} We are looking at large numbers of electron micrographs on which we want to } quantitate the number of gold particles. Does anyone know of any program } that can take an electron micrograph (or a scanned TIFF image of a } micrograph) and count the number of gold particles? If there is such a } program, can it be used to count gold particles of two different sizes? }
NIH Image (Mac Version) by Wayne Rasband would be IMHO ideal for you. It will count the particles in a field of view as well give you their relative sizes (areas). Documentation is excellent and also available. NIH Image is public domain and free.
You can download by FTP a copy from the following sites:
zippy.nimh.nih.gov (site maintained by NIH & best place to look for current info) www.amc.anl.gov (site maintained by Nestor for Microscopy & Microanalysis usually a version behind but accessible)
log into either specifying a user name of
anonymous
and a password of
youremailaddress-at-yourhostcomputerdomain
at either site you will have to search the directory structure to find the right place but it will be obvious.
on Zippy look under /pub/nih-image
on Nestor look under /ANLSoftwareLibrary/4-MacShareware/Imaging/NIH-Image & SpinOffs
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Would you not have to set the size of the bins to be finer than the magnitude of the noise in your signal in order to benefit from the image averaging? Our SEM images are collected at 256 gray levels and the noise is typically several gray levels in amplitude. I think you would want to do the averaging before the thresholding or segmentation or binning. Otherwise you would be "taking a vote" as to whether a given pixel should be of this level or that. Maybe that will produce the same result mathematically, but it seems counter-intuitive. You would still need to accurately set you bin boundaries (or thresholds) to properly split the gray level contrast.
Unless you are really hard pressed for disk or RAM, I would think it easier just to leave the image at full gray level resolution until the very end. I think most commercial image processors represent an image using 256 gray levels internally, even if they contain less than 256 gray levels. So you really wouldn't be helping yourself out much. You could benefit from using image compression when storing your files. But then, you would have to spend some CPU time to uncompress the files, but that should be quite fast except on the slowest processors. But it will be harder to visually judge the imporvement if you bin the images early on.
I would leave the images at 256 gray levels until the end if possible.
At 11:52 AM 7/1/96 +0200, you wrote: } a recipient wrote: } } } } What is binning? } } } I'm sorry, I might have misused a term reserved for histograms. } By binning I mean assigning an integer to a certain range of grey levels } on the micrograph. Everything from black to dark grey would be represented } as 0 in the digital image, dark grey to middle grey as 1 etc.. The finer } the binning the more numbers (bins) you need to represent all the grey } levels present on the micrograph. } } My original question was: } } } I'm looking for some theory (or experience) on the effect of binning on } } image quality. Given an image with a certain noise level, how fine does } } the binning need to be to get the full information content? } } (especially if I'm going to average the digitized images to reduce } } noise) } } Philip ---------------------------------------------------- Warren E. Straszheim 270 Metals Development, Ames Lab/ISU, Ames IA, 50011 Phone: 515-294-8187 FAX: 515-294-3091
I'm sorry, I might have misused a term reserved for histograms. By binning I mean assigning an integer to a certain range of grey levels on the micrograph. Everything from black to dark grey would be represented as 0 in the digital image, dark grey to middle grey as 1 etc.. The finer the binning the more numbers (bins) you need to represent all the grey levels present on the micrograph.
My original question was:
} I'm looking for some theory (or experience) on the effect of binning on } image quality. Given an image with a certain noise level, how fine does } the binning need to be to get the full information content? } (especially if I'm going to average the digitized images to reduce } noise)
Philip
-- Philip Koeck Karolinska Institutet Dept. of Bioscience Novum S-14157 Huddinge Sweden Tel.: +46-8-608 91 93 Fax.: +46-8-608 92 90 Email: Philip.Koeck-at-csb.ki.se
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We are looking at large numbers of electron micrographs on which we want to quantitate the number of gold particles. Does anyone know of any program that can take an electron micrograph (or a scanned TIFF image of a micrograph) and count the number of gold particles? If there is such a program, can it be used to count gold particles of two different sizes?
Thanks, Sanford Simon Sanford M. Simon Laboratory of Cellular Biophysics Box 304 Rockefeller University 1230 York Avenue New York, N.Y. 10021 212-327-8130 (voice) 212-327-8022 (fax) simon-at-rockvax.rockefeller.edu (e-mail)
I have a working Hitachi 450 available. It was under contract until last year. Must move it quickly to make way for another microscope. If you are interested, please contact me at my e mail address. I had saved it to put in one of our technical high schools which it would be great for, however I simply don't have enough of me to go around and must uncomplicate my life a bit. Thanks Judy Murphy
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
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Simon,
At 14:50 30.06.96 -0400, Sanford Simon {simon-at-rockvax.rockefeller.edu} wrote: } We are looking at large numbers of electron micrographs on which we want to } quantitate the number of gold particles. Does anyone know of any program } that can take an electron micrograph (or a scanned TIFF image of a } micrograph) and count the number of gold particles?
Under DOS/Windows, "analySIS" connects to Philips, and possibly other, TEMs and has functions for evaluating images of immunogold labelled sections. It is sold by Soft Imaging Software, Gmbh, Germany, att. Vasant Desai, Tel +49 251 798 000, Fax +49 251 798 00 99. SIS just announced a website at http://www.soft-imaging-web.de
} If there is such a } program, can it be used to count gold particles of two different sizes? }
Yes.
Best regards, Finn-Mogens.
***************************************************************** Finn-Mogens Haug University of Oslo, Inst.bas.med.sci. Department of anatomy E-mail: f.m.haug-at-basalmed.uio.no Box 1105 Blindern Phone : +47 22 85 12 67 N-0317 Oslo, NORWAY Fax : +47 22 85 12 78
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #3.0 ] --
Randy Stoter posted the following: ================================================= I think that you will find that a number of suppliers of carbon evaporation } equipment now recommend carbon 'string', in preference to carbon rods. This } 'string' is a multi filament braid and avoids precisely the type of problem you } mention. Additionally, I think you will find that using 'string' the whole } evaporation process is rather more controllable - does anyone know of any } disadvantage of carbon string (apart from requiring a different evaporator } head)? ================================================== There are really two "kinds" of carbon "fiber", one that is more of a "braid" and another that is much thinner in diameter, sometimes referred to as carbon "string" or "thead". SPI produces both products, and the "basic fiber" is the same for both products. I will refer to both as carbon "fiber" but I am speaking of both forms of the product. Individual carbon coaters are designed to take either one or the other or both. The SPI Supplies carbon coaters can use either.
It has been our own experience that under the best of circumstances, the granularity of a carbon coating deposited with carbon rods (but in a soft and not a diffusion pumped) vacuum is a bit smaller (e.g. slightly smaller grain size) than what is possible using either of the two mentioned carbon fibers. The only people who we have encountered over the years who seem to possibly find this smaller grain size to be beneficial are those looking at submicron particle size particles on membrane filters. There is a point where the grain size of the evaporated carbon starts to get confused with particles of interest collected on the filter membrane. Another advantage is that the process, at least in the SPI carbon coaters is much faster, almost as a "flash" evaporation, that is, it all happens within a time frame of a second (or less), and therefore neither the sample or the "head" itself tends to have any "heating" problems. Using the carbon rods, however, it is a slower kind of process, exposing the sample to much more radiant heat as evidenced by the much higher temperatures taken on by the head. And because one has to wait for the carbon rod head and posts to "cool down" where as the carbon fiber head never really does get that hot, the "throughput" of samples when carbon fiber coating is employed, tends to be much faster.
The degree of control of what we call the "flash evaporation", and the ability to reproduce coating thicknesses, sample to sample, is related to the homogeneity along the carbon fiber, probably the most difficult parameter to control in the manufacture of these two particular products. However, the control is more than adequate, at least with our particular products, that coating reproducibility does not seem to be an issue.
There is another (apparent only) disadvantage and that is the difficulty imparting ultra high purity to the carbon fiber. With the carbon rods, the standard purity (from SPI at least) is 5 ppm ash. However, we have never been able to produce carbon fiber or braid down to that level and have it still retain its desirable mechanical properties. So the purity of the fiber tends to be on the order of 10 ppm ash, perhaps even as high as 12-13 ppm ash. However this is still well below the level that the typical EDS user will detect any of the impurities. So in the end, although the best purity of the carbon fiber tends to not be quite as good as the best purity carbon rods, a "reality check" would suggest that in the end it still would not matter to most persons. And forgive me if this now sounds like a commerical statement, but all carbon fiber does not come from the same place!
Further information about the two different SPI carbon fiber products can be found in the SPI "electronic catalog" at our web site given below. Look up in the catalog table of contents and then under "sample preparation" and then under "evaporation supplies".
Chuck
====================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: GVKM07A-at-prodigy.com West Chester, PA 19381-0656 USA Customer Service: spi2spi-at-2spi.com
Take a look! ########################## WWW: http://www.2spi.com ########################## ======================================================
} } } Another question. What will Fuji price policy be in the future. At the } } } moment their plates seem to be rather expensive, so that if we want to } } } equip 5 microscopes with this system we might think twice... } } } } } Since the plates are reusable, it is only a one-time expense. } } this is not perfectly true. Fuji claims their plates are reusable 500 } times, so you may have to buy new ones every few years. It comes to be } a bit less expensive than photo plates, but you need to add the reader's } price. Therefore it is more expensive actually.
Can anyone with experience comfirm that the plates are reusable 500 times? Is this also true if one wishes to quantitate the response-- such as for ED intensity measurements? } } } } Yet another one. I would be happy to be able to get the plate out of the } } } microscope just after taking the picture. } } } I elaborate: with photographic plates I understand that we have to work in } } } batches because plates are light sensitive. } } } } IP's are also light-sensitive--you should not expose them to fluor- } } escent light when taking them to the reader. } } One of the argument of Fuji is precisely that dark room is not necessary } any more. So here is something that I do not understand clearly. On the } other hand I repeat that I have never seen any number printed on any IP, } therefore it must be quite less sensitive than photo plates. } They are correct that a darkroom is not necessary, but it is best to transport the IP to the reader with minimal exposure to light--espe- cially the shorter wavelengths. I have to agree with you that the sen- sitivity of IP's to light is less than photo's--I think I equated the erasability of IP's by UV to sensitivity. This can be used to number the IP's by exposing a corner, which had been previously "darkened", to a number mask. The numbers would show up as "undark".
} } } As a consequence one has to } } } wait till the plate is processed in order to know if it is good or not. } } } For this reason camera makers do have a great argument telling that the } } } image can be computer stored instantaneously. } } } } } } If you can get the IP out of the microscope and have it processed within } } } minutes this would be a great improvement. It "just" takes a modification } } } of the photo box, and I imagine that a good engineer could do that. By } } } solving these two problems I believe that Fuji would clear any arguments } } } against their system. } } } } } This is as easy as removing a single piece of film--no problem on } } our scope. The readers I've seen take on the order of a few minutes to } } process an IP. } } Right. But if you make high resolution images, you may want to know } quickly if the photo was successful or not (focus, drift). Therefore being } able to see the result just after taking the picture can be a real } improvement versus normal photo plates. On a Philips microscope, removing } one plate is the same as removing 36: you HAVE to switch off the HT. this } means that when working in high resolution it is not so easy. The system } I imagine would allow you to get the plate out the microscope within } seconds, without generating any perturbation in the vacuum system. } I see your problem. All we have to do is air the camera. On a good day, when the camera window is well-sealed, we could even leave the beam on for this--except, of course, the camera door interlock won't allow it. If we were foolish enough to defeat the interlock, we could observe the image on our video system while the IP was removed and read. In any case, we can turn the beam off, remove the IP, then turn the beam on again, and the image should not have changed. If the specimen were not suseptible to drift or radiation damage, we would be able to procede as you want to. Do you take a through-focus series when you want hi-res? Yours, Bill Tivol
For the many of you that have asked about a PC Version of NIH Image. It has been ported to the PC by the Scion Corporation. You may download the alpha version 5 copy from the general NIH Image Download WWW page. The URL is:
http://rsb.info.nih.gov/nih-image/download.html
Sorry, but I've not used this version so I have no comments on how well it works, but the people at Scion have good things in the past to upgrade their hardware (frame grabbers for the Mac) and modify the software to be current with NIH Image. So I would expect this to be similiar in quality.
The NIH Image Home Page is:
http://128.231.98.16/nih-image/
Just to keep on the up and up you should all know that I have no financial interests in Scion Corp. and just happen to be a satisifed user of their Mac Frame Grabber Hardware.
I'll upload a copy of the PC software to the ANL Microscopy & Microanalysis Library in the next week or so. It will be there before the MSA/MAS/MSC-SMC meeting in Minneapolis (August 11-15th).
} Randy Nessler wrote: } } ... } While in the tour host mode, I was } explaining thermionic emission at the TEM. One student asked the question, } what } happens to the tungsten nucleus after the electrons are freed and removed? } ...
Put in simple words, I believe the answer is like this:
The Tungsten atoms are part of a "metallic" crystal, which implies that part of the electrons are moving freely throughout the crystal. One could envisage this as the nuclei remaining at a fixed position while being surrounded by a "cloud" of electrons. The electrons escaping from the tip and as such being removed from the crystal, are simply replaced by other ones from from the power supply. So what happens to the nuclei: nothing.
Hope this frees the mind of your students, if so maybe you can trouble them by telling them that electrons can also be seen as electromagnetic waves instead of particles ...
The technique of fiber evaporation as well as a comparison of granularity of rod versus fiber depositions can be found in:
D. Vesely and S. Woodisse (1982). Carbon coating with carbon fiber filaments, Proc. RMS 17 (3):137-139.
"Precise and reproducible deposition of thin and ultrathin carbon films by flash evaporation of carbon yarn in high vacuum, J. Microsc., 133: 17-25 (1984),
For granularity of carbon films, please refer to H. K. Koelbel (1976). Carbon supporting films for high resolution electron microscopy- improvement of quality and preparation technique. Mikroskopie 31,1.
In summary: Granularity depends in high-vacuum deposition on the predegasing conditions. If you predegase rods and yarn only shortly to dark red (to remove organics), you do not loose internally adsorbed gas molecules. These gas molecules are radicalized when evaporated with the carbon and will adsorb in flight to the carbon atoms preventing their crystallization. Therefore, yarn can produce as fine structured films as rods that are used only once. However, if predegased carefully, yarn will more consistently produce very fine structured films since the amount of deposited carbon can very accurately be controlled by the lenght the amount of yarn and the distance to the object.
Best regards Klaus
********************************** } Randy Stoter posted the following: } ================================================= } I think that you will find that a number of suppliers of carbon evaporation } } equipment now recommend carbon 'string', in preference to carbon rods. This } } 'string' is a multi filament braid and avoids precisely the type of problem } you } } mention. Additionally, I think you will find that using 'string' the whole } } evaporation process is rather more controllable - does anyone know of any } } disadvantage of carbon string (apart from requiring a different evaporator } } head)? } ==================================================
Charles A. Garber, Ph. D. posted
} It has been our own experience that under the best of circumstances, the } granularity of a carbon coating deposited with carbon rods (but in a soft and } not a diffusion pumped) vacuum is a bit smaller (e.g. slightly smaller grain } size) than what is possible using either of the two mentioned carbon fibers.
} The degree of control of what we call the "flash evaporation", and the ability } to reproduce coating thicknesses, ==============================
****************************************************************************** * Klaus-Ruediger Peters, Ph.D. :Molecular Imaging Laboratory * * Director, Molecular Imaging Laboratgory : http://panda.uchc.edu/ * * Biomolecular Structure Analysis Center : htklaus/index.html * * University of Connecticut Health Center : * * 263 Farmington Ave. :F r e e Access to Differential * * Farmington, CT 06030-2017; U.S.A :Contrast Software at * * e-Mail: Peters-at-BSAC.UCHC.EDU : http://panda.uchc.edu/ * * Tel: (860) 679-3977; Fax: (860) 679-1989 : htklaus/DHP-Img.html#Software* ******************************************************************************
Has anybody experience with Model 2000 Specimen Prep System by E.A. Fischione ?
I need to prepare TEM foils from hard and brittle materials, Ti-V nitrides and carbides at the moment. I have been looking for the best spherical grinder for TEM discs preparation prior to ion milling and I have got offer on Model 2000 Specimen Prep System by E.A. Fischione. Description of it sounds great. It looks like it do the same as spherical grinder does and much more.
Thanks Milan ------------------------------------------- Milan Svoboda Institute of Physics of Materials Zizkova 22, 616 62 Brno, Czech Republic e-mail: svobm-at-ipm.cz -------------------------------------------
Someone once wrote about putting molecular sieves into dialysis tubing to keep the "dust" down. This sounds like a great idea so I decided to try it. My question, however, is: how the heck do you get the tubing open in a non-aqueous environment. I've been soaking a piece of tubing in abs. EtOH for an hour now and it just doesn't want to open up. Any clues would be greatly appreciated.
TIA
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Biomedical Research | | 222 Maple Avenue | | Shrewsbury, MA 01545 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfbr.edu | | | |_________________________________________________|
Is there a method to demonstrate lipid in botanical tissue fixed in formaldehyde, dehydrated to 70% ETOH, embedded in LR White and cold cured. "TIA"
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
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Hello,
Thanks to all who replied to my request for assistance for my dead TN5500.
After many phone calls from Nick Dmytryshyn from Canberra Packard, we managed to narrow down the problem to a dead todd power supply and a loose board. The power supply was a surprise since we had replaced the power supply a year and a half ago. We will also be replacing the computer fans to increase the cooling in the system, which is most likely the reason why our power supply died so quickly.
Tania Jones Laboratory Manager DynaMotive Technologies
} Hello folks, } } Someone once wrote about putting molecular sieves into dialysis tubing to keep } the "dust" down. This sounds like a great idea so I decided to try it. My } question, however, is: how the heck do you get the tubing open in a non-aqueous } environment. I've been soaking a piece of tubing in abs. EtOH for an hour now } and it just doesn't want to open up. Any clues would be greatly appreciated. } } TIA } } John } } ___________________________________________________ } | | } | John G. Aghajanian, Ph.D. | } | Worcester Foundation for Biomedical Research | } | 222 Maple Avenue | } | Shrewsbury, MA 01545 | } | | } | Tel: 508 842-8921 ext. 147, 161 | } | Fax: 598 842-9632 | } | JOHNA-at-sci.wfbr.edu | } | | } |_________________________________________________| } } ******************************************************* Greg Erdos Phone: 352-392-1295 Scientific Director, ICBR Electron Microscopy Core Lab 218 Carr Hall Fax: 352-846-0251 University of Florida E-mail: gwe-at-biotech.ufl.edu Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/
A regular full time position is open in The Jackson Laboratory Biological Imaging Department - Histology Laboratory. The Jackson Laboratory is a non-profit independent laboratory founded in 1929 on the premise that the causes of cancer and other diseases could be discovered through Mammalian genetic research. The Laboratory specializes in mammalian genetics using inbred laboratory mice as model systems to study health problems such as cancer diabetes, anemia, heart disease and aging. Located on a large island in the gulf of Maine and surrounded by Acadia National Park, The Jackson Laboratory is currently undergoing a major expansion of its scientific staff and its research facilities.
There is a regular, full-time position available in the Biological Imaging Department for the Histology Laboratory. The position includes histological techniques such as paraffin embedding, single and serial sectioning and cryotomy in conjunction with immunohistochemistry techniques, straining using heavy metals, as well as other special stains. Applicants must posses a Bachelors degree or equivalent, with experience in the above techniques. Two years related laboratory experience working with Murine specimens is preferred. The successful candidate must be a self starter, pay attention to detail and be able to work independently with little supervision. This individual will be responsible for providing services in support of numerous diverse research projects, must interact well with multiple users and work productively in a team environment. Position will be filled at Biomedical Technologist or Senior Biomedical Technologist level depending on background and experience of successful applicant.
Salary range is mid to high $20,000 plus benefits and is negotiable depending on level of experience.
Interested applicants should send CV to:
Joanne Bradt Employment Specialist The Jackson Laboratory 600 Main Street Bar Harbor Maine 04609 (207) 288-3371 ext. 1281 (207) 288-3371 ext. 1082 FAX jcb-at-aretha.jax.org
} Is there a method to demonstrate lipid in botanical tissue fixed in } formaldehyde, dehydrated to 70% ETOH, embedded in LR White and cold } cured. } "TIA" } } *************************************** } David Garrett "DGARRETT-at-GAB.UNT.EDU" } University of North Texas } Dept. Biological Sciences } (817)565-3964 Fax (817)565-4136 } *************************************** } At the LM level there is as long as there is lipid remaining in the tissue. You can demonstrate it using several lipid dyes. Nile Red is my dye of choice at the moment. It penetrates into all of the tissue but only flouresces in a neutral environment (i.e. neutral lipids). Stain sections for 1-5 minutes and then view using a flourescein filter pack on a flourescent microscope.
If the lipid has been dissolved out then of course there is no way to detect it. One method for maintaining lipid in tissue is to complex it so it is not removed. We have found the OTAP method particularly good for this. Check out papers by John Guyton and Keith Klemp for details.
I hope this is useful.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-bgsm.edu * **************************************************************
I would like to have information about software imaging analysis, concerning asbestos applications. I work with a Philips system. Dr Luigi Gobbi Istituto di Scienze Fisiche FAX (39)-71-2204605 e-mail fismed-at-anvax2.unian.it
VG Microtech, as a manufacturer of carbon evaporation equipment does "recommend carbon 'string', in preference to carbon rods," but only for reasons of ease of use.
Actually, carbon rod evaporation should be more controllable. You try to gauge the thickness of carbon laid down by string, but with the carbon rod method it is possible to control the length of burn and, therefore, the deposition.
The main reason we have come across for the rods breaking is when the rod has been formed by sharpening, which introduces micro cracks. The carbon rods should be ground to shape.
As Chuck Garber mentioned, "under the best of circumstances, the granularity of a carbon coating deposited with carbon rods (but in a soft and not a diffusion pumped) vacuum is a bit smaller (e.g. slightly smaller grain size) than what is possible using either of the two mentioned carbon fibers." We believe this is due to the massive deterioration in vacuum when the fibre is flashed. The process time with rods should only be 1-2 seconds longer (at least in the Polaron carbon coaters). If you push enough power through the carbon rod, it will also flash instantly as the fibre appears to do.
Chuck continues, "Using the carbon rods, however, it is a slower kind of process, exposing the sample to much more radiant heat as evidenced by the much higher temperatures taken on by the head." This is a debatable point, as the heating is by transmission through the metal. To evaporate, the carbon must reach, I think, about 2700 degrees C. Mass for mass, the same amount of energy in watts will be used. The Polaron systems have a shutter to protect the sample during outgassing. They also have switchable power supplies for the use of any fibre or rod.
As for the issue of relative purity, there are often higher peaks of sulpher in the fibre (no statement toward any individual commercial product is being made here).
Also, note that the fibre can fall as spindle-like shards onto the sample surface if handling is rough.
} Regards, } } Tony King } Product specialist } VG Microtech/ Polaron range } } Tel: +44 (0)1825 746251 } Fax: +44 (0)1825 768343 } } Disclaimer: } The views and opinions expressed are not necessarily } those of VG Microtech. }
Further information about the Polaron carbon coaters can be found at the Energy Beam Sciences web site (http://www.ebsciences.com/). Steven Slap
******************************** Energy Beam Sciences, Inc. Adding Brilliance To Your Vision ebs-at-ebsciences.com http://www.ebsciences.com/ ********************************
While in no way wishing to offend ANY of the vendors on the listserver I have had a few inquiries from my users into EdgeCraft Diamond knives. I have no experience with EdgeCraft, and I am unable to provide any answer to my users (I have used many other manufacturers knives and as with everyone else in ultramicrotomy I have my personal favorites without any hard evidence supporting my choice).
Does anyone out there have any experience and comments for or against EdgeCraft knives (my interested users will doing roomtemp, ultrathinsectioning of biological material)? Please e-mail me directly DO NOT respond to the listserver, as I do not think it would be fair to EdgeCraft or any other manufacturer to do so.
[ I have no personal or fianicial ties with ANY diamond knife manufacturers or vendors]
Thank you.
Richard E. Edelmann Electron Microscopy Facility Supervisor 352 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513-529-5712 Fax: 513-529-4243 E-mail: edelmare-at-muohio.edu
I would appreciate any ideas about foundations or other sources we might approach to obtain matching funds for a multi-user SEM proposal -specifically an environmental SEM- that will be submitted to NSF. The research that will be done using this machine is wide-ranging, including marine science, earth science and material science problems. We are aware of Keck, and that is a possibility, but there are other proposals in line for Keck at our university, so we can't count on that. I would be very grateful for any suggestions! Thanks alot Pam Reid
__________ Dr. Pamela Reid Research Associate Professor University of Miami/RSMAS-MGG 4600 Rickenbacker Causeway Miami, Fl 33149
Has any one heard of a Krumdieck tissue slicer. We have found a reference to it but can find no info on it. Any help would be greatly appreciated. Thanks
Mark Elliott, PhD UBC-Pulmonary Research Lab Vancouver BC
I am about to begin a study of whether certain intercellular spaces are continuous with other spaces in crabs. All of my references suggest using horseraddish peroxidase with DAB and H202, but they are 20+ years old. Are there any more recent modifications of this technique? How does this compare to Lanthanum or Ruthenium? Has anyone tried an organism with hemocyannin? I would greatly appreciate any advice or updates.
Thank you.
______________________________________________________________________ Donald L. Lovett e-mail: lovett-at-trenton.edu Assoc. Professor, Dept. of Biology voice: (609) 771-2876 Trenton State College, NJ 08650-4700 fax: (609) 771-2674
Information, both positive and negative on diamond knives and other products, is helpful to users. The most reliable information about a product is from someone who has used it in the field. I, for one, would welcome comments on products I may have occasion to purchase in the future. I have no experience with EdgeCraft.
On Wed, 3 Jul 1996, Richard E. Edelmann wrote:
} Date: Wed, 3 Jul 1996 09:07:24 -500 } From: Richard E. Edelmann {edelmare-at-CASMAIL.MUOHIO.EDU} } To: microscopy-at-Sparc5.Microscopy.Com } Subject: EdgeCraft Diamond Knives } } While in no way wishing to offend ANY of the vendors on the } listserver I have had a few inquiries from my users into EdgeCraft } Diamond knives. I have no experience with EdgeCraft, and I am } unable to provide any answer to my users (I have used many other } manufacturers knives and as with everyone else in ultramicrotomy I } have my personal favorites without any hard evidence supporting my } choice). } } Does anyone out there have any experience and comments for or } against EdgeCraft knives (my interested users will doing roomtemp, } ultrathinsectioning of biological material)? Please e-mail me } directly DO NOT respond to the listserver, as I do not think it would } be fair to EdgeCraft or any other manufacturer to do so. } } } [ I have no personal or fianicial ties with ANY diamond knife } manufacturers or vendors] } } Thank you. } } } } } Richard E. Edelmann } Electron Microscopy Facility Supervisor } 352 Pearson Hall } Miami University, Oxford, OH 45056 } Ph: 513-529-5712 Fax: 513-529-4243 } E-mail: edelmare-at-muohio.edu }
Sara E. Miller, Ph. D. P. O. Box 3020 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-8735
} Is there a method to demonstrate lipid in botanical tissue fixed in } formaldehyde, dehydrated to 70% ETOH, embedded in LR White and cold } cured. ******************* Every EM user knows that high atomic number atoms can be used to increase contrast and therefore "stain". Lower atomic number atoms when packed into large molecules are also denser and can be used as "stains" in EM. That is true for quite a number of stains, including the common lipid stain Sudan Black B. This particular stain will in fact show as completely opaque in TEM.
Also the opposite, the loss of lipids can be demonstrated by using a lipase to digest these. Use gold grids if incubating the sections on the grid.
The Product Engineering, Physical Failure Analysis Laboratory of Siemens = Microelectronics Ltd, North Tyneside, UK invites applicants for a TEM = Specialist position currently available. Ideally candidates must have a = PhD degree, Physics or Materials Science and 5-10 years experience in = the field of TEM. Knowledge and experience in Si semiconductors and Si = IC Processing would be preferred but not essential. The candidate should = be a good team worker with excellent problem solving skills and able to = work in a high pressure manufacturing environment. The candidate should = have the legal right to work in the UK.
The successful applicant will form part of an analytical problem solving = team that will support the manufacturing of 200mm processed Si Wafers = for the Microelectronics industry through materials and process = analysis. The new wafer fab is a 1.1bn pound investment by Siemens and = will fabricate 16MB DRAM's on 200mm Si Wafers initially, moving on to = newer generations of DRAM and Logic Products utilising 0.25-0.35um = technology.
Interested parties please email your CV to me at=20
alan.leslie-at-nts.hl.siemens.de
or mail or fax them to=20
Siemens Microelectronics Ltd 3 Kingfisher Way Silverlink Business Park Silverlink Wallsend Tyne and Wear NE28 9ND UK
Tel : (44)-191-295-0300 Fax : (44)-191-295-0400
Alan Leslie PE PFA Section Head Siemens Microelectronics Ltd
The Product Engineering, Physical Failure Analysis Laboratory of Siemens = Microelectronics Ltd, North Tyneside, UK invites applicants for an Auger = Analyst position currently available. Ideally candidates must have an = MSc or PhD degree, Physics or Materials Science and 1-5 years experience = in the field of Auger. Experience in other areas of Surface Science and = Analytical Techniques is desired. Knowledge and experience in Si = semiconductors and Si IC Processing would be preferred but not = essential. The candidate should be a good team worker with excellent = problem solving skills and able to work in a high pressure manufacturing = environment. The candidate should have the legal right to work in the = UK.
The successful applicant will form part of an analytical problem solving = team that will support the manufacturing of 200mm processed Si Wafers = for the Microelectronics industry through materials and process = analysis. The new wafer fab is a 1.1bn pound investment by Siemens and = will fabricate 16MB DRAM's on 200mm Si Wafers initially, moving on to = newer generations of DRAM and Logic Products utilising 0.25-0.35um = technology.
Interested parties please email your CV to me at=20
alan.leslie-at-nts.hl.siemens.de
or mail or fax them to=20
Siemens Microelectronics Ltd 3 Kingfisher Way Silverlink Business Park Silverlink Wallsend Tyne and Wear NE28 9ND UK
Tel : (44)-191-295-0300 Fax : (44)-191-295-0400
Alan Leslie PE PFA Section Head Siemens Microelectronics Ltd
Ian Harrowfield - CSIRO _--_|\ Ian.Harrowfield-at-minerals.csiro.au Division of Minerals / \ tel +61 3 9647 0295 PO Box 124, Port Melbourne \_.--._/ fax +61 3 9646 3223 3207 AUSTRALIA
Ian Harrowfield - CSIRO _--_|\ Ian.Harrowfield-at-minerals.csiro.au Division of Minerals / \ tel +61 3 9647 0295 PO Box 124, Port Melbourne \_.--._/ fax +61 3 9646 3223 3207 AUSTRALIA
It seem the debate centers around the assumptions of resolution and coating flat polished samples.
We still have a need to carbon coat topographic samples for EDS. In which case resolution does not enter the image when one is using barge pole probe diameters to get enough signal. It is difficult enough having to view samples with the relatively poor carbon coating and this is made worse if there is high topography. Directional coating results in a preferential coating of the surfaces facing the carbon source. The solution is to have a rotating sample holder that effectively presents all aspects of the sample to the source at some point in time. This thus requires the carbon source to evaporate over a reasonably long period of time and can not be achieved by "flash" evaporation. So rods remain the best.
I agree with Tony King that rods are more controllable, and, can suit more applications - from making very thin carbon films to topographic coating. If one is only coating flat polished samples for microanalysis and wish to reduce operator and other variables then carbon string may the better method for standardization.
Regards
Dane Gerneke E M Unit (Southern most EMU in Africa) University of Cape Town Tel + 27 21 650 2819 Fax + 27 21 6891528 e-mail IN%"dane-at-uctvms.uct.ac.za"
I am seeking assistance from those who have interfaced analog SEMs to IBM PCs for the purpose of digital image acquisition for under $5,000.
My instrument is an ARL SEMQ electron microprobe. It has easily accessed blanking signals as well as external X and Y scan drive inputs.
Numerous manufacturers offer suitable hardware solutions, but the major obstacle to my implementation is development of a Windows 95 software interface. I need 3 drop down menus: resolution, scan speed and file save.
I am not a programmer. Has anyone developed code for the interface to a particular board? Would someone with prior expertise be willing to consult for a fee for me?
Thank you very much.
Bart Cannon Cannon Microprobe 1041 NE 100th Street Seattle, WA 98125
I have a rough rock (Silicate) which I wish to polish and put in the SEM. Is there a method for polishing, either mechanical or chemical? Is there a lab which does this type of work routinely to which I might send my sample for preparation? I would appreciate hearing any of your ideas.
Papers from the 22nd Annual Meeting of the Microscopical Society of Canada (Ottawa, 1995) are published in Volume 26, Issue #6 of MICRON. This includes review papers on structure determination of proteins (D. L. Dorset) and on low-energy point-source microscopy (H.J. Kreuzer) + 10 research papers.
The 24th Annual Meeting of MSC will be held in Edmonton, 4 - 7 June 1997, and will include symposia on developments in SEM, SPM, confocal microscopy, energy-filtered TEM and microscopy of dynamic processes. For further information, see web page http://www.ualberta.ca/~mmid.mschome.html or contact me at the address below.
Ray Egerton, Physics Department, University of Alberta, Canada T6G 2J1 Phone: 403-492-5095, FAX: 403-492-0714, e-mail: egerton-at-phys.ualberta.ca ------------------------------------------------------------------------
Hello to Everyone: I am in need of an oil difusion pump. The "tree" in the older one was destroyed, but the "housing" remains in good condition. It is highly unlikely that only a "tree" will be found, even though that is all I need. Therefore I am looking for the complete unit. It goes on a Hitachi HUS-4 vacuum evaporator. The pump is model #DPF 25- 7-37 Chome Minami Synamachi - Koto-ku Tokoyo, Japan.
Some specs on the machine include: (1) from bottom of unit to top of flange 12-1/4" high (2) flange is 1-1/4" thick (probably not very important) (3) inside diameter is 2-1/2"
Writings on side of the pump: (1) Final vol. = 3 x 10-7 mm hg (2) Speed, pumping = 15 l/sec (3) Power = 350 watt (4) Oil change = 60 cc
If anyone out there knows where I might be able to find one of these, your response will be greatly appreciated. It is also possible that I may be able to substitute another pump of similar size and capacity. ******************* George Smith, Ph.D. Union College Schenectady, NY 12302 smithg-at-gar.union.edu (518)374-4907 ******************
George W. Smith, Ph.D. Dept. of Biology Union College Schenectady, NY 12308
I would like to know if anyone out there have experience from video time lapse tape recorders, especially in conjunction w video microscopy. I4m interested in buying such a system but don't know all alternatives and their pros and cons. All information of interest.
Thanks in advance
============================================= Mikael Gustafsson MD, PhD Dept Med. Microbiology and Dept Internal Medicine, Cardiology section University Hospital of Linkoping S 581 85 LINKOPING SWEDEN
} I am about to begin a study of whether certain intercellular spaces are } continuous with other spaces in crabs. All of my references suggest } using horseraddish peroxidase with DAB and H202, but they are 20+ years } old. Are there any more recent modifications of this technique? How } does this compare to Lanthanum or Ruthenium? Has anyone tried an } organism with hemocyannin? I would greatly appreciate any advice or updates. } } Thank you. } } ______________________________________________________________________ } Donald L. Lovett e-mail: lovett-at-trenton.edu
LaNO3 worked in tracing intercellular spaces in amphipods, but also appeared to penetrate into the (sensory receptor) cell, near the ciliary roots. The reference is buried in boxes, but it was VJ Steele, Journal of Morphology, on the anatomy of the Organ of Bellonci in _Gammarus_ (I forget the exact title), year was around 1985, definitely between '84-'87. Sorry I can't do better on the reference. Phil
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Philip Oshel University of Illinois Rm 74 Bevier Hall 905 S. Goodwin Ave. Urbana, IL 61801 (217) 244-3145 oshel-at-ux1.cso.uiuc.edu
} Directional coating results in a preferential } coating of the surfaces facing the carbon source. The solution is to have a } rotating sample holder that effectively presents all aspects of the sample } to the source at some point in time. This thus requires the carbon source to } evaporate over a reasonably long period of time and can not be achieved by } "flash" evaporation. So rods remain the best. } } Dane Gerneke
I disagree that carbon string can only be flash-evaporated. We use carbon cord (braided string, fairly thick), and this glows for long enough to evenly cover a rotating sample of complex topography. The only trick is to have to sample rotating before you start applying current to the cord, and to apply the current moderately slowly ("slow, but a little faster than not-too-slow", the usual sort of quantitative method). This lets the cord glow white-hot for some seconds before burning through, long enough that the current can be turned down before burn-throug. The coating thickness can be controlled this way. We haven't used carbon rods for some while now, and our users seem pleased with their results Phil
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Philip Oshel University of Illinois Rm 74 Bevier Hall 905 S. Goodwin Ave. Urbana, IL 61801 (217) 244-3145 oshel-at-ux1.cso.uiuc.edu
} I have a rough rock (Silicate) which I wish to polish and put in the SEM. } Is there a method for polishing, either mechanical or chemical? } Is there a lab which does this type of work routinely to which I might } send my sample for preparation? } I would appreciate hearing any of your ideas. } } Elaine Mohrbach } Cell Imaging } Univ. of VT }
There are two approaches, depending on who you know: 1) Go over to the geology department, they ought to have rock saws, flat laps, and everything else you need. 2) Try an amateur rockhound or lapidary--they will also have the equipment needed. There is probably a lapidary club in town. Either way, whoever does the sawing and grinding will need to end up with an extra-fine (or finer) grit diamond lap, *probably*. With some minerals, other saws/laps are better. The soft minerals give the most trouble, but in Vermont (if I read my screen font right), you should be dealing with hard minerals. Geologists also usually embed rock samples in epoxy for the final polish, and to hold the specimen in the scope, but if you don't mind smearing the sides with silver or carbon paint/paste, an vise-type holder for irregular specimens works fine. Phil
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Philip Oshel University of Illinois Rm 74 Bevier Hall 905 S. Goodwin Ave. Urbana, IL 61801 (217) 244-3145 oshel-at-ux1.cso.uiuc.edu
To: Microscopy ListServer Subscribers and All Society Members of MSA, MAS, MSC/SMC
The Microscopy & Microanalysis 96 Program Booklet will be mailed to all individuals registered for the meeting about July 15th, 1996. This booklet will contain the final program information about the meeting, dates and times of all sessions and other related information.
For those of you who wish on-line assess the entire program database (Authors, Paper Titles, Session Dates, Times and Rooms) is now also a searchable index on the MSA WWW site at the URL:
http://WWW.MSA.Microscopy.Com
Look for the Hot Link called "M&M96 Program Search Engine"
You may search the M&M 96 Program by various keywords, Day of the Week or Authors name and the relevant portion of the database will be delivered to your screen in a concise tabular format (requires NetScape V 2.0 or Tables compatible browser).
As this mailing reaches more than just the Microscopy Listserver database, those receipents who wish to subscribe to the Microscopy Listserver in order to be kept abreast of developments in the program the subscription information is given below.
Please feel free to forward this information to others whom you think may also be interested.
Cheers.... Nestor Your Friendly Neighborhood SysOp ----------------------------------------------------------
******************************** * FAQ's File * (Frequently Asked Questions) *Microscopy/Microanalysis Listserver: ******************************** * Last Updated June 28, 1996 ********************************
What is the purpose of the Microscopy Listserver? ---------------------------------------------
The purpose of this system is to give the scientific community a centralized Internet (Email) address to which questions/comments/answers in the various fields of Microscopy & Microanalysis can be rapidly distributed to a list of interested individuals and organizations.
The Microscopy Listserver is sponsored and hosted by:
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as a free service to Microscopists WorldWide.
What type of Microscopy/Microanalysis does this List address? -------------------------------------------------------------
For the purposes of this discussion forum, Microscopy & Microanalysis should be considered to include all techniques which employ a probe such as: photons (including x-rays), electrons, ions, mechanical and/or electromagnetic radiation to form a representation or characterization of the morphology, crystallography, elemental, chemical or electronic structure of any material in either physical and/or life sciences applications.
Some of the more common techniques which are associated with these fields include the following: optical microscopy, x-ray microscopy, scanning electron microscopy, transmission electron microscopy, atomic force microscopy, scanning tunneling microscopy, scanning ion microscopy, analytical electron microscopy, electron microprobe, x-ray energy dispersive spectroscopy, electron energy loss spectroscopy, electron diffraction, convergent beam diffraction, high resolution electron microscopy, high voltage electron microscopy, etc. (the list continues...ad infinitum.. )
How do I subscribe? -------------------
Send an Email Message to "Listserver-at-MSA.Microscopy.Com" in the body of your message include the lines
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Optional: Real Name Optional: Real (Surface Mail) Address Optional: Microscopy/Microanalysis Interests
Replace the "USERID-at-EMailAddress" with your Email address. You may also append your Real Name, and Address etc.. to the end of the message as a courtesy. I'd be interested to know who you are and where you're from, and what you do.
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I need to buy some mouse or rabbit antibodies against bovine lymphocytes (CD3, CD4, etc.)for immunohistochemistry purposes. We have just received the money to buy it by direct importation. We will be very grateful for any information. ============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-spider.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| r. 278 Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 618606 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
You may use horse spleen ferritin. It may be observer with LM (revealed by Perls reaction) and with EM.
On Wed, 3 Jul 1996, Donald Lovett wrote:
} } I am about to begin a study of whether certain intercellular spaces are } continuous with other spaces in crabs. All of my references suggest } using horseraddish peroxidase with DAB and H202, but they are 20+ years } old. Are there any more recent modifications of this technique? How } does this compare to Lanthanum or Ruthenium? Has anyone tried an } organism with hemocyannin? I would greatly appreciate any advice or updates. } } Thank you. } } ______________________________________________________________________ } Donald L. Lovett e-mail: lovett-at-trenton.edu } Assoc. Professor, Dept. of Biology voice: (609) 771-2876 } Trenton State College, NJ 08650-4700 fax: (609) 771-2674 } } }
unsubscribe ****************************************************************************** * * * Michael Bosma Telephone: +46 418 670 62 * * The Swedish University of Fax: + 46 418 670 81 * * Agricultural Sciences E-mail: Michael Bosma-at-vf.slu.se * * Dept of Plant Breeding Research * * S-26831 Svaloev, Sweden * * * ******************************************************************************
Thank your for your kindness in reply my mail. I have already hunt the Serotec address and phone but I failed all times I've tried.
On Mon, 8 Jul 1996 bruyntjes-at-hvvc03.voeding.tno.nl wrote:
} Hi } } There is a comany called Serotec in Europe, Canada and/or USA who } deliveres antibodies against bovine lymphocytes (CD1, CD2, CD4, CD6, CD8, } CD14, CD45. } I hope this will help you, } } Joost Bruijntjes } TNO Zeist } Holland } E mail: bruyntjes-at-voeding.tno.nl } }
Message-Id: {199607091530.IAA14196-at-cynic.ns.uvic.ca} X-Sender: krensing-at-uvaix.uvic.ca X-Mailer: Windows Eudora Light Version 1.5.2 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
I find the multiple messages sent by calarco-at-bu.edu very close to spamming. I am not a U.S. citizen and subscribe to this listserver to keep abreast on microscopy. Please refrain from this in the future. Thanks.
Bill , I fully agree with you, let's try and keep it to micrscopy!
Robert Schoonhoven Laboratory of Molecular Carcinogenesis and Mutagenesis University of North Carolina CB#7400, Rosenau Hall Chapel Hill, NC 27599-7400 Ph. 919-966-6343 Fax 919-966-6123
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hello,
as i mentioned before, we had trouble booting up our TN5500 system. we replaced the power supply and reseated one of the boards and our problems looked like they were solved.... however, it looks like i'm back at it, trying to get the system to boot again. the power supply is working fine and i have cleaned the contacts and reseated several boards with no luck. keyboard lights come on and the drives are being accessed, so i figure the problem probably lies in the floppy disks. i've tried using several of the bootable disks i have, but no luck.
if you have any suggestions (and if it is a problem with the disks, do you know where i can get 8 inch floppy disk versions of "new" disks) i would be very grateful.
thank you in advance,
tania jones laboratory manager dynamotive technologies corp.
Buehler Polishing Equipt. (3 platter table) in fair condition Dark Room Equipt. (plate and print processor) print processor in good condition, plate processor in fair condition Buehler cut-off saw and grinder belt - old but functioning LKB Ultramicrotome - in great condition Philips Diffractometer (eucentric goniometer, warhaus camera, laue camera and track, debye-scherrer camera (may need new generator and x-ray tube)
Pls. Contact Lynne Garone (GARONEL-at-Polaroid.com) or 617 386-1446.
I am sure that you are well-meaning in your efforts to move the affected people to action regarding the NSF Academic Research Infrastructure Program budget, but I, for one, do not like having my electronic mailbox filled with redundant and/or irrelevant messages. One message with a list of all affected states and contacts would have been plenty. They consume bandwidth and disk space, irrespective of the ease of deletion. I cannot speak for the rest of the Microscopy list, but please do not send any more of these state-by-state messages to me, either personally or through the Microscopy list. Let's stick to microscopy and related technology.
Bill ========================== Bill Heeschen/Analyt. Sci.- Mat. Char. ----- 1897-F Building / The Dow Chemical Co. --- --- Midland, MI 48667 U.S.A. --- D O W --- phone: (517)636-4005 fax: (517)636-5453 --- --- Email: waheeschen-at-dow.com ----- ========================== R
Thank you all for your inquiries. I had so many, I decided to write a general note. Also so everyone knows that it is no longer available. It is packed up and going out the door Friday, which met my time schedule to move another instrument in. It is going to be used for an outreach program, which I am happy about as that was my original intention for the instrument. Thank you again for all those that responded. Have a happy summer. Perhaps I will see you at MSA if you will be there.
Judy Murphy
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
The Analytical and Materials Characterization Laboratory at Polaroid Corp. has a need for a microscopist with a strong background in polymeric and crystalline materials to join our Microscopy and Surface Analysis Group. Experience in transmission electron microscope operation and sample preparation is essential. Experience in scanning electron microscopy, light microscopy, x-ray photoelectron spectroscopy (XPS) and x-ray diffraction is desirable. Strong interpersonal, documentation and communication skills are necessary. Strong computer skills with application to digital imaging and image analysis are desirable.
This position requires a Ph.D. in chemistry, materials science, physics or engineering with training in polymer science or a M.S. with equivalent training.
Supervisor : Lynne Garone, e. mail: GaroneL-at-Polaroid.com
Location: W4-1D 1265 Main St. Waltham, Ma. 02254 (617) 386-1446
I am all for lobbying congress for increased funds but perhaps you could limit your postings to "Attention United States" rather than sending 50 individual copies of the same message. It is a little annoying especially for those of us getting legislative alerts via other sources.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Being over the other side of the world, this message will probably hit you a little out of phase and after much has been said and the matter probably put to rest (Nestor is on the ball and pretty quick at sorting this sort of stuff - Hi Nestor!).
It is very tempting to suggest that all on the Microscopy Listserver simply hit the Reply icon on all these messages to demonstrate how effectively email to a listserver can amplify a single message. But this would be irresponsible too, and I could not endorse such actions :-).
We have been using a Brdu cell proliferation kit which utilises an FITC labelled antibody on paraformaldehyde fixed parrafin sections.We are looking for a counterstain so that we can view the same sections under brightfield conditions, particularly the nucleii. Haematoxylins seem to strip the FITC from the antiobody, any suggestions would be most welcome.
Peter Smith AgResearch Wallaceville Upper Hutt New Zealand
I am all for lobbying congress for increased funds but perhaps you could limit your postings to "Attention United States" rather than sending 50 individual copies of the same message. It is a little annoying especially for those of us getting legislative alerts via other sources.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
We are looking for chromium foil or thin wire for evaporative coating on biological specimens with our cryo-SEM. However, companies we talked to only sell chromium chips. If anyone has any information regarding this, please let us know. Thank you very much in advance. } } There are a couple of sources for the types of evaporation materials you are looking to acquire. R.D. Mathis Co. offers chrome plated tungsten rods (Part# CRW-1). These rods are widely used in the optics and electronics industry. This would be the economical solution if your evaporation power supply has the correct voltage current relationship for the evaporation of this form of the material. The telephone number for RD Mathis is 310-426-7049. Your second source of chromium foil is Goodfellow. The foils are very expensive; however, if they work they may be what you need. Goodfellow's telephone number is 1-800-821-2870. One other option you may want to look into is adapting you cryo-SEM stage for the sputter deposition of these materials. You did not indicate the stage you are using, however, I know Oxford is achieving some excellent results with this approach.
best regards, Jim Campbell Denton Vacuum, Inc 609-439-9100
It's a quandry. I agree with the people who think that the list should remain for microscopy & microscopy-related items. But then funding cuts that eliminate money for microscopy (equipment or otherwise) *are* microscopy related. But then, this is an international list, so why should people in other countries have to be bothered by our government's actions? But then, collaboration in science is international, and if a scientist in the US can't get $$ to buy/replace equipment, a scientist in Germany/Japan, etc. may not get the research done. Etc. There must be a means to get scientists to act as a *community* on issues such as funding, support of education by governments, and all the rest of the messy stuff we don't like to get involved with. And therefore suffer from *because* of that lack of involvement. This must be done both on a national and international basis. Mail servers such as the microscopy list must be kept free of politics, or they will get buried in the swill of politics. So maybe there should be two microscopy servers, "A" & "B", one strictly for microscopy as now, the other for other issues, such as funding, legislation, education, and the other things that go into making a community. I'm } sure { Nestor's looking for something else to do... Phil
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Philip Oshel University of Illinois Rm 74 Bevier Hall 905 S. Goodwin Ave. Urbana, IL 61801 (217) 244-3145 oshel-at-ux1.cso.uiuc.edu
Can any one tell me if my MacII versions of Diffract 1.3 and Crystal 2.29 (stereographic projection stuff) will work on a PowerMac when we get one? Regardless, who currently distributes these?? Thanks in advance for your help.
I have recorded the motion of motile hormogonia of the thermophilic cyanobacterium Mastidocladis laminosus with a GYYR Time Lapse VCR Model #TLC 2051-232. According to the machine's manual, the signal recorded on the T-120 cassette is NTSC.
I request advice about how I can take individual frames from the VCR tape and transform them into publishable quality glossy or half-tone prints.
R. Howard Berg, Ph.D. Biology Department University of Memphis Memphis, TN, 38152 E mail: bergrh-at-cc.memphis.edu phone: 901-678-4449 fax: 901-678-4457
Simple solution: Set up a web home page for lobbying and post the URL here, instead of sending 50 messages to the list--one for each state. Regards, Ram Devanathan ramd-at-lanl.gov
Microscopists, a microbiologist came by today and asked if I knew anything about "goniometric eyepieces". He wants to determine the hydrophobicity of bacterial colonies by measuring the contact angle of water droplets ricocheting off the surface of the colonies using this eyepiece on a monocular microscope. Is anyone familar with this technic and/or knows where one can get such an eyeiece? Also, are there other methods to determine hydrophobicity? Any help or leads would be appreciated.
Thanks, Hank Adams EML, NMSU las Cruces NM THE DROUGHT IS OVER IN NEW MEXICO!! YEH
Best regards, Prof. Ivani de S. Bott (Organizing Committe Member)
PS please note the capital letter "M" in the address (.../Micromat96/..) some people have had problems getting through because the address IS case-sensitive and they hadn't noticed this detail.
My 2 cents on "keeping to microscopy": It *is* appropriate (in my opinion) to alert list members if some really monumental crisis endangers funding for most facilities. The key is (1) a brief alert, with reference to a web site or another server to get details, (2) only if the circumstances are really unusual. What is *not* appropriate is to clog the pipeline with a multiplicity of nearly identical messages, regardless of whether they are political or histological. Perhaps it should be a rule of thumb that the more remote a topic is from miscroscopy, the shorter the message.
----------------------------------------- Alfred Kracher Geological Sciences Iowa State University Ames, IA 50011-3212 akracher-at-iastate.edu http://www.public.iastate.edu/~akracher vox:515 294 5439 fax:515 294 6049 -----------------------------------------
Message-Id: {199607101921.AA207486462-at-pigseye.mmm.com} X-Mailer: Windows Eudora Version 1.4.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
I agree with Philip Oshel that perhaps there should be one forum for scientific/technical microscopy issues, and a second for political/social microscopy issues:
} . . . So maybe there should be two microscopy servers, "A" & "B", one } strictly for microscopy as now, the other for other issues, such as } funding, legislation, education, and the other things that go into making a } community. . . .
Also my 2 cents worth on lobbying: I would like to think that as scientists we would not let ourselves become so self-interested that we would just jump on the funding bandwagon whenever the issue came up. After all there is only so much money to go around (less and less all the time!!) and people need to work together to figure out where it is MOST needed. [Pause for group hug] Ok, so I don't have a clue where that is (neither do I want to get too preachy) but it bothers me to be TOLD to complain about funding cuts without giving any thought to the issues.
Karen
Life Sciences Sector Lab Reply: kszaruba-at-mmm.com 3M Company 3M Center 270-1S-01 Phone: 612-737-2971 St. Paul, MN 55144-1000
These opinions are my own and may not represent those of 3M.
Message-Id: {199607101921.AA207486462-at-pigseye.mmm.com} X-Mailer: Windows Eudora Version 1.4.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
I agree with Philip Oshel that perhaps there should be one forum for scientific/technical microscopy issues, and a second for political/social microscopy issues:
} . . . So maybe there should be two microscopy servers, "A" & "B", one } strictly for microscopy as now, the other for other issues, such as } funding, legislation, education, and the other things that go into making a } community. . . .
Also my 2 cents worth on lobbying: I would like to think that as scientists we would not let ourselves become so self-interested that we would just jump on the funding bandwagon whenever the issue came up. After all there is only so much money to go around (less and less all the time!!) and people need to work together to figure out where it is MOST needed. [Pause for group hug] Ok, so I don't have a clue where that is (neither do I want to get too preachy) but it bothers me to be TOLD to complain about funding cuts without giving any thought to the issues.
Karen
Life Sciences Sector Lab Reply: kszaruba-at-mmm.com 3M Company 3M Center 270-1S-01 Phone: 612-737-2971 St. Paul, MN 55144-1000
These opinions are my own and may not represent those of 3M.
I was asked by someone in our lab to post the following.
Mark Elliott
Recently, I have experienced two kinds of staining problems:
1. background staining & 2. peppery lead precipiate staining on cytoplasm.
The routine post-stains that we use are uranyl acetate and Sato's lead. This method never used to give any background staining. Occasionally we get peppery lead precipitate on cytoplasm of epon sections, as described in textbooks.
Meanwhile, this kind of background staining is most prominent with epon sections and not as bad with Spurr's sections. So far, the LR White sections from human biopsies have not shown much of this problem.
I have checked for all possible causes including staining duration, cleanliness of boat, of washing distilled water, and of tools. If I were to suspect some of the Epon ingredients that might have gone out-dated, same might have happened to the Spurr's.
I have tried some standard methods described in textbooks for removing stain contamination but they did not seem to work that well.
Can anyone offer any advice on how I can go about solving this problem or removing background stains?
Thank you!
Fanny Chu Pulmonary Research Lab St. Paul's Hospital Vancouver, BC Canada
Message-Id: {199607101840.AA151664024-at-pigseye.mmm.com} X-Mailer: Windows Eudora Version 1.4.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
I would like to request that replies to Peter Smith's question:
} We have been using a Brdu cell proliferation kit which utilises an FITC } labelled antibody on paraformaldehyde fixed parrafin sections.We are looking } for a counterstain so that we can view the same sections under brightfield } conditions, particularly the nucleii. Haematoxylins seem to strip the FITC } from the antiobody, any suggestions would be most welcome. . . .
be posted to the whole group as I am interested, too.
Also, I would be interested in a MOUNTING MEDIUM which neither fades the fluorescence, nor the counterstain. While I have not tried this test with fluorescence, I have tried a number of aqueous mounting media ("Mount-Quick Aqueous", "Fluoromount G", low-temp. agarose and even "Brite" floor polish) on stained 1 micron epoxy sections. All of the above mountants have faded all of the counterstains which I tried (too many to list). This fading happens before the mountant even has time to set. In past fluorescence experiments I thought my counterstains were not working, but now I think the 10% glycerol I was using to mount the coverslips simply leached them out.
Just as an aside I have had good results using Zymed methyl green stain (Zymed Laboratories, South San Francisco, CA, USA (800) 874-4494; no affiliation with myself) on 1 um epoxy tissue sections. This is sold as a counterstain for immunoperoxidase/DAB staining; I don't know its effect on fluorescence.
TIA,
Karen Zaruba
Life Sciences Sector Lab Reply: kszaruba-at-mmm.com 3M Company 3M Center 270-1S-01 Phone: 612-737-2971 St. Paul, MN 55144-1000
These opinions are my own and may not represent those of 3M.
We had our new filament on our Amray FEG 1910 on April 12 this year, the extraction current ( Iext) was high (about 120 micro A) until few days ago. The current dropped to about 55 micro A and we have decided to stop using it untill we know the way to solve it. (The vaccum system works well, 0.4x10-10 torr at gun)
Question: What is the normal life of a filament such as ours? How to make the filament life longer? We only have our machine for a little over one year, we need more experiences.
Thanks in advance.
Zhen Quan Liu zqliu-at-pku.edu.cn
--------------------------------------------------------------------- Zhen Quan Liu (Ph.D) Tel:(86) 10 6275 1427(Office) Physics Building (86) 10 6275 3727(Home) EM Lab. Email: zqliu-at-pku.edu.cn Physics Building Email(home) wl-at-ibmstone.pku.edu.cn Peking University Fax (office): (86) 10 6275 1615 Beijing 100871, China
I was asked by someone in our lab to post the following.
Mark Elliott
Recently, I have experienced two kinds of staining problems:
1. background staining & 2. peppery lead precipiate staining on cytoplasm.
The routine post-stains that we use are uranyl acetate and Sato's lead. This method never used to give any background staining. Occasionally we get peppery lead precipitate on cytoplasm of epon sections, as described in textbooks.
Meanwhile, this kind of background staining is most prominent with epon sections and not as bad with Spurr's sections. So far, the LR White sections from human biopsies have not shown much of this problem.
I have checked for all possible causes including staining duration, cleanliness of boat, of washing distilled water, and of tools. If I were to suspect some of the Epon ingredients that might have gone out-dated, same might have happened to the Spurr's.
I have tried some standard methods described in textbooks for removing stain contamination but they did not seem to work that well.
Can anyone offer any advice on how I can go about solving this problem or removing background stains?
Thank you!
Fanny Chu Pulmonary Research Lab St. Paul's Hospital Vancouver, BC Canada
Where can we buy tungsten wire of 0.012 inch diameter? } } Goodfellow offers a wide variety of tungsten wire in various sizes. They are located in Berwyn, PA and the toll free number is 1-800-821-2870 or via fax 1-800-821-2020. Denton Vacuum has no financial interest in this company.
James Campbell Marketing Manager Denton Vacuum, Inc 609-439-9100 Fax 609-439-9111 campbell36-at-aol.com July 11, 1996 8:19 am
This listserver (in my opinion) should not allow political comment and discussion. While science must have a political component and awareness this is not the place to discuss it. Where do you draw the line? If you allow dissuasion of funding will you allow my concern about CCW on the university campus? If I disagree a proposed topic and want to lecture about my concerns about "non-Christian value" would you want to hear about it?
I sort of agree with Alfred Kracher, set up your own web site and forward one note about it. Or go one better, set up your own listserver to deal with your agenda.
These opinions are mine alone and have no relationship to my employer. Thank you.
Frank Karl
They that give up essential liberty to obtain a little temporary safety deserve neither liberty nor safety. Benjamin Franklin
Subscribe microscopy Halcrow-at-unbsj.ca Dr Kevin Halcrow, Telephone (506)-658-5567 Professor of Biology, Fax (506)-658-5650 University of New Brunswick, EMail Halcrow-at-unbsj.ca Saint John, NB Canada E2L 4L5
} We have been using a Brdu cell proliferation kit which utilises an FITC 9+} labelled antibody on paraformaldehyde fixed parrafin sections.We are looking } for a counterstain so that we can view the same sections under brightfield } conditions, particularly the nucleii. Haematoxylins seem to strip the FITC } from the antiobody, any suggestions would be most welcome. . . .
I would suggest a 1% aqueous solution of Methyl Green, staining time approx. 1 minute (or less). It worked fine with my tissues although it is a pain to make up as the Methyl Green has a mind of it's own as to what the powder will land on.
} Also, I would be interested in a MOUNTING MEDIUM which neither fades the fluorescence, nor the counterstain. {
Innovex Biosciences sells Advantge Whch is a permanent aqueous mounting media (to be used with coverslips). I have used it and it works exceptionally well. (the usual disclaimer). Phone - (510) 222-7800
regards, Robert Schoonhoven Laboratory of Molecular Carcinogenesis and Mutagenesis University of North Carolina CB#7400, Rosenau Hall Chapel Hill, NC 27599-7400 Ph. 919-966-6343 Fax 919-966-6123
Microscopists, a microbiologist came by today and asked if I knew anything about "goniometric eyepieces". He wants to determine the hydrophobicity of bacterial colonies by measuring the contact angle of water droplets ricocheting off the surface of the colonies using this eyepiece on a monocular microscope. Is anyone familar with this technic and/or knows where one can get such an eyeiece? Also, are there other methods to determine hydrophobicity? Any help or leads would be appreciated.
Thanks, Hank Adams EML, NMSU las Cruces NM THE DROUGHT IS OVER IN NEW MEXICO!! YEH
} Applied Image Inc in Rochester, NY, (715)482-0300. I dialed that and got a recording. I went to http://www.bigbook.com/, entered "Applied" in business name, "Rochester" in city, NY in state, and found out that the correct phone number is
Applied Image Inc
Category: Electrical Fittings & Cons Material Circuit Breakers Electric Electrical Apparatus & Equip Address: 1653 Main St E Location: Rochester, NY 14609 Phone: (716) 482-0300
The display includes a street map showing how to get there.
The yellow pages categories look a little fishy...
I have seen this background stain on blocks that were left in the oven for more than a week or on blocks that were polymerized in a humid environment. Usually, the background stains on the semithin sections that I mount on a slide and stain with toluidine blue as well.
I don't have a remedey for the blocks that already show this though. Just try polyimerizing the new blocks in the dessicator.
Hope this helps. Karen P.
On Wed, 10 Jul 1996 melliott-at-prl.pulmonary.ubc.ca wrote:
} I was asked by someone in our lab to post the following. } } Mark Elliott } } } Recently, I have experienced two kinds of staining problems: } } 1. background staining & } 2. peppery lead precipiate staining on cytoplasm. } } The routine post-stains that we use are uranyl acetate and Sato's } lead. This method never used to give any background staining. } Occasionally we get peppery lead precipitate on cytoplasm of epon } sections, as described in textbooks. } } Meanwhile, this kind of background staining is most prominent with } epon sections and not as bad with Spurr's sections. So far, the LR } White sections from human biopsies have not shown much of this } problem. } } I have checked for all possible causes including staining duration, } cleanliness of boat, of washing distilled water, and of tools. If I } were to suspect some of the Epon ingredients that might have gone } out-dated, same might have happened to the Spurr's. } } I have tried some standard methods described in textbooks for removing } stain contamination but they did not seem to work that well. } } Can anyone offer any advice on how I can go about solving this problem } or removing background stains? } } Thank you! } } } Fanny Chu } Pulmonary Research Lab } St. Paul's Hospital } Vancouver, BC } Canada }
Greetings, Frank Karl suggests no politics on the list because you can't draw the line. But I think the line can be drawn, at least to the same degree that the line can be drawn between microscopy topics and other techno stuff. I wonder how many EM centers whose staff participate on this list have had equiptment paid for by the NSF's equipment money. I bet its most of them (at least those in the USA). The loss of this money should be of interest to at least as many readers as are interested in, say, pepper artifacts. Clearly the fault of the messages that set off this thread was the multiple posting, 1 per state. But a single message about the alert serves the interest of many on the list and should be allowed.
If some religous group decided that e.m. was immoral (imagine an artist took a homoerotic picture that somehow involved an electron microscope) and were fulminating to pass a law banning e.m., such a circumstance would obviously also be appropriate political material for this list. The usual random fulminations of religous groups omit electron microscopy and so are obviously off limits. I don't see what the problem with line drawing is.
Just my two, well three or four, cents, Tobias Baskin
Pat Calarco, as member of the MSA Public Policy Committe was told by the Society (please remember that the Microscopy Society of America -MSA both Hosts and funds the Microscopy Listserver) to use all it's resources (including the Listserver) to get the information out to the membership.
In her zeal, she did go overboard, and Pat and I have touched base and worked out a method to more succinctly get this information out to both Society members, as well as the microscopy community, when either might be impacted.
Certainly there are many of our subscribers who don't care about the NSF funding for instrumentation, but there are also a significant number that do. This is a double edged sword and there were clearly errors made in the posting, and we have dealt with the problem. So please consider it closed.
If anyone of our subscribers has similiar needs in their global area (the listserver has subscribers from over 50 countries) , please touch base with me first. Allow me to advise you on the most appropriate method of getting information out .
Cheers... Nestor Your Friendly Neighborhood SysOP
P.S. All other comments on lobbying can be address to my assistant at the address
Message-Id: {199607112044.PAA02282-at-Sparc5.Microscopy.Com} MIME-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: Microscopy-at-Sparc5.Microscopy.Com
We have an emscope SC 500 sputter coater and SB 250 carbon head, but no instruction manuals. I have heard that this company was aquired by another. I would appreciate any information/suggestions on how we may be able to track down some manuals for these instruments. Thanks.
************************************************************ - - ************************************************************ Heather Owen Department of Biology Western Kentucky University 1 Big Red Way Bowling Green, KY 42101-3576
Does anyone have detailed protocols for oxygen scavanger systems to reduce bleaching in fluorescence microscopy?
Do I want the thymol free catalase? Do I want dry powder or=20 the crystal suspension in water? How long is the enzyme solution good for? (The catalase assay procedure says "use these dilute solutions promptly"). Is there any way I can make up a 100X stock of the two enzymes and be able to keep it for a while, like say a week?
In Nature 380: 451-453 (1996)[96180121] R. D. Vale, T. Funatsu,=20 D. W. Pierce, L. Romberg, Y. Harada & T. Yanagida, I see "...0.5% mercaptoethanol and an oxygen scavanger system[20]..." where note 20 is J Mol Biol 216: 49-68 (1990)[91039337] Y. Harada,=20 K. Sakurada, T. Aoki, D. D. Thomas & T. Yanagida.
In that paper, (page 51) they give only the following information:
I am planning to try this system, but I'm not sure about enzyme stability, exactly which kind to use, etc.
(People here have been using 30% 2-mercaptoethanol instead, but it has some fluorescent impurities, we don't want to distill it, it is difficult to filter, and I hope to find that the oxygen scavanger system gives longer exposure times without bleaching).
I have verified that the amounts make sense: this should be enough to eliminate 5 times saturation with oxygen. (The enzyme system uses 2 moles of glucose to remove 1 mole oxygen).=20
I have concluded that there is not much point in using beta-D-glucose rather than just D-glucose. The optical rotation of beta-D-glucose changes to that of D-glucose with a half life of 48 minutes, so to get any benefit of using the beta anomer, you would have to use it immediately after dissolving. Also regular D-glucose is 63% beta anyway. (Methods in Enzymology, 63:371 note a).
I want to avoid fluorescent impurities and the Sigma beta anomer=20 is only 97+% pure while the plain D glucose is 99.5% pure.
Solubility of Oxygen in water: CRC Handbook Chemistry & Physics (1987) = pB112.=20 3.16 mL of O2 dissolves in 100 mL of water at 25=B0C.=20 (1 Atm)(3.16 mL)/((0.082LAtm/Kmol)(298K)) =3D 0.129 mmol of O2 in 100 mL = =20 The solubility of Oxygen is 1.29 mmol/L, under 2mM.=20
} I was asked by someone in our lab to post the following. } } Mark Elliott } } } Recently, I have experienced two kinds of staining problems: } } 1. background staining & } 2. peppery lead precipiate staining on cytoplasm. } } Fanny Chu
I don't know about the background (will ask), but the lead pepper could be a pH problem--check that the Pb stain is } =ph *12*, not less. Phil
&&&&&&&&&&& Illigitmi non carborundum &&&&&&&&&&&
Philip Oshel University of Illinois Rm 74 Bevier Hall 905 S. Goodwin Ave. Urbana, IL 61801 (217) 244-3145 oshel-at-ux1.cso.uiuc.edu
It is a sad commentary on how apathetic our professional community has become when a call for action by this community from the society that funds the operation of this listserver can elicit such a volume of negative criticisms. No wonder we are looked upon with such disdain and cynicism by the public. Our apathy and pettiness has seen the dismantlement of most of the shared instrumentation programs at the federal level save those from the Department of Defense. It is great to sit about and discuss all the fantastic new technologies available to the microscopy community but what will happen when the avenues to acquiring these new technologies is slammed permanently shut for many of us. Most private sources of funding require a commitment FIRST from the institution and other public sources before funds are granted for such ventures. I wonder how many of you realize that the request which was the subject of this discussion was requested by the senate sub-committee considering these appropriations and that previous input had been greatly appreciated and recognized. Our silence to them and vindictive attitude toward those trying to take a positive step toward providing these committees with their requested input sends the wrong message to everyone. If we are unwilling to support each other through the sharing of our knowledge with those on this listserver as well as those outside the listserver when requested, we shall definitely deserve the fate that awaits us, "......a slow fade to black". Some of the criticisms I have seen were constructive, i.e. A single alert message rather than 6 or 7 (not 50 as has been incorrectly stated, those went out for states where senators sit on the sub-committee making these decisions). But divorcing solicited input required for making INFORMED political decisions can only hurt this community. Could we all be a little more tolerant, there are not many of these requests each year (2-3 I believe).
Foster Findlay Associates Ltd is one of the leading image processing software developers for microscopists and general medical applications. Below is a short description of three software packages which should be of interest to you as an active micrsocopist. These packages are C_Images 3D (a unique library of true 3D image processing functions with AVS/Express visualisation), PC_Image and C_Images 2D. For further information about these products and a demo copy of the software contact me Dominique Miller or visit our WWW site.
PC_Image 2.2 The Latest High Performance Imaging Solution From Foster Findlay Associates Ltd.
July sees Foster Findlay Associates launch the most powerful version of their best selling software yet - PC_Image 2.2. This comprehensive, multi-purpose image processing and analysis solution, now offers a new, intuitive user interface and the capacity to add your own modules. This new facility allows you to easily integrate your own tailor-made mini- applications into PC_Image, giving added flexibility, speed, almost endless functionality and can now also be linked to our new microscope stage control software - WinStage.
PC_Image 2.2 combined with our C_Images library, of over 350 functions, is an unbeatable partnership wherever there is a need for analytical image processing. PC_Image not only provides the ease of use and familiarity associated with a Windows based application, but it places a unique development package of processing and analysis functions at your finger tips, quickly enabling you to develop full blown applications that can be inserted directly into PC_Image. Custom applications can be developed by us, or purchased off-the-shelf, for example, modules already exist for grain sizing or three colour component capture.
PC_Image features calibration, LUT transforms, morphology, various convolutions and 6 non-linear filters, FFT, thresholding, a comprehensive selections of measurements, shade correction and densitometry. PC_Image can process true 24 bit colour and images of 1024x1024 are easily supported, input can be from CCD cameras or a variety of image formats such as TIFF, Windows Bitmap, Biorad, Kontron, PGT, Seescan, SFI, Visionetrics, Data Translation and many more. PC_Image 2.2 is now available for Windows 3.1and '95 and continues to support an ever increasing number of framegrabbers including several PCI boards.
C_Images 3D is a comprehensive image processing and analysis library. Accessible at different levels, C_Images 3D is a powerful environment for building end-user applications or implementing your own complex algorithms. These include more accurate results and measurements, some of which can only be obtained in 3D, such as surface area, length and orientation of principal axes of an object. The unique capability of C_Images 3D for handling images provides the means to process datasets much larger than available RAM. C_Images 3D makes use of AVS, which provides an easy to use interface with interactive modular programming and visualisation capabilities.
For further information contact: Mr Dominique Miller Newcastle Technopole Kings Manor Newcastle upon Tyne NE1 6PA United Kingdom Tel: +44 (0) 191 201 2180 Fax: +44 (0) 191 201 2190 Email: Dominique-at-ffaltd.demon.co.uk
---------------------------------------------------------------------------- |From: Dominique Miller | | |Marketing Executive | | |Foster Findlay Associates | Phone: National (0191) 201 2180 | |Newcastle Technopole | International +44 191 201 2180 | |Kings Manor | Fax: National (0191) 201 2190 | |Newcastle upon Tyne | International +44 191 201 2190 | |NE1 6PA |E-Mail: Dominique-at-ffaltd.demon.co.uk | |UK | WWW: http://www.demon.co.uk/ffaltd/ | ----------------------------------------------------------------------------
Just announced - Philips Electronics North America Corporation has acquired the assets of ElectroScan. According to the press release, ElectroScan employees will be offered employment with Philips. Don Grimes, Microscopy Today
Message-Id: {1.5.4.32.19960712115434.00678cdc-at-alf.zfn.uni-bremen.de} X-Sender: heibueck-at-alf.zfn.uni-bremen.de X-Mailer: Windows Eudora Light Version 1.5.4 (32) Mime-Version: 1.0 Content-Type: text/plain; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable
Dear colleagues,
Our institute is working especially with x-ray microanalytical investigations of nutrients (for example phosphorus, potassium) in different plant materials. Our previous analytical unit was an Philips TEM EM 420 with an EDAX 9100.=20 Now we are planning to buy a new SEM with a x-ray microanalytical unit. We have seen one new equipment from ZEISS (GEMINI) with a cryo-unit, made by Oxford Instruments and a Si/Li detector (LINK). The demonstration, we=B4ve got, was not convincing and we are not sure, that this instrument is suitable for our question. Who have experiences with this equipment and the detection of light elements and who is working with other analytical units and a similar questions.
I presently use Pizzaz Plus for handling images on the PC. On the Mac I use Graphic Converter. Someone told me that Hijack and U-Pal for the PC are comparable to the mac Graphic Converter. Anyone knows other reasonably priced PC soft for converting formats (e.g, PICT to TIFF, etc) out there. Photoshop is out of my price range now. Please respond to me directly not to the list {Fermin-at-tmc.tulane.edu} . Thanks.
********************************************************************* *Cesar D. Fermin, Ph.D \|T|/ Fax (504) 587-7389 * *Tulane Medical School /|M|\ Voice Mail (504) 584-2618 * *Pathology/SL79 \|C|/ Secretary (504) 584-2436 * *New Orleans La 70112-2699 /|*|\ Lab/Techn. (504) 584-2521 * * {Fermin-at-tmc.tulane.edu} ----} Prof. Pathology & Otolaryngology * *http://www1.omi.tulane.edu/departments/pathology/fermin/cdftop.html* *********************************************************************
Hi, EMSCOPE was bought by the VG Microtech (Polaron) division of Fisons a few years ago. VG Microtech's distributor in U.S.A. is Energy Beam Sciences. Give them a call at 413-786-9322 (Steve Slap or Jeff Balou) or email at ebs-at-ebsciences.com. I am sure they can assist you.
Good luck
Jean-Pierre Slakmon
} We have an emscope SC 500 sputter coater and SB 250 carbon head, } but no instruction manuals. I have heard that this company was } aquired by another. I would appreciate any information/suggestions } on how we may be able to track down some manuals for these instruments. Thanks. } } ************************************************************ } - - } ************************************************************ } Heather Owen } Department of Biology } Western Kentucky University } 1 Big Red Way } Bowling Green, KY 42101-3576 } } (502) 745-6501 voice, (502) 745-6856 fax, Owenha-at-WKUVX1.WKU.EDU } } } } ________________________________________________________________
To: Microscopy ListSer {Microscopy-at-MSA.Microscopy.Com} cc: VANHART --ENDVM5 Dan VanHart 7-1262 EMMI --ENDVM5 F. Emmi SHURBAN --ENDVM1 Hurban, S. CONROW --ENDVM5 K.M. (Karen) Conro JUNGDY --ENDVM5 Dae-Young Jung RICKM --ENDVM1 Musa, R.
We've evaluated video thermal printing on several of our SEM's as an inexpensive replacement for instant film. It's OK for low resolution needs, as long as the image is not very noisy. Our Cambridge S-250 has an image store, so I can beat the noise problem in that case. Our Phillips can not "build" a video image, so the use of thermal prints is less.
We'd like to implement a SONY 890 or similar video printer on an AMRAY 1600 a 1610 and a JEOL 733 probe. My impression is that the cost of the hardware and software needed to interface to these would wipe out years of savings. I heard the Amray TV cards are even out of production.
I have a service company for microscopes. My stock is over $250 000.00 in parts and accessories for any type of microscope.
If I would want to make a parts and price list, I would need two persons wotking full time on it, because this come and goes, and it never comes twice with the same cost.
I recommend you let me know specifically, what you need, and I will look if i can help you.
If there are anyone among the exhibitors who will take part in Microscopy & Microanalysis annual meeting, please contact me. I would like to make a business talk before going there. Our company will be able to be your business partner in Korea.
At 13:54 12-07-96 +0200, you wrote: } Dear colleagues, } } Our institute is working especially with x-ray microanalytical } investigations of nutrients (for example phosphorus, potassium) in= different } plant materials. Our previous analytical unit was an Philips TEM EM 420= with } an EDAX 9100.=20 } Now we are planning to buy a new SEM with a x-ray microanalytical unit. We } have seen one new equipment from ZEISS (GEMINI) with a cryo-unit, made by } Oxford Instruments and a Si/Li detector (LINK). The demonstration, we=B4ve } got, was not convincing and we are not sure, that this instrument is } suitable for our question. Who have experiences with this equipment and the } detection of light elements and who is working with other analytical units } and a similar questions. } } Heike Buecking } Dr. Heike Buecking } Universitaet Bremen } Physiologische Pflanzenanatomie } UFT } Leobener Str. } 28359 Bremen } FRG } Tel: 0049-421-2182954 } Fax: 0049-421-2183737 } e-mail: heibueck-at-uft.uni-bremen.de **************************************** Dear Heike: The largest difference between thick (SEM/Microprobe) and thin sections (TEM) EDS analysis is the size of the "penetration envelope". That is the depths and the area from which the X-rays originate. In thin sections that area is effectively the beam diameter. =20
In sections too thick for beam penetration (SEM) the area from which X-rays originate, with the instrument at a very high magnification, depends mostly on kV and the average atomic number of the specimen. For average rock that may be around 3 microns diameter. For biological materials this tends to be around 20 microns. You could certainly analyse those elements, but you could not tell, for most biologist's purposes the location of higher concentrations.=20
Furthermore, if the higher concentrations ocurred in, for instance, part of chloroplasts at 5%, this would result in a sizeable peak. The accross the cell percentage may be only 0.2% and perhaps not detectable in EDS.=20
There are always exceptions, but EDS on TEM is more useful to biologists and EDS on SEM is generally more useful for the material scientist. Es ist wirklich ganz einfach. } Jim Darley
In a message dated 96-07-12 23:41:39 EDT, you write:
{ { They use a Nachet optics at this time. Can you assist? } }
I am not very familiar with Nachet optic. I deal mainly with Carl Zeiss, Leica, Nikon, Olympus. But a trinocular head could always be adapted if necessary.
Do you have more optical information about Nachet optic ?
Binocular, trinocular have to be adapted to the particulary optic. It is either infinity corrected or a particular focal distance. We need to know, what this focal distance is.
In a message dated 96-07-13 00:58:26 EDT, you write:
{ { Do you also sell used microscopes and other equipment? Right now I am looking for a used stereomicroscope, with zoom (or at least covering a wide range of magnifications, like x5-25), no cameraports needed, a "boomstand" would be a great plus. { {
Dear Laszlo,
I mainly service and repair microscopes. But good service does not have limit. Selling is also a service. I do help and sell sometimes microscopes. I actually serviced several microscopes at your location.
Reference: Tony Piazza 415/750-2167 Bldg2 room 436B
I can help you find a stereo microscope, with zoom on a boom stand.
} } Also, could you give some insigth about really cheap I mean low priced stereomicroscopes? I have seen several supply companies selling stereomicroscopes from Korea and from Russia for about $500. Are they any good? } }
Microscopes are like anything else. Cars, for example. You can buy a car for $500 and another for $100 000. You cannot expect from the $500 car the same result as from the other one !
If you were sombody buying a microscope to use it twice a year for ten minutes, I would sell you a $500 microscope.
However, if you think to use this microscope for routine work altmost every day, I would think, that a better and more expensive microscope might be a good investment.
Let me know, what do you think of it.
best regards, Emile Meylan SERCO Technical Services, Inc. 800/483-0508
In a message dated 96-07-13 10:14:54 EDT, you write:
{ { Are you involved exclusively with optical scopes, or do you service SEM's as well? If you service SEM's, I'd like to know more about your company. } }
Dear John,
I am only involved with Light microscopes. To service SEM's you would need the 100% support of the vendor of this microscope. Making my own company, I do also compete with this vendors, and some of them do not like it very much.
However, I have been working with the Company Carl Zeiss for many years. I still have a very good and very close relationship with them. If your service or parts problem is related to Carl Zeiss, let me know. I could maybe help you with the right contact.
I would be grateful if someone could suggest a suitable embedding medium (preferably alternatives to paraffin) to section plant leaf tissue for light microscopy.
Thank you.
Greg Forbes Greg Forbes, plant pathologist, International Potato Center (CIP) Quito, Ecuador
A client has several units in a warehouse, out of service, complete, but offered "as is". Not items we would normally buy and warehouse, though.
Any fellow listmember is invited to please send your best bid, remembering that they are "as is" units, though guaranteed complete, and I will present it to the owner.
1 AMRAY SEM 1500
- - with a handler for 6" wafers (although it is not necessary to use it)
I would like to address a specific problem we have encountered. We have been taking mouse eyes through to wax and have been having trouble keeping the vitreous intact. Results have been inconsistant - sometimes the vitreous cuts beautifully and others not. We have used a range of protocols varying dehydration and infiltration times as well as using toluene as the clearing agent (we routinely use xylene). Has anyone had experience cutting eyes? Any suggestions will be welcomed.
Message-Id: {199607150741.DAA05924-at-thomas.ge.com} X-Authentication-Warning: thomas.ge.com: Host [3.52.8.39] didn't use HELO protocol
John, Reicher-Jung is now a part of Leica ph. 800-248-0123. They should have a copy they can send you. regards,
Robert Schoonhoven Laboratory of Molecular Carcinogenesis and Mutagenesis University of North Carolina CB#7400, Rosenau Hall Chapel Hill, NC 27599-7400 Ph. 919-966-6343 Fax 919-966-6123
Comments: Authenticated sender is {t.g_reed-at-fs2.mt.umist.ac.uk}
In the past there have been discussions here concerning (or making reference to) the NSF's Important Notice 91, concerning use of NSF-sponsored instrumentation by Universities for a fee. I have not been able to find an on-line copy of this document, so I have had it transcribed and made it available at http://18.82.0.42/nsf.in91/in91.html, subject to the understanding that neither MITor myself are liable for any transcription errors.
Tony Garratt-Reed
******************************* ** ** ** Anthony J. Garratt-Reed ** ** tonygr-at-mit.edu ** ** ** *******************************
Bob, I don't have a stain right to hand, but (assuming you mean LM stains) I suggest you get the following books for your library: "Conn's Biological Stains", 9th edition or later--chemistry, reactions, and properties of stains. "Staining Procedures", 4th edition or later. Recipes. both by the Biological Stain Commission, published by Williams & Wilkens (Baltimore) Kiernan, J.A."Histological & Histochemical Methods: Theory & Practice." 1990, 2nd edition (may be a later one). Pergamon (NY). Excellent reference/text. Info you want is on pp. 288-298 for amines. Other pages for other "amine-like" groups. (Keirnan may reply himself to your question; this would be most useful.) Phil
} Fellow Microscopists; } } Once again I am in need of a specific stain, and being unfamiliar with } stains in general, I ask your assistance. I am looking for a stain that is } specific to a compound which is cationic, w/amine-type functional groups. } I would like to be able to visualize the homogeneity of a coating on a } substrate, (preferably by light microscopy, but we do have SEM and EDX). } Someone recommended the use of TNBS (trinitrobenzenesulfonic acid, or } picrylsulfonic acid), but I would like to think that there is something a } little less hazardous to use (as well as a little less expensive to } obtain). } } TIA, } } Bob } ******************************* } Bob Citron } Chiron Vision } 555 W. Arrow Hwy } Claremont, CA 91711 } USA } ph: (909)399-1311 } email: Bob_Citron-at-cc.chiron.com } ********************************
&&&&&&&&&&& Illigitmi non carborundum &&&&&&&&&&&
Philip Oshel University of Illinois Rm 74 Bevier Hall 905 S. Goodwin Ave. Urbana, IL 61801 (217) 244-3145 oshel-at-ux1.cso.uiuc.edu
I have heard differing opinions as to what temperature to set buffer washes and fixatives for animal perfusion. We prepare brain tissue for immunocytochemical localization of membrane proteins. When we perfuse rats, we wash out the blood with room temperature phosphate buffer, and then use cold fixative (4% paraformaldehyde in phosphate buffer with or without 0.1% glutaraldehyde). I am interested in anyone's opinion on the best temperature for these solutions as well as the logic behind the choice.
A related question: the phosphate buffer we use for perfusion is 0.12 M. Does anyone wish to share their opinions on the best molarity for good ultrastructure?
Ron Petralia
Ronald S. Petralia NIDCD/NIH 36/5D08 36 CONVENT DR MSC 4162 BETHESDA MD 20892-4162 Tel: 301-496-3804 Fax: 301-480-3242 petralia-at-pop.nidcd.nih.gov
Message-Id: {9607151524.AA1203-at-worldcom-37.worldcom.com} To: Microscopy {Microscopy-at-Sparc5.Microscopy.Com}
I am casting the interior of a cylinder with an epoxy. The epoxy I am using cures in 8 hours at room temperature and reaches a peak temperature of 82F. The manufacturer quotes the shrinkage of this epoxy as .001in./in. I have the following questions regarding shrinkage: Does epoxy shrink the same in all directions (x&y vs z)? Is there a good method for measuring shrinkage? Would shrinkage alter the dimensions of a flexible material (draw in the sides of my cylinder)? Any references on this subject would be most appreciated. Thank you for your help,
Diana Kittleson Pillsbury Technology Center 330 University Avenue S.E. Mpls., Mn 55414 612-330-1898 Diana_L_Kittleson-at-pbtc.com
We use the acrylic resin LR White for a lot of our light microscopy work of plant materials. It is a forgiving resin and you only need to dehydrate to 80-90% EtOH. Iy sections very well at2-5um. Some of the stains and histochemical agents don't work in quite the same way ie phloroglucinol for lignin but it does give good infiltration and very nice thin sections
Patrick Echlin University of Cambridge UKOn Sat, 13 Jul 1996, Gregory Forbes wrote:
} I would be grateful if someone could suggest a suitable embedding medium } (preferably alternatives to paraffin) to section plant leaf tissue for light } microscopy. } } Thank you. } } Greg Forbes } Greg Forbes, plant pathologist, International Potato Center (CIP) } Quito, Ecuador } } Email: gaf2-at-cornell.edu } } CURRENTLY: Dept. Plant Pathology, Cornell University, } 334 Plant Science Bldg. Ithaca, NY 14853 } Fax: +1(607)2554471 Tel: +1(607)2553188 } }
We use the acrylic resin LR White for a lot of our light microscopy work of plant materials. It is a forgiving resin and you only need to dehydrate to 80-90% EtOH. Iy sections very well at2-5um. Some of the stains and histochemical agents don't work in quite the same way ie phloroglucinol for lignin but it does give good infiltration and very nice thin sections
Patrick Echlin University of Cambridge UKOn Sat, 13 Jul 1996, Gregory Forbes wrote:
} I would be grateful if someone could suggest a suitable embedding medium } (preferably alternatives to paraffin) to section plant leaf tissue for light } microscopy. } } Thank you. } } Greg Forbes } Greg Forbes, plant pathologist, International Potato Center (CIP) } Quito, Ecuador } } Email: gaf2-at-cornell.edu } } CURRENTLY: Dept. Plant Pathology, Cornell University, } 334 Plant Science Bldg. Ithaca, NY 14853 } Fax: +1(607)2554471 Tel: +1(607)2553188 } }
} OK, so confocal and deconvolution methodologies have their place } for achieving the ultimate in resolution when imaging certain kinds of specimens. } But what about in real time on an everyday basis when you are sitting } at the 'scope scanning through a new specimen just trying to get a sense of what's } going on. Is deconvolution useful for these situations, or must I wait for the } computer to digest and regurgitate my image before I know whether I have } something interesting? In more direct terms, is deconvolution practical for rapidly .} and easily evaluating new specimens where the major purpose is getting } few informative pix, not the ultimate in 3D resolution?
and Aryeh Weiss wrote:
} I think it would be useful if users of wide field deconvolution } systems post some processing times for "typical" dataset, } specifying the computer configuration (hardware and software) } which they use
At the risk of restarting the deconvolution thread again, I would like to respond to these two points.
VayTek's goal from the beginning has been to develop a practical deconvolution "toolbox". The toolbox approach means a complete integrated system with several algorithms, each appropriate for various situations and applications.
We know it is important to have rapid feedback in real-time situations. To do this with deconvolution means integrating the data acquisition system with a rapid deconvolution algorithm. We have done this on both the Macintosh and the PC.
With our system, the user can easily move the stage, view a live image, capture a single image, and apply a single image nearest neighbor deconvolution. This process takes about 5 to 10 seconds. Color images (which cannot be captured on a confocal system) take a few seconds longer.
If the user wants a slightly better deconvolved image, he/she can use a nearest neighbor algorithm with three consecutive optical slices. It takes only slightly longer to capture the three images.
There will be objections by others about the value of nearest neighbor vs. constrained iterative and that this approach is not valid. (Or perhaps they have a vested interest in pushing the constrained iterative.) However, we know from experience, (many of our users will verify this), that usually there are small difference in images deconvolved with nearest neighbor and constrained iterative. The nearest neighbor algorithm is very useful for determining what the image will look like when deconvolved with the constrained iterative - and doing it quickly.
After a specimen has been previewed with the nearest neighbor and the proper settings have been determined, a full stack can be captured and deconvolved with the constrained iterative.
In other words, the answer is yes, there is a way to use deconvolution in a practical, real time situation. It may not be as fast as the confocal, but it is pretty close. There are trade-offs however, one being flexibility.
Our integrated system does make a good "first line" system for some researchers with some specimens.
Regarding typical times:
This a little more complex since there are several variables involved. But I'll try.
In general, the times for Macintosh and PC are pretty comparable. All these times are for VayTek's imaging systems.
1) 512 x 512 grayscale image with a DSP board - never more than 4 seconds/image using nearest neighbor - any size Psf
2) 512 x 512 grayscale image without DSP board - depends on CPU speed - for Pentium 150 - no DSP board - 2 to 10 seconds - nearest neighbor - with a small PSF; longer with large PSF
3) 512 x 512 color image with DSP board - 16 Mb memory - nearest neighbor - 10 to 15 seconds
4) stack of 25 512 x 512 grayscale images - with DSP board - sufficient memory - small PSF - optimal number of iterations - constrained iterative - 5 to 15 minutes
5) Data acquisition - live image - real time; capture and store a single 512 x 512 image to disk - 1 to 2 seconds
6) Time to capture a single image and deconvolve using a single image nearest neighbor algorithm - 5 to 10 seconds
7) Time to capture a stack of 25 images - 512 x 512 grayscale and write to disk - 25 to 30 seconds
8 ) Time to deconvolve a stack of 25 images 512 x 512 grayscale using nearest neighbor and DSP board - about 2 minutes
I hope this answers your questions
John Kesterson, Ph.D. VayTek, Inc. http://www.vaytek.com
We are looking for a TEM with (P)EELS mapping capability to study initiation of calcification if soft biological tissues. We need to detect calcium and map its distribution. EDS analysis does not seem to be feasible for us.
If you have or know of a lab in the continental USA that has this capability please get in direct contact with:
Barbara Ellingorth, (612)481-7562 St. Jude Medical, St. Paul, MN
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #3.0 ] --
Bob Citron wrote the following: ================================================== I am looking for a stain that is specific to a compound which is cationic, w/amine-type functional groups. I would like to be able to visualize the homogeneity of a coating on a substrate, (preferably by light microscopy, but we do have SEM and EDX). Someone recommended the use of TNBS (trinitrobenzenesulfonic acid, or picrylsulfonic acid), but I would like to think that there is something a little less hazardous to use (as well as a little less expensive to obtain). ============================================================= You would have to be a bit more specific as to the functionality of the "amine type functional groups". Are you talking about a good old fashioned "quat" (e.g. "Gaf-quat")? If you are talking about substrates like human hair or skin, which are the only substrates I have ever heard of quats being applied, generally the amount of coating, when realistically applied, there does not seem to be enough there to give any kind of "effect". We have only been successful in visualizing quats either by "Before" vs. "After" microscopy (SEM) on the same identical area (directly on hair or using replicas if on skin). And then it is not the coating itself but the effect of the coating that is being resolved. In some instances, you can see the deposition in cross- section TEM. These techniques were published in the early 1970's in the J. Society of Cosmet. Chemists. I don't have the exact references at my finger tips but I am sure you could find them. But the first work on hair was authored by DiBianca and the work on skin, by the undersigned.
Chuck
====================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: GVKM07A-at-prodigy. com West Chester, PA 19381-0656 USA Customer Service: spi2spi-at-2spi. com
Look for us! ############################ WWW: http://www.2spi.com ############################ =====================================================
---------- } From: Donald Lovett {lovett-at-trenton.edu} } To: Microscopy-at-Sparc5.Microscopy.Com } Subject: Horseraddish peroxidase/DAB } Date: Wednesday, July 03, 1996 3:13 PM } } } I am about to begin a study of whether certain intercellular spaces are } continuous with other spaces in crabs. All of my references suggest } using horseraddish peroxidase with DAB and H202, but they are 20+ years } old. Are there any more recent modifications of this technique? How } does this compare to Lanthanum or Ruthenium? Has anyone tried an } organism with hemocyannin? I would greatly appreciate any advice or updates. } } Thank you. } } ______________________________________________________________________ } Donald L. Lovett e-mail: lovett-at-trenton.edu } Assoc. Professor, Dept. of Biology voice: (609) 771-2876 } Trenton State College, NJ 08650-4700 fax: (609) 771-2674 }
Can anybody answer some technical questions about an Eikonix scanner.
Specifically: How does the calibration work? With our scanner it doesn't seem to make any difference how You run the CALIB program, with lights first on then off or vice versa, or on (off) both times.
Does anybody know whom to call for technical support? The company in Bedford MA doesn't exist anymore.
Philip -- Philip Koeck Karolinska Institutet Dept. of Bioscience Novum S-14157 Huddinge Sweden Tel.: +46-8-608 91 93 Fax.: +46-8-608 92 90 Email: Philip.Koeck-at-csb.ki.se
Message-Id: {199607161513.KAA14550-at-mailhub.iastate.edu} X-Sender: wes-at-pop.ameslab.gov X-Mailer: Windows Eudora Pro Version 2.1.2 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
Sounds like your shrinkage is rather small, 1/10th of 1%. That could very well affect the dimensions of your sample. But it is negligible compared to the precision of most measurements that I am familiar with. You probably get bigger errors elsewhere. A one pixel error measuring a feature with a 100 pixel diameter in a 1024 pixel-wide image will give you 1% error.
The shrinkage would ideally be the same in all directions, but can vary depending on what kind of constraints your samplke is under. We use 1" bakelite rings to embed samples and pour in about a 1/4" of epoxy. The rings don't move much and because the epoxy is thin in the vertical direction and because the top is a free surface, all the shrinkage (which isn't much) takes place in the vertical direction. (Some of my engineering mechanics background coming through here.) Like I said, it depends on your mold.
At 10:02 AM 7/15/96 +0000, you wrote: } I am casting the interior of a cylinder with an epoxy. The epoxy I am using } cures in 8 hours at room temperature and reaches a peak temperature of 82F. } The manufacturer quotes the shrinkage of this epoxy as .001in./in. I have the } following questions regarding shrinkage: } Does epoxy shrink the same in all directions (x&y vs z)? } Is there a good method for measuring shrinkage? } Would shrinkage alter the dimensions of a flexible material (draw in the sides } of my cylinder)? } Any references on this subject would be most appreciated. } Thank you for your help, ---------------------------------------------------- Warren E. Straszheim 270 Metals Development, Ames Lab/ISU, Ames IA, 50011 Phone: 515-294-8187 FAX: 515-294-3091
Dear Laszlo, I replied to you 7/13/96, but got my email back from the internet. Could not find your email address. I copy the microscopy forum in case you dont get this one. Emile
Subj: Re: No Subject
In a message dated 96-07-13 00:58:26 EDT, you write:
{ { Do you also sell used microscopes and other equipment? Right now I am looking for a used stereomicroscope, with zoom (or at least covering a wide range of magnifications, like x5-25), no cameraports needed, a "boomstand" would be a great plus. { {
Dear Laszlo,
I mainly service and repair microscopes. But good service does not have limit. Selling is also a service. I do help and sell sometimes microscopes. I actually serviced several microscopes at your location.
Reference: Tony Piazza 415/750-2167 Bldg2 room 436B
I can help you find a stereo microscope, with zoom on a boom stand.
} } Also, could you give some insigth about really cheap I mean low priced stereomicroscopes? I have seen several supply companies selling stereomicroscopes from Korea and from Russia for about $500. Are they any good? } }
Microscopes are like anything else. Cars, for example. You can buy a car for $500 and another for $100 000. You cannot expect from the $500 car the same result as from the other one !
If you were sombody buying a microscope to use it twice a year for ten minutes, I would sell you a $500 microscope.
However, if you think to use this microscope for routine work altmost every day, I would think, that a better and more expensive microscope might be a good investment.
Let me know, what do you think of it.
best regards, Emile Meylan SERCO Technical Services, Inc. 800/483-0508
In a message dated 96-07-16 09:22:11 EDT, you write:
{ { A reader wants to sell some equipment including a Zeiss oil immersion objective. Your guidance is appreciated. } }
Dear Elinor,
Ask your customer for a little more details. Or have him contact me. A Carl Zeiss immersion objective is very vague. It could be an objective of $300 or $4500.
Very often, the Carl Zeiss objectives have the part nr. written on iy.
Last fall I posted a message indicating my interest in purchasing a used Hitachi 600 TEM. The scope I had hoped to purchase was taken by another group before we could get funds approved. Please respond directly to me if you know of anyone wishing to sell their TEM. I really do not want other brands.
Thank you.
______________________________________________________________________ Donald L. Lovett e-mail: lovett-at-trenton.edu Assoc. Professor, Dept. of Biology voice: (609) 771-2876 Trenton State College, NJ 08650-4700 fax: (609) 771-2674
The Internet did not accept your email address. I send a copy to the microscope forum, in case you do not get this one. best regards, Emile
Subj: Re: LM - Need parts for Zeiss scope
In a message dated 96-07-13 10:14:54 EDT, you write:
{ { Are you involved exclusively with optical scopes, or do you service SEM's as well? If you service SEM's, I'd like to know more about your company. } }
Dear John,
I am only involved with Light microscopes. To service SEM's you would need the 100% support of the vendor of this microscope. Making my own company, I do also compete with this vendors, and some of them do not like it very much.
However, I have been working with the Company Carl Zeiss for many years. I still have a very good and very close relationship with them. If your service or parts problem is related to Carl Zeiss, let me know. I could maybe help you with the right contact.
The question has come up here at my facility as to whether anyone knows if there is anything in the literature or if anyone knows - if anything has been done to or with crosslinking formvar. Replies can be sent to me at the above address and if there is anything I will make a summary for the bulletin board. thank you in advance. Mei Lie Wong
Mei Lie Wong Department of Biochemistry HHMI-UCSF Ph. 415-476-4441 Fax 415-476-1902 email wong-at-msg.ucsf.edu
During the installation of a new instrument we had 60 cycle magnetic field problems. This was especially bad at the longer working distances.
The source turned out to be the electronics control unit of a cold cathode discharge (Penning) guage (Varian) which was located right next to the electron optical column.
Needless to say, we now operate the instrument with the guage off.
Joe Geller Geller MicroAnalytical Lab 426e Boston St. Topsfield, MA 01983-1216 508 887-7000
I consult to a place that does SEM for forensis and failure analyses. We are looking for an educational video on SEM and EDS that we can show visiting clients and use during technical demonstrations and presentations.
Does anyone have, know, or can sell a video along these lines. The audience will be for the most part college educated but in non-technical subjects. Videos from manufacturers are acceptable.
Thanks and please answer to email address: "robertco2-at-aol.com"
I have a couple of clients who want to coverslip some ground bone specimens. The bones were labeled in vivo with fluorescent compounds that incorporate into the matrix. During the specimen prep, the alcohol fixed bones are washed in xylene or acetone. Is there any reason not to use Permount to coverslip? In addition, if anyone has any experience looking at oxytetracycline, alizarin or calcein blue labeled bones and is willing to talk about it, I would appreciate if they sent me a direct reply and I will contact them. Thanks in advance.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
The Education Committee of MSA has a large collection of videos on microscopy. Among the videos are two fine videos on EDS by Dale Newberry. Information on the videos is part of the MSA website at www.msa.microscopy.com.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-bgsm.edu * **************************************************************
On Wed, 17 Jul 1996 RobertCO2-at-aol.com wrote:
} I consult to a place that does SEM for forensis and failure analyses. We are } looking for an educational video on SEM and EDS that we can show visiting } clients and use during technical demonstrations and presentations. } } Does anyone have, know, or can sell a video along these lines. The audience } will be for the most part college educated but in non-technical subjects. } Videos from manufacturers are acceptable. } } Thanks and please answer to email address: "robertco2-at-aol.com" } } Robert Sherman }
The Education Committee of MSA has a large collection of videos on microscopy. Among the videos are two fine videos on EDS by Dale Newberry. Information on the videos is part of the MSA website at www.msa.microscopy.com.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-bgsm.edu * **************************************************************
On Wed, 17 Jul 1996 RobertCO2-at-aol.com wrote:
} I consult to a place that does SEM for forensis and failure analyses. We are } looking for an educational video on SEM and EDS that we can show visiting } clients and use during technical demonstrations and presentations. } } Does anyone have, know, or can sell a video along these lines. The audience } will be for the most part college educated but in non-technical subjects. } Videos from manufacturers are acceptable. } } Thanks and please answer to email address: "robertco2-at-aol.com" } } Robert Sherman }
We ae contemplating the replacement of the tungsten filament on our SEM (Cambridge Stereoscan 360) by a LaB6 source. I met several users at the Summer School of SEM and Micronalysis at Louvain-la-Neuve (Belgium) who expressed different views. The "pro-LaB6" are delighted to use it. The "anti-LaB6" people emphasize on the poor stability of the electron beam and the mechanical brittleness of the source. Some have returned to the "old" tungsten filament. So, ex- or new LaB6 user, what is your opinion ?
Best regards,
Philippe Drouillon Solvay Research and Technology Brussels (Belgium)
Extra X400 information begins: Originator Name: Philippe (PDU) DROUILLON Org Units: AC : NOH : LC-AN001 Organisation: NOHX400DEC Domain: BE/RTT/SOLVAY Node.Userid: IBMX400.201986
Message Id: 75511181706991/348287 MHS Importance: Normal Sent by Name: Philippe (PDU) DROUILLON Org Units: AC : NOH : LC-AN001 Organisation: NOHX400DEC Domain: BE/RTT/SOLVAY Node.Userid: IBMX400.201986 Free Fmt Name: Philippe DROUILLON Phone Number: 3218 Subject: LaB6 filament on a SEM (message corrected) Recipients Name: INTERNET INTERNET Domain: GB/IBMX400/IBMMAIL Node.Userid: IBMMAIL.INTERNET Free Fmt Name: INTERNET INTERNET
We ae contemplating the replacement of the tungsten filament on our SEM (Cambridge Stereoscan 360) by a LaB6 source. I met several users at the Summer School of SEM and micronalysis at Louvain-la-Neuve (Belgium) who expressed different views. The "pro-LaB6" are delighted to use it. The "anti-LaB6" people emphasize on the poor stability of the electron beam and the mehanical brittleness of the source. Some have returned to the "old" tungsten filament. So, ex- or new LaB6 user, what is your opinion ?
Best regards,
Philippe Drouillon Solvay Research and Technology Brussels (Belgium)
Extra X400 information begins: Originator Name: Philippe (PDU) DROUILLON Org Units: AC : NOH : LC-AN001 Organisation: NOHX400DEC Domain: BE/RTT/SOLVAY Node.Userid: IBMX400.201986
Message Id: 34511181706991/348285 MHS Importance: Normal Sent by Name: Philippe (PDU) DROUILLON Org Units: AC : NOH : LC-AN001 Organisation: NOHX400DEC Domain: BE/RTT/SOLVAY Node.Userid: IBMX400.201986 Free Fmt Name: Philippe DROUILLON Phone Number: 3218 Subject: LaB6 filament on a SEM Recipients Name: INTERNET INTERNET Domain: GB/IBMX400/IBMMAIL Node.Userid: IBMMAIL.INTERNET Free Fmt Name: INTERNET INTERNET
I am looking to purchase a Bausch & Lomb eyepiece reticle which is no longer in production. It is described as a micrometer disc, PN 31-16-08. It has 100 lines divided into half units with longer lines at every 10 units. It is used to measure 0.001" at 100X.
If you are a source or know of a source, I would appreciate some help. Thanks in advance.
Do somebody have an expirience in a cutting the hard tissues by material science diamond knifes? Do they give an appropriate quality of the sections and how long they live? Thank you in advance, Elia Beniash
B&L microscopy was purcased by Cambridg Instruments which in turn was bought/merged with Leitz which chaned its name to Leica (they've had a major indentity thing over there since the American Optical, AO, Reichert Jung days). Any way their tel # is 800-248-0123. best of luck
Robert Schoonhoven Laboratory of Molecular Carcinogenesis and Mutagenesis University of North Carolina CB#7400, Rosenau Hall Chapel Hill, NC 27599-7400 Ph. 919-966-6343 Fax 919-966-6123
you asked } I have a couple of clients who want to coverslip some ground bone specimens. The bones were labeled in vivo with fluorescent compounds that incorporate into the matrix. During the specimen prep, the alcohol fixed bones are washed in xylene or acetone. Is there any reason not to use Permount to coverslip? {
A good reason not to use Permount is that in contains flourescent materials. Use one of the mounting medias that were developed for flourescent techniques. I cant help you with the request on bone staine but Cathy Sanderson (e-mail Histology-at-aol.com) has been working with a multitude of bone techniques for many years and you might want to e-mail her. regards,
Robert Schoonhoven Laboratory of Molecular Carcinogenesis and Mutagenesis University of North Carolina CB#7400, Rosenau Hall Chapel Hill, NC 27599-7400 Ph. 919-966-6343 Fax 919-966-6123
Hi, i'm using latex beads (polystyrene) as test objects for scattering experiments in EM. For comparing Monte Carlo calculations i need the exact composition data (in at%). My current data are 50% H, 50% C, density 1.049 g/ccm. Does anyone have different or more precise values?
does anyone know if there is a US representative and contact number for the manufacturer of Riber ion pumps?
thanks in advance
steve
Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University 5500 Campanile Dr. San Diego, CA 92182-4614 phone: (619)594-4523 fax:(619)594-5676 email:sbarlow-at-sunstroke.sdsu.edu
You may want to contact Dr. Charles Garber from SPI/Structure Probe. I have seen him comment many times on the listserver about materials science diamond knives and I believe he had also provided the type of information you are looking for. Unfortunately, I did not save his comments. You can reach him at:
spi2spi-at-2spi.com or you can get some good information from the SPI web site at http://www.2spi.com.
Good luck!
Best regards-
David Henriks TEL: 800-728-2233 (toll-free in USA) South Bay Technology, Inc. 714-492-2600 1120 Via Callejon FAX: 714-492-1499 San Clemente, CA 92673 USA e-mail: henriks-at-southbaytech.com http://www.southbaytech.com
Manufacturers of Precision Sample Preparation Equipment and Supplies for Metallography, Crystallography and Electron Microscopy.
Alan, Call Klarmann Rulings, Inc., (800) 252-2401. They make all kinds of eyepiece reticles. Measure the inside diameter of your eyepiece. This is who our Leica dealer usually orders eyepiece reticles from. These usually cost in the $50 range. We've purchased several different styles for Zeiss, Wild, Nikon and Leitz eyepieces.
Glen MacDonald Research Scientist Hearing Research Laboratories of the Virginia Merrill Bloedel Hearing Research Center Box 35-7923 University of Washington Seattle, WA 98195-7923 (206) 616-4156 glenmac-at-u.washington.edu
On Thu, 18 Jul 1996, Alan Stone wrote:
} I am looking to purchase a Bausch & Lomb eyepiece reticle which is no longer } in production. It is described as a micrometer disc, PN 31-16-08. It has } 100 lines divided into half units with longer lines at every 10 units. It } is used to measure 0.001" at 100X. } } If you are a source or know of a source, I would appreciate some help. } Thanks in advance. } } Alan Stone } Alan Stone } }
To: Microscopy ListSer {Microscopy-at-MSA.Microscopy.Com} *** Reply to note of 07/18/96 06:37
We have a Cambridge S-250, and have run LaB6 for about 14 years. We used to have some trouble with the "carbon leg" designed mount. I burned a couple out in a few hundred hours (tip still looked like new). Since going to Denka M3's, we've gotten 1 to 3K hours per x-stal. I just took out or 1st M7, and it was OK, but the x-stal bent over (way odd!), but that was after about 2500 hours. Our gun vacuum is an indicated 7x10-7 torr.
We like the high brightness and long life. We heat them up slowly, and recently began using the 1st saturation, not the 2nd saturation point, like we always have. Reportedly, that'll make for even longer life. The current stability looks good, so we'll stick with it.
W is a mess to clean up after, the life is short (~40 hours?), and the brightness is low. For about $700/LaB6 tip, there's no contest.
In message {n1374416961.90105-at-wiccmail.weizmann.ac.il} "csbeneas" writes: } Do somebody have an expirience in a cutting the hard tissues by material } science diamond knifes? Do they give an appropriate quality of the sections } and how long they live? Thank you in advance, Elia Beniash
I have used a materials science diamond knife, with a 55 degree cutting edge angle, to section epoxy resin embedded eggshell from chicken, turtle and platypus. I vacuum infiltrated the resin into the shell fragments and was able to get reasonably good ultrathin sections of about 80 nm. The chicken shell was the most difficult to section.
The larger knife edge angles, about 55 degrees, are important to give the edge more durability to increase their useful life, they are less likely to chip, even if it is diamond. Its difficult to give you a typical lifetime for these knives, as it all depends on how much it is used, with what care and how it is maintained. Usually, one does not pass a diamond knife around, just reserve it for yourself or a specific highly skilled individual who knows how to use it properly.
I've not sectioned bone, but would be interested to hear from anyone who has, whether for LM or EM, and what staning methods were used.
Gib Ahlstrand, MMS Newsletter Editor Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.crl.umn.edu
"MICROSCOPY & MICROANALYSIS 96" in Minneapolis, Minnesota, Aug.11-15, 1996
Could the following be put on your mailing list / bulletin board? Thanks! - Owen
TEM Automation: Post-doctoral position --------------------------------------
We have a two-year post-doctoral position available for a project involving the automation of TEM.
The EPSRC-funded project "Comprehensive automation of TEM" involves developing a computer-based image acquisition and control system for TEM, addressing not only the very high resolution mode but also other modes together with start-up and shut-down procedures, condenser stigmating, tilt-shift purity adjustment etc. as well as remote computing. We envisage 1024sq CCD as well as conventional TV image pickup, using 200kV and 400kV JEOL microscopes at the Departments of Materials Science & Metallurgy and of Chemistry, in a PC/Windows environment, probably using C++, perhaps Synoptics new IO package, and perhaps a high speed array processor.
The ideal person would have experience both of TEM and of programming; the latter is the more important requirement however. The post is available from 1st October 1996, but the start date could be deferred some months if necessary; salary on UK academic scales according to age and experience, up to point 7 (15,154 pounds pa currently).
Please contact any of the project investigators for more information: Dr W Owen Saxton Dept Materials Science & Metallurgy, Pembroke St, CB2 3QZ email wos1-at-cam.ac.uk Dr David M Holburn Dept Engineering, Trumpington St, Cambridge email dmh-at-eng.cam.ac.uk Dr Angus I Kirkland Dept Chemistry, Lensfield Rd, Cambridge, and JEOL UK Ltd, Silvercourt, Watchmead, Welwyn Garden City, AL7 1LT email aik10-at-cam.ac.uk
Those interested in the position should send a CV, list of publications, and covering letter to Owen Saxton by 31st August; electronic and paper submissions are both welcome.
Mr-Received: by mta SRVR05.MUAS; Relayed; Thu, 18 Jul 1996 14:54:00 -0400 Mr-Received: by mta SRVR05; Relayed; Thu, 18 Jul 1996 14:54:02 -0400 Mr-Received: by mta SRVR01; Relayed; Thu, 18 Jul 1996 14:58:05 -0400 Disclose-Recipients: prohibited
Senior Assistant/Associate Scientist
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In article drouillon-solvay-at-e-mail.com writes: } From: drouillon-solvay-at-e-mail.com } Date: Thu, 18 Jul 1996 05:16:54 EDT } Subject: LaB6 filament on a SEM } Hello, } We are contemplating the replacement of the tungsten filament on our SEM } (Cambridge Stereoscan 360) by a LaB6 source. } I met several users at the Summer School of SEM and micronalysis at } Louvain-la-Neuve (Belgium) who expressed different views. } The "pro-LaB6" are delighted to use it. } The "anti-LaB6" people emphasize on the poor stability of the electron beam and } the mehanical brittleness of the source. Some have returned to the "old" } tungsten filament. } So, ex- or new LaB6 user, what is your opinion ?
We have been using LaB6 off and on in our S360 for 5-6 years. Enough to say we have mixed feelings, not all positive by any means.
1. The ion pump system is fiddly to use, especially to get it started once air has got into it. The column isolation valve over the objective lens is prone to leaks on the O ring on the shaft and the shaft needs careful polishing. The O ring on the valve itself has atmospheric pressure working against it and needs to be precisely located to avoid leaks.
2. Careless users are especially prone to opening the isolation valve before the chamber has pumped down. That puts the S360 out of action for the rest of the day including the time it takes to repump and restart the ion pump.
3. The LaB6 emitter SEEMS to be rather mobile in the S360 gun. We had bad experiences with Denka M3 emitters. The beam would drift out of line over about 5 minutes. Entirely useless for EDS and pretty exasperating for capturing an image. Cambridge suggest cleaning the Wehnelt aperture every friday to reduce this drift but thats very fiddly indeed and actually doesn't help that much. Oddly enough the Cambridge Electron Beam Litho writer in Sydney uses Denka M3 emitters and they are superbly stable in that machine.
We have been using Kimball Lab6 mounts with better results but after a time drift will still occur. Right now we have a tungsten emitter in but we still use the Ion pump system so as to retain a consistent operating procedure, and to try for better emitter life.
Very similar results were noted in the S-360 at the Australian National University so it looks like a generic problem.
Kimball have done a lot of work with their LaB6 emitters in S-360 microscopes so I reckon they have the best backup if you have problems.
In the end we bypassed the problem by going to FESEMs and we no longer try to push the S-360 to the limit of performance.
====================================================== Weida Qian Department of Materials Science & Engineering Northwestern University Evanston, IL 60208
Hello Steve, There is a company in California which rebuilds Getter Pumps. They do great work and it costs less than buying a new pump. If you cannot wait for the rebuild, they have used pumps for sale. Here is the address:
Dunaway Stockroom Corp. 1305 Space Park Way Mountain View California 94043
Phone 800/446-8811 FAX 415/965-0764
Personally, I think you would do well using a better quality pump than Riber.
Good Luck. Alex Greene Scientific Instrumentation Services, Inc. Austin, Texas
At 10:17 AM 7/18/96 -0800, you wrote: } greetings all } } does anyone know if there is a US representative and contact number for the } manufacturer of Riber ion pumps? } } thanks in advance } } steve } } Dr. Steven Barlow } EM Facility/Biology Dept. } San Diego State University } 5500 Campanile Dr. } San Diego, CA 92182-4614 } phone: (619)594-4523 } fax:(619)594-5676 } email:sbarlow-at-sunstroke.sdsu.edu } } } }
Hi, i'm using latex beads (polystyrene) as test objects for scattering experiments in EM. For comparing Monte Carlo calculations i need the exact composition data (in at%). My current data are 50% H, 50% C, density 1.049 g/ccm. Does anyone have different or more precise values?
Thanks, Bernd ***********************
Interesting, you may not have to contend with only H & C. To avoid clumping of the particles the solution used is not superpure water. A little conductivity is required and usually sulfate groups are used to act on the surface of the latex particles.
Perhaps this will not affect your results, perhaps you can wash the particles with superpure water and accept some clumping. I suggest that you contact Duke Scientific Corp, in California; I only have their fax number, which is: 1 415 424 1158. They have done a lot of work on calibration processes using latex particles and should have all required information.
Jim Darley Probing & Structure Microscopy Supplies & Accessories
Phone +61 77 740 370 Fax: +61 77 892 313 A great microscopy site http://www.ultra.net.au/~pns/ ***********************
I want to add a few comments to the post from Dave King. Brightness of LaB6 tips is estimated at about 10 times that of tungsten. Normal lifetime should exceed 500 hours (not everyone gets the lifetimes Dave reported in his post, mainly because his vacuum is better than most).
We always recommend that LaB6 cathodes be operated at just "below" the second saturation for best performance.
We have a binder full of technical information on the Denka LaB6 cathodes which we sell which we would be happy to send to anyone (please e-mail me with your complete s-mail address).
Best regards, Steven E. Slap, Vice-President ******************************** Energy Beam Sciences, Inc. Adding Brilliance To Your Vision ebs-at-ebsciences.com http://www.ebsciences.com/ ********************************
Melvyn Dickson reported some problems with the use of LaB6 cathodes which I would like to address.
LaB6 cathode designs using tungsten wire for mounting the LaB6 crystal, like the Denka Model 3, will "drift" as the tungsten reacts to heat. For EDS, critical dimension analysis or line width measurement (or any other application where the beam needs to stay in one place for an extended period of time), I would recommend using an LaB6 cathode where the crystal is mounted on fixed molybdenum posts, such as the Denka Model 7. This design offers superior mechanical stability.
For most other SEM applications, the Model 3 should work well, and is, in fact, what LEO presently supplies installed in their new LaB6 instruments.
Best regards, Steven E. Slap, Vice-President ******************************** Energy Beam Sciences, Inc. Adding Brilliance To Your Vision ebs-at-ebsciences.com http://www.ebsciences.com/ ********************************
On your question reguarding LM or EM sectioning and staining of bone, I've done several procedures for LM with decalcified temporal bones of humans and rodents. I've embedded in medcast, and JB-4 and sectioned with a glass knife and I've embedded in parafin and parlodion (celloidin) and sectioned on a steel knife. For staining, toluidine blue gives a nice, metachromatic stain in JB-4, but not in medcast and hematoxylin/eosin gives a nice metachromatic stain in both parafin and celloidin [the hematoxylin formula that works the best is for Harris' hematoxolyn]. The medcast tissue was for EM study, but we were mainly interested in the inner ear tissue embedded in the bone, so I can't comment on how well the bone stained. We had no problem sectioning it on standard diamond knives, as it was decalcified. I've tried embedding the bones in medcast without decalcification (no vacuum) and sectioning on glass knives, but only the outermost millimeter of bone was infiltrated with resin, so the center portion just crumbled. Usually, we are interested in the tissue embedded in the bone and not the bone itself, so decalcification is the easiest way to go.
APB, I'm trying to fix, dehydrate and embed in paraffin, cells in a suspension of collagen and agarose. The agarose is giving me fits. I'm trying different concentrations of formalin, and have tried xylene and ethlyne glycol as transitions to wax. Two problems are occurring. 1) the center of the tissue is not embedding. 2) When sectioning the agarose shears and comes out of the paraffin. Any suggestions would be helpful, TIA
Steve Hendrix Wright State University s002swh-at-desire.wright.edu
Light microscopists, I need your help! Our lab has a Zeiss Axioplan microscope with DIC capability. The DIC has been used with transmitted light, but are the optics such that this will work with transmitted light as well? Also, can you Kohler illuminate in the reflected light mode?
Let me say in advance, that I appreciate your help in providing information regarding these questions. Thanks!
David McClellan Eli Lilly & Co. e-mail: DM-at-lilly.com
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In response to the recent string: ================================================================ } i'm using latex beads (polystyrene) as test objects for scattering experiments } in EM. For comparing Monte Carlo calculations i need the exact composition data } (in at%). My current data are 50% H, 50% C, density 1.049 g/ccm. Does anyone } have different or more precise values? (Bernd) ==========================================================} } Interesting, you may not have to contend with only H & C. } To avoid clumping of the particles the solution used is not superpure water. A } little conductivity is required and usually sulfate groups are used to act on } the surface of the latex particles. (Jim Darley) ========================================================= What is being referred to is the surfactant used during the polymerization of the latex emulsion. In the past, the surfactant was sodium lauryl sulfate, or (almost) plain ordinary "soap". Some emulsion chemists even refered to it not as a surfactant, but as the "soap". Today, there are more sophisticated surfactants used, but in any case, on a mass basis, the amount present is very low, down to fractions of a percent in composition. After all, it is was more than that, it would not be a surfactant. I have never heard of anyone having problems with their calculations with such small amounts of surfactant present in the suspension.
Of course, there are all kinds of latex emulsions, ones that DO have larger amounts of surfactant present, but the suspensions typically sold by those offering EM supplies, such as SPI, select suspensions that are very low in terms of surfactant concentration, in order to have "clean" looking latex spheres when viewed by TEM or SEM.
Chuck
====================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: GVKM07A-at-prodigy. com West Chester, PA 19381-0656 USA Customer Service: spi2spi-at-2spi. com
Look for us! ############################ WWW: http://www.2spi.com ############################ =====================================================
On December 12 and 13, 1996, the Belgian and Dutch Societies for Microscopy organise a joint meeting in Gent. You can find general information and the program at our wedsite at
(1) Zeiss West dry darkfield condenser with holder Z (0.75/0.85) (2) Zeiss West planopo 25x phase 3, or 25x planapo bright field objective (160mm) (3) Leitz Ortholux nosepiece
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It sounds to me as if incomplete infiltration is the problem, and the most likely cause of this is incomplete dehydration. Agarose is hygroscopic and will pick up water from "anhydrous" reagents that have taken on water from atmospheric humidity. You may want to add an additional absolute ethanol step and/or xylene step in your paraffin processing routine. Also, use fresh ethanol for the last absolute step and fresh xylene for the last xylene step. Using a desiccant like "t.h.e Desiccant" in your bottles of solvents will keep them water-free longer.
We would like to prepare cross section of metallic multilayers deposited on a MgO substrate using the tripode polishing technique. We are beginners in the tripode technique and our first attempts were not successfull due to the cleavage of MgO. Can anybody help us ?
We are interested in embedding a variety of connective tissue matrices, most from tissues including skin and cartilage, in OCT in preparation for cryostat sectioning. Our protocol in the past has been to freeze in "hexanes" cooled to just above its freezing point of -94 C in liquid nitrogen, store in hexanes at -70C, then embed in OCT and cool to -20 C prior to cryostat sectioning. We are concerned that there may be a freezing protocol resulting in more favorable structure. We are concerned that the freeze-thaw-freeze occuring as the tissue is frozen, then placed in OCT at ambient temp., then frozen again may not be optimum, even for LM.
Recently, we have considered the use of other freezing media, including isopentane. We've noticed that some other laboratories are embedding fresh tissue in OCT, then freezing the entire block in isopentane prior to cryostat sectioning.
As we make our choice in deciding how to proceed, perhaps others with experience in freezing tissue in preparation for LM immunocytochemistry might contribute opinions based on their experience. Our goal is for adequate stabilization, long term storage of unembeddedd tissue, and good adherence of tissue to OCT.
We are planning to buy a new cryostat soon and would like some input from current users of newer systems. (We are replacing a 1963 crystat!) Specifically, we are inclined toward the Zeiss HM505N, but have not found anyone in St. Louis using one that we could look at or talk to. People using the Zeiss HM500 can't say enough nice things about it, but it costs about $10K more than the HM505.
We are a research lab where it will get significant use, but not like in a path lab where it might be used around the clock. We cut mostly cochleas and eyecups, both of which are small, but suffer from mixed consistency-- part boney tissue, part soft tissue, part hollow spaces. Most of our sectioning is for immunohistochemistry, requiring 6-12 micron sections. St. Louis is a very humid city, so frost on the blade can be a problem for cryostats.
We are inclined toward a mechanical system since the motorized ones just sound like one more thing to go wrong. Most of the repairs we have heard about in newer systems have involved the electronics. Also the vacuum system looks like another "bell & whistle." Has anyone out there found it to worth an extra $2-3,000?
Most people we talked to said they liked the cryostat they have, but they would probably be happy with whatever they got used to using. The only exception was people from the lab with the Zeiss HM500 who said they liked it better than anything else they had tried and that they got consistently bettter sections. So, are there any users of Zeiss HM505's out there? If so, how do you feel about it?
I'm looking for a economically priced SEM to be located at a facility in the Boston area. The SEM would be used for 'general purpose' work, nothing special. I would like to attach my EDS system to it such that I can perform x-ray work. Should anyone know of something that might be available, please contact me.
David, I am not sure what the setup on your axiplan is but if you have the reflected light insert you can Kohler in reflected light but most transmitted light setups would be sold with the fluoresence insert which can not be kohlered as it lacks an aperture diaphram, you would also need to get the reflected light polarizer and 50% mirror reflector that into slot where the fluoresence filter slider may already be, again I would need to know more of your exact setup to comment, keep in mind that the optics for transmitted light are usually corrected for a coverslip, reflected light optics generally are not, this is more of a problem at 40x and higher as optical quality suffers and I am not sure how DIC quality would be, what are you trying to do and what exactly is on the microscope.
Scott E. Berman Advanced Imaging Concepts, Inc. (609) 921-3629
Some comments from Roger Heady, who runs the Cambridge S360 here:
We have used LaB6 exclusively for many years, we had some trouble in the early years but have consistently good results now. Our latest filament has 1372 hours on the clock and is still first class, not drifting at all. The aperture was cleaned at about 700 hours.
Some tips - We use Kimball Physics type ES 423E, style 90-20 These have carbon mounts, not tungsten wire. We use a 1500 micron aperture rather than the 1000 micron as recommended by Cambridge. When the filament image starts shifting, clean the aperture with acid: 1 part conc (36%) HCL to 4 parts water. Immerse, shaking, for 60 sec. Water rinse. Rinse in weal alkali (ammonia or NAOH), water rinse, alcohol rinse, dry uand use. No polishing.
Set the Kimball tip using an epi-illumination microscope to 125 micron below the front of the aperture plate, and take great care with the centring. ---------------------------------------------------------------------- Sally Stowe |Email: stowe-at-rsbs.anu.edu.au Facility Coordinator |Post: ANU Electron Microscopy Unit |ANUEMU (RSBS) Ph 61 6 249 2743 |Australian National Univ. FAX 61 6 249 4891 |Canberra, http://online.anu.edu.au/EMU/home.htm |AUSTRALIA 0200
Hello Doug, this is Bob Underwood with whom you freeze fractured with at the University of Wash, in Dr Holbrooks lab.
We routinely place fresh skin samples directly into OCT as soon as possible and freeze them in either a isopentane or ethanol and dry ice slush. Isopentane works better. The ethanol can make the OCT turn to rubber if it comes in contact with it. The blocks are stored in -70 freezer and some have been cut periodicly for 10 years. When using desposable knives, however, the tissue can detach from surrounding OCT. I haven't found anything better easier yet. But if you can cool the isopentane in LN2 it is better but sometimes unavailable.
Sorry to ask again, but I really need an EDAX 9900 keyboard! Just hoping someone out there that didn't see this message previously might know of someone who's retired their 9900.
Also, I'm interested in a small ISI scope. Miniscan 7, SX-30, SX-40, etc.
We routinely freeze brain tissue in our EM/Imaging lab. We take fixed tissue and run it through a sucrose series (10%, 20%, and 30%) to cryoprotect. We then place the tissue in OCT in a foil cup and place that on dry ice cooled with acetone. We then store that at -20'c until we section.
If you would like more details, let me know. We are having pretty good success.
Cheri Owen Detroit Neurotrauma Institute Detroit, Mi 48201 313-285-4027
On Mon, 22 Jul 1996, Doug Keene wrote:
} Fellow Microscopists: } } We are interested in embedding a variety of connective tissue matrices, most } from tissues including skin and cartilage, in OCT in preparation for cryostat } sectioning. Our protocol in the past has been to freeze in "hexanes" cooled to } just above its freezing point of -94 C in liquid nitrogen, store in hexanes } at -70C, then embed in OCT and cool to -20 C prior to cryostat sectioning. We } are concerned that there may be a freezing protocol resulting in more favorable } structure. We are concerned that the freeze-thaw-freeze occuring as the tissue } is frozen, then placed in OCT at ambient temp., then frozen again may not be } optimum, even for LM. } } Recently, we have considered the use of other freezing media, including } isopentane. We've noticed that some other laboratories are embedding fresh } tissue in OCT, then freezing the entire block in isopentane prior to cryostat } sectioning. } } As we make our choice in deciding how to proceed, perhaps others with } experience in freezing tissue in preparation for LM immunocytochemistry might } contribute opinions based on their experience. Our goal is for adequate } stabilization, long term storage of unembeddedd tissue, and good adherence of } tissue to OCT. } } Many Thanks, } } Doug Keenee } }
Is there a secret for embedding ligaments to look at collagen fibers? do I have to do vacuum infiltration and curing? Oh, by the way, this is for TEM. In all of my 30 years of doing EM I've never had a real infilteration problem with any type of tissue until now. I'm goin' nuts. I had 9 blocks to cut thin sections on and some of the blocks were O.K. meaning not the best but could get some type of section to look at. After facing on a Pyramitome, the blocks had a nice,smooth, glassy face. Some of the blocks seemed to disintigrate as they were being sectioned. Thicker sections (1.5u) didn't look that bad. Only the thin sections stank! I'm using Spurr embedding resin which I've never had a problem with. Maybe the gods of EM weren't smiling on me the day I embedded them. I did try extending infiltration time over a couple of days. All chemicals were fresh including alcohols. The only difference is I used ETOH for my 50:50, etc. I usually use propylene oxide. That was on order and hadn't come in yet. ETOH is supposed to good with Spurr embedding resin. Right? At least that's what I was told. I used ETOH in the past with no problem. Is P.O. better for ligaments? Is there a good procedure for embedding ligaments? Heeeeeeeeeeeeeeeeeeeeeeelp!
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plan-view and crosss-sectional specimen preparation from blanket thin films and buried layers on mostly Si and GaAs substrates; basic and high resolution imaging of the films and layers; feature measurements from the images; and printing / labeling of the images.
We expect a fast turn-around along with dependable and high quality service.
Please respond to me directly. I will provide a summary of the responses to the interested third parties. Thank you.
} Fellow Microscopists: } } We are interested in embedding a variety of connective tissue matrices, most } from tissues including skin and cartilage, in OCT in preparation for cryostat } sectioning. Our protocol in the past has been to freeze in "hexanes" cooled to } just above its freezing point of -94 C in liquid nitrogen, store in hexanes } at -70C, then embed in OCT and cool to -20 C prior to cryostat sectioning. We } are concerned that there may be a freezing protocol resulting in more favorable } structure. We are concerned that the freeze-thaw-freeze occuring as the tissue } is frozen, then placed in OCT at ambient temp., then frozen again may not be } optimum, even for LM. } } Recently, we have considered the use of other freezing media, including } isopentane. We've noticed that some other laboratories are embedding fresh } tissue in OCT, then freezing the entire block in isopentane prior to cryostat } sectioning. } } As we make our choice in deciding how to proceed, perhaps others with } experience in freezing tissue in preparation for LM immunocytochemistry might } contribute opinions based on their experience. Our goal is for adequate } stabilization, long term storage of unembeddedd tissue, and good adherence of } tissue to OCT. } } Many Thanks, } } Doug Keenee } Doug, I have been freezing skin equivalents made of collagen I and III in OCT (Miles) using isopentane for some time and have found storage of tissue to be the major obstacle to good histology. If you know someone with some cryobottle space or a cryofreezer try to store there. Typically my preps have good overall structure up to 3-4 months at -80, and now about a year in storage in a cryofreezer. If you have many different stains to run on the tissue you may want to section twice as many slides as you think you might need, fix in methanol or acetone for 10 min. at 4 C and then store the slides at -70 to -80 C. I found this method easier than freezing and thawing my original tissue each time I wanted to stain for something. Hope it Helps, Steve Hendrix s002swh-at-desire.wright.edu Rogosin Institute Ohio Branch }
I have received a letter from a VCE student needing some help for her= extended essay, here is her request - "I am a year 12 student studying the International Baccalaureate. For my= extended essay, I have chosed Canine Hip Dysplasia, and require either= electron micrographs or schematic diagrams of pectineal muscle hypotrophy,= or any myofibre hypotrophy, showing the difference between type 1 and type= 11 myofibres Thank you=20 Kristine Batchelor"
If anyone out there can help it would be much appreciated. I will forward= any information on to her Regards Joan Clark
} } To:microscopy-at-MSA.microscopy.com } } From:joan.clark-at-sci.monash.edu.au (Joan Clark) } } Subject:Help needed from VCE student } } } } I have received a letter from a VCE student needing some help for her } } extended essay, here is her request - } } "I am a year 12 student studying the International Baccalaureate. For my } } extended essay, I have chosed Canine Hip Dysplasia, and require either } } electron micrographs or schematic diagrams of pectineal muscle } } hypotrophy, or any myofibre hypotrophy, showing the difference between } } type 1 and type 11 myofibres } } Thank you } } Kristine Batchelor" } } } } If anyone out there can help it would be much appreciated. I will forward } } any information on to her } } Regards } } Joan Clark } } }
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} INSIDE SALES/TECHNICAL SUPPORT } } Diagnostic Instruments, a 29 yr. old supplier of high quality instruments } to the international microscope market, is looking for a professional, } self-motivated person with good communication skills for inside sales and } technical support. The ideal candidate will possess a BS in Biology, } Chemistry, or other technical degree. Our rapidly growing company will } present opportunities for advancement to outside sales and marketing } positions. Please send or e-mail your resume to: } } Diagnostic Instruments } Attn: Sales Dept. } 6540 Burroughs Sterling Hts., Mi 48314 } rpatten-at-diaginc.com Susanne Pignolet Brandom, Ph.D. MC Services 847-548-6522
MicroWorld Resources and News http://www.mwrn.com/
} To: Microscopy List } From: delannoy-at-welchlink.welch.jhu.edu (Michael Delannoy) } Subject: E-mail legalities } } } To: zaluzec-at-sparc5.microscopy.com } } From: delannoy-at-welchlink.welch.jhu.edu (Michael Delannoy) } } Subject: E-mail legalities } } } } } To: zaluzec-at-Microscopy.Com (Nestor J. Zaluzec) } } } From: delannoy-at-welchlink.welch.jhu.edu (Michael Delannoy) } } } Subject: E-mail legalities } } } } } } Nestor, } } } I am ready to submit the confocal vs deconvolution demo summary, and } } } am concerned about liabilities. Can I critique company products (we looked at } } } five different systems) and reveal our final decision without getting sued? } } } What are the guidelines and disclaimers to avoid legal harassment? I would } } } appreciate any info you have or sources I should contact. } } } Thank you. } } } } } } Mike D. } } } } }
If infiltration was the problem I'd expect to see something resembling a rubber erasor or jelly bean. Acetone works better for infiltration than ethanol, if you need to work without p.o.
We've had ocassional instances of Spurr's shattering like this. The problem seems to be bad bottles of DMAE. Our policy is to throw out the DMAE with each empty bottle of DER 736.
Good luck,
Glen MacDonald Research Scientist Hearing Research Laboratories of the Virginia Merrill Bloedel Hearing Research Center Box 35-7923 University of Washington Seattle, WA 98195-7923 (206) 616-4156 glenmac-at-u.washington.edu
I wish to understand the design and usage of the various grids available for TEM today, and so I would like to ask if anyone could direct me to references in the literature that pertain to the grids themselves, and the methods of preparing them for a specimen, whether the references be for common or specialized specimen supports, and in any area of TEM application.
I want to know things like: why they are the way they are (some aspects are intuitively understandable, but perhaps there are some interesting subtleties in the reasons); how they are typically used in practice; how they perform in the TEM (such as how they affect the results); and what the ideal specimen support in any given application might be. If anyone has information related to these questions that is not embodied in a published source, I would be happy to learn of it, too.
Cheers,
Cam
____________________________________________________________________________ Box 1932 Main Station T: 1 403 435 2167 Cameron Sorlie, President Edmonton, AB T5J 2P3 Canada F: 1 403 433 9376 General Microdevices, Inc. ----------------------------------------------- generalmicro-at-ccinet.ab.ca ______________________________________________________ Microtechnology products for science and industry
Dear All I am looking for an image intensified camera to fit a Philips 400/420. Does anyone have one that they don't use and would be willing to sell/donate
Many thanks
Chris
Chris Gilpin Biological Sciences Electron Microscope Unit G452 Stopford Building Oxford Road Manchester M13 9PT phone +44 161 275 5170 fax +44 161 275 5171
The last few years I have been involved in research on localized (near nanometer resolution) strain characterization using two-beam electron diffraction contrast imaging (an operational mode of transmission electron microscopy). See any book on transmission electron microscopy for the basics.
Part of this project was to develop a software package allowing for the analysis of strain fields of arbitrary geometry. This software (SIMCON) allows one to model a microscopic strain field with various mathematical methods (including finite elements) and subsequently verify this model by comparison with experimental observations in the TEM.
As of today we are releasing SIMCON as freeware, you can find out all details at the following address:
I am currently using DeltaGraph Professional for ternary plots of pyroxene endmember compositons. The software does not allow one to change the axis scale, thus tighly grouped points on a plot are unsightly at best. Is anyone aware of a softwar e program that will run in Windows 95 allowing one to manipulate the ternary plot axis?
We are beginning to look at a new computer and software. (The 286 just can't keep up!) Can anyone suggest software or strategies to record and manage data; ie cutomers, case ID, inventory, billing, grid location, etc.?
We would like to be able to find all information that we already have for a specific animal/tissue when they come in, for comparative reasons.
Does anyone know where I can purchase some phosphor powder? My last batch was from Levy West Laboratories Enfield and was purchased in 1978. It is marked "Luminescent material" and "Type 4CS70F". Does Levy West still exist and if so does anyone have a phone or fax number. Is this type of phosphor still available somewhere???? Thanks for any help you can give me.
Anne Esposito E.M. Connection e-mail: Silverstf-at-aol.com
When I use my modem for an internet connection my TX speed is about 1K/sec ave rate. Does anyone have insight into the reason for this speed reduction? My modem is 14.4K
Thank you Robb ============================================= \\\\|//// | ~ ~ | |(-at-)-(-at-)| Have a Great Day! | 0 | / | [___] | ooo0---- { } ---0ooo------------------------------- /*\ Robb Westby NORAN Instruments /*//\ 800.691.4610 voice mail pager \//*/ 608.828.4428 voice mail box \*/ 608.831.4461 fax internet robbwestby-at-noran.com http:\\www.noran.com -----------------------------------------------
I have TEM experience only with cells and tissue. Someone has asked me if I could show the structure of a pneumococcal polysacharide using TEM. I understand that proteins, DNA and viruses are studied using negative staining. Does anyone have information about the possibility of seeing a sugar molecule for the purpose of comparing two structural forms of it? Is there a negative stain? Thanks for any comments. Pat Masarachia e-mail pat_masarachia-at-merck.com Merck Research Labs West Point, Pa 19486 phone 215-652-7999
Regarding the recent postings about diffraction contrast simulations, one of the more recently developed contrast simulation programs around is MaComis, developed by Rene Rasmussen at Comisoft. It has a user friendly Macintosh interface, but I don't think it has the several advanced features that Alwyn Eades is looking for. I have an advanced beta version of the program but I am not sure if it ever made it out of beta and into the "marketplace". I have Comisoft's address as of early 1993, but I don't know if Rene is still running it or has moved on to other places and things. I would be happy to pass on my information regarding about MaComis and Comisoft to whomever is interested. However, I have no personal or commercial connection to McComis or Comisoft.
Roy Christoffersen Texas Center for Superconductivity 3201 Cullen Houston, TX 77204-5932 roy-at-bayou.uh.edu (713) 743-8273 FAX: (713) 743-2787
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A baud is one bit per second. A data byte is about 10 bits (7 or 8 data plus some start and stop bits).
Therefore, 1 K-byte per second is about 10 k-bit per second or 10K baud. And remember you have to leave some room for acknowledgements from the other end, response delays, etc. You actually seem to be doing pretty well.
At 03:00 PM 7/25/96 -0500, you wrote: } } When I use my modem for an internet connection my TX speed is about 1K/sec ave } rate. } Does anyone have insight into the reason for this speed reduction? } My modem is 14.4K ---------------------------------------------------- Warren E. Straszheim 270 Metals Development, Ames Lab/ISU, Ames IA, 50011 Phone: 515-294-8187 FAX: 515-294-3091
To: Microscopy Listserver Subscribers & MSA Members
Re: Late Breaking Poster Session for Microscopy & Microanalysis 96 August 11-15, 1996 Minneapolis, Minnesota
The deadline for receipt of abstracts for the Late Breaking Posters session of the Microscopy & Microanalysis -96 Meeting was set to be Friday, July 26 .
As I have completed processing of all the received applications, I have decided to extend the deadline a few more days to allow any individuals still interested in submitting a late breaking poster a last chance to present their results at the meeting. I will still accept applications and abstracts through 5pm local time next Tuesday (July 30th). You will be informed by Thursday August 1 of poster acceptance or rejection.
Information on submission is given below as well as on the Microscopy Society of America WWW Site
To submit a late breaking poster you must fill out a Data Form (available via the WWW http://www.msa.microscopy.com ) and prepare an Abstract as described in the Call for Papers ( or contact the Microscopy & Microanalysis office). Express Mail the completed Data Form and Abstract to:
Dr. Nestor J. Zaluzec, Program Chair Microscopy & Microanalysis '96 797 Bonnie Brae Ct Bolingbrook, IL 60440 USA
After your submission is reviewed, you will be contacted about acceptance and with instructions on when and where to display your poster. The EXTENDED deadline for receipt is: Tuesday JULY 30, 1996 5 pm local time.
Although Abstracts accepted as Late Breaking Posters cannot be published in the Proceedings, they will be listed on a special Meeting handout and in daily Meeting newsletters.
Hello all subscribers, Duodenal brush border vesicles - DBBV- and negative staining. We do negative staining of duodenal content of ostriches regularly as well as faeces samples of dogs, calves, etc to detect the presence of any possible virus particles eg. parvo, corona, picorna, rota, etc. However we experience some difficulties in diagnosing paramyxovirus (orthomyxo?) particles beyond doubt. The problem arises when we encounter paramyxovirus-like particles from low numbers to masses of them in a specimen BUT no sign of any helical nucleoprotein strands. How can we break up these particles to release any nucleocapsid strands if they are virus particles? We centrifuge 15ml suspension of samples at low speed (3000g, 15min) and then supernatant at high speed (20000g, 60min) and perform neg. staining on pellet as standard procedure. I have one reference on DBBV: Arch Virol 1987, 97:309-323 by Schnagl RD. et al but their one small micrograph is not adequate to compare with myxoviruses. Can anyone supply me with a micrograph of x100-150K magnification of DBBV (or any other reference) to compare with our own known micrographs of paramyxoviruses or give info on how to obtain DBBV easily(?) for negative staining. Q: What happens to the duodenal epithelial lining during a viral (bacterial) infection; can it shred structures (vesicles) in such vast numbers and do they have a viral-like appearance with negative staining? Any suggestions would be appreciated very much. Friendly greetings, Hercules Els EM Unit, Fav Vet Sci, Univ of Pretoria, Onderstepoort 0110 Rep of South Africa hjels-at-op1.up.ac.za
} I have TEM experience only with cells and tissue. Someone has asked me } if I could show the structure of a pneumococcal polysacharide using TEM. } I understand that proteins, DNA and viruses are studied using negative } staining. Does anyone have information about the possibility of seeing } a sugar molecule for the purpose of comparing two structural forms of it? } Is there a negative stain? Thanks for any comments. } Pat Masarachia } e-mail pat_masarachia-at-merck.com } Merck Research Labs } West Point, Pa 19486 } phone 215-652-7999 } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } Negative staining might give you some information. Just start trying the different ones and see how they look. Low angle metal shadowing of a thin dispersion of the material on a carbon or formvar coated grid should also show some structure or you may need to go alll the way to making carbon-Pt replicas.
If you could get access to a Zeiss energy filtering instrument with a cryo holder you might be able to view it in thin films of vitreous ice. ******************************************************* Greg Erdos Phone: 352-392-1295 Scientific Director, ICBR Electron Microscopy Core Lab 218 Carr Hall Fax: 352-846-0251 University of Florida E-mail: gwe-at-biotech.ufl.edu Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/
{} I have TEM experience only with cells and tissue. Someone has asked me {} if I could show the structure of a pneumococcal polysacharide using TEM. } } I understand that proteins, DNA and viruses are studied using negative
{Negative staining might give you some information. Just start trying the
{If you could get access to a Zeiss energy filtering instrument with a cryo {holder you might be able to view it in thin films of vitreous ice.
A GOOD negative stain prep is the way to start but vitreous ice and a cryo holder, as the above author suggests, is definitely the way to go if you can manage it. Although contrast with cryo is much less than that obtained with negative stain you don't generally have to worry about the effects of drying. I would, however, suggest a different scope than a Zeiss. Some of the other scopes on the market have a much better track record with cryo.
Sorry!! I am resending this message because something was incorrect in my Eudora configuration and I did not receive the replies. I would appreciate hearing again from the people that did respond.
I am currently using DeltaGraph Professional for ternary plots of pyroxene endmember compositons. The software does not allow one to change the axis scale, thus tighly grouped points on a plot are unsightly at best. Is anyone aware of a softwar e program that will run in Windows 95 allowing one to manipulate the ternary plot axis?
Some time ago someone asked about where to buy diffusion pump heaters. I have just come across a good source, and am giving it here for everyone who may have an interest in such info. It is: Dalton Electric Heating Co. 28 Hayward Street Ipswich, MA 01938-9978 Dalton advertises producing over 100 standard types of heaters, and a willingness to make custom heaters for obsolete or foreign-made pumps.
On Thu, 25 Jul 1996, EMLAB-at-vet.ksu.edu wrote: } We are beginning to look at a new computer and software. (The 286 } just can't keep up!) Can anyone suggest software or strategies to } record and manage data; ie cutomers, case ID, inventory, billing, grid } location, etc.? } } We would like to be able to find all information that we already have } for a specific animal/tissue when they come in, for comparative } reasons. } } ka } } } } ============================================= \\\\|//// | ~ ~ | |(-at-)-(-at-)| Have a Great Day! | 0 | / | [___] | ooo0---- { } ---0ooo------------------------------- /*\ Robb Westby NORAN Instruments /*//\ 800.691.4610 voice mail pager \//*/ 608.828.4428 voice mail box \*/ 608.831.4461 fax internet robbwestby-at-noran.com http://www.noran.com -----------------------------------------------
Yes, DeltaGraph Prof is capable to change the axis scale for ternary diagram. The procedure for this is
a. active the figure that you want to modify b. go to Chart (manu), then Axis, Value, Axis Attributes C. assign length and units for axis
} I am currently using DeltaGraph Professional for ternary plots of } } pyroxene endmember compositons. The software does not allow one to } change the } axis scale, thus tighly grouped points on a plot are unsightly } at best. Is } anyone aware of a softwar
Xiaogang
*********************************** * Xiaogang Xie * * SEM & Microprobe lab * * Dept. of Geology and Geophysics * * Louisiana State University * * Baton Rouge, LA 70803 * * Office (504)388-2240 * * Fax (504)388-2302 * ***********************************
Postdoctoral position available immediately to study the 3-D structure of insect flight muscle. The research project, which is funded through 1999, offers a unique opportunity to correlate 3-D electron microscopy, mechanics, X-ray diffraction and atomic modeling of different physiological states including AMPPNP and AMPPNP-glycol treated muscle, freeze-substituted quick-frozen contracting muscle, Drosophila wild-type and mutant flight muscle, relaxed muscle, etc. Several experimental approaches including electron microscope tomography and oblique section reconstruction for producing 3-D images, alignment and classification of 3-D crossbridge structures and fitting of atomic coordinates of actin and myosin S1 into the envelope of 3-D images obtained by 3DEM. The position involves primarily computer processing of electron micrographs and molecular modeling. Salary dependent on years of relevent postdoctoral experience.
Our laboratory is part of the Structural Biology Program at Florida State University. The Institute of Molecular Biophysics is strategically located between the Biology and Chemistry Buildings. Abundant opportunities are available to develop new collaborations and investigate new scientific questions. For more information check out the institutes web page at http://www.sb.fsu.edu. Our laboratory has Silicon Graphics workstations and a Perkin-Elmer PDS 1010M microdensitometer. Electron microscope facilities include a Philips CM300-FEG and CM120 electron microscopes equipped with Gatan and Oxford Instruments cryostages. Interested applicants should send their CV and names, addresses and phone numbers of 3 references to Dr. Kenneth A. Taylor, Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-3015, USA. My E-mail address is taylor-at-sb.fsu.edu. Phone number 1-904-644-3357, FAX 1-904-561-1406.
Kenneth A. Taylor, Ph.D. Phone: 904-644-3357 Institute of Molecular Biophysics Fax: 904-561-1406 Florida State University E-mail: taylor-at-sb.fsu.edu Tallahassee, FL 32306-3015 Home pages: http://www.sb.fsu.edu/~taylor/ http://www.fsu.edu/~biology/faculty/kat.html
Just to follow up on my previous comments, and to add to a couple of the others, some work has been done on staining of cryo-fixed specimens, which helps with the low contrast of cryo-fixed material. I can't remember the details of the technique - I guess you actually have to stain the specimen before cryo-fixation - but I think that a tin compound is used.
Some type of electron spectroscopic imaging would also help with the contrast, the options are either a bolt-on imaging spectrometer, such as the one Gatan produce, or the in-coloumn energy filter. Until recently, Zeiss were the only option here. However, Philips have just launched a version of their CM120 which has an in-column filter. I'm sure they would like some nice applications problems for promoting it - I'd give them a call and see if you can get some free microscope time!
Comments: Authenticated sender is {vaihihbt-at-mailhost.rz.ruhr-uni-bochum.de}
Dear Microscopists
We are only just beginning to use LR White as embedding medium for use in EM- immunocytochemistry. A manual by AGAR SCIENTIFIC LTD. distributed through PLANO recommends to bring the specimen from 70 % EtOH into LR White/70 % EtOH (2:1) before embedding in pure LR White. We find that this mixture very quickly separates into two phases. How shall we handle this? Furthermore the specimen seems to be cured insuffiently even after 24 hours of polymerisation at 55 C. Any comments on this?
Thanks for any inputs
************************************************************** Hans-Martin Vaihinger Ruhr-University of Bochum Comparative Endocrinology Research Section Building ND 5/37 44780 Bochum GERMANY ********************************************************* phone ++49 234 700 4329 fax ++49 234 709 4551 email Hans-Martin.Vaihinger-at-RZ.Ruhr-Uni-Bochum.de
I would like to thank all of you who responded about my problem with the infiltration of ligaments. It's good to here that others are having problems with the Spurr embedding kits. I used fresh chemicals and this was the first problem I've ever encounterd using this resin. Araldite 502 has never let me down, maybe I'll continue to use 502 and wait until the problems are sorted out with Spurr's embedding resin. Again..... THANKS!
We have used microsoft access for a number of years and have found it to be excellent for data management, storage of block information, etc etc.Its compatibility with other software packages also makes it useful, its worth having a look at.
} Dear Microscopists } } We are only just beginning to use LR White as embedding medium for } use in EM- immunocytochemistry. A manual by AGAR SCIENTIFIC LTD. } distributed through PLANO recommends to bring the specimen from } 70 % EtOH into LR White/70 % EtOH (2:1) before embedding in pure LR } White. We find that this mixture very quickly separates into two } phases. How shall we handle this? } Furthermore the specimen seems to be cured insuffiently even after } 24 hours of polymerisation at 55 C. Any comments on this?
The 70% EtOH needs to be made up freshly just before use. Even stored in a tightly stoppered bottle, the concentration of the EtOH seems to decrease. This solved my similar problems.
Diana van Driel Dept Ophthalmology Sydney University C09 AUSTRALIA 2006
As part of our organization's efforts to attain ISO9000 certification, I will soon be faced with the task of preparing our electron microprobe laboratory for a preliminary audit.
I would like to correspond with anyone who has been throught this experience.
On Fri, 19 Jul 1996 s002swh-at-desire.wright.edu wrote:
} Date: Fri, 19 Jul 1996 19:12:30 -0500 } From: s002swh-at-desire.wright.edu } To: Microscopy-at-Sparc5.Microscopy.Com } Subject: RE: LM Tissue Embedding } } APB, } I'm trying to fix, dehydrate and embed in paraffin, cells in a } suspension of collagen and agarose. The agarose is giving me fits. I'm } trying different concentrations of formalin, and have tried xylene and } ethlyne glycol as transitions to wax. Two problems are occurring. 1) the } center of the tissue is not embedding. 2) When sectioning the agarose } shears and comes out of the paraffin. } Any suggestions would be helpful, } TIA } } Steve Hendrix } Wright State University } s002swh-at-desire.wright.edu } Infiltration into cells that have been embedded in agar and then aldehyde-fixed is difficult to impossible. We don't do wax, rather epoxiy and acryllic for electron microscopy and have run into this problem. We get around it by fixing the cells in glutaraldehyde, pelleting them, then encasing them in agar, and finally going through the other fixes and dehydrants.} } }
Sara E. Miller, Ph. D. P. O. Box 3020 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-8735
Oxford Instruments, Microanalysis Group, a leading manufacturer of x-ray microanalysis instrumentation, has an immediate opening for an Applications Engineer experienced in Wavelength Dispersive X-ray Spectrometry to join our support team. This exciting position is based in our West Coast facility in Fremont, CA.
The varied role will provide: customer demonstrations and applications, customer training, technical support of the sales teams, product testing and all other aspects of effective customer support. Some domestic and foreign travel will be required.
The successful candidate will have a minimum of 2 years experience in wavelength dispersive spectrometry (microanalysis or XRF) and a degree in the physical sciences. Excellent verbal and written communication skills, and a professional, outgoing personality are a must. Additional experience working with SEMs, EDS analysis, PCs and Microscopt Windows is desirable.
Please contact hr-at-ca.oxford.usa.com for an application or fax your resume to:
Oxford Instruments, Inc. Microanalysis Group Attn: Marketing Dept. 45950 Hotchkiss Street Fremont, CA 94539 Fax: 510/656-8944
We are beginning to look at a new computer and software. (The 286 just can't keep up!) Can anyone suggest software or strategies to record and manage data; ie cutomers, case ID, inventory, billing, grid location, etc.?
We would like to be able to find all information that we already have for a specific animal/tissue when they come in, for comparative reasons.
On Thu, 25 Jul 1996 generalmicro-at-CCINET.AB.CA wrote:
} Date: Thu, 25 Jul 1996 03:44:25 -0600 } From: generalmicro-at-CCINET.AB.CA } To: microscopy-at-Sparc5.Microscopy.Com } Subject: TEM - grids/specimen supports } } } Hello, } } I wish to understand the design and usage of the various grids available } for TEM today, and so I would like to ask if anyone could direct me to } references in the literature that pertain to the grids themselves, and the } methods of preparing them for a specimen, whether the references be for } common or specialized specimen supports, and in any area of TEM } application. } } I want to know things like: why they are the way they are (some aspects are } intuitively understandable, but perhaps there are some interesting } subtleties in the reasons); how they are typically used in practice; how } they perform in the TEM (such as how they affect the results); and what the } ideal specimen support in any given application might be. If anyone has } information related to these questions that is not embodied in a published } source, I would be happy to learn of it, too. } } Cheers, } } Cam } } ____________________________________________________________________________ } Box 1932 Main Station T: 1 403 435 2167 } Cameron Sorlie, President Edmonton, AB T5J 2P3 Canada F: 1 403 433 9376 } General Microdevices, Inc. ----------------------------------------------- } generalmicro-at-ccinet.ab.ca } ______________________________________________________ } Microtechnology products for science and industry } Cam, } There is information on grids, support films, preparation/use of, etc. in "Negative Staining" by MA Hayat and SE Miller, Mc Graw-Hill Publishing Co., Hew York, 1990.
Sara E. Miller, Ph. D. P. O. Box 3020 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-8735
} Date: Tue, 23 Jul 1996 16:14:36 -0400 (EDT) } From: rutledge phil {prutle1-at-gl.umbc.edu} } To: microscopy {Microscopy-at-Sparc5.Microscopy.Com} } Subject: collagen } } Microscopists: } } Is there a secret for embedding ligaments to look at collagen fibers? } do I have to do vacuum infiltration and curing? Oh, by the way, this is } for TEM. In all of my 30 years of doing EM I've never had a real } infilteration problem with any type of tissue until now. I'm goin' nuts. } I had 9 blocks to cut thin sections on and some of the blocks were O.K. } meaning not the best but could get some type of section to look at. After } facing on a Pyramitome, the blocks had a nice,smooth, glassy face. } Some of the blocks seemed to disintigrate as they were being sectioned. } Thicker sections (1.5u) didn't look that bad. Only the thin sections } stank! I'm using Spurr embedding resin which I've never had a problem } with. Maybe the gods of EM weren't smiling on me the day I embedded them. } I did try extending infiltration time over a couple of days. All } chemicals were fresh including alcohols. The only difference is I used ETOH } for my 50:50, etc. I usually use propylene oxide. That was on order and } hadn't come in yet. ETOH is supposed to good with Spurr embedding resin. } Right? At least that's what I was told. I used ETOH in the past with no } problem. Is P.O. better for ligaments? Is there a good procedure for } embedding ligaments? Heeeeeeeeeeeeeeeeeeeeeeelp! } } Thanks, } } Peace, } } Phil 8-{( } I don't know anything about ligaments, so I may be all wet, but we've cut some pretty rubbery stuff. I can answer only some of your questions. Yes, ethanol works fine with Spurr. After a graded series, we use 95% 2 X, then 100% 3 X. The secret is that the ethanol must be DRY. If it's not a new bottle, you should put in some drying beads (molecular sieves, made of diatomaceous earth, I think). We leave the beads in the bottle, and every time we empty it ~200 ml), we pour out the beads and bake them. CAUTION: Make sure the ethanol has evaporated completely before baking, or the whole lot will explode, sending beads everywhere! (Experience speaking.) One variable I can't comment on is the length of time in each change. We use 10 min for cell monolayers and 20-30 min for tissues and agar-embedded cells. You might want to try a little longer, if you're having trouble with dehydration. Then we use the anhydrous ethanol mixed 50:50 with resin, followed by 100 % resin 2 X. Again, the time will vary with the tissue. For cells, we use 30 min, and for tissues, 30-60 min, depending on the tissue and its size.
To salvage your already-embedded tissue, you might want to bake it some more (another 3-6 hr), try cutting a little thicker sections (100-120 u), and picking them up on Formvar-coated grids. If the sections fall apart in the boat after floating a while, cut only 1 or 2 sections and then pick them up on film-coated grids. Finally, carbon-coat the sections in a vacuum evaporator to stabilize them in the beam.
Good luck. Sara
Sara E. Miller, Ph. D. P. O. Box 3020 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-8735
Mime-Version: 1.0 Larry Stoter {LPS-at-teknesis.demon.co.uk} Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit Content-Description: cc:Mail note part
Dear All, The information Larry gives is not quite correct; Philips have announced the launch of a "Biofilter" microscope, however it is not an in-column filter. For those of you interested please attend the Philips booth at both Eurem and MSA for demo's and "free microscope time". Suffices to say that the concept is quite revolutionary!
Sincerely, Jan Ringnalda, Sr. Application Specialist, Philips Electron Optics, Mahwah, NJ 07430
Just to follow up on my previous comments, and to add to a couple of the others, some work has been done on staining of cryo-fixed specimens, which helps with the low contrast of cryo-fixed material. I can't remember the details of the technique - I guess you actually have to stain the specimen before cryo-fixation - but I think that a tin compound is used.
Some type of electron spectroscopic imaging would also help with the contrast, the options are either a bolt-on imaging spectrometer, such as the one Gatan produce, or the in-coloumn energy filter. Until recently, Zeiss were the only option here. However, Philips have just launched a version of their CM120 which has an in-column filter. I'm sure they would like some nice applications problems for promoting it - I'd give them a call and see if you can get some free microscope time!
We have a Hitachi H9000NAR TEM that we need to sell. It is a 300kV, 1.8A resolution, +/-15deg sample tilt high-resolution scope.
Included with the scope are * single-tilt sample rod, * double-tilt sample rod, * single-tilt heating stage, * turbo-pumped sample rod storage device, * Gatan 622 video camera, * Haskris water chiller.
The scope has been continuously under service contract and is in excellent condition. Asking price is $420,000.
Henk Colijn colijn.1-at-osu.edu OSU Campus Electron Optics Facility ------------------------------------------------------------------- Murphy's Law: If anything can go wrong, it will. Commentary: Murphy was an optimist.
Know anyone who would enjoy some practice at focusing and astigmating a high resolution TEM, but doesn't have one at hand? Want to show someone in your office or classroom what happens to the CTF as a function of defocus? Our browser-interactive simulator at {http://www.umsl.edu/~fraundor/epc/index.html} is now up.
You might also contribute to others' learning, by finding Scherzer defocus or modeling the CTF from images, and then sharing your strategy and results with others who access the page. You even vote with your feet, since use patterns as well as your comments will be monitored to help decide what new "specimens" and/or "scope models" we might host in days ahead.
As part of our organization's efforts to attain ISO9000 certification, I will soon be faced with the task of preparing our electron microprobe laboratory for a preliminary audit.
I would like to correspond with anyone who has been throught this experience.
unsubscribe -- dr. Marc D'Olieslaeger Coordinator Analysis Department Materials Physics Division Institute for Materials Research Limburgs Universitair Centrum Wetenschapspark 1 3590 Diepenbeek Belgium tel. +32-11-26.88.26. tel +32-11-26.88.15. (direct line) fax +32-11-26.88.99. email : mdoliesl-at-luc.ac.be
I am getting confused and nervous (about price) of deconvolution. We are putting together a deconvolution system for a Nikon SA upright, some type of cooled CCD monochrome camera (haven't decided which) on a PowerMac platform.
We already have the microscope and are committed to PowerMac. We have contacted several companies that can provide the whole system, therefore:
1. I would love to hear from anyone who has had experience in putting such a system together, with warnings and recommendations.
2. I'm confused about the various terms describing deconvolution:
EPR algorythem Nearest Nieghbor Inverse fourier analysis constrained iterative Poisson point model PSF deconvolution 3D blind deconvolution
Is there somone who could simplify this or knows of a good reference that clarifies the methods?
I am a grad student working on the development of a technique to local ize "antifreeze" gene expression in winter flounder gill epithelium. So far, I have been successful in describing the spatial dynamics of expression using non-isotopic digoxigen probes and a HRP/DAB marker. My problem lies in attempts to pin point the specific cell types that are expressing. In the development of my protocoal, I took whole gill filaments and processed them for insitu hybridization, getting a nice pattern of expression. My next step was to embed this tissue in resin and cut semi-thin and ultra-thin sections which were subsequently scoped to theoretically reveal staining in association with the involved cells. Unfortunately, I cannot detect any diff erences between experimentals and controls, infact no staining is detectable at all. The question is, what is happening to the DAB staining product and why can't I see it in section? Any ideas??
I found that without calibration we had dramatic lines running through the images. These lines seemed to be from the chip; they were in fixed locations. The calibration on a blank piece of film and without saturation solved this problem. Compared to background subtraction, the images appeared the same. -Michael Cammer
On Tue, 16 Jul 1996, Philip Koeck wrote:
} Can anybody answer some technical questions about an Eikonix scanner. } } Specifically: How does the calibration work? } With our scanner it doesn't seem to make any difference how You run } the CALIB program, with lights first on then off or vice versa, } or on (off) both times. } } Does anybody know whom to call for technical support? } The company in Bedford MA doesn't exist anymore. } } Philip } -- } Philip Koeck } Karolinska Institutet } Dept. of Bioscience } Novum } S-14157 Huddinge } Sweden } Tel.: +46-8-608 91 93 } Fax.: +46-8-608 92 90 } Email: Philip.Koeck-at-csb.ki.se }
You can find brief discussions of the design of TEM specimen grids in: Techniques for Electron Microscopy, D. H. Kay, Editor. 2nd. Ed, Blackwell Scientific Pubs. (and F. A. Davis) 1965, Ch. 3, and in The Principles & Practice of Electron Microscopy, by Ian M. Watt. Cambridge Univ. Press 1985, p. 82.
} I have TEM experience only with cells and tissue. Someone has asked me } if I could show the structure of a pneumococcal polysacharide using TEM. } I understand that proteins, DNA and viruses are studied using negative } staining. Does anyone have information about the possibility of seeing } a sugar molecule for the purpose of comparing two structural forms of it? } Is there a negative stain? Thanks for any comments.
If the polysacharride is extracellular, ie as a bacterial capsule, then preparative technique is critical. Bacterial surface capsules, slime layers, etc. are highly hydrated, and when negative stained and dried, they condense down into an amorphous mass. This even happens during dehydration for embedment and sectioning. There are several methods for stabilizing capsules to quantitatively assess their distribution, extensiveness, etc. The most straightforward that I have used uses cationized ferritin to stabilize it prior to fixation and embedment. Ruthenium red also may be used, but in my experience is not as satisfactory. A good starting reference is:
Jacques et al. 1988. J. Bacteriol. 170(7). pp 3314-18.
Good luck.
-=W.L. Steffens=- Department of Veterinary Pathology College of Veterinary Medicine University of Georgia
Hi All. I have been having a bit of a problem with some stray EM (?) fields on our SEM. The problem has been traced to the power feeding the wall outlets. Anything plugged into these outlets and the microscope will affect the images produced. Specifically the backscatter detector and the PC we use to capture images digitally. The results are the same for images captured photographically if either the backscatter or PC is plugged into the wall. They are not the cause of the problem but simply feed the noise to the microscope via any cables connected to it. If I unplug all from the outlets or unplug cables leading to the microscope the problem is corrected. It doesn't matter if they are on or not, it still feeds the noise.
We have so far simply run extension cords to other outlets in other rooms and on other circuits in order to solve this problem but it has worsened and I am running out of different outlets. I recently purchased an APC line conditioner which is supposed to continuously condition the power and give out a fresh signal but needless to say it hasn't worked or I wouldn't be asking for your help.
If you would like to see an image with the problem go to the web address at the bottom of this message and look in "What's New". You will notice the vertical banding pattern produced.
Your help and expertise will be greatly appreciated. Vendors please jump in with any advise you may have.
Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 904-392-1295 ICBR EM Core Lab fax 904-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
} } Help, } } I am a grad student working on the development of a technique to local } ize "antifreeze" gene expression in winter flounder gill epithelium. So far, } I have been successful in describing the spatial dynamics of expression using } non-isotopic digoxigen probes and a HRP/DAB marker. My problem lies in } attempts to pin point the specific cell types that are expressing. } In the development of my protocoal, I took whole gill filaments and processed } them for insitu hybridization, getting a nice pattern of expression. My next } step was to embed this tissue in resin and cut semi-thin and ultra-thin } sections which were subsequently scoped to theoretically reveal staining in } association with the involved cells. Unfortunately, I cannot detect any diff } erences between experimentals and controls, infact no staining is } detectable at all. The question is, what is happening to the DAB staining } product and why can't I see it in section? Any ideas?? } } H. Murray } } Is the tissue osmicated? Are the sections counterstained? If so the DAB may be lost visually. You may try a thicker section and veiw them with a lower accellerating voltage at the scope in order to increase the contrast between any electron dense DAB and tissue. Without counterstaining.
} Duodenal brush border vesicles - DBBV- and negative staining. } We do negative staining of duodenal content of ostriches regularly as } well as faeces samples of dogs, calves, etc to detect the } presence of any possible virus particles eg. parvo, corona, picorna, } rota, etc. However we experience some difficulties in diagnosing } paramyxovirus (orthomyxo?) particles beyond doubt. The problem arises } when we encounter paramyxovirus-like particles from low numbers to } masses of them in a specimen BUT no sign of any helical nucleoprotein } strands. How can we break up these particles to release any } nucleocapsid strands if they are virus particles?
These "virus-like" particles that you describe are a source of much debate among those of us who look at negative-stained fecal preps. I find them routinely in ostriches, emus, and turkeys, both ill and clinically healthy birds. If you suspect infection with a pleomorphic, enveloped virus, such as coronavirus or paramyxovirus, these things really are a nuisance. I don't believe that there is any known way of getting rid of them, short of lysis in distilled water. This is not completely effective, as most of them are already in lysis to some extent.
} Can anyone supply me with a micrograph of x100-150K magnification of } DBBV (or any other reference) to compare with our own known } micrographs of paramyxoviruses or give info on how to obtain DBBV } easily(?) for negative staining.
See Goodwin et al. 1995. Avian Pathology 24(3) pp 497-505.
} Q: What happens to the duodenal epithelial lining during a viral } (bacterial) infection; can it shred structures (vesicles) in such vast } numbers and do they have a viral-like appearance with negative } staining?
Gut is constantly turning over enterocytes, with the old ones being shed as "effete" enterocytes. These are basically membrane ghosts, which explains why you often see them in both healthy and ill animals. The glycocalyx on the membrane surface when negative stained often has a fuzzy or spiked appearance, and is very easily confused with enveloped viruses. Their wide range in size and surface projection morphology is what I use to distinguish them from real viruses.
-=W.L. Steffens=- Department of Veterinary Pathology College of Veterinary Medicine University of Georgia
I have a new Critical Pt. Dryer from Denton Vacuum. I have not used a critical point dryer before and the configuration of this one is confusing. I would appreciate hearing from anyone with a similar model who would be willing to talk me through a first run.
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hans-martin.vaihinger-at-rz.ruhr-uni-bochum.de wrote: } } Dear Microscopists } } We are only just beginning to use LR White as embedding medium for } use in EM- immunocytochemistry. A manual by AGAR SCIENTIFIC LTD. } distributed through PLANO recommends to bring the specimen from } 70 % EtOH into LR White/70 % EtOH (2:1) before embedding in pure LR } White. We find that this mixture very quickly separates into two } phases. How shall we handle this? } Furthermore the specimen seems to be cured insuffiently even after } 24 hours of polymerisation at 55 C. Any comments on this? }
I am having exactly the same experience with LR White and would appreciate seeing the replies. (I have started going to 95% ethanol in order to get good mixing).
Thanks!
Karen Zaruba E-mail: kszaruba-at-mmm.com 3M Company Phone: (612) 737-2971 St.Paul, MN USA
Subject: Time: 3:55 PM OFFICE MEMO RE:Vac Mail Date: 7/30/96
Mary: I have faced the problem you refer to rather frequently when I have to be out of town on consulting trips and vacation. To a large extent what you do will depend on the situation for your local computing system, which is the one that has to handle incoming mail while you are away. If the system has the capacity to do so, you can simply let the messages accumulate in your local receiver system, and then go over them when you get back. I have done this when I have been away for as much as a month, and I have only gotten warning messages saying I had exceeded the allowed number of stored messages, but our system is large enough so that nothing ever seemed to get discarded. If your system has limited capacity it will probably become overloaded and start discarding incoming messages (but then you'll lose them anyway if you follow the suggestion in the next paragraph). I suggest you check with your local system operator on this matter. There may be some optimum protocol you can follow to minimize stress all around.
If you don't like this kind of an approach you can unsubscribe from the listserver before you leave, and then subscribe again when you get back. Basicaly, however, the problem is not one of concern to the listserver, but one involving what goes on at the receiving end of the listserver operation in your own institution.
On Thu, 25 Jul 1996 generalmicro-at-CCINET.AB.CA wrote:
} Date: Thu, 25 Jul 1996 03:44:25 -0600 } From: generalmicro-at-CCINET.AB.CA } To: microscopy-at-Sparc5.Microscopy.Com } Subject: TEM - grids/specimen supports } } } Hello, } } I wish to understand the design and usage of the various grids available } for TEM today, and so I would like to ask if anyone could direct me to } references in the literature that pertain to the grids themselves, and the } methods of preparing them for a specimen, whether the references be for } common or specialized specimen supports, and in any area of TEM } application. } } I want to know things like: why they are the way they are (some aspects are } intuitively understandable, but perhaps there are some interesting } subtleties in the reasons); how they are typically used in practice; how } they perform in the TEM (such as how they affect the results); and what the } ideal specimen support in any given application might be. If anyone has } information related to these questions that is not embodied in a published } source, I would be happy to learn of it, too. } } Cheers, } } Cam } } ____________________________________________________________________________ } Box 1932 Main Station T: 1 403 435 2167 } Cameron Sorlie, President Edmonton, AB T5J 2P3 Canada F: 1 403 433 9376 } General Microdevices, Inc. ----------------------------------------------- } generalmicro-at-ccinet.ab.ca } ______________________________________________________ } Microtechnology products for science and industry } Cam, }
You can read this article that have helped me in prepearing the grids for TEM:
Fahrenbach, W.H., Continuous Serial Thin Sectioning for Electron Microscopy, J. of electron Microscopy ,Technique 1:387-398 (1984).
smithp-at-agresearch.cri.nz wrote: } } We have used microsoft access for a number of years and have found it to be } excellent for data management, storage of block information, etc etc.Its } compatibility with other software packages also makes it useful, its worth } having a look at. }
Regarding Microsoft Access, how does it handle large text fields? And most importantly, can it search for any part of the text in a field? (Example: in a search for "skin" would it bring up a field containing "porcine ligament and skin samples"?)
A couple years ago I spent quite a bit of time trying to set up a database of all the information that we keep in our "Histopathology" logbook, including specimen ID, description, experimental/surgical parameters, contact names, project name, etc. etc. The hope was that this would include all samples for LM, TEM or SEM and would not only be useful to us in tracking samples and generating reports, but also be accessible by our managers/primary investigators to check on progress and results.
Needless to say this was a bit of a pipe dream. At the time I needed PC/Mac cross-platform compatibility and the only "easy" choice was Filemaker Pro by Claris. This software was extremely easy to use and could have worked well for the initial stage (reproducing the logbook). However, it was limited in ability to "share" fields and their information from one file to another. Eventually these software limitations and the difficulty of cross-platform compatibility forced us to cancel the whole endeavor. :(
However, if Access or some other software would be capable of handling searches of large text fields then I would be interested to hear of it!
Karen Zaruba E-mail: kszaruba-at-mmm.com 3M Company Phone: 612-737-2971 St. Paul, MN USA
Disclaimer: These opinions are my own and may not represent those of 3M.
I am almost absolutely certain that your problem is associated with an earthing (or grounding) loop. It is very important to have only one single earth path. If you have separate earth path as supposedly exists with your additional backscatter detector and/or PC (the microscope is connected to the earth terminal, the PC is connected too, and both instruments are connected to each other through their grounds), there are alternative, physically separated earth paths, which will result in noise currents, and almost certainly impair the performance of any electron microscope. Some years ago we had similar problems with our computer and EDS system, but there is a remedy. It would be too long and sketches are also needed to explain. The best I can propose is to check a good textbook such as "Design of the Electron Microscope Laboratory" by Ronald H. Anderson (North Holland, 1985, p61-66) or consult an expert electrician as a final solution. In case you cannot obtain quickly the book I can fax you these pages.
Good luck!
Kris
Kristof KOVACS Associate Professor University of Veszprem, Central Laboratory P.O.Box 158, Veszprem, HUNGARY H-8201 Phone: +36-(88)-421-684
Sounds like you're describing structures that I found associated with the glycocalyx of enterocytes during duodenal ulceration. They have been called glycocalyceal bodies or R-bodies. No one seems to know what they do or precicely where they come from. A good review article on glyc. bodies is
P.B.Marcus "Glycocalyceal bodies and their role in tumour typing" J. Submicroscop. Cytol: 1981: 13: (3) 483-500.
Incidently, if anyone out there has any more recent references or knows what glyc. bodies are or where they come from I'd be interested in the information
Regards
Mike Gregory
On Tue, 30 Jul 1996, Buddy Steffens wrote:
} } Duodenal brush border vesicles - DBBV- and negative staining. } } We do negative staining of duodenal content of ostriches regularly as } } well as faeces samples of dogs, calves, etc to detect the } } presence of any possible virus particles eg. parvo, corona, picorna, } } rota, etc. However we experience some difficulties in diagnosing } } paramyxovirus (orthomyxo?) particles beyond doubt. The problem arises } } when we encounter paramyxovirus-like particles from low numbers to } } masses of them in a specimen BUT no sign of any helical nucleoprotein } } strands. How can we break up these particles to release any } } nucleocapsid strands if they are virus particles? } } These "virus-like" particles that you describe are a source of much } debate among those of us who look at negative-stained fecal preps. I } find them routinely in ostriches, emus, and turkeys, both ill and } clinically healthy birds. If you suspect infection with a } pleomorphic, enveloped virus, such as coronavirus or paramyxovirus, } these things really are a nuisance. I don't believe that there is } any known way of getting rid of them, short of lysis in distilled } water. This is not completely effective, as most of them are already } in lysis to some extent. } } } Can anyone supply me with a micrograph of x100-150K magnification of } } DBBV (or any other reference) to compare with our own known } } micrographs of paramyxoviruses or give info on how to obtain DBBV } } easily(?) for negative staining. } } See Goodwin et al. 1995. Avian Pathology 24(3) pp 497-505. } } } Q: What happens to the duodenal epithelial lining during a viral } } (bacterial) infection; can it shred structures (vesicles) in such vast } } numbers and do they have a viral-like appearance with negative } } staining? } } Gut is constantly turning over enterocytes, with the old ones being } shed as "effete" enterocytes. These are basically membrane ghosts, } which explains why you often see them in both healthy and ill } animals. The glycocalyx on the membrane surface when negative } stained often has a fuzzy or spiked appearance, and is very easily } confused with enveloped viruses. Their wide range in size and } surface projection morphology is what I use to distinguish them from } real viruses. } } } } -=W.L. Steffens=- } Department of Veterinary Pathology } College of Veterinary Medicine } University of Georgia }
} If you don't like this kind of an approach you can unsubscribe from the } listserver before you leave, and then subscribe again when you get back. } Basicaly, however, the problem is not one of concern to the listserver, but } one involving what goes on at the receiving end of the listserver operation } in your own institution. } In case you are on any other lists, be aware that some listowners unsubscribe people if too many messages get bounced back once the receivers mailbox is full (happens to one list I am on every so often and usually takes me a month to notice :-)
Keith --- Interface Analysis Centre, University of Bristol, Oldbury House, 121, St. Michael's Hill, Bristol, BS2 8BS, England Telephone: +44 (0)117 925 5666 | Facsimile: +44 (0)117 925 5646 | URL: http://www.phy.bris.ac.uk/research/iac/home.html
This is an odd image. Typically, with periodic EM or mechanical interference, you'll see jagged edges. The amplitude of the "jagginess" increases proportionally with magnification.
I assume in this image your referring to the banding on the substrate. Is this correct?
My best guess at this time if that the scan rotation was at 90 degrees and you've left the SEM's ABC circuit on during image collection.
I would like to contact microscopist operating IRFTmicrocopy (reflected light). My interest in in ageing of polymeric (electrical insulation) materials. Perhaps anyone would care to meet at the MSA meeting in Minneapolis (notice on the bulletin board?) Thank you Prof.Walter A.Mannheimer Metallurgy and Materials Engineering Federal University of Rio de Janeiro POBox 68505 21945 Rio de Janeiro, Brazil Voice +5521 280-7443 (Dept.office) +5521 590-0579 (direct) Fax +5521 290-6626 Email: wamann-at-metalmat.ufrj.br
} Hi All. } I have been having a bit of a problem with some stray EM (?) fields } on our SEM. The problem has been traced to the power feeding the wall } outlets. Anything plugged into these outlets and the microscope will affect } the images produced. Specifically the backscatter detector and the PC we use
snips...
} Your help and expertise will be greatly appreciated. Vendors please } jump in with any advise you may have.
Scott,
You are almost certainly setting up ground loops. ALL your equipment must go back to a common, local ground. You need to go back to the mains power source for your SEM and run extensions to all auxillary equipment from this point. Be careful not to make these extensions too long, or you may suffer pick-up from other sources. There should be some auxillary power outputs in the back of your SEM and these are the prefered power source. However, if you have a lot of auxillary equipment, you may risk overloading these.
Ideally, you should have a mains matching trasnformer between your SEM and your local power supply - this helps to provide protection agianst power surges, and some isolation from interference. You should tap in at this point to provide additional power sources.
To Scott Whittaker: Normally I'd agree with Kris Kovacs assesmmment, but usually noise from ground loops is much more periodic, as the dominant ground currents are usually 60 Hz.
A proper analysis of the instrumentation should be performed, but this is often impractical. Often floating the 110VAC inputs to the PC and BSE (using "cheater plugs") and grounding their frames directly to the instrumentation ground of the SEM (via very low impedance braided cables) will solve this problem. However, this approach is more of a diagnostic procedure than a permanent remedy. Extreme care must be taken if this solution is employed, as uninsulated ground cables running about in your SEM can cause havoc if they come in contact with the wrong thing. Also, although this may be a better instrumentation practice, it isn't the best for safety reasons, as your depending on the ground cables (and the SEM earth ground) for safety grounding. If you choose to employ this approach, DISCUSS IT WITH YOUR LOCAL MAINTENANCE PEOPLE. I assume no responsibility for the situation.
Kris Kovacs Wrote: } I am almost absolutely certain that your problem is associated with anearthing (or grounding) loop. It is very important to have only one single earth path. If you have separate earth path as supposedly exists with your additional backscatter detector and/or PC (the microscope is connected to the earth terminal, the PC is connected too, and both instruments are connected to each other through their grounds), there are alternative, physically separated earth paths, which will result in noise currents, and almost certainly impair the performance of any electron microscope. Some years ago we had similar problems with our computer and EDS system, but there is a remedy. It would be too long and sketches are also needed to explain. The best I can propose is to check a good textbook such as "Design of the Electron Microscope Laboratory" by Ronald H. Anderson (North Holland, 1985, p61-66) or consult an expert elctrician as a final solution. In case you cannot obtain quickly the book I can fax you these pages. } End of Kris Kovacs reply
Hi there, I am trying to locate someone from St. Louis going to MSA. I have submitted an abstract, but will not be able to make it to Minneapolis on monday. I am trying to find someone to hang it up for me.
thanks
Lucio
ps - please reply to me directly at one of the emails below.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Lucio Mule'Stagno MEMC Electronic Materials Inc University of Missouri -St.Louis Silicon Materials Research Group Physics Dept., 501 Pearl Dr., 8001 Natural Bridge rd., St.Peters, St.Louis MO 63376 MO 63121 tel: 314 279 5338 314 516 5931/3 fax: 314 279 5363 314 516 6152 email: lmulestagno-at-memc.com luciom-at-newton.umsl.edu URL:http://www.newton.umsl.edu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
I'm not sure if this is what your looking for, but alcian blue 8GX can be added to glutaraldehyde fixative to enhance preservation and contrast of cell coats and, if part of the coat contains glycosaminoglycans, alcian blue is better at maintaining the thickness of the coat than ruthenium red. Electron Microscopy Sciences has it (Ft. Washington, PA), we have an old jar of it from Sigma Chem Co (St. Louis, MO) and I'm sure there are other suppliers. The reference EMS gives on fixation is Schofield, et al., Histochem J. 7:139 (1975).
We are looking for a buffer for snake eyes. Not snake oil. Someone sugested a Mckenzie Buffer. If anyone has a recipe for this or an additional buffer for snakes we would like to know.
thanks
Mike
=========================================================== Michael Dunlap lab (916) 752-0284 Facility For Advanced Instrumentation fax (510) 422-2282 University of California mrdunlap-at-ucdavis.edu Davis CA, 95616 http://carbon.ucdavis.edu ============================================================
Comments: Authenticated sender is {wesleysm-at-biology.und.ac.za}
I would like to add another 'complaint' to the list. Plant tissues embedded in LR White show signs of severe plasmolysis. I have broken down the infiltration into 25, 50, 75 and 100% resin/75% EtOH and prolonged the infiltration stages (+- 2 hrs each) but to no avail.
By the way, the mixture tends to separate as well! Maybe this is the reason for the shrinkage...?
################################################### James Wesley-Smith Electron Microscope Unit George Campbell Building University of Natal Durban, South Africa http://www.und.ac.za/und/emu/emunit.html ###################################################
Greetings, We have been using some capsules that are like "beem" capsules, 8mm diameter and with a snap-on cap, only instead of having a cone or a pyramid, they are simply flat on the bottom. They are made from polyethylene (polypropylene ones are made too). These are great for flat embedding. We got ours from TAAB in England, but we have used them up nearly. Does anyone know if these are for sale in the USA? A quick perusal of catalogs from several major USA supply houses failed to find 'em. Thanks for any leads. Tobias Baskin
I've used (or rather TRIED to use)LR White for LM but with very little success. I found that the blocks were brittle and didn't stain well with LM stains. Needless to say, I'm not impressed and have stuck to GMA embedding. I understand that the shelf-life for LR White is very short; perhaps that was part of my problem as well as part of yours???
On Tue, 30 Jul 1996 kszaruba-at-mmm.com wrote:
} hans-martin.vaihinger-at-rz.ruhr-uni-bochum.de wrote: } } } } Dear Microscopists } } } } We are only just beginning to use LR White as embedding medium for } } use in EM- immunocytochemistry. A manual by AGAR SCIENTIFIC LTD. } } distributed through PLANO recommends to bring the specimen from } } 70 % EtOH into LR White/70 % EtOH (2:1) before embedding in pure LR } } White. We find that this mixture very quickly separates into two } } phases. How shall we handle this? } } Furthermore the specimen seems to be cured insuffiently even after } } 24 hours of polymerisation at 55 C. Any comments on this? } } } } } I am having exactly the same experience with LR White and would appreciate } seeing the replies. (I have started going to 95% ethanol in order to get } good mixing). } } Thanks! } } Karen Zaruba E-mail: kszaruba-at-mmm.com } 3M Company Phone: (612) 737-2971 } St.Paul, MN USA }
Delilah W. Irving tel: 510-559-5653 USDA - ARS - WRRC fax: 510-559-5777 800 Buchanan St. email: dirving-at-pw.usda.gov Albany, CA 94710
Listmembers, I don't know if this works on every list, but a technique I've used to stop emailbox overflow when I'm out of town is to use the following command:
the TO: address should be LISTSERV-at-MSA.MICROSCOPY.COM in the text of the message put the line (no signature or footers): SET MICROSOPY NOMAIL
When you're ready to get your mail again, send the following message to the listserv address (LISTSERV-at-MSA.MICROSCOPY.COM): SET MICROSCOPY MAIL
It's probably about as effective as unsubscribing and resubscribing (provided you remember how to do that without sending your request to all of us list readers). It does have the advantage of avoiding the welcome messages that come when you resubscribe (not that we mind Nestor's eloquent prose...).
Have a safe vacation.
Doug ..................................................................... : Douglas W. Cromey, M.S. Dept. of Cell Biology & Anatomy : : Sr. Research Specialist University of Arizona : : (office: AHSC 4212A) P.O. Box 245044 : : (voice: 520-626-2824) Tucson, AZ 85724-5044 USA : : (FAX: 520-626-2097) (email: dcromey-at-ccit.arizona.edu) : :...................................................................: http://www.pharm.arizona.edu/exp_path.html
} From: Robert Underwood {underwoo-at-u.washington.edu} } To: Microscopy List {Microscopy-at-Sparc5.Microscopy.Com} } Subject: Help on Deconvolution } Date: Tuesday, July 30, 1996 10:52 AM } } I am getting confused and nervous (about price) of deconvolution. We are } putting together a deconvolution system for a Nikon SA upright, some type } of cooled CCD monochrome camera (haven't decided which) on a PowerMac } platform. } } We already have the microscope and are committed to PowerMac. We have } contacted several companies that can provide the whole system, } therefore: } } 1. I would love to hear from anyone who has had experience in putting such } a system together, with warnings and recommendations. } } 2. I'm confused about the various terms describing deconvolution: } } EPR algorythem } Nearest Nieghbor } Inverse fourier analysis } constrained iterative } Poisson point model } PSF deconvolution } 3D blind deconvolution } } Is there somone who could simplify this or knows of a good reference } that clarifies the methods? } } Bob } University of Washington
I've written deconvolution software in the past (although I'm not working with it right now), and perhaps I can answer a few things...
The Point Spread Function (Optical Transfer Function, Modulation Transfer Function, all different forms of the same thing) describes how a 'point object', smaller than the resolution of the scope, blurs in 3-D. As large objects can be considered as clouds of little points, this gives all the information about how any complex object gets blurred.
This blur is reversed to sharpen the data, providing a reconstruction of what the original object was. The relationship is:
D = P*O; P(-1)*D = P(-1)*P*O ~ O
The data is the object convolved with the point spread function, reconstruction consists of convolving the data with the inverse point spread function to approximate the object. This is approximate, as some parts of the PSF are zeros, and you lose that information permanently. In addition, 1/0 is infinity, and tends to be badly behaved mathematically.
Fourier transforms are used to simply these calculations - a multiplication in the Fourier domain is equivalent to convolution in a normal image.
Full inverse deconvolution involves taking the 3-D Fourier of the PSF and data, inverting the PSF (with math tricks to handle zeros), multiplying it times the data and inverse Fourier transforming the result. Lots of number crunching.
Constrained iterative runs multiple cycles of this, 'constraining' the result at each pass with limits such as non-negativity or frequency limits (things you _know_ about the object), to minimize the errors from the lost data and contaminating noise. Lots of number crunching, repeated 25-50 times, rather better results than a single pass.
Nearest Neighbor is an approximation of full single inverse deconvolution (fairly good, actually), that uses the data planes above and below each plane to estimate out of focus blur, and output an object approximation. Less accurate, but a _lot_ less computation. This is also more appropriate for _thin_ specimens, as you really need a fair number of planes for decent full deconvolution. Full iterative deconvolution surpasses nearest neighbor accuracy at about iteration 10 to 15 out of 50.
EPR I'm not so certain of - that sounds like one of the maximum entropy techniques. If it is, it has computation on the order of constrained iterative, with some mathematical pluses and minuses to differentiate it.
Poisson point model; that term sounds like a noise model. When handling places where the PSF approaches zero you have to have a good model of the data SNR (signal to noise ratio) to see how close you can cut matters. Poisson, Gaussian, and white noise are all different models that have been used, depending on acquisition methods, experimental knowledge, and the bias of the experimentor.
3-D blind deconvolution refers to techniques of estimating the PSF in the absence of exact knowledge. I have not used such algorithms, and I can't tell you how good they are. In general, if you can get a good estimate of the PSF (using analytic techniques if you have low abberations or experimental PSF's if not) you will probably have a better reconstruction than if your program guesses at it from the characteristics of your data.
Warnings and recomendations:
Match your immersion fluid to your embedding media in regards to refractive index. Otherwise changing relative path lengths at different depths and refractive indexes in your specimen result in depth dependent spherical abberations, meaning that your PSF is highly non-uniform and non-linear, and your reconstruction suffers accordingly. The best results I've seen are from water immersion and no cover slip, the second best from water or glycerin immersion and wet mounts with cover slips. Good objectives are equally important, particularly in regards to Plan (flat field of view) qualities.
Consider time: iterative techniques are ones where you go away for lunch or the evening, and look at results the next day. Nearest neighbor can be done in a few minutes at most. Confocal scopes give you immediate results (at the cost of massive photobleaching and longer scan times).
Get good data, with a high SNR. I once had someone come in with some data that had a dynamic range of 35 grey values on 20 grey values of noise (SNR of ~1.5), and ask for a reconstruction. Results were, shall we say, poor. Since you're amplifying edges, salt and pepper noise will have a major effect compared with the smooth blurred object edges. Starting with a high signal to noise ration mitigates this.
Correct for non-linearities in your imaging prior to deconvolution!!! This means background subtraction, flat field division, and scaling the images in your stack to account for photobleaching, in that order. [For bleaching, you can just assume that all full field images of the same area, regardless of depth, will have the same integrated intensity, and scale accordingly.] The PSF division technique is based on assumptions of linear modulations in the object, and non-linear effects such as these _will_ distort your results.
Cost: not much that can be said here. It will likely cost you on the order of a low to medium grade confocal microscope to implement deconvolution.
Most importantly - data acquisition is really only a minor piece of the bag. Deconvolution in its various forms, confocal, whatever; you'll get 3-D data. Data analysis, rendering and the extremely difficult 3-D measurements of position, area, and volume: these are what will pay off in the long run. I won't make product recommendations, but I _strongly_ suggest that you consider your analysis software (integrated or separate) as a major item in your system design. Review these with as critical an eye as the acquisition method. Great data only pays off if you can derive answers from it.
Hope this is helpful!
-- Kevin Ryan Media Cybernetics kevin-at-mediacy.com
We have used LR White for over 15 years. The biggest problem that we have is when there is EtOH left in the material before it goes to 100% LRWhite. To take care of that problem we do NOT put a mixture of alcohol and LR Whitae before going to pure. We definitely do go to 100%, then 3 changes of pure LR White, then into capsules. The gelatinous mess that results from the EtOH/LR White was never found to polymerize even after 2 yrs., even after try ing to dry it out and all sorts of other manipulations to save a valuable sample. Good Luck, Judy Murphy
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
hans-martin.vaihinger-at-rz.ruhr-uni-bochum.de wrote: } } Dear Microscopists } } We are only just beginning to use LR White as embedding medium for } use in EM- immunocytochemistry. A manual by AGAR SCIENTIFIC LTD. } distributed through PLANO recommends to bring the specimen from } 70 % EtOH into LR White/70 % EtOH (2:1) before embedding in pure LR } White. We find that this mixture very quickly separates into two } phases. How shall we handle this? } Furthermore the specimen seems to be cured insuffiently even after } 24 hours of polymerisation at 55 C. Any comments on this? }
I am having exactly the same experience with LR White and would appreciate seeing the replies. (I have started going to 95% ethanol in order to get good mixing).
Thanks!
Karen Zaruba E-mail: kszaruba-at-mmm.com 3M Company Phone: (612) 737-2971 St.Paul, MN USA
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We have used LR White routinely for a number of years. We find the best results are obtained by dehydrating to 100% Ethanol. The main things to watch out for are keeping the temperature well controlled and excluding oxygen (which can penetrate many commonly used moulds such as BEEM capsules).
Excessive brittleness seems to be caused by the embedding temperature being too high (perhaps only a degree or two above 60C).
Fresh LR White has a shelf life of 1 year at 4C though we have used older batches occasionally. The major problem with old batches can be premature polymerising. This is not a great problem if it occurs in the bottle but more so if it occurs whilst infiltrating the sample. Uncatalysed resin, which comes with a powder catalyst, has an almost indefinite shelf life and we make up a fresh bottle of active resin just before the old one runs out.
Ian
Ian Hallett HortResearch Mt Albert Research Centre Private Bag 92 169 Auckland, New Zealand Fax 64-9-815 4201 Telephone 64-9-849 3660 EMail ihallett-at-hort.cri.nz
In my experience, the "high mag jaggies" are most often caused by a mechanical vibration. Often the roughing pump is the problem. Sometimes a couple of tennis balls under the roughing pump will decrease the effect substantially. It's not always this simple though. Also check for cables touching the vacuum line from the RP. They can transmit these vibes.
} Dear Microscopists } } We are only just beginning to use LR White as embedding medium for } use in EM- immunocytochemistry. A manual by AGAR SCIENTIFIC LTD. } distributed through PLANO recommends to bring the specimen from } 70 % EtOH into LR White/70 % EtOH (2:1) before embedding in pure LR } White. We find that this mixture very quickly separates into two } phases. How shall we handle this? } Furthermore the specimen seems to be cured insuffiently even after } 24 hours of polymerisation at 55 C. Any comments on this?
I replied to this a few days ago, but am not sure that it arrived. So, again...
The 70% EtOH needs to be made up freshly just before use from 100%. Even stored in a tightly stoppered bottle, the concentration of the EtOH seems to decrease. This solved my similar problems.
70% EtOH seems to be the bare mininum that LR White will mix with; as soon as the concentration drops below this there are problems with infiltration, curing and the final blocks are brittle. I keep the LR White in the fridge and use it without warming. All vials are rotated continuously during processing. I embed the tissue in gelatin capsules in a normal embedding oven. I rarely have problems with the blocks, though I must admit I would only use LR White for immuno, never as a replacement for resin.
Diana van Driel Dept Ophthalmology Sydney University C09 AUSTRALIA 2006
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