clinical Histo- and Cytopathology Laboratory.
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I highly recommend the small angle cleavage technique for III-V materials. It is a very easy technique to learn, produces superior samples to any other technique, and it is very quick, approx. 9 samples per hour. Out of that 9 samples, about 5-7 will be usable and 2-3 of those will be fantastic. Go to the South Bay Technology web page and check out their microcleave kit. There are some examples of a MQW GaAs-InGaAs structure that I prepared in their document section along with some other examples. Send me your address, and I can send you a disk with a detailed poster on how to do it. You can also go to the #4 TEM sample prep book from MRS proceedings. John McCaffrey and I have a detailed discussion on how to do it.
-Scott
Scott D. Walck, Ph.D. PPG Industries, Inc. Glass Technology Center P. O. Box 11472 (letters) Guys Run Rd. (packages) Pittsburgh, PA 15238-0472
Walck-at-PPG.com
(412) 820-8651 (office) (412) 820-8515 (fax)
-----Original Message----- } From: Bruce Brinson [mailto:brinson-at-rice.edu] Sent: Monday, November 11, 2002 5:07 PM To: MSA Listserver
Hello TEM Microscopist and sample prep masters,
I've spent a humbling amount of time trying to learn to prepare TEM cross section samples from GaAs substrates. I should also point out that I do not have mentor for this project. For the moment I am working with dummy samples. When I start working with the real material of interest (InSb AlAs quantum wells), we will typically have enough material for one attempt... so I need to get the yield (to the TEM) up to or atleast close to 1. I am able to block, polish and dimple the material easily enough but loose many samples transferring from the dimpling block (glass post) to the Cu support.
My procedure is .... then 1. dimple on a glass post. 2. I then put a piece of filter paper and stack 2 u-scope slides in a dish. I put the post, sample down, with one edge of the post on the u-scope slides allowing a space for the dimpled bit to fall off then immerse in acetone . To this point my yield is ~1
Mounting to the Cu support is where I often loose the samples due to fracturing. Use fine tipped tweezes typical of those use with 3mm TEM grids and all the caution I can muster. I lift the sample out and attempt to lay it on a glue bearing support. Some times I get there in 1 piece, sometimes I don't.
Since we need a yield of 1 for 30+ samples this is a real problem.
Any advice on the sample prep. of this material would be greatly appreciated.
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Alan,
You've made a great point that would be of considerable interest to many microscopists.
I would love to see this discussion ensue and then a final wrap-up written for publication in Microscopy Today!
Ron Anderson, Editor Microscopy Today
-----Original Message----- } From: Allan Mitchell [mailto:allan.mitchell-at-stonebow.otago.ac.nz] Sent: Sunday, November 10, 2002 2:42 PM To: Microscopy-at-sparc5.microscopy.com Cc: phillipst-at-missouri.edu
Hi Tom, and others
You have raised an interesting point that I have never really been able to get a handle on satisfactorily, that is how do you choose the appropriate acrylic resin to use for a new project,
eg,
should I choose LR White or LR Gold for project X, should I chemically polymerise or perhaps use UV ?
or maybe I should try Unicryl, or Lowicryl, but which one HM20, K4M etc.
Are there others I should be considering?
I would be interested in seeing a discussion on how different people go about choosing a particular acrylic resin for a new project they may have to undertake.
Regards
Allan
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Can anyone maybe help with this question from one of our post graduate students?
Hello,
My name is Susanne Schmidt, am an Austrian Veterinarian and I am doing my post grad studies at Oderstepoort university. I do research on the histology of the uterus of non pregnant and pregnant cows. For fixation of the uterus/placenta I want to use a perfusion method to make sure that maternal and fetal membranes stay well together. As a fixative I will use Glutaraldehyde 2,5% in 0,013M Millonig's buffer and I will perfuse the organ through the uterine arteries, leaving the veins open. At the same time I will fill the amniotic cavity with fixative to insure immersion through the fetal placental membranes. The physiological blood pressure in a cow is +/- 145 mmHg (=0,15bar). I already tried to perfuse the uterus (after perfusing with saline to get rid of blood...) , using a infusion bag filled with gluytaraldehyde hanging up at a height of 2 m above the organ. (I perfused through both arteries for about 15-20 min) I used tubes which are normaly used for a drip in horses and 18G or 20 G needles,depending on the thickness of the arteries. The result was not as I had expected and I have the feeling that the "blood"-pressure was not heigh enough . My question now is: How can I ensure that the fixative runs through the organ with a pressure of more or less 0,2 bar! (or do I need more ??)
Are there any other possibilities to make sure that maternal and fetal membranes stay together when I take my samples so that I can have a good picture of the foeto-maternal junction??
Thank you very much for you advice and help!!! Please answer to following adress: Susanne-at-op.up.ac.za
Thanks to those who responded to my posting. There were all kinds of suggestions: from vibration, field interference, faulty ground. Some are more specific e.g. chilled water pulsing, bad turbo pump, faulty digital system, electrical motor or monitor nearby.
I am glad that my colleague, Milos Kalab has found the cause of the problem. It was the multiple stage he had used being loose. It explans why the fault was not there all the time. In addition, thanks to the service rep, Frank Shapiro who found the vibration caused by pulsing chilled water at a higher mag.
Milos Kalab is very please with all the suggestions and would like to put them on his website. If any one of you do not want to be identified, please let me know. I will provide the ERL when it is ready.
AnnFook Yang EM Unit, Eastern Cereal and Oilseed Research Centre, Room 2091, Bldg. 20, Central Experimental Farm, Ottawa, Ontario Canada K1A 0C6
RMC-Boeckeler is still in business in Tucson Arizona. The 6000xl was declared obsolete by RMC-Ventana in 1999 because lack of spare parts. Limited service might be available from RMC depending what is wrong with the instrument.
Please contact: Dave Roberts or Greg Becker at 520-745-0001 Email: Dave-at-boeckeler.com or Greg-at-boeckeler.com
Best,
Al Coritz, Sales Manager Electron Microscopy Sciences & Diatome
----- Original Message ----- } From: "Sergey Ryazantsev" {sryazant-at-ucla.edu} To: {Microscopy-at-sparc5.microscopy.com} Sent: Tuesday, November 12, 2002 9:19 PM
Thanks for all the help with working with gradients.
I ended up using adobe gaussian blurr function with 2 pixel to get rid of fine data and 50 pixels to generate a background image. I then down loaded imagej (http://rsb.info.nih.gov/ij/) to do the image math. I ended up adding the inverted background to the original image and was than able to then enhance the contrast of the subtle pattern. I also cropped edges and annotation out before I did the routine. I have to say imagej is amazing.
The 36th Annual Fall Symposium of the New England Society for Microscopy (NESM) will be held on Friday, December 6th at Gordon College in Wenham, MA.
The meeting begins at 12Noon with registration in the Lane Student Center. There are 3 Sessions: Session 1 is devoted to four 15-minute student presentations. Session 2, the Biological Session will feature 2 talks on Confocal Microscopy and Session 3, the Materials Science Session will feature 2 talks, one of which is by Lucille Giannuzzi, the MAS Tour Speaker.
Following the technical talks, NESM will hold it's annual business meeting. Dinner will follow at 6:00pm and then Debora Mayer, from the Mayer Conservation Studio in Portsmouth, NH will speak on "Fiber Analysis and Art".
Details regarding registration, speakers/topics, and directions to Gordon College can be found on NESM's website: http://prism.mit.edu:8083. Further information on Gordon College (including a map of the campus) can be found on it's website: http://www.gordon.edu.
The deadline for advance registration (and dinner) for this meeting is Friday, November 29th.
} } } Hello everybody } } I need service for my RMC MT6000-XL ultramicrotome. It's located at } UCLA, Los Angeles, CA. If you could recommend real person/company I } would really appreciate. Thanks. Sergey } } _____________________________________
Sergey, RMC is now part of BoeckelerInstruments in Tucson AZ. You can contact Dave Roberts or Greg Becker at 502-745-0001. They may still be supporting the 6000 series. Lee
-- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy Core Facility Manager, Optical Microscopy Core Facility Joan & Sanford I. Weill Medical College of Cornell University voice (212)746-6146 fax (212)746-8175
I have been using a CCD which I purchased from a company called TVIPS. Their cameras are very easy to install and easy to use. You can contact Hans Tietz, the president of the company, at the address below:
hans.tietz-at-tvips.com
Regards, Thearith
------------------
Thearith Ung
Quantum Dot Corporation 26118 Research Road Hayward, CA 94545, USA Tel: 510-887-8775 (Ext 4125) Fax: 510-783-9729 Webiste: www.qdots.com
--------------------
-----Original Message----- } From: Jensen, Karen [mailto:Karen_Jensen-at-urmc.rochester.edu] Sent: Monday, November 11, 2002 1:43 PM To: 'microscopy-at-msa.microscopy.com'
Dear Listers:
We are planning to upgrade our TEM digital camera purchased 9 years ago from. We do alot of kidney biopsies as well as various research specimens. Does anyone have any experience with the higher resolution TEM digital cameras? We are looking for something around 2-4 megapixels.
Thanks
Karen
Karen L. Bentley, M.S.(previously Jensen) Associate Scientist & Project Manager Electron Microscope Research Core University of Rochester Medical Center Rochester, NY 14642 585-275-1954
I have to buy a critical point dryer for my new lab in Argentina, and thought that the EMS 850 could be a good option. I will very much appreciate any tip from anyone who knows that CPD, or another model that could recommend instead. Please reply to my personal address {ramirez-at-amnh.org} . Thanks! Martin
Martin J. Ramirez Division of Invertebrate Zoology American Museum of Natural History Central Park West at 79th Street New York 10024, N.Y. USA Tel: (212) 769 5609 Fax: (212) 769 5277 email: ramirez-at-amnh.org
} Hello everybody } } I need service for my RMC MT6000-XL ultramicrotome. It's located at UCLA, } Los Angeles, CA. If you could recommend real person/company I would really } appreciate. Thanks. Sergey } } _____________________________________ } } Sergey -
Contact RMC! dave-at-boeckeler.com
Caroline
Caroline Schooley Project MICRO Coordinator Microscopy Society of America Box 117, 45301 Caspar Point Road Caspar, CA 95420 Phone/FAX (707)964-9460 Project MICRO: http://www.msa.microscopy.com/ProjectMicro/PMHomePage.html Intertidal invertebrates: http://www.fortbragg.k12.ca.us/AG/marinelab.html
Many thanks for all help I got from this list. Thanks!!!
Actually I have been trying all different kind of resins for my immunolabeling experiments. I have same question as Allan has. Which one is right resin to use? A book I received today is very informative about choosing acrylic resins. The title is : Resin Microsocpy and On-Section Immuno-ctyochemistry, second edition, by G. R. Newman and J. A. Hobot, 2001, Springer, (ISBN 3-540-67277-X).
Shanling
-----Original Message----- } From: Allan Mitchell [SMTP:allan.mitchell-at-stonebow.otago.ac.nz] Sent: Sunday, November 10, 2002 2:52 PM To: Microscopy-at-sparc5.microscopy.com Cc: phillipst-at-missouri.edu
Hi Tom, and others
You have raised an interesting point that I have never really been able to get a handle on satisfactorily, that is how do you choose the appropriate acrylic resin to use for a new project,
eg,
should I choose LR White or LR Gold for project X, should I chemically polymerise or perhaps use UV ?
or maybe I should try Unicryl, or Lowicryl, but which one HM20, K4M etc.
Are there others I should be considering?
I would be interested in seeing a discussion on how different people go about choosing a particular acrylic resin for a new project they may have to undertake.
Regards
Allan
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
At 10:41 AM 11/13/2002 -0500, Leona Cohen-Gould wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
They do not support the MT6000 series. When we had a problem we were told it was obsolete and we would need to but a new microtome.
Alan W Nicholls, PhD Director of Research Service Facility (Electron Microscopy) Research Resources Center - East (M/C 337) Room 100 Science and Engineering South Building The University of Illinois at Chicago 845 West Taylor St Chicago, IL 60607-7058
I have to buy a critical point dryer for my new lab in Argentina, and thought that the EMS 850 could be a good option. I will very much appreciate any tip from anyone who knows that CPD, or another model that could recommend instead. Please reply to my personal address {ramirez-at-amnh.org} . Thanks! Martin
Martin J. Ramirez Division of Invertebrate Zoology American Museum of Natural History Central Park West at 79th Street New York 10024, N.Y. USA Tel: (212) 769 5609 Fax: (212) 769 5277 email: ramirez-at-amnh.org
I'm trying to work out the method for embedding glutaraldehyde fixed centrifuged urine to embed in 4% Noble Agar, such that the specimen can be blocked in Epon-Araldite for sectioning to hunt for Human Polyoma Virus infection in transplant recipients.
} From the paper that I've referred to, they fail to mention the details of blocking in the 4% Agar embedding, and I can't remember those critical temperatures to get the agar into just the right condition for embedding - ie: not too hot, and not too cold.
I've been asked to see if I can find the following for a Zeiss Axioplan fluorescent microscope ASAP. Power supply, collector lens, lamp housing and socket. Does anyone have any leads? Thanks.
Mary
Mary McKee MGH Renal Unit Bldg. 149, Rm. 8113 149 13th St. Charlestown, MA 02129
(617)726-3696 (phone) (617)726-5669 (fax)
Fees for EM Services: $20.00/hr* $30.00/hr, if you watch $40.00/hr, if you help $50.00/hr, if you tried to do it first, and couldn't
If anyone can help, please respond to Carlos Salcedo [mailto:centurion1_2000-at-yahoo.com]
Thank you
Susanne Brandom microscopy.info
-----Original Message----- } From: Carlos Salcedo [mailto:centurion1_2000-at-yahoo.com] Sent: Friday, November 08, 2002 1:26 PM To: spb-at-mwrn.com
I work at American University in the documentary film dept. We are looking ofr someone that can do video microscopy in the WDC area. We are trying to fotograph moving images of cell replication in the microscope environment for a cancer documentary.
Do you know anyone/agency/service provider that can handle this type of request?
We have a budget for this. We just can't find the service. NIH has it but they don't accept outside requests?
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Why doesn't the NIH do a CRADA (Cooperative Research and Development Agreement) with you under the Stevenson-Wydler Technology Transfer Act of 1986?
If they wanted to, it seems to me that the work could likely be fit in.
gary g.
At 05:08 PM 11/13/2002, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
We use Agarose Type 1X which sets slowly at room temperature. This gives you more time to add your cells before it sets. My protocol is given below.
Dave
Enrobing cells in Agarose
When dehydrating cells one needs to centrifuge between exchanges. Cells can be lost at each stage. A solution is to enrobe the cells in agarose and thereafter treat them like tissue samples.
Fix, rinse in buffer and osmicate cells
Rinse 3 x 1m in distilled water
Dissolve 0.3g agarose (type IX ultra-low gelling temperature, Sigma, A-5030) in 10ml distilled water. Use hotplate and magnetic stirrer
Centifuge agarose and cells
Place in fridge for 15m (agarose will gel below 15 degrees C)
Cut end off Eppendorf with razor blade
Place remaining cone upright and cut vertically
Scoop out sample with small spatula and mounted needle
Place in distilled water and start dehydration
On Wed, 13 Nov 2002 15:45:53 -0600 Garry Burgess {GBurgess-at-exchange.hsc.mb.ca} wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } I'm trying to work out the method for embedding glutaraldehyde fixed } centrifuged urine to embed in 4% Noble Agar, such that the specimen can be } blocked in Epon-Araldite for sectioning to hunt for Human Polyoma Virus } infection in transplant recipients. } } } From the paper that I've referred to, they fail to mention the details of } blocking in the 4% Agar embedding, and I can't remember those critical } temperatures to get the agar into just the right condition for embedding - } ie: not too hot, and not too cold. } } Can anyone remind me of this Agar procedure? } } Garry } }
---------------------------------------- Patton, David Email: David.Patton-at-uwe.ac.uk "University of the West of England"
Dear Sir: You are right, it is not easy to find references about the OTO method, but it works very well in transmission electronmicroscopy studies. It was a method used by people who studied intestinal absorption in fishes in the 70´s and 80´s as it was my case. I suggest that you look at the original paper of Seligman et al., 1966, Journal of Cell Biology 30:424-432. I´ve used this method in my associate professor thesis and I was succesful at first try I just followed the recipe of the paper. My objective was to demonstrate quilomicra and lipid droplets in the enterocytes of an antarctic fish. This paper was not yet published.
Prof. Dr. Francisco Javier Hernandez Blazquez Departament of Surgery - Sector of Anatomy Faculty of Veterinary Medicine and Animal Sciences University of São Paulo Av. Prof. Dr. Orlando Marques de Paiva, 87 05508-000 - São Paulo (SP) - Brazil Tel..55 (11) 3091 1374 Fax 55 (11) 3091 7805 email: fjhblazq-at-usp.br
At 11:09 12/11/02 -0600, Tom Phillips wrote:
} I have just read Willingham & Rutherford (1984) J. Histochem Cytochm 32(4):455-460 paper on the use of ferrocyanide reduced osmium with the osmium-thiocarbohydrazide-osmium (OTO) technique of Seligman. I thought the conclusion that R-OTO gave a superior preservation and contrast of membranes in TEM was a good one based on their images. But when I did a Medline search for papers using either the OTO or R-OTO technique, the limited number of papers was essentially limited to SEM work. Perhaps the method is buried in papers and not coming up in a keyword search. Or maybe no one uses it or there are problems with it?? Has any one used the R-OTO (or even OTO) technique with tissue biopsies (Willingham & Rutherford use cell cultures so it is tough to extrapolate the correct osmication times)? Are there hidden pitfalls and disadvantages to this technique? Any comments welcome. Tom } } } Thomas E. Phillips, PhD } Associate Professor of Biological Sciences } Director, Molecular Cytology Core } 3 Tucker Hall } University of Missouri } Columbia, MO 65211-7400 } } 573-882-4712 (office) } 573-882-0123 (fax) } PhillipsT-at-missouri.edu
I use the OTO method on about half of my specimens. I have only had one time that things didn't work and I got a bad precipatate on the cells. I follow the Willingham technique. The temp of the solutions does seem to be very important. If I can help please let me know.
Nancy Smythe, Research Specialist Medical University of South Carolina Charleston, SC
Colleagues: I am interesting in cameras that are compatible in the UHV. Do they exist? Most of SEMs today equipped with a camera inside of vacuum chamber, however you don't see these cameras inside of the UHV system. Any solution??? Thank you.
Joseph Fu National Institute of Standards & Technology 100 Bureau drive Stop 8212 Gaithersburg, MD. 20899-8212 Tel: 301-975-3495 Fax: 301-869-0822 Email: jofu-at-nist.gov
Hi group - anybody out there working on layers of paint samples? Looking to distinguish layers by wavelength. Apparently this party has already used SEM and optic scopes without success. Guess density is to close to distinguish layers. We have been told that a PanaCL could do the trick - would appreciate any suggestions. Thanks Barb
You can try calling the Zeiss Spare Parts Dept. in NY: 1-800 633 6610 and then the Spare Parts extension.
Good luck, Holly
Holly L. Aaron CRL Molecular Imaging Center http://imaging.berkeley.edu
} -----Original Message----- } From: Mary McKee (by way of MicroscopyListserver) } [mailto:mckee-at-helix.mgh.harvard.edu] } Sent: Wednesday, November 13, 2002 5:08 PM } To: microscopy-at-sparc5.microscopy.com } Subject: Zeiss Axioplan replacement parts } } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } Help! } } I've been asked to see if I can find the following for a Zeiss Axioplan } fluorescent microscope ASAP. } Power supply, collector lens, lamp housing and socket. Does } anyone have any } leads? Thanks. } } Mary } } Mary McKee } MGH Renal Unit } Bldg. 149, Rm. 8113 } 149 13th St. } Charlestown, MA 02129 } } (617)726-3696 (phone) } (617)726-5669 (fax) } } Fees for EM Services: $20.00/hr* } $30.00/hr, if you watch } $40.00/hr, if you help } $50.00/hr, if you tried to do } it first, and couldn't } }
Hi I am looking at developmental stages of oogenesis in sharks and rays. For part of this work I want to document stages using light microscopy, particularly looking at distribution and amounts of collagen around the oocytes. Samples are fixed in ~ 4% para/2%glut. I have tried Mallorys triple (mordant HgCl), Pollocks (HgCl) and recently Gomori triple (Bouins). I have yet to get any other colours than pinks and reds for the Mallory and Gomori, the Pollocks dissolves my sections of the slides. I have used JB4 & Technovite, sections ~ 2-3 um dried on glass slides at 70C for 20-30 minutes. Any suggestions as to why the collagen is proving so elusive? It is there I have in TEM`s.
Thanks Ian
Ian R. Davenport Ph.D. candidate Clemson University Department of Biological Sciences 132 Long Hall Clemson, SC 29634-0326 Phone no. 864-656-3598 Fax no. 864-656-0435
I have a number of references to use of the OTO method that includes TEM. The one caveat I remember (having primarily used the technique for SEM) is the quality of the thiocarbohydrazide. One paper (and it was for SEM)recommended washing the TCH to remove some impurities that could/would compromise the results. I can't find my protocols right now (I'll look again later) but here are some references that include TEM in the results.
Aoki, M. & Tavassoli, M. OTO method for preservation of actin filaments in electron microscopy. J Histochem Cytochem 29:682-3, 1981
Vriend, R.A. & Geissinger, H.D. An improved direct intermicroscopic (LM} SEM} TEM) correlative procedure for the examination of mammalian skeletal muscle. J Microscopy 120:53-64, 1980
Geissinger, H.D. et al. Osmium-thiocarbohydrazide-osmium versus tannic acid- osmium staining of skeletal muscle for scanning electron microscopy and correlative microscopy. Trans Amer Microsc Soc 102:390-8. 1983
Birn, H., Christensen, E.I., & Nielsen, S. Kinetics of endocytosis in renal proximal tubules studied with ruthenium red as membrane marker. Am J Physiol 264:F239-F250, 1993
Lemke, C. & Linss, W. The employment of the OTO-method for presentation of cytoskeletal components in enucleating erythroblasts. Acta Histochem 33, Suppl: 69-72, 1986
Miyai, K. et. al. Scanning electron microscopy of hepatic ultrastructure. Secondary, backscattered, and transmitted electron imaging. Lab Invest 35:369- 376, 1976
Plus the Willingham paper, of course.
If/when I find my protocol (haven't done this for several years now, so everything is filed real deep) and reference I will send the info.
Roger Moretz, Ph.D. Dept of Toxicology BI Pharmaceuticals -- Where the world is only slightly less weird than it actually is. } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } On-Line Help http://www.msa.microscopy.com/MicroscopyListserver/FAQ.html } -----------------------------------------------------------------------. } } } } } Dear Sir: } You are right, it is not easy to find references about the OTO method, but it } works very well in transmission electronmicroscopy studies. It was a method used } by people who studied intestinal absorption in fishes in the 70´s and 80´s as it } was my case. I suggest that you look at the original paper of Seligman et al., } 1966, Journal of Cell Biology 30:424-432. I´ve used this method in my associate } professor thesis and I was succesful at first try I just followed the recipe of } the paper. } My objective was to demonstrate quilomicra and lipid droplets in the enterocytes } of an antarctic fish. This paper was not yet published. } } } Prof. Dr. Francisco Javier Hernandez Blazquez } Departament of Surgery - Sector of Anatomy } Faculty of Veterinary Medicine and Animal Sciences } University of São Paulo } Av. Prof. Dr. Orlando Marques de Paiva, 87 } 05508-000 - São Paulo (SP) - Brazil } Tel..55 (11) 3091 1374 Fax 55 (11) 3091 7805 } email: fjhblazq-at-usp.br } } } } At 11:09 12/11/02 -0600, Tom Phillips wrote: } } } I have just read Willingham & Rutherford (1984) J. Histochem Cytochm } 32(4):455-460 paper on the use of ferrocyanide reduced osmium with the } osmium-thiocarbohydrazide-osmium (OTO) technique of Seligman. I thought the } conclusion that R-OTO gave a superior preservation and contrast of membranes in } TEM was a good one based on their images. But when I did a Medline search for } papers using either the OTO or R-OTO technique, the limited number of papers was } essentially limited to SEM work. Perhaps the method is buried in papers and not } coming up in a keyword search. Or maybe no one uses it or there are problems } with it?? Has any one used the R-OTO (or even OTO) technique with tissue } biopsies (Willingham & Rutherford use cell cultures so it is tough to } extrapolate the correct osmication times)? Are there hidden pitfalls and } disadvantages to this technique? Any comments welcome. Tom } } } } } } Thomas E. Phillips, PhD } } Associate Professor of Biological Sciences } } Director, Molecular Cytology Core } } 3 Tucker Hall } } University of Missouri } } Columbia, MO 65211-7400 } } } } 573-882-4712 (office) } } 573-882-0123 (fax) } } PhillipsT-at-missouri.edu } } }
On Friday, November 8, 2002, at 07:07 AM, by way of MicroscopyListserver wrote: }
} Name: ramki kalyanaraman } } Organization: Washington University in St. Louis } } Education: Graduate College } } Location: St. Louis, MO, USA } } Question: Is there a way to statistically justify the number of TEM } samples that must be prepared to be confident. One answer to this may } be on the basis of the feature size beining invesitgated. There are } probably two extreme cases to base the answer upon: In one case we } have numerous independent prior measurements of the sample properties } while in the other we do not. If there are any good journal paper/Book } chapter that answers this question, I would appreciate knowing about } it. } thank you
Dear Ramki, Suppose you can describe the state of your images by the values of one or more parameters, and that each parameter takes on a range of values within your set of images. For parameters that do not vary significantly from one image to the next, few images would be necessary to have confidence, but for parameters whose value differs, you would have to measure the distribution of values, requiring many images. At the very least, you would need two images to have any idea whether a parameter varied. Examples of the extremes are the number of lipid layers in, say, outer mitochondrial membrane, or the number of faces on an icosahedral virus, versus the volume of a cell or the density of receptors on its surface. In the first cases, a few images combined with prior knowledge would be enough, but in the second cases, many more measurements would be necessary (although, if ten measurements indicated that the parameters had a Gaussian distribution, you could extract mean and SD values). Similar considerations for non-quantitative parameters are less certain, but you would at least have to show that independent preparations of your sample gave the same results for the feature of interest. I seem to remember this discussion happening previously on this list, so you may wish to look through the archives. Yours, Bill Tivol EM Scientist and Manager Cryo-Electron Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
Good Afternoon, } From the volume of e-mail exchanged on the subject of polycontrast paper it seems there are many that continue to make use of their darkrooms. We have recently sold our enlarger and would also be happy to part with a Kodak Dektomatic 65 Print Processor. If you're interested we are willing to sell it for Cdn.$1000.00. Contact me off-line if you'd like more information.
Paul
Paul J. Gerroir Microscopy Materials Characterization Xerox Research Centre of Canada 2660 Speakman Drive Mississauga, Ontario L5K 2L1
1. How does glutaraldehyde fixation affect the stains you are using? Glut fixation makes many tissues "overly" eosinophilic. Can you try Bouin's or even buffered formalin? 2. Why use a trichrome if you are just looking for collagen? How about a specific collagen (only) stain (van Gieson, picro-sirius red with polarized light) to keep things simple. 3. Are you sure the stain you are using penetrates the plastic you are using? Got any paraffin-embedded specimens? 4. Is the collagen you see in the TEM 'bundled' like type I and II or more like the reticular fibers of type III?
ian davenport wrote:
} Hi } I am looking at developmental stages of oogenesis in sharks and rays. For } part of this work I want to document stages using light microscopy, } particularly looking at distribution and amounts of collagen around the } oocytes. Samples are fixed in ~ 4% para/2%glut. I have tried Mallorys } triple (mordant HgCl), Pollocks (HgCl) and recently Gomori triple (Bouins). } I have yet to get any other colours than pinks and reds for the Mallory and } Gomori, the Pollocks dissolves my sections of the slides. } I have used JB4 & Technovite, sections ~ 2-3 um dried on glass slides at } 70C for 20-30 minutes. } Any suggestions as to why the collagen is proving so elusive? It is there I } have in TEM`s. } } Thanks Ian } } Ian R. Davenport } Ph.D. candidate } Clemson University } Department of Biological Sciences } 132 Long Hall } Clemson, SC 29634-0326 } Phone no. 864-656-3598 } Fax no. 864-656-0435
Geoff -- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
Barbara Malony wrote: ========================================================== Hi group - anybody out there working on layers of paint samples? Looking to distinguish layers by wavelength. Apparently this party has already used SEM and optic scopes without success. Guess density is to close to distinguish layers. We have been told that a PanaCL could do the trick - would appreciate any suggestions. ========================================================== Generally speaking, paint layers are pigmented and each different layer has a somewhat different pigment system, so on the basis of morphology alone, different layers are easily discerned via diamond knife ultramicrotomed thin sections and TEM. While the temptation