On Thu, 28 Oct 1993, Nestor J. Zaluzec (708)-252-5075, -4964 wrote:
} } } Steve Barlow asked } } Tissue cryosections and immunofluorescence. } } Can anyone suggest some treatments for reducing background } } fluorescence in rabbit muscle tissue?.........(text deleted) } } } I have absolutely no experience in LM Fluorescence problems } but an obvious solution to me (which probably means I'm wrong) } is to change the wavelength of the light source. If the illumination
I tried to avoid autofluorescence by changing the wavelength. I suppose that might work in some situations where a single specific component with a narrow band of excitation or emission is present. But some things, like the lipofuscin granules in gut will fluoresce at any wavelength. my hypothesis is that most biological autofluoresce is due to a number of molecules (and processing distortions) that will glow over a range of frequencies.
For what its worth, my recent experience with whole vestibular organs showed that FITC, AMCA and Cascade Blue wash out quickly, leaving little backgound. But Texas Red required an overnight wash.
} Some general questions for discussion: } } 1. what does one use for a control when using a monoclonal Ab--there is, } in this case, no 'pre-immune'? Is a monoclonal Ab known not to be expresed } in the sample the best alternative? } } 2. what does one use for a control when using a polyclonal rabbit Ab } (commercial or otherwise) for which no pre-immune is available? My 2 cents: I had gotten by with finding the IgG concentration of the antibody preparation. Then I dilute a normal (non-immune) serum, or purified IgG, from the appropriate species to a similar concentration. IgG and IgM estimates for the normal sera of several species may be found in R. Lindmark, et al, J. Immunmol. Meth., 62:1-13, 1983. Of course, not all vendors know that much about their products, and most homemade antibodies are relatively uncharacterized. Sometimes the IgG content of an immune serum or monoclonal prep. can be estimated by comparing the total protein to that of non-immune serum for that species, or control culture/ascites fluids. I have encountered commercial "non-immune" sera that gave a better anti-cytokeratin label than the monoclonals being evaluated.
I've seen alot of discussion about staining lately which is interesting, but now I have a simple question. We deal mainly with metals/ceramics/semiconductoring materials hence, we do no staining etc.. So for the "novice in me" can someone tell me what IgG and IgM are? I'm not able to deduce the meaning from the context of the discussion. It would help me (and probably a few others) to follow the discussion a little better.
Remember this list has a whole slew of different readers and not all of us know each others "standard" abbreviations from the different fields of microscopy (or even the same field but in different applications). Try to keep this in mind when posting things.
IgG and IgM stand for immunoglubulin G and immunoglobulin M, and in this context refer to different types of antibodies used for immunohistochemistry. For most applications, primary antisera are of the IgG type, but for some applications, and for antibodies derived from certain sources and by different procedures, they may be IgM. This is important to know, since the secondary antibody, which is directed against the primary antibody and usually carries the tag which will allow visualization, must be directed against the appropriate immunoglobulin and the right host species.
There is an excellent review, both for its information content and its historical value, on the basics of indirect immunohistochemiostry by Ludwig Sternberger, 1974. I don't have the reference handy, but I will track it down and report back.
Hope this has been useful...
David Morilak Dept of Psychiatry Stanford university morilak-at-cmgm.stanford.edu -------
IgG and IgM stand for immunoglubulin G and immunoglobulin M, and in this context refer to different types of antibodies used for immunohistochemistry. For most applications, primary antisera are of the IgG type, but for some applications, and for antibodies derived from certain sources and by different procedures, they may be IgM. This is important to know, since the secondary antibody, which is directed against the primary antibody and usually carries the tag which will allow visualization, must be directed against the appropriate immunoglobulin and the right host species.
There is an excellent review, both for its information content and its historical value, on the basics of indirect immunohistochemiostry by Ludwig Sternberger, 1974. I don't have the reference handy, but I will track it down and report back.
Hope this has been useful...
David Morilak Dept of Psychiatry Stanford university morilak-at-cmgm.stanford.edu -------
I've had some real problems trying to send this message to the list, so I hope it gets through this time, and please forgive me if it gets sent a few times:
Nestor:
IgG and IgM stand for immunoglubulin G and immunoglobulin M, and in this context refer to different types of antibodies used for immunohistochemistry. For most applications, primary antisera are of the IgG type, but for some applications, and for antibodies derived from certain sources and by different procedures, they may be IgM. This is important to know, since the secondary antibody, which is directed against the primary antibody and usually carries the tag which will allow visualization, must be directed against the appropriate immunoglobulin and the right host species.
There is an excellent review, both for its information content and its historical value, on the basics of indirect immunohistochemiostry by Ludwig Sternberger, 1974. I don't have the reference handy, but I will track it down and report back.
Hope this has been useful...
David Morilak Dept of Psychiatry Stanford university morilak-at-cmgm.stanford.edu -------
Message-Id: {m0ouTk2-000dBZC-at-unpsun1.cc.unp.ac.za} To: microscopy-at-ANLEMC.MSD.ANL.GOV
Following up my earlier reply, here is the citation for Sternbergers classic reference on immunocytochemical techniques:
Sternberger, Ludwig A. IMMUNOCYTOCHEMISTRY (Englewood Cliffs, N.J. : Prentice-Hall, 1974)
Sternberger, Ludwig A. IMMUNOCYTOCHEMISTRY. 2nd ed. (New York : Wiley, 1979)
Sternberger, Ludwig A. IMMUNOCYTOCHEMISTRY. 3rd ed. (New York : Wiley, 1986)
If anyone is interested, it's a great introduction to immunocytochemistry, and for anyone who is doing it, it is a great look into how the technique has been developed.
Thanks to all for the general info on IgG & IgM. Just goes to show you that us materials types are interested in lots of things too!
BTW I smiled at Michael Herron's closing comment:
} Hey, it ain't no cruise missle but it what we do :^)
Remind me in a less busy time to describe how the ANL EM Center proposed and developed a protocol for using an SEM on Cruise and other Missles (US & Russian) as a nondestructive means for nuclear arms verification. It was actually a small ($20K) but funded program for a year by the START program! The funny thing is the procedure worked. It got killed in the end because anything that inexpensive couldn't work (at least one of the explaination I got)
Message-Id: {m0ouTLM-000dBTC-at-unpsun1.cc.unp.ac.za} To: microscopy-at-ANLEMC.MSD.ANL.GOV
On Wed, 3 Nov 1993, Michael Cammer wrote:
} } We are looking for a system for gross storage and easy retrieval of a } large number of images for both teaching and research. Does anybody have } any suggestions? } } Basically, we have a large (microscope) slide library that we would like } to scan in color. We need the images available for playback on a video } monitor and for photography. Should we use a computer and, if so, with
} what software (e.g. database) and what storage device (for speed, quanity, } compatibility w/ other systems, and an eye towards support in the future)? } The initial application is for a slide library for teaching, but, perhaps, } this could be turned into an interactive system available to students. } Also, I have been thinking that such a system might be useful for storing } our images for research; even without this specific query, we need more } and more compatible storage. } } Any suggestions for specific parts or for whole integrated systems (e.g. } is there something smilar for other fields?) would be welcome. } } Thanks- } } Michael cammer-at-aecom.yu.edu } } }
Cost aside, one alternative is HDTV for capturing the images. I'm not sure what the current state of the art is, but several years ago, Sony offered complete HDTV systems (cameras, analog and digital storage, post-processing hardware, optical fiber closed-circuit transmission, etc.) specifically aimed at the pathology market. I saw the system demonstrated in the Sony showroom in Tokyo; the image quality was extraordinary. As I recall, there are several sites in the US that offer HDTV-based remote consulting.
Apart from cost, one reason to hold off on the Sony system is that their HDTV standard is different than the one that three US developers have decided (at long last) to develop jointly: You could end up with a pricey white elephant.
Storage of massive amounts of data will depend on a number of factors that include required speed of retrieval, number of users who need simultaneous access, available hardware, money. Running all the images onto CD-ROM is not a bad alternative; costs are not terribly bad (if you want to make your own, you'll have too pay around $4K) although speed of retrieval may be frustrating.
I'm quite interested in hearing of ways people have developed archives of microscopy data. Having representative images on-line in conjunction with other data can make a very powerful tool.
Dave Coder Dept. of Immunology Internet: dcoder-at-u.washington.edu
Related to the current discussion thread on imarge archiving - what systems and software do people use for cataloging and retrieving images? We are accumulating stacks of images from calcium ratioing, confocal, serial images from convential microscopy (lm and SEM) and stuff that has been converted to projection slides and Linotronic files.
There are both commercial and shareware programs offered.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206) 543-8360
John Mansfield North Campus Electron Microbeam Analysis Lab University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 17:27
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On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote:
} Date:11/3/93 } NC EMAL } Subject: Critical Point Dryer } I have a colleague who is in the market for a critical point drying system. } Not knowing anything about such things or exactly how they work, I want to ask } if there is a manufacturer or model that is preferred by the majority of people } and if it is possible to dry material that is wet with a liquid other than } water. } Any help or info would be appreciated. } Thanks. } John Mansfield
The idea of critical point drying is to change the liquid to a suitable liquified gas, which has a low enough critical point to change directly fron liquid phase to gas phase. It does not matter wether the change is made directly or via a series of liquids. The only thing which matters is to maintain the sample as intact as possible while changing the liquids. So all you need to know is the solubility of different liquids in each other and finally in the liquified gas.
Hopefully this helps, others can give you more exact advice.
Best regards,
Jouko Mki
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Mki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote:
} Date:11/3/93 } NC EMAL } Subject: Critical Point Dryer } I have a colleague who is in the market for a critical point drying system. } Not knowing anything about such things or exactly how they work, I want to ask } if there is a manufacturer or model that is preferred by the majority of people } and if it is possible to dry material that is wet with a liquid other than } water. } Any help or info would be appreciated. } Thanks. } John Mansfield
The idea of critical point drying is to change the liquid to a suitable liquified gas, which has a low enough critical point to change directly fron liquid phase to gas phase. It does not matter wether the change is made directly or via a series of liquids. The only thing which matters is to maintain the sample as intact as possible while changing the liquids. So all you need to know is the solubility of different liquids in each other and finally in the liquified gas.
Hopefully this helps, others can give you more exact advice.
Best regards,
Jouko Maki
P.S. Sorry for repeating this message. The first sending contained 8-bit characters causing errors. I hope this goes through properly. jm
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Maki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
Received: From ANLVM(MAILER) by ANLEMC(ANJE5.40) for ZALUZEC-at-ANLEMC; Wed, 3 Nov 93 20:42 CST Received: from ANLVM by ANLVM (Mailer R2.07B) with BSMTP id 6784; Wed, 03 Nov 93 19:43:46 CST Received: from nx1.soils.umn.edu by ANLVM.CTD.ANL.GOV (IBM VM SMTP R1.2.2ANL-MX) with TCP; Wed, 03 Nov 93 19:43:44 CST Received: from (localhost) by nx1.soils.umn.edu (5.65c) id AA12448; Wed, 3 Nov 1993 19:40:03 -0600 Goldsmith) To: Multiple recipients of list {nih-image-at-nx1.soils.umn.edu}
} We are looking for a system for gross storage and easy retrieval of a } large number of images for both teaching and research. Does anybody have } any suggestions? } } Basically, we have a large (microscope) slide library that we would like } to scan in color. We need the images available for playback on a video } monitor and for photography. Should we use a computer and, if so, with } what software (e.g. database) and what storage device (for speed, quanity, } compatibility w/ other systems, and an eye towards support in the future)? } The initial application is for a slide library for teaching, but, perhaps, } this could be turned into an interactive system available to students. } Also, I have been thinking that such a system might be useful for storing } our images for research; even without this specific query, we need more } and more compatible storage. } } Any suggestions for specific parts or for whole integrated systems (e.g. } is there something smilar for other fields?) would be welcome. } } Thanks- } } Michael cammer-at-aecom.yu.edu Michael, We are also looking at alternatives for the long term archiving and easy retrieval of digital images. Many of our images are directly aquired using Perceptics HR 24 RGB frame grabber with a SONY DXC930P (P for Pal) which gives about 1Mb per image of real 24 bit colour with low noise if the lighting is good. Or we use a Videk (Kodak) MegaVision with a Perceptics MegaGrabber for a 1.4Mb Monochrome image, or we use the Data Translation DT 2555 Quik Capture for a 400Kmonochrome image. These images are used for illustration, measurement etc. We have a Richoh read-write optical disc which is useful for storage, and works well. We have now started to worry about back-ups of our now extensive files of images. We are going to make a Photo CD, which will require the production of films or slides from digital images. although next year Kodak will offer a service called portfolio which will allow the production of a CD containing digital only material. The idea of a photo cd is attractive because it is becoming a well accepted plateform for images, and is likely to be durable in the physical and temporal sense. The other advantages are the availability of "juke boxes" for CD's allowing the use of six or so in one machine. This access might not be as quick as from a hard disc, but it will be better than scanning in the original again. Because photo-cd has the "thumbnails", rapid data base access becomes a reality, software is already available, eg shoebox. So we will report tto the list as we learn. Noel T Goldsmith DSTO Aeronautical Research Laboratory 506 Lorimer Street Port Melbourne Vic 3207 Australia
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} } } } } On Wed, 3 Nov 1993 18:25:54 -0600, kartenh-at-Sdsc.Edu said:
} } Cost aside, one alternative is HDTV for capturing the images.
kartenh} Another alternative is the Kodak digital camera back on a kartenh} Nikon 8008. This directly dumps data into a Mac through kartenh} the SCSI port. It provides a color (24 bit?) image with kartenh} about 1500 pixel horizontal resolution X about 1000 kartenh} pixel. It grabs the image in about the same time that kartenh} film does. But it is slow in dumping it into the kartenh} computer. (Each image is about 4.5 MB). They provide a kartenh} plug-in driver for Adobe Photoshop, so you can directly kartenh} manipulate the image. H. Karten
I believe this is the very same DCS2000 I used earlier this year. It is most emphatically *not* a 24 bit scanner, although all the product lit seems to imply it very strongly. Relevant fragments from old sci.image-processing articles follow. As I wrote before, the PhotoCD images I just got were *much* preferable to the DCS2000 images.
|} From: sasrer-at-unx.sas.com (Rodney Radford) |} Subject: Re: Digital Cameras - Suggestions? |} Date: Wed, 9 Jun 1993 23:27:46 GMT
|} John Peterson {jp-at-taligent.com} writes: |} } Kodak DCS. This is a Nikon 35MM with a new back containing a |} } 1024x1024x24 CCD array. Old models required you to lug around a |} } suitcase, newer ones have a box about the size of the camera body |} } attached to the bottom of the Nikon. Suitcase stores about 1-200 |} } photos (depends on compression), smaller one can store photos in |} } small strap on SCSI disk. Mondo expensive, $8-20K. Smaller |} } cameras only take 20-30 photos on a battery charge; suitcase |} } models go longer. Suitcase has an LCD screen to review images. |} |} Most of your description was very accurate, and very |} informative. However, I don't know of any 24bit DCS cameras |} available (someone correct me if I am wrong, and give me the model |} number). We currently have a DCS200 camera, which is a Nikon F3 |} camera with a strap on hard drive capable of storing 50 images. |} However, it is only an 8bit CCD array with an RGB color mask in |} front so that each pixel can be EITHER red, green, or blue, such |} as: |} |} R G R G R G R G .... |} G B G B G B G B .... |} R G R G R G R G .... |} G B G B G B G B .... |} |} The resulting image on the DCS200 is a 1024x1536 image and takes |} 1.5MB (data is NOT compressed in the camera/disk). After this image |} is downloaded into the computer, it is interpolated into a full |} 24bit image by calculating 'suitable' values of red, green, and |} blue for each pixel. The result is pretty good, but not as good as |} a real 24bit image!
|} From: sasrer-at-unx.sas.com (Rodney Radford) |} Subject: Re: Digital Cameras - Suggestions? |} Date: Thu, 10 Jun 1993 13:05:40 GMT |} |} ledwards-at-leland.Stanford.EDU (Laurence James Edwards) writes: |} |} } Not to quibble, but in standard usage (at least on the output |} } side) a 24bit image means 8bits in rgb ... each "pixel" is |} } composed of an rgb triad. So I would say John had the depth right |} } but the resolution wrong ... that is, the unprocessed image is |} } really 512x768x24. Do they use linear interpolation, or something |} } a little more sophisticated?
|} Except that the interpolation algorithm yields a 1024x1536x24 bit |} image, not the 512x768x24 image you mention above. So they 'fake' |} the other 16bits in EVERY pixel location. So, I still stand by my |} original 1024x1536x8 bit description of the CCD array. Below is a |} paragraph from the DCS200 Programmer's Specifications:
|} "The CCD sensor for the DCS200 Digital Camera does not capture all |} three RGB colors for each pixel. Instead, it captures one color for |} each pixel, so that interpolation is required to construct three |} full RGB planes for the image. The grid below represents the pixel |} grid before interpolation. Green pixels predominate the grid because |} Green captures the luminance levels which can translate across to the |} Red and Blue planes."
|} The interpolation algorithm consists of three seperate passes over |} the image, interpolating the green, red and blue values. The Green |} interpolation algorithm basically looks at the 4 nearest neighbors |} as two pairs (up/down, left/right) and calculates the two delta |} values. It then uses the average of the pair with the largest delta |} value. The red/blue algorithms are very similar, except that there |} is no test for largest delta values - instead some pixels always |} use the up/down pair, some use the left/right pair, and others use |} a 4way average all all neighbors.
|} There is also a black-level color correction pass (enhances |} contrast), and a gamma correction pass. There is also a list of |} 'defective' CCD rows in the camera, and how to adjust for them |} (replace with a neighbor row, or average with two other rows).
|} We have received 3 seperate copies of the KCS200 manual, and in |} EVERY one, the algorithms have changed, so I guess they are not |} fixed in stone, and Kodak is still experimenting. Note that it is |} also possible to just use the interpolated Green plane as a B&W |} image.
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} We are looking for a system for gross storage and easy retrieval of a } large number of images for both teaching and research. Does anybody have } any suggestions? ............ } Michael cammer-at-aecom.yu.edu
to which Harvey Karten replied:
} There are several options. Low to medium resolution, use Video. File size } is modest (circa 300KB per image). Resolution is low = despite what your } television salesman tells you, you won't do better than about 350 lines of } resolution. You can then archive them for database using Aldus Fetch or } Multi-User's Search 2.0 image database. } Alternately, use intermediate photos, and send them off to Kodak for } Photo-CD ROMs. They provide a few different levels of resolution (from } video levels up to about 2,000 lines), depending on your display } capability. Increasing amounts of software to access Kodak Photo-CDs. } } For the user demanding higher levels of resolution, you will have to go to } a Scitex or Leaf scanner (costs about $15,000) which will give youj about } 5,000x4,000 pixel resolution. This is slightly better than a sharply } focussed image on Kodachrome 35 mm (also about 20 megapixels, or 4K x 5K.) } } Image database programs for the Mac were reviewed in the Sept. '93 MacUser, p190, a good starting point for comparisons. "Aldus Fetch" (mentioned by Harvey) received a four-mice review. Our graphics group here has set up a very usable multiuser database using Fetch, for drafted figures and scanned images. It is used to organize and access their archive and active files.
But, how to acquire the digital files? Capture by CCD camera directly from the microscope will be a problem regardless of it's CCD array size if you want to capture a large area (ie. low magnification). If you shoot film at low magnification, you can then scan the transparencies with a film scanner (2000-4000dpi,big files !), your own (your time and $$) or Kodak's PhotoCD ($$). You do have control of cropping, resolution, color or exposure control if you do it yourself. If your images have a narrow color range, you can reduce the file size by x3 by transforming it to 8-bit indexed color without much perceptible loss. "Photoshop" or "Fast Eddie" can do this for you. I have had some discussions with people in our photo library (chemical), who want to establish a digital equivalent. They have over 50,000 negatives and slides in the freezers. Let Kodak handle it! (As per Harvey's recommendation). PhotoCD's provide enough resolution (2000x3000 pixels) for direct 300dpi dye-sublimation output of 6.67"x10" prints. Discussion of the adequacy of dye-sub output can be found in the earlier record of this forum. A review of Kodak's image manipulation program, "Kodak PhotoEdge", can also be found in Sept'93 MacUser. Kodak PhotoCD's can even be viewed on the home TV, a nice feature for cramming students, even if not high-res.
I have been playing with a Nikon LS-3510AF film scanner (on loan from my local Nikon representative, S & M Microscopes, Colorado Springs, CO --A "thank you" plug). You know what? You can stick a slide (40mm x40mm scan area) directly in the 35mm slide holder and scan it. No microscope! At the maximum scanner resolution of 3165 dpi that is a resolution of 0.124 micrometers/pixel which is about what I get at x100 on my microscope set-up. It does a nice job with a slide sandwiched between crossed polarizers (we geologists do this a lot). I assume your slides are however of animal or vegetable, not mineral subjects. You wouldn't be able to do fluorescence stains without major, but not impossible, modifications to the scanner ( Is there a market out there?). Will microscopes be obsolete soon? Within the last year there was an article in "Advanced Imaging" about the development of a digital microscope. I can not remember the exact issue off-hand. In that set up the sample is stepped across a high density (4000-6000 pixel) linear array CCD, just like most film scanners do ( but theirs still looked like a microscope).
Good luck with your project. Maybe some of this will help.
============================================================= Robert Zimmermann rzimm-at-usgs.gov (Internet) U.S. Geological Survey 303-236-5626 MS 905 Box 25046 Federal Center Denver CO 80225
} On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote: } } } Date:11/3/93 } } NC EMAL } } Subject: Critical Point Dryer } } I have a colleague who is in the market for a critical point drying system. } } Not knowing anything about such things or exactly how they work, I want to ask } } if there is a manufacturer or model that is preferred by the majority of } } people } } and if it is possible to dry material that is wet with a liquid other than } } water. } } Any help or info would be appreciated. } } Thanks. } } John Mansfield } } The idea of critical point drying is to change the liquid to a suitable } liquified gas, which has a low enough critical point to change directly } fron liquid phase to gas phase. It does not matter wether the change is } made directly or via a series of liquids. The only thing which matters is } to maintain the sample as intact as possible while changing the liquids. So } all you need to know is the solubility of different liquids in each other } and finally in the liquified gas. } } Hopefully this helps, others can give you more exact advice. } } Best regards, } } Jouko Maki
The most common transition fluid used these days is liquid carbon dioxide. Specimens are typically dehydrated through a graded series of organic solvent, ethanol or acetone, then placed in the cooled critical point dryer (CPD). Ethanol and acetone are both miscible with CO2. Once the specimens are in the chamber, liquid CO2 is introduced, and, through several exchanges (filling and draining with CO2 under pressure from the tank), the organic solvent is completely replaced by CO2.
The chamber is kept cool to keep the CO2 in liquid phase. After exchange of liquids, the temperature is increased and the pressure inside the vessel increases also. Once the critical point is passed, 1073 psi and 31.1 deg C for CO2, the vessel is slowly vented. After atmospheric pressure is reached, the chamber is opened and the specimens removed. They are warm and dry, and they tend to rehydrate pretty fast, so having a dessicator handy is a good idea.
The typical CPD device is a high pressure bomb that has valves (3) for introducing liquid CO2 and venting liquid (bottom of chamber) and gas (top of chamber). There is a mechanism for heating and cooling, too. One temperature control system has electronic heating and cooling devices (can't think of the name now) and sensors for temp control. These can be automated. The other kind, which I prefer, has a water jacket around the pressure vessel. You run ice water in to cool and warm/hot water to heat. You have to keep an eye on the chamber to make sure you don't heat too fast. But I watch the chamber, even when it's automated.
I like the water controled units because (1) they are cheaper (2) there are fewer parts to go bad (3) they are smaller. The one I use now is made by Polaron (model 3200) and is sold by Ted Pella. The last price I have is about $3000. Another consideration is the size of the chamber. If you run only a few specimens you might want a smaller one.
Hope this helps, John (and others),
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
If I may add my two bits worth concerning Kodak's Photo CD system: (- and aplogies in advance if this information has already been covered in previous discussions) KODAK offer an Product Information Kit re Photo CD and their image processing covering o Pro Photo CD (6000 by 4000 pixels of resolution) o Lite Box image library system o Photo CD Catalogue (6000 image storage) o Portfolio Photo CD (variable resolution from base allowing 800 images or 1 hour of sound, or any combination in between) and more
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-d
=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= David L. Hirschberg bnhirsch-at-dapsas1.weizmann.ac.il Department of Neurobiology (972) (0)834-2127 (0)834-2412 work Weizmann Institute of Science (972) 847-4805 home Rehovot 76100 Israel (972) 834-4131 fax
} On Thu, 4 Nov 1993 04:29:55 +0200, John Mansfield wrote: } } } Date:11/3/93 } } NC EMAL } } Subject: Critical Point Dryer } } I have a colleague who is in the market for a critical point drying system. } } Not knowing anything about such things or exactly how they work, I want to ask } } if there is a manufacturer or model that is preferred by the majority of } } people } } and if it is possible to dry material that is wet with a liquid other than } } water. } } Any help or info would be appreciated. } } Thanks. } } John Mansfield } } The idea of critical point drying is to change the liquid to a suitable } liquified gas, which has a low enough critical point to change directly } fron liquid phase to gas phase. It does not matter wether the change is } made directly or via a series of liquids. The only thing which matters is } to maintain the sample as intact as possible while changing the liquids. So } all you need to know is the solubility of different liquids in each other } and finally in the liquified gas. } } Hopefully this helps, others can give you more exact advice. } } Best regards, } } Jouko Maki
The most common transition fluid used these days is liquid carbon dioxide. Specimens are typically dehydrated through a graded series of organic solvent, ethanol or acetone, then placed in the cooled critical point dryer (CPD). Ethanol and acetone are both miscible with CO2. Once the specimens are in the chamber, liquid CO2 is introduced, and, through several exchanges (filling and draining with CO2 under pressure from the tank), the organic solvent is completely replaced by CO2.
The chamber is kept cool to keep the CO2 in liquid phase. After exchange of liquids, the temperature is increased and the pressure inside the vessel increases also. Once the critical point is passed, 1073 psi and 31.1 deg C for CO2, the vessel is slowly vented. After atmospheric pressure is reached, the chamber is opened and the specimens removed. They are warm and dry, and they tend to rehydrate pretty fast, so having a dessicator handy is a good idea.
The typical CPD device is a high pressure bomb that has valves (3) for introducing liquid CO2 and venting liquid (bottom of chamber) and gas (top of chamber). There is a mechanism for heating and cooling, too. One temperature control system has electronic heating and cooling devices (can't think of the name now) and sensors for temp control. These can be automated. The other kind, which I prefer, has a water jacket around the pressure vessel. You run ice water in to cool and warm/hot water to heat. You have to keep an eye on the chamber to make sure you don't heat too fast. But I watch the chamber, even when it's automated.
I like the water controled units because (1) they are cheaper (2) there are fewer parts to go bad (3) they are smaller. The one I use now is made by Polaron (model 3200) and is sold by Ted Pella. The last price I have is about $3000. Another consideration is the size of the chamber. If you run only a few specimens you might want a smaller one.
Hope this helps, John (and others),
John chandler-at-lamar.ColoState.EDU Fort Collins, CO
I need several options for storage of fixed tissues that might be used later for E.M. Currently I store tissues in the original fixative. Any other options would be appreciated.
I tried posting a message to the list server and got the following reply. Any thoughts?
} Bad address -- {MICROLISTUSA} } Error -- } %MAIL-E-NOSUCHUSR, no such user MICROLISTUSA } } Start of returned message } } Date: Wed, 10 Nov 1993 16:47:40 -0500 } Message-Id: {199311102147.AA09853-at-mtu.edu} } To: microscopy-at-anlemc.msd.anl.gov } From: llsutter-at-mtu.edu (Larry Sutter) } Subject: ESEM } } We are in the initial stages of evaluating an environmental SEM. } I have heard from some people that a field emission SEM with a cryo stage } will provide very comparable performance. This is an important distinction } to make early because we definitely have application for a field emission } SEM for work on photo catalysts, polymers, and materials research. The } primary interest in an environmental SEM is for examining sludges and } soils. If this work can be done on a FE/SEM with a cryo stage, our opinion } is that the FE/SEM would ultimately be more versatile. } } I would greatly appreciate any input on this matter especially from } (but not limited to) people that have used both types of instruments. } } Thanks... } } } End of returned message
In case you haven't noticed there have been some glitches in the system the last few days. The most common problem reported was either NOMAIL or BAD Addresses. I think the system is fixed, however, I'm not 100% sure. The mailing list has grown to ~ 400 users now and I've had to reconfigure the system to handle 2 groups USA vs. the rest of the World. Because of this it was necessary to manually edit system subscription files. I hope no one was erroneously deleted, however, some of you may be receiving 2 copies of messages. In a 2nd mailing I will attempt to identify duplicate addresses. For the time being please excuse the headaches, multiple copies and various test messages.
Please report any errors directly to me at
zaluzec-at-anlemc.msd.anl.gov
Nestor Zaluzec ANL EM Center
======================= BTW here is a message which I believe only reached part of the mailing list. It was originally sent out on Nov. 7th...
========================
ROYAL MICROSCOPICAL SOCIETY MEETINGS CALENDAR
17 March 1994 ANNUAL IMMUNOCYTOCHEMISTRY MEETING
29 March 1994 ANNUAL LIGHT MICROSCOPY MEETING, London
11 - 13 April 1994 MICROSCOPY OF COMPOSITE MATERIALS II, University of Oxford
13 April 1994 ANNUAL CYTOMETRY AND HISTOCHEMISTRY MEETING, Birmingham Organizers: Dr Gillian Lawrence and Dr B. K Shenton
Outline Programme 10.30 am Joint Session, Cytometry and Histochemistry in Pathology 2.00 pm Parallel Sessions A Histochemistry - Viviane Maggi Prize Competition B Cytometry 3.45 pm Pearse Prize Lecture - Professor S. J. Holt 4.30 pm Bingo Meyer Memorial Lecture
Contributed papers are requested for the joint session, the cytometry session and the Viviane Maggi Prize Competition for beginners. The closing date for receipt of abstracts will be 31 January 1994.
18 - 22 April 1994 EM SPRING SCHOOL, University of Sheffield
28 June 1994 FLOW CYTOMETRIC IMMUNOPHENOTYPING OF LEUKAEMIAS AND LYMPHOMAS MEETING, London
27 - 30 June 1994 COMPUTERS IN MICROSCOPY COURSE, University of Cambridge
13 - 14 July 1994 BENCH-TOP FLOW CYTOMETRY WORKSHOP, Newcastle University
17 - 22 July 1994 LM SUMMER SCHOOL, University of Leeds
5 - 9 September 1994 IMMUNOCYTOCHEMISTRY COURSE, Oxford Polytechnic
12 - 15 September 1994 MICRO 94 EXHIBITION AND CONFERENCE, London
12 September 1994 AGM AND PRESIDENTIAL LECTURE, London
19 - 23 September 1994 FLOW CYTOMETRY COURSE, University of Cambridge
October 1994 MICROSCOPY OF CATALYSIS, Joint Royal Microscopical Society/Royal Society of Chemistry Meeting, London
October 1994 FUNCTION TESTING BY FLOW CYTOMETRY WORKSHOP
November 1994 ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY COURSE, Sutton
Details of all meetings are available from the Royal Microscopical Society, 37-38 St Clements, Oxford, OX4 1AJ, United Kingdom. Telephone +44 (0)865 248768, fax +44 (0)865 791237, email RMS-at-UK.AC.OX.VAX.
MICRO 94 FIRST CIRCULAR
MICRO 94, International Microscopy and Image Analysis Conference and Exhibition 12-15 September 1994
Earls Court Park Inn International, Lillie Road, London, SW6
Description of Conference
The Conference will consist of tutorial and review lectures, technical lectures organized by the trade, and posters. Experts in the field of instrumentation, analytical methodology in physics and materials sciences, cytometry, cytochemistry, cytology, image analysis and image processing are invited to give the lectures.
Each speaker will provide an overview of the particular topic in question and ample time for discussion will be provided. Seven half-day sessions of tutorial and review lectures and six half-day sessions of technical lectures will be organized.
Full length papers of the invited tutorial lectures will be considered for publication in the Journal of Microscopy.
Contributed papers are welcomed and will appear in poster sessions. Abstracts will appear in the Proceedings of the Royal Microscopical Society.
The Physiological Society and the Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) will organize separate lectures within the scope of the conference.
Outline Scientific Programme
At the MICRO 94 Conference and Exhibition, emphasis will be placed on Image Processing and Analysis. The launch of a new Special Interest Group within the Royal Microscopical Society will also be taking place.
Review and Tutorial Lecture topics are expected to include:-
Image processing and analysis Computer manipulations of images Scanning probe microscopy in materials sciences and in life sciences Near field optical microscopy 3-D microscopy and reconstruction Confocal scanning laser microscopy Materials Microscopy of composite materials Microscopy of electronic materials Nanostructures Cytometry and Cytochemistry Study of cell proliferation with flow cytometry Tumour markers Probes for in situ hybridisation Probes for immunocytochemistry (p53, BrdU, Ki67, PCNA) Molecular probes for analysis of living cells (co-sponsored by The Physiological Society) Proteases in pathological processes Invasion and metastasis of cancer cells Arthritis and rheumatism Infections Environmental SEM
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited.
Furthermore, on the Monday, a special session on how to use the light microscope will take place.
Exhibition
An Exhibition of the latest microscopes, ancillary instrumentation and technology, of material needed for preparation of microscopic specimens (fluorescent probes, antibodies and dyes) and soft and hardware for image processing and image analysis will be held at the Earls Court Park Inn International. Admission to the exhibition is free by conference badge or exhibition only badge which will be obtainable at the registration desk. Exhibition tickets will also be available well before the exhibition takes place.
There will be approximately 50 companies exhibiting, providing an extensive range of equipment for you to view.
If you would like further details of the MICRO 94 Conference and Exhibition, please contact the Royal Microscopical Society, 37-38 St Clements, Oxford, OX4 1AJ, United Kingdom. Telephone +44 (0)865 248768, fax +44 (0)865 791237, email RMS-at-UK.AC.OX.VAX. Conference enquiries should be addressed to Miss Karen Hale, Exhibition enquiries should be addressed to Mrs Allison Winton.
-------------------end of message----------------------
} Details of all meetings are available from the Royal Microscopical Society, } 37-38 St Clements, Oxford, OX4 1AJ, United Kingdom. Telephone +44 } (0)865 248768, fax +44 (0)865 791237, email RMS-at-UK.AC.OX.VAX.
I believe, the Email address of the RMS is written in the wrong order. To my knowledge, it should read rms-at-vax.ox.ac.uk
Please, correct me if I am wrong.
Jouko Maki
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jouko K. Maki Navigare necesse est... Laboratory Manager, Ph.D. Laboratory of Electron Microscopy Kiinamyllynkatu 10 FIN-20520 TURKU FINLAND University of Turku Tel.: + 358 21 633 7318 INTERNET: jouko.maki-at-utu.fi FAX.: + 358 21 633 7380
The "small" problem I thought I solved earlier has turned out to be just the tip of an iceberg. The server will keep running however, it will be intermittent while I try to debug the system.
Part of the problem appears to be in "Multinet" the Internet gateway I'm using here at ANL. Some of the messages you will be receiving in the next few days will have double headers while I sort out the works. The first header will likely always show my name, while the second one will be the true sender.
Please keep me (Zaluzec-at-ANLEMC.MSD.ANL.GOV )informed of problems that you see so that I can continue debugging.
MAIL FROM: {ZALUZEC} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 9-NOV-1993 22:37:57
} Kayton asks: I need several options for storage of fixed tissues that might be used later for E.M. Currently I store tissues in the original fixative. Any other options would be appreciated.
====== What is the time scale you need to store your samples? Are they already EM (?TEM/SEM?) specimens?
MAIL FROM: {ZALUZEC} RCPT TO: {MICROSCOPYLISTUSA} ARRIVAL_TIME: 11-NOV-1993 14:44:27
48 hr. post-germination pea root tips and segments through differentiation zone were fixed in 2.5% glutaraldehyde in 0.15 M cacodylate buffer (pH 6.8), followed by 2% OsO4 in same buffer, then dehydrated and embedded in a resin mixture of equal parts epon (substitute=Epox 812): Spurr. 1-2 micron sections do not stain with 1% toulidine blue with varied times and temps. Am interested in good general stain which will also reveal mitotic figures__ANY SUGGESTIONS?
MAIL FROM: {GWERDOS-at-gnv.ifas.ufl.edu} RCPT TO: {MICROSCOPYLIST} ARRIVAL_TIME: 11-NOV-1993 08:20:35
Robert Kayton asked about fixed tissue storage. We generally advise the use of Trumps fixative as it has bee repoted good for this purpose. (McDowell & Trump, 1976, Arch. Pathol Lab. Med. 100:405)
Last year there was a paper by Sopsi et al. (Ultrastructural Pathol. 16:351) that reported good results storing fixed tissue frozen at -70 C after cryo- protection with sucrose (10% I think). They report good immunoreactivity after 2 years and it looked pretty good.
Greg Erdos Univ. of Florida gwerdos-at-gnv.ifas.ufl.edu
It's solved. The problem now definitely appears cured.
You can delete the rest of this message now, unless you want to know what happened.
========================
Symptom: Mailserver lockup, the batch server takes } 2 days to deliver mail that previously took 1/2 hour to process.
Cause:
Ultimately the mailserver lockup was caused by a faulty DNS (Domain Name Server) Address. The subscriber provided an address which checked out initially, remember that the instructions ask you to verify your address! However, after being added to the subscription list, something went haywire with one host node. In effect the mailserver would spend ~ 1-2 days searching & eventually finding the location of the subscriber. Unfortunately the way this software runs each subscriber is accessed sequentially and thus the mail would "appear" to hang doing nothing for a day (or more) while this one host is identified using god know how many secondary, teriary, DNS's in effect bouncing it's way around internet.
Solution: I've removed the problem address from the list and will contact the user seperately. Since the mail actually makes it through to the "lost" host I had no direct traceback feature to locate him and it took about a week to find. Some of you may not have noticed a problem since your subscription would have been entered and cleared before the problem one. In effect the only way to find the problem was to manually test the addresses, something I do not recommend that anyone does.
So... sorry that some of you had to put up with: 1.) multiple copies of repeated messages: 2.) excessively long headers with 1/2 the worlds email addresses 3.) multiple test messages
Many of you will not have received all of the messages posted to the listserver during this last week. If I see one that looks very important that in my infinite wisdom should be reposted I'll do so, otherwise it will just sit until the topic becomes posted again by the original author. I have an archive of everything and eventually will setup some way for anyone to search that archive but I've got alot of catching up to do and this is extremely low on my priority list of things to do.
That's all Folks! :-)
Nestor Z. ANL EM Center
Some how I just knew this listserver would become masochistic
In our standard H600 TEM, we have been getting comparable tungsten filament life to that in some of our other machines configured with sputter pumps, for use with LaB6 cathodes. Emboldened by this, we are fitting a Lab6 to this system to see what happens - anybody out there who has already done this, or has any comments? The cathode we have is a Denka, although we are generally moving to Kimball.
---------------------------------------------------------------------- Sally Stowe | Facility Coordinator ANU Electron Microscopy Unit | Australian National University Canberra, AUSTRALIA | Ph 61 6 249 2743 FAX 61 6 249 4891 | Email stowe-at-rsbs-central.anu.edu.au -------------------------------------|-------------------------------- -
John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu
I just sent off a posting suggesting the creation of a micrscopy newsgroup on the UseNet News system.
It's in the following newsgroups: sci.chem,sci.engr.chem,sci.geo.geology,sci.polymers,sci.materials,sci.misc,sci.p hysics,sci.research,sci.techniques.xtallography,sci.optics,sci.archaeology,sci. engr.mech,sci.geo.geology,sci.med
The following text is the full body of the posting, I encourage comments from the microscopy and imaging community. Please feel free to add to the necessary areas of interest, I will post follwo-ups when I have some response. I only posted this about two hours ago and I have had two messages of support already.
This post could equally well be titled: pre-RFD : sci.techniques.microscopy
Following is a proposed RFD (request for discussion) to create a microscopy newsgroup on the internet
Would potential users of this proposed newsgroup like to give their opinions on this (YES or NO) either by following on to this post (which should appear in sci.materials) or as email to (John.F.Mansfield-at-umich.edu).
Would people also consider passing this proposal onto colleagues who may be interested in participating in a microscopy newsgroup. Potential users of this newsgroup might like to consider adding their names to the RFD as co-proposers. It is hoped that at least one-hundred people can give support to this newsgroup before the RFD is officially posted to news.groups so as to show there is decent support for a microscopy newsgroup on the internet. If it looks like this will take a while to do, summaries of responses (who will remain anonymous) will be posted regularly to keep people informed what has been happening.
The reason for the name sci.techniques.microscopy is that the Usenet administrators have created the "techniques" hierarchy for newsgroups such as microscopy. Going with this name has the best hope of avoiding hair splitting arguments on newsgroup "names" that seem to plague the creation of other science newsgroups. The above name does not affect the aims or the charter of what this microscopy newsgroup is trying to achieve.
Please note if a particular topic of microscopy becomes very popular or there is a perceived need to create a subset of this newsgroup such as "SEM", "AFM" or "TEM", etc - it is relatively easy to create a
sci.techniques.microscopy.sem or sci.techniques.microscopy.tem, etc,
Pre - Request For Discussion (RFD) of sci.techniques.microscopy (unmoderated).
Proposers :- John Mansfield (John_Mansfield-at-mse.engin.umich.edu) Lachlan Cranswick (lachlan-at-dmp.csiro.au)
Please note that this proposed newsgroup is intended to be an open forum for discussion of microscopy. Thus relevant topics for this newsgroup should only be limited to what the participants in this proposed newsgroup regard as microscopy.
--------------------------------------------
We propose a new unmoderated newsgroup called sci.techniques.microscopy. The main aim of sci.techniques.microscopy is to provide an open forum for the discussion of microscopy and related fields on the internet.
(Could people reading this post mention it to colleagues who might be interested and benefit from a microscopy group but not normally use the internet newsgroups.)
Proposed Name of Group ------------------------- sci.techniques.microscopy (unmoderated)
Purpose of New Newsgroup ---------------------------
The purpose of sci.techniques.microscopy is to provide an open discussion forum for the microscopy community on the internet. The newsgroups allow the rapid and timely discussion of opinions and information that would take months or years (or not at all) on conventional paper journals.
Topics for discussion would include :- --------------------------------------- Optical Microscopy Confocal Microscopy Scanning Probe/Force Microscopy (formerly Atomic Force Microscopy) - SPM, SFM, AFM Scanning Tunnelling Microscopy - STM Scanning Electron Microscopy - SEM Transmission Electron Microscopy - TEM High Resolution Electron Microscopy - HREM Analytical Electron Microscopy - AEM Scanning Transmission Electron Microscopy - STEM High Voltage Electron Microscopy - HVEM X-ray Energy Dispersive Spectroscopy - XEDS Electron Energy Loss Spectroscopy - EELS Diffraction Contrast Imaging Phase Contrast Imaging Selected Area Electron Diffraction - SAED or SAD Convergent Beam Electron Diffraction - CBED Image Filtering Specimen Preparation (Electropolishing, Ion Milling, Ultramicrotoming, etc.) Software Data formats Databases Hardware/Equipment - specs, opinions, etc. Applications Announcements/reviews of papers/conferences. Preparation techniques. Non-ambient techniques General Discussion/opinions/questions. Positions vacant
Anything else that is relevant to microscopy in general.
What is the Process of Creating a Newsgroup? --------------------------------------------
} (a) RFD: Discussion, i.e., public hearing to take place in the newsgroup news.groups for approximately one month (b) CFV: Call for votes (the voting period will be about 25 days) (c) Counting of votes and public display of votes (d) Announcement of new newsgroup
(a)--} (b) assumes no major disagreements about this newsgroup during discussion. (c)--} (d) assumes that the vote is favourable., i.e., Y } N+100 .and. Y } (2/3)(Y+N) Y being the number of YES votes, N being the number of NO votes for the creation of the proposed newsgroup. =============================================
Please comment/criticise/suggest by followons to this post or email to John.F.Mansfield-at-umich.edu
John Mansfield, North Campus Electron Microbeam Analysis Laboratory, 2455 Hayward, Ann Arbor, Michigan 48109-2143. Ph: (313)-936-3352. __________ YYURYYUBICURYY4ME.
Hitachi HU-11E Parts for free: If you are nursing along an HU-11E TEM, and would like some spares for it, please contact me directly (please don't respond to the list!) --Forepumps and DP are spoken for --Most other parts go to anyone who can use them. I'll ship light stuff inside the continental US for free; you'll have to pay shipping on the heavy stuff.
If I haven't heard from someone within two weeks, it goes in the dumpster. (TBAITW; as in "too big and in the way").
Julian Smith III Winthrop University 803-323-2111 (Vox) 803-323-2246 (Fax)
} Sally Stowe asks about swapping W/LaB6 in a H600.
Sorry but I'm geting old and forget full so I can't remember the details of an H600, but, the appropriate question to ask is why are you attempting to put LaB6 in the H600? not if it will work! The LaB6 filaments when operated properly and under *CORRECT* conditions will give you a greater Brightness and hence more electrons in electron-optically equivalent probes. Thus for small probe/analytical work or for imaging conditions which will improve with a more coherent source (HREM) and in good vacuum systems they work very well. It will improve your uscope work assuming that you are short on electrons in the configuration your are setting up your electron probe (the argument applies to both AEM and/or HREM modes), however, if you poison the filament by using it in a poor vacuum system (i.e form an oxide or carbide on the surface), then the work function will decrease and you will actually get worse performance than a standard W filament and at ten to twenty times the cost of W. So why do you want to do this?
Assuming you have appropriate reasons and a good vacuum (and students who do not dump the vacuum system to air with a hot filament running), then the next question to ask is your uscope equipped with a filament power supply that can be modified to drive the tip?
} Sally STowe asks about using LaB6 under "tungsten" vacuum configuration - ie without sputter pump.
} Nestor Zaluzec asks 1. Why? 2. Can the filament power supply be modified to drive Lab6 tip?
In answer to (1) - we want to get the best resolution we can over the next few months, and at the moment, on smallest condenser aperture, we cant make best use of the "spot size" adjustment because of lack of electrons, although the resolution does seem to be inproving sharply as far as can be seen. This microscope does seem to be particularly prone to charge effects or whatever if the beam is not well spread. Vacuum in the specimen area is 8 x 10-6mbar. The last tungsten filament lasted 330ish hours, so reckon we have some reasonable users at the moment.Touch wood. (2) - yes, filament supply can be switched.
---------------------------------------------------------------------- Sally Stowe | Australian National Univ. Facility Coordinator Canberra, AUSTRALIA ANU Electron Microscopy Unit | Ph 61 6 249 2743 Email stowe-at-rsbs-central.anu.edu.au | FAX 61 6 249 4891 -------------------------------------|-------------------------------- -
We use a Park Scientific Universal BD2 Scanning Probe Microscope, controlled by a HP 382 workstation. Does anyone know of any software which will easily (and reliably) extract the images into a format which can be read by PC's? It is possible to export a TIFF file but the format appears (according to the operator) to be slighly different to other TIFF formats.
Any help would be appreciated.
Colin V.
##################################################################### ********************** * Between the idea * 0------* And the reality * } ---|--- { * Between the motion * | * And the act * / \ * Falls the Shadow * _/ \_ * T.S. Eliot * ********************** Colin Veitch Tel + 61 (0)52 47 2611 CSIRO Division of Wool Technology Tel + 61 (0)52 47 2891 (dir.) P.O. Box 21 Fax + 61 (0)52 47 2657 BELMONT Vic 3216 Australia
} Colin V. writes.... } We use a Park Scientific Universal BD2 Scanning Probe Microscope, } controlled by a HP 382 workstation. Does anyone know of any software } which will easily (and reliably) extract the images into a format which } can be read by PC's? It is possible to export a TIFF file but the format
Colin it sounds like Park is at fault. We routine transfer TIFF files from one computer Vax, Mac, PC, Sun... to another an access TIFF files with no difficulty. My guess is that Park took short cuts and did not properly implement TIFF (this happens alot from what I can tell). You should demand that Park rewrite their code to properly implement TIFF which is no at Version 6.0. T
There are lots of programs which can read TIFF files and display them on the PC.
Yes, 1.% Toluidine Blue in 1% sodium borate will stain sodium ethoxide etched Spurr epoxy.
On Fri, 12 Nov 1993, Nestor J. Zaluzec (708)-252-5075, -4964 wrote:
} From: SMTP%"-at-cc1.kuleuven.ac.be:desclinj-at-ulb.ac.be" 12-NOV-1993 02:02:51.52 } To: ZALUZEC } CC: } Subj: Re: LM - Need help on staining epoxy/Spurr embedded plant tissues_ } } From: desclinj-at-ulb.ac.be (Desclin Jean) } Message-Id: {9311120706.AA00745-at-is1e.vub.ac.be} } Subject: Re: LM - Need help on staining epoxy/Spurr embedded plant tissues_ } To: ZALUZEC-at-anlemc.msd.anl.gov (Nestor J. Zaluzec) } Date: Fri, 12 Nov 93 8:06:14 MET } In-Reply-To: {931111144412.202000ba-at-anlemc.msd.anl.gov} ; from "Nestor J. Zaluzec" at Nov 11, 93 2:44 pm } X-Mailer: ELM [version 2.3 PL11] } } Hello! } what is the pH of your toluidine blue solution? It might help } trying out a higher pH than usual (I am afraid I am not familiar } with plant tissues :-(...). } If you are not re-using the semi-thin sections afterwards (i.e. } resectioning for TEM), you might try treating them with NaOH } dissolved in absolute ethanol (to etch the plastic somewhat), such } as used prior to some immunohistochemical procedures. I believe } toluidine blue might work after that. } Good luck! } John } } } *********************************************************** } * Jean C. Desclin (John), Associate Prof. of Histology * } * Laboratory of Histology - Faculty of Medicine * } * Brussels Free University (U.L.B.) * } * e-mail: desclinj-at-ulb.ac.be (internet) * } * snail mail: route de Lennik 808 * } * B - 1070 Brussels Belgium * } *********************************************************** }
Many thanks to those who offered information regrding my query. All the information has been most illuminating (and useful).
Colin V.
##################################################################### ******************************* * Logic is invincible because * 0------* in order to combat logic it * } ---|--- { * is necessary to use logic. * | * P.Boutroux * / \ ******************************* _/ \_
Colin Veitch Tel + 61 (0)52 47 2611 CSIRO Division of Wool Technology Tel + 61 (0)52 47 2891 (dir.) P.O. Box 21 Fax + 61 (0)52 47 2657 BELMONT Vic 3216 Australia
} Colin V. writes.... } We use a Park Scientific Universal BD2 Scanning Probe Microscope, } controlled by a HP 382 workstation. Does anyone know of any software } which will easily (and reliably) extract the images into a format which } can be read by PC's? It is possible to export a TIFF file but the format
Colin it sounds like Park is at fault. We routine transfer TIFF files from one computer Vax, Mac, PC, Sun... to another an access TIFF files with no difficulty. My guess is that Park took short cuts and did not properly implement TIFF (this happens alot from what I can tell). You should demand that Park rewrite their code to properly implement TIFF which is no at Version 6.0. T
There are lots of programs which can read TIFF files and display them on the PC.
I'm starting to do gold labeling of protein in yeast cells and I'm having problems with infiltration. This is my first time in 25 years I've had to work with yeast. Any helpful hints?
Yes, 1.% Toluidine Blue in 1% sodium borate will stain sodium ethoxide etched Spurr epoxy. I've only had toluidine fail in Spurr's if the pH was off (someone didn't make up the correct borate } . For autoradiography and nuclear anntigen immunocytochemistry, it can be diluted to 0.1% or even 0.01% in 1% sodium borate to keep from obscuring the label.
} } If you are not re-using the semi-thin sections afterwards (i.e. } } resectioning for TEM), you might try treating them with NaOH } } dissolved in absolute ethanol (to etch the plastic somewhat), such } } as used prior to some immunohistochemical procedures. I believe } } toluidine blue might work after that. } } Good luck! } } John } } } } } } *********************************************************** } } * Jean C. Desclin (John), Associate Prof. of Histology * } } * Laboratory of Histology - Faculty of Medicine * } } * Brussels Free University (U.L.B.) * } } * e-mail: desclinj-at-ulb.ac.be (internet) * } } * snail mail: route de Lennik 808 * } } * B - 1070 Brussels Belgium * } } *********************************************************** } } } }
"Nestor J. Zaluzec (708)-252-50" {ZALUZEC-at-anlemc.msd.anl.gov} CC: ZALUZEC-at-anlemc.msd.anl.gov
Reply_ RE} AFM: Imaging Software Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu We have a Nanoscope 3 and Digital Instruments have a TIFF export that NIH-Image doesnt like. When we import the images into image we lose the LUT information. I have found that the MOST useful piece of software for manipulating images, except NIH-Image of course, is Photoshop. If you are at a Univ you can get really good prices on it and it happily reads almost anything I try and read into it.
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Colin it sounds like Park is at fault. We routine transfer TIFF files from one computer Vax, Mac, PC, Sun... to another an access TIFF files with no difficulty. My guess is that Park took short cuts and did not properly implement TIFF (this happens alot from what I can tell). You should demand that Park rewrite their code to properly implement TIFF which is no at Version 6.0. T
There are lots of programs which can read TIFF files and display them on the PC.
Nestor Z. ANL EM Center -------------
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"I need several options for storage of fixed tissues that might be used later for E.M. Currently I store tissues in the original fixative. Any other options would be appreciated."
One alternative is to put the tissue into the buffer originally used with the fixative. The only drawback here is that after a period of time (} 1 year) the buffer usually tends to become acidic, and there goes the tissue preservation. Alternatively, I used this when working years ago in another lab so have no recent knowledge of the pros and cons, but tissue that you expect to use for TEM, process it through osmium and up to 90% alcohol and then store. I know that I did this and then subsequently finished processing the tissue into epon several months later and the ultrastructural preservation was very good. The best option to maintain good quality morphology is of course to process the tissue into resin then you have a fossil which can not deteriorate.
Hope this is of some help,
Gerry Little.
Dr Gerald Little | Ph (049) 215618 The Neuroscience Group | Discipline of Anatomy | Fax (049) 216903 Faculty of Medicine | The University of Newcastle | Email ANGJL-at-medicine.newcastle.edu.au Australia, 2308 |
I passed Daniel Callahan's query to Richard Lee at Argonne. This is his reply:
Our experience with carbon is mostly on diamond using conventional (tungsten) filaments in an SEM. We typically resolve sub-micron filaments and crystallites dowm to 30 to 50 nm with 10 kV operation. Fullerene crystals were difficult for us because of charging--even at 10kV.
Our experience with an high resolution FEG-SEM was also on diamond and was most impressive! They excel at low kV operation with no charging problems. We have taken photos on diamond nanophase materials (coatings) with 3 nm or better resolution.at 5kV using a new Leica-Cambridge 360FE.
You can try conventional SEM's at 5kV, but you will be limited in magnification. If you go much above 10kV you probably will have charging problems and loss of surface detail. The lower the kV, the better the surface detail with a material of low Z.
I passed Daniel Callahan's query to Richard Lee at Argonne. This is his reply:
Our experience with carbon is mostly on diamond using conventional (tungsten) filaments in an SEM. We typically resolve sub-micron filaments and crystallites dowm to 30 to 50 nm with 10 kV operation. Fullerene crystals were difficult for us because of charging--even at 10kV.
Our experience with an high resolution FEG-SEM was also on diamond and was most impressive! They excel at low kV operation with no charging problems. We have taken photos on diamond nanophase materials (coatings) with 3 nm or better resolution.at 5kV using a new Leica-Cambridge 360FE.
You can try conventional SEM's at 5kV, but you will be limited in magnification. If you go much above 10kV you probably will have charging problems and loss of surface detail. The lower the kV, the better the surface detail with a material of low Z.
Reply to: RE} hex. kikuchi maps The program DIFFRACT will do this on a Macintosh computer very nicely. This program is available from Virtual Laboratories, 37 Highland Court., Ukiah, Calif., USA (Attn: Janet Schlinger) FAX: 707-462-5275.
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Does anyone know a programm for calculating hexagonal kikuchi maps on PC and with/without the possibility to vary the c/a-ratio?
Heinrich Kestler Institut fuer Werkstoffwissenscahften Lehrstuhl I Martensstrasse 5 D-91058 Erlangen, Germany
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The are also public domain programs in the EMMPDL for calculating Diffraction Patterns/Kikuchi Maps. They are not all elegant but they work. To access information about the EMMPDL send an Email message to:
EMMPDL-at-anlemc.msd.anl.gov
In the Email message include the text line
SEND HELP EMMPDL
You will get instructions on how to get abstract listings file names and how to get copies of code for the programs.
We are considering the use of a TEM-style cryo stage for some x-ray diffraction experiments on frozen hydrated specimens. Our stability requirements are not at all high (drift is not a problem at the 0.1 micron/hour level), so we could probably do with a older model cryo stage. We would custom-build an airlock around whatever cryo holder, so we are not limited to a stage for e.g., JEOL or Philips. However, we do need a specimen loading and transfer system to go with the stage. We do not need much in the line of tilt control, and copper or other metals are fine (no need for beryllium).
If anyone has a cryo specimen holder that they are willing to loan or sell at modest cost, please contact me.
Thanks! Chris Jacobsen Department of Physics SUNY at Stony Brook Stony Brook NY 11794-3800 USA fax 516-632-8101 cjacobsen-at-ccmail.sunysb.edu CJACOBSEN-at-SBCCMAIL.bitnet
Reply_ RE} AFM: NanoIII to NIH Image And more... Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu If I label my Nanoscope III images as "test.tif" or something similar then I can open the images into NIH-Image without losing the LUT. If I use just "test" without the tiff extension then I get gray images. I do not have AccessPC set to automatically open .tif files are NIH-Image files and so I am unsure as to why this is happening. I can drag and drop a filename.tif file onto NIH-Image and it opens just fine. (We have also use the raw data import option but sometimes want to retain the colors of the original image for presenation purposes.) By the way I am getting my images off the Nanoscope by using Farallon's Appletalk PC software, it works OK but it seems a little slow. Has anyone else had experience with this kind of setup? I also cannot get the ethernet card I am using (a 3COM) to work correctly with NCSA Telnet and FTP, which we would like to use to transfer data to workstations and other computers than Macs. --------------------------------------
Regarding John Mansfield's comment, it looks to me as though the Nanoscope III stores raw data with a greyscale LUT. I base this on the fact that changing color table parameters in Top View mode and then selecting another file does not trigger a "Current file has been modified, save it?" query the way changing the
Z scale does.
Libby Shaw MIT CMSE Surface Analysis Facility
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Reply_ RE} TEM: Re: hex. kikuchi maps Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu You do have to be wary of Diffract, it is buggy, even the latest version. There is a new product from those guys called Desktop Microscopist it is suppoedly a complete rewrite. However, after playing with it for 10 minutes at MSA I managed to crash it! Treat all software with care if you havent written and debugged it yourself!!
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Chris Boothroyd
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Dear Colleagues
I have started a new teaching/research position, to find that there is a very nice Jeol 1200, but no ultramicrotome! We have no funds for one in the near future, and such machines in Japan are very expensive, at least twice the price elsewhere.
However, we may be able to find enough funds for a second hand machine. I also need a 'tome like the old JB4 for semi-thin sections, it must have a retracting arm and be able to use glass knives. I don't care how old the machines are as long as they work.
If anyone has an old microtome that they wish to dispose of for YEN, we would be very interested. We will pay all shipping charges etc, of course.
Look forward to many offers!
Robert W. Ridge Biology Department ICU 10-2 Osawa 3-Chome Mitaka-shi Tokyo 181 Japan
Tel: +81-422-33-3484 (0422-33-3484 for callers within Japan) Fax: +81-422-33-1449 Email ridge-at-icu.ac.jp
Phil: there were two errors in the protocol I sent you. 1. You should start out using LR White HARD grade, not medium grade; polymerize this at 50 degrees C for 24 hrs. There may be a small of amount of fluid LR white at the top of the capsule. These block section fine, also. 2. You may need to block free aldehydes to prevent background problems; so you can include a step between the buffer wash and dehydration, adding 1%ammonium chloride to your buffer solution. -Louisa
Reply_ RE} } AFM- NanoIII to NIH Ima Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu Re: the message below that seems to be replyinmg to my gripes about slow data transfer and printing from our Nanoscope connected to the Appletalk net at the U of M. Sorry, I didnt make myself clear about our physical transport layer! We are using the Appletalk PC package on a 3-COM Ethernet card. We have mostly Macs and several color and greyscale capable printers on the network and so it pays us to have Appletalk connectivity. The major problem with the networking and the Nanoscope is that the AFM software walks over the Network software and vice versa. Digital Instruments have fixed the AFM software so that it runs in a reduced memory mode when the Appletalk stuff is loaded. Anyone know if I can fix this by adding more memory? I am a PC neophyte, Macs are my computer of choice. Plus, does anyone know why Digital Instruments used such and slow stodgy 486 for the platform for their instrument (other that it was cheap)?
OK, Opinions are mine and not the U's! Jfm.
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Hi!
Appletalk IS notoriously slow for transferring images, the maximum speed is just over 200 Kb/sec. Compare with ethernets 10Mb/sec. Put ethernet into the MACs... We have a fairly well working connection between PC's and UNIX workstations (Silicon Graphics). We use Walker Richer Quinns reflection software using TCP/IP as communication protocol. There are some very conveniant solutions though. One includes the Lantastic network (Editirs choice in PC-magazin for a number of years). Now there is a TCP/IP module for this network. It supports NFS and other goodies as well. In practice, I think this will provide more of an invisible network than ever before. Lantastic is also available for the MAC. Moreover windows for workgroups has TCP/IP options at very low prices.
Mikael Gustafsson dept medical microbiology Univ. Link0ping MikGu-at-mme.liu.se FAX 046/13/224789 SWEDEN
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Reply_ AFM- NanoIII Reply from: John Mansfield North Campus Electron Microbeam Analysis Laboratory University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 jfmjfm-at-engin.umich.edu or John_Mansfield-at-mse.engin.umich.edu Apparently I am wrong in assuming that the computer that is used by Digital Instruments for their Nanscope III is a cheap model. Apparently it is all Intel built. For those of you who have an older model with a 33Mhz processor and want a little more speed, call Mark Lean at Digital and get a price for an Overdrive processor to take it up to 66Mhz. (or go third party). Has anyone put a Pentium in a Nanoscope III yet? :-)
Pat Robinson asks...... ....................stuff deleted.............
} this 8500 is quite a different beastie. I'm especially interested in } ways and means of moving images to our network of Digital (DEC) PC's } running desktop publishing software, via TIFF or otherwise.
If you really want to work efficiently. Toss the TN buy yourself a Mac and framegrabber board. Then get a copy of the Public Domain program NIH-Image. It will grab images off your Sony, do some reasonable processing and save files as Tiff which you can then send via your network (use Pathworks, AppleTalk, TCP, just chooze your protocol & hardware) to your DEC machines.
In reference to } From jkelly-at-pruffle.nist.gov Fri Nov 19 15:58:12 1993 } To: microscopy-at-anlemc.msd.anl.gov } Subject: Image Printing } } I am working with image files on PC's - usually TIFF format } on IBM compatibles. I would like to get good quality B & W } plain paper hardcopy of the images. What are the good } combinations of print software and printers for either the PC's } or Mac environment? Thanks in advance.
For computer image printing, two popular solutions are laser printers (dithered black and white) and dye sublimation printers. We use IDL from Research Systems Inc. (Boulder CO) to do many many things, one of which is to generate PostScript files of images. It runs on PC and Mac (and Unix and VMS). We then get pretty good and very cheap B&W prints on any old PostScript laser printer. For the final print, we have a Tektronix Phaser IIsd (the "sd" or "sdx" is important) dye sublimation printer which takes color or B&W PostScript files. Kodak also now sells a similar product. Codonics sells a dye sublimation printer which takes TIFF files directly.
-- ******************* Chris Jacobsen, Asst. Prof., Department of Physics, SUNY at Stony Brook Phone (516) 632-8093, FAX -8101 Bitnet: cjacobsen-at-sbccmail jacobsen-at-xray1.physics.sunysb.edu ALL-IN_ONE: CJACOBSEN *******************
I have some synthetically prepared mineral powders, grain size in the 1-30 micron range, which I would like to get some EDS analyses of. This would mean embedding the microcrystals in epoxy and polishing them to get nice cross sections -- would someone with experience in this kind of sample prep be willing to share some tips/hints?
Thanks,
Bernhardt Saini-Eidukat Dept. of Geosciences North Dakota State University Fargo, ND 58105
We have been transferring files from the Tracor 8500 to PC and Mac formats using the following steps:
1. IBM-compatible computer connected from serial port 7 of 8500 to com2 of IBM. 2. On 8500 convert file to ".tif" format and re-save the file to the EXPORT directory. 3. Files are transferred to an IBM-compatible image handling application, such as Procomm. If using Procomm, open Procomm and input the following file specification: "/u3/export/name.tif". As the file is transferred, a byte count is tallied which should match that read on the 8500. 4. Copy image from Procomm directory to an IBM-formatted diskette.
Cheers, Charles E. Lyman
Department of Materials Science and Engineering Lehigh University, 5 East Packer Avenue Bethlehem, PA 18015-3195 E-mail: cel1-at-Lehigh.edu Tel: 215-758-4249 FAX: 215-758-4244
Message-Id: {9311221558.AA20274-at-sifon.cc.mcgill.ca} To: Bernhardt Sainieidukat {sainieid-at-badlands.NoDak.edu}
Bernhardt, I've mounted small grains for use in the microprobe on several occasions. Usually the material I work with is a bit larger, but the techniques should still apply if you work carefully. If you have a large sample and are not concerned with individual grains, the simplest way to work is to set them in in epoxy on a standard thin section blank. If you have a reliable thin-section technician he should have no problen grinding it down to a 15 um thickness. Alternatively, if you have a small number of grains or you are interested in specific grains you can mount them in a plexiglass holder. First cut out a piece of plexiglass to fit into your sample holder (the thinner the better, I find 3 mm is about right). Next drill a number of small holes in the holder (I use a 2mm diameter). I mount these holders on a cleaned thin section blank using double sided tape. You should be very careful that there are no bubbles between the tape and the blank. Fill a hole with epoxy (I use Struers Epofix, it's nice and thin). You probably will want to carry out the next steps under a microscope. Using very fine forceps drop a grain into a hole and stir the epxoy with a fine wire to wet the grain and cause it to settle into the epoxy. If necessary you can adjust the grain so that it's resting on its most stable surface. Depending on the grain size I can usually get 10-15 grains in a mount before they start interfering with each other. Once the epoxy has set you can remove the blank slide on the bottom (usually I have to break it off). Polishing these mounts is crucial, if you take to much material off you lose your sample (This can really ruin your day if that was the only sample you had). I generally start with a very light brush with 300 grit SiC paper, just enough to remove any excess epoxy. This is followed by 600 grit paper. At this stage I polish for 30 seconds to a minute and then look at the result in a microscope to see how much exposed material I have. When sufficient material is exposed I do the fine polishing using 3, 1 and then 0.3 um Al2O3 either by hand or on a lap, depending how nervous I am about losing the material. Again I use short polishing periods followed by observation in the microscope. I've generally had good results with this technique and have rarely lost any material. One problem I have with larger grains is that it is sometimes difficult to get a large polished surface when the grain is sitting on it edges. I hope this helps you. Call me back if any of this is not clear
********************************************************************** * Glenn Poirier glenn_p-at-geosci.lan.mcgill.ca * * Electron Microprobe Lab Phone: (514) 398 6774 * * Earth and Planetary Sciences Fax: (514) 398 4680 * * McGill University THERE ARE THREE SIDES TO EVERY STORY; * * Montreal, Quebec YOUR SIDE MY SIDE AND THE TRUTH * **********************************************************************
I'm looking to buy a set of ultra long working distance objectives to fit onto an Olympus BHSM microscope. The longest WDs I've found so far belong to Olympus' own objectives (20x with 11mm and 50x with 8mm). Anyone know of any alternatives?
I'm looking to buy a set of ultra long working distance objectives to fit onto an Olympus BHSM microscope. The longest WDs I've found so far belong to Olympus' own objectives (20x with 11mm and 50x with 8mm). Anyone know of any alternatives?
} I'm looking to buy a set of ultra long working distance objectives to } fit onto an Olympus BHSM microscope. The longest WDs I've found so far } belong to Olympus' own objectives (20x with 11mm and 50x with 8mm). } Anyone know of any alternatives?
One trick is to look for U-stage lenses. These are designed for universal-stage mineralogical microscopes where the stage tilts as well as rotates. These lenses have a separate hemispherical glass element that is placed over the thin-section. Used without the hemisphere they have very long working distances.
Another trick is to use reflecting objectives.
I don't know the Olympus BHSM. Assuming it has standard threads (.8in 38tpi) and the equivalent of a 160mm tube length... We use a Leitz UT40 which is a U-stage lens. Without the hemisphere it has a magnification of 25x and a working distance of 14mm. Leitz has a few other U-stage lenses that are parfocal with this lens. Chromatic aberration can be a problem for refracting objectives with these long working distances.
We also use a reflecting objective from Ealing (15x, 21mm or 24mm working distance). Ealing sells several other reflecting objectives with higher magnification. The 15x is identical to the 15x lens made by Beck as far as I can tell. You may be able to purchase the Ealing lens corrected for infinity focus or a 210mm tube length, if that is what the Olympus requires. These reflecting objectives are just the ticket if you can tolerate their large size and the central obscuration.
Jeff Sweeney sweeney-at-dionheinz.uchicago.edu
Some good references are:
%0 Journal Article %A Burch, C. R. %D 1943 %T On aspheric anastigmatic systems %B Proceedings of the Physical Society %V 55 %N 312 %P 433-444 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1943 %T Reflecting microscopes %B Proceedings of the Physical Society %V 59 %P 41-46 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1945 %T Flat-field singlet aplanats %B Proceedings of the Physical Society %V 57 %P 567-576 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1947 %T Semi-aplanat reflecting microscopes %B Proceedings of the Physical Society %V 57 %P 47-49 %K reflecting objective
A couple of things I forgot to mention in my original mailing - the Olympus has infinity corrected optics (I had found that Nikon had a nifty lens, but it turned out not to be infinity corrected) and the microscope is part of a Renishaw imaging Raman spectrometer. I have been told by others that reflecting objectives would be no use since the Raman laser would either be reflected straight back into the spectrometer without reaching my sample or would have to be defocussed, preventing me doing any small area analysis.
Richard Lee of the Energy Technology Division at Argonne National Laboratory answers Daniel Callahan's question of 11 Nov 93:
Our experience with carbon is mostly on diamond with tungsten filaments in a SEM. We typically resolve sub-micron filaments and crystallites down to 30 to 50 nm at 10kV. Fullerene crystal were difficult for us because of charging, even at 10kV.
Our experience with an high resolution FEG-SEM was with diamond and the results were impressive. The FEG-SEM excels at low kV with no charging problems. We have taken photos of diamond nanophase materials (coatings) with 3 nm or better resolution at 5 kV using a new Leica-Cambridge 360FE.
You can try conventional SEM's at 5 kV but you will be limited in magnification. If you go much above 10 kV you probably will have charging problems and loss of surface detail. The lower the kV the better the surface detail with a material of low Z.
} A couple of things I forgot to mention in my original mailing - the } Olympus has infinity corrected optics (I had found that Nikon had a } nifty lens, but it turned out not to be infinity corrected) and the } microscope is part of a Renishaw imaging Raman spectrometer. I have } been told by others that reflecting objectives would be no use since } the Raman laser would either be reflected straight back into the } spectrometer without reaching my sample or would have to be } defocussed, preventing me doing any small area analysis.
Geez, you're making this more difficult :-) Chromatic aberration is not a problem with Ramam since you are working so near the Rayleigh peak, so look for a U-stage lens with infinity focus. You can use a reflecting objective but you must send the probe laser into the lens off-axis. Your imaging system may not accomodate this (probably not, huh). The focus of the laser can be adjusted with the beam expander if your beam expander is adjustable (probable not, huh). Alternatively, the laser can be prefocused with a long focal-length plano-convex lens. You might consider coating the reflecting objective for the laser wavelength. This would help aviod beam damage (not really a problem) and would give better signal in general.
I would like to hear recommendations for text books and review articles on the subject of light microscopy system design. I am interested in both basic information and the most recent technology. I would specifically be interested in hearing about those "desk reference" type books or review articles you always keep near by, and articles that have good bibliographies.
I am working on designing a photoluminescence imaging system (not confocal).
Please send references to me and I will compile a list. Also, if anyone would like a copy of the completed list, just let me know. Thanks.
Erik Reuter, e-reuter-at-uiuc.edu, 217-244-6378 phone, 217-244-6375 fax
And I haven't yet mentioned the high temperatures I want to have my samples at! Also, one of our third year students wants to Raman his diamond coatings at 400 C - What will that do to either ULWD or standard objectives???
Chromatic aberration is not } a problem with Ramam since you are working so near the Rayleigh peak, so } look for a U-stage lens with infinity focus. You can use a reflecting } objective but you must send the probe laser into the lens off-axis. } Your imaging system may not accomodate this (probably not, huh).
I'll go and ask.
The } focus of the laser can be adjusted with the beam expander if your beam } expander is adjustable (probable not, huh).
I already know this is, but you might be able to tell we haven't had the system long and are all still coming to terms with it. Someone here suggested using reflecting objectives with the beam expanded and with a blanking-widget in the lens to prevent direct reflection of the laser back into the spectrometer.
Alternatively, the laser } can be prefocused with a long focal-length plano-convex lens. You might } consider coating the reflecting objective for the laser wavelength. } This would help aviod beam damage (not really a problem) and would give } better signal in general. } } Jeff Sweeney sweeney-at-dionheinz.uchicago.edu } iThankyou,
"Photography Through The Microscope", P-2, cat.# 152-8371 "Kodak Scientific Imaging Products", L-10, cat. # 813-9321
The first has loads of basic information about objectives, condensers, setting-up illumination, flourescence, phase contrast, etc. Both have bibliographies.
The scope is 10 years old but in very good condition. It has been continuously maintained under service contract and is still in daily use. The components and accessories are listed below. All documentation and records are included. We would like to sell this as a complete package.
JEM-200CX TEM - S/N EM132031-86 200CX-SEGZ JEOL 200CX side entry goniometer HM-PP 3.5A, +30o SEG polepiece HA-PP 4.5A, +60o SEG polepiece 200CX-SQH-2 High Mag single tilt holder 200CX-SCSH Std. SEG quick change single tilt holer 200CX-BST double tilt holder (w/ one Gatan Be cup) 200CX-SHH sample heating holder 200CX-SCH single tilt cooling holder 200CX-SHU heating holder control unit 200CX-SEH single tilt straining holder 200CX-SFH faraday cup Haskris recirculating water chiller 200CX-ASID-3D STEM/SEM system 200CX -BEI-3 BSE detector (needs new Si crystals) 200CX-BF/DF STEM BF/DF option 200CX-FLC Free lens control 35-GMC Gamma control HA-EDS high angle EDS interface 200CX-HXS hard x-ray aperture 200CX-IMS-2 Image selector switch 35-MDD Multiple image display 200CX-SRT Scan Rotation & tilt correction 200CX-UHR UHR (photo) CRT w/ Polaroid 545 film back 35-VCA Video control amp. (derivative/filtering) 200CX-WFM-B Tek 501 waveform monitor 200CX-WFM-M lens current readout 35-YMD Y modulation device EM-THG Top entry Ultrahigh resolution goniometer TN-2000 Tracor-Northern TN-2000 EDS system (PDP-11/23 w/ 128kB RAM) SpectraChrome 512 Color monitor Horizontal Be window EDS detector High Takeoff angle (72o) Be window EDS detector EDS preamplifiers (x2) Digital beam control and interface EDS and Image acquisition software DEC LA-120 printer Honeywell Video Graphics recorder JEOL ASD system (projector lens scan coil) with OSU mods for scanning the TEM image) OSU built hollow cone illumination device Manuals and notes misc spare parts and filaments, including pole pieces and defl. coils HP 7221C 8 pen plotter operator's chair Gatan DT cryo low bkg holder Gatan cryo transfer holder
colijn.1-at-osu.edu OSU Campus Electron Optics Facility 292-0674 ------------------------------------------------------------------- Assumption is the mother of all screwups.
Regarding J. Kelly's (jkelly-at-enh.nist.gov) question:
} I am working with image files on PC's - usually TIFF format } on IBM compatibles. I would like to get good quality B & W } plain paper hardcopy of the images. What are the good } combinations of print software and printers for either the PC's } or Mac environment? Thanks in advance.
Have you considered videoprinters? You can videoprint any analog signal: RS-170 (NTSC), PAL (Europe), VGA... at 1200 dots/inch, 64 grey-levels, and it's *fast* (30 sec) compared to digital printing. You get near Polaroid quality for 40 cents a shot (one 8x10 inch, or two different 3x5 inch images can be printed together). I know you specified "plain paper," but the thermal paper is easily handled. Our Seikosha VP-3500 has been so reliable and popular with our customers, we put another on a Sun workstation based SEM. Capital investment is about $7500. One drawback: the images are not as stable. When I put a cup of hot coffee down on a print, it "developed" a black circle. ******************* Craig A. Smith, Honeywell Solid State Electronics Center, MN14-2C25 12001 State Highway 55, Plymouth, MN 55441-4799 USA Phone (612) 954-2895, FAX (612) 954-2040 smithc-at-ccsvax.ssec.honeywell.com *******************
Like J. Kelly, we are interested in printers. Specifically we are interested in purchasing a high-resolution gray scale printer to print TIF files from both PC's and from Silicon Graphic computers. Our files contain light microscopic images of hippocampal dendrites visualized at high magnification using a variety of staining methods (Golgi, biocytin, neurobiotin). I have information from Harris on their PhotoPro2000 and from Alden on their 9315 continuous tone printer. They range in price from $7500-10,000. What has been your experience & what would you recommend? We need a high-res image, and I am a bit concerned about the longevity of the thermal paper images. Many thanks.
Nancy L Desmond Dept. of Neurosurgery Univ. of Virginia Health Sciences Center Charlottesville, VA 22908 804.924.5607 (phone) NLD-at-GALEN.MED.VIRGINIA.EDU
} I have information from Harris on their PhotoPro2000 and from Alden } on their 9315 continuous tone printer. They range in price from } $7500-10,000.
This is the first I've hear of these brands. So I have no opinion, except that they sound expense for thermal printers. Lots of systems were exhibited at the MSA annual meeting for continuous tone printing. You may want to get a copy of the Exhibitors Guide from the MSA meeting and call up a few of the vendors for more information. Try contacting Bill Gunning Dept. of Path. Medical College of Ohio. 3000 Arlington Ave. Toledo, Ohio 43699-0008. He runs the Advertising for the MSA EXPO and can probably tell you how to get a copy. (419) 381-3484
} We need a high-res image, and I am a bit concerned } about the longevity of the thermal paper images.
High res means different things to different people. Do you mean pixel resolution, gray scale resolution, Image size or what? It also depends upon you digitization source (TV, CCD, Scanner....) and it's "pixel/gray scale" resolution. Also what is the purpose of your prints? Publication reports, records, or what.. If you have them on the computer it's certainly archived there resonably well at least as good as the lifetime of data on disks. I've got floppy disks with data on them that are now approaching 15 & 20 years old and to my surprize the data was still readable even though the disks at the time were only supposed to be good for ~ 10 years! Of course that implies you still have the computer to read all that "good" old data :-)
I would not trust thermal paper for archiving but only for a quick look see and maybe a lab notebook record. The best thing to do here is get the manufacturer's your interested in to give you copies of their prints and lay them out in the sunlight and shade for a few days/weeks. See how long they survive and then decide. The different brands will give different results, some are suprizingly good.
Here's a question for those of you that used to use the old Celli tape popular in England about 10+ years ago.
In the past I had some of this tape which I used to use to prepare specimens of graphite for TEM. Here I would use the old trick of just continually touching the surface of the graphite with fresh tape to delaminate the graphite gradually until I made a nice thin & optically transparent section, which stuck to the tape and was usually perfect for TEM. The nice thing about this old type of scotch tape was that the adhesive used would disolve beautifully in Acetone and the backing for that tape would just float away as it was not soluablein the acetone. Unfortuantely, the current type of "SCOTCH BRAND" transparent tape completely dissolves in Acetone and most other solvents I tried.
I'm back to trying to make more graphite samples as my old ones have finally bit the dust so to speak. Since the new scotch tape failed, I tried extraction replica tape which is available from most EM supply vendors, however it does not have the adhearance of either the old Celli tape or the newer scotch tapes. (BTW the new scotch tape very nicely delaminated the graphite and gives beautifully thin sections if I could ever remove them from the adhesive). Does anyone know of a solvent which works on the newer scotch brand tapes or have another/similar idea/procedure for layered structures which are not strongly bound?.
I'd prefer to avoid ion milling the graphite although I may have to resort to this in the end.
Forwarded message: From K.R.Hallam-at-bristol.ac.uk Mon Nov 29 12:21:47 1993 Message-Id: {9815.9311291221-at-irix.bris.ac.uk}
Dear All,
I was asked if I would post a summary of the replies received after my queation over ULWD objectives to attach to an Olympus BHSM / Renishaw imaging Raman system. Here goes....
} From jacobsen-at-xray1.physics.sunysb.edu Tue Nov 23 14:22:40 1993
Nikon makes a toolmaker's lens which can be fitted onto a Nikon Optiphot. It has 10x mag and a working distance of several cm. Very good images...
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 15:26:16 1993
One trick is to look for U-stage lenses. These are designed for universal-stage mineralogical microscopes where the stage tilts as well as rotates. These lenses have a separate hemispherical glass element that is placed over the thin-section. Used without the hemisphere they have very long working distances.
Another trick is to use reflecting objectives.
I don't know the Olympus BHSM. Assuming it has standard threads (.8in 38tpi) and the equivalent of a 160mm tube length... We use a Leitz UT40 which is a U-stage lens. Without the hemisphere it has a magnification of 25x and a working distance of 14mm. Leitz has a few other U-stage lenses that are parfocal with this lens. Chromatic aberration can be a problem for refracting objectives with these long working distances.
We also use a reflecting objective from Ealing (15x, 21mm or 24mm working distance). Ealing sells several other reflecting objectives with higher magnification. The 15x is identical to the 15x lens made by Beck as far as I can tell. You may be able to purchase the Ealing lens corrected for infinity focus or a 210mm tube length, if that is what the Olympus requires. These reflecting objectives are just the ticket if you can tolerate their large size and the central obscuration.
Some good references are:
%0 Journal Article %A Burch, C. R. %D 1943 %T On aspheric anastigmatic systems %B Proceedings of the Physical Society %V 55 %N 312 %P 433-444 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1943 %T Reflecting microscopes %B Proceedings of the Physical Society %V 59 %P 41-46 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1945 %T Flat-field singlet aplanats %B Proceedings of the Physical Society %V 57 %P 567-576 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1947 %T Semi-aplanat reflecting microscopes %B Proceedings of the Physical Society %V 57 %P 47-49 %K reflecting objective
} From treado+-at-pitt.edu Tue Nov 23 21:03:41 1993
In response to your question posted to the Microscopy discussion list November 23, we use Leica ultra long working distance infinity-corrected objectives on our Olympus BHS microscope.
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 21:30:36 1993
Geez, you're making this more difficult :-) Chromatic aberration is not a problem with Ramam since you are working so near the Rayleigh peak, so look for a U-stage lens with infinity focus. You can use a reflecting objective but you must send the probe laser into the lens off-axis. Your imaging system may not accomodate this (probably not, huh). The focus of the laser can be adjusted with the beam expander if your beam expander is adjustable (probable not, huh). Alternatively, the laser can be prefocused with a long focal-length plano-convex lens. You might consider coating the reflecting objective for the laser wavelength. This would help aviod beam damage (not really a problem) and would give better signal in general.
} From e-reuter-at-uiuc.edu Wed Nov 24 00:20:00 1993
I am also interested in learning about ultra long working distance objective. Would you please summarize to the list or to me what you find out in a few days?
} From timonf-at-earth.ruu.nl Wed Nov 24 07:41:09 1993
The problem with U-stage lenses is that the highest magnification I know which is available is a 30 x, and the optical quality is not too good (some distortion of the image and loosing a loss of brightness)
} From muepf-at-iff067.iff.kfa-juelich.de Wed Nov 24 07:52:31 1993
One of the --- expensive --- alternatives is to get a Questar long
[Sorry - I've lost part of the original here because someone picked up the telephone next door while my modem was connected - the message went on to mention a 3000 ECU alternative - Could you please resend the details to me - Thank you]
} From treado+-at-pitt.edu Wed Nov 24 17:14:22 1993
If you are only interested in doing Raman microprobing the reflective objective may be suitable by defocusing and illuminating off-axis. If you want to perform imaging I would use refractive objectives. The imaging quality is far superior with refractive optics.
We use Leica objectives: 5X, 0.10 NA, 50 mm WD and 50X, 0.45 NA, 20.6 mm WD.
We operate at elevated temperatures (} 200 oC) and jacket the objective in a water cooled housing. According to Leica the optical cements fail above 110 degrees.
Forwarded message: From K.R.Hallam-at-bristol.ac.uk Mon Nov 29 12:21:47 1993 Message-Id: {9815.9311291221-at-irix.bris.ac.uk}
Dear All,
I was asked if I would post a summary of the replies received after my queation over ULWD objectives to attach to an Olympus BHSM / Renishaw imaging Raman system. Here goes....
} From jacobsen-at-xray1.physics.sunysb.edu Tue Nov 23 14:22:40 1993
Nikon makes a toolmaker's lens which can be fitted onto a Nikon Optiphot. It has 10x mag and a working distance of several cm. Very good images...
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 15:26:16 1993
One trick is to look for U-stage lenses. These are designed for universal-stage mineralogical microscopes where the stage tilts as well as rotates. These lenses have a separate hemispherical glass element that is placed over the thin-section. Used without the hemisphere they have very long working distances.
Another trick is to use reflecting objectives.
I don't know the Olympus BHSM. Assuming it has standard threads (.8in 38tpi) and the equivalent of a 160mm tube length... We use a Leitz UT40 which is a U-stage lens. Without the hemisphere it has a magnification of 25x and a working distance of 14mm. Leitz has a few other U-stage lenses that are parfocal with this lens. Chromatic aberration can be a problem for refracting objectives with these long working distances.
We also use a reflecting objective from Ealing (15x, 21mm or 24mm working distance). Ealing sells several other reflecting objectives with higher magnification. The 15x is identical to the 15x lens made by Beck as far as I can tell. You may be able to purchase the Ealing lens corrected for infinity focus or a 210mm tube length, if that is what the Olympus requires. These reflecting objectives are just the ticket if you can tolerate their large size and the central obscuration.
Some good references are:
%0 Journal Article %A Burch, C. R. %D 1943 %T On aspheric anastigmatic systems %B Proceedings of the Physical Society %V 55 %N 312 %P 433-444 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1943 %T Reflecting microscopes %B Proceedings of the Physical Society %V 59 %P 41-46 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1945 %T Flat-field singlet aplanats %B Proceedings of the Physical Society %V 57 %P 567-576 %K reflecting objective
%0 Journal Article %A Burch, C. R. %D 1947 %T Semi-aplanat reflecting microscopes %B Proceedings of the Physical Society %V 57 %P 47-49 %K reflecting objective
} From treado+-at-pitt.edu Tue Nov 23 21:03:41 1993
In response to your question posted to the Microscopy discussion list November 23, we use Leica ultra long working distance infinity-corrected objectives on our Olympus BHS microscope.
} From sweeney-at-dionheinz.uchicago.edu Tue Nov 23 21:30:36 1993
Geez, you're making this more difficult :-) Chromatic aberration is not a problem with Ramam since you are working so near the Rayleigh peak, so look for a U-stage lens with infinity focus. You can use a reflecting objective but you must send the probe laser into the lens off-axis. Your imaging system may not accomodate this (probably not, huh). The focus of the laser can be adjusted with the beam expander if your beam expander is adjustable (probable not, huh). Alternatively, the laser can be prefocused with a long focal-length plano-convex lens. You might consider coating the reflecting objective for the laser wavelength. This would help aviod beam damage (not really a problem) and would give better signal in general.
} From e-reuter-at-uiuc.edu Wed Nov 24 00:20:00 1993
I am also interested in learning about ultra long working distance objective. Would you please summarize to the list or to me what you find out in a few days?
} From timonf-at-earth.ruu.nl Wed Nov 24 07:41:09 1993
The problem with U-stage lenses is that the highest magnification I know which is available is a 30 x, and the optical quality is not too good (some distortion of the image and loosing a loss of brightness)
} From muepf-at-iff067.iff.kfa-juelich.de Wed Nov 24 07:52:31 1993
One of the --- expensive --- alternatives is to get a Questar long
[Sorry - I've lost part of the original here because someone picked up the telephone next door while my modem was connected - the message went on to mention a 3000 ECU alternative - Could you please resend the details to me - Thank you]
} From treado+-at-pitt.edu Wed Nov 24 17:14:22 1993
If you are only interested in doing Raman microprobing the reflective objective may be suitable by defocusing and illuminating off-axis. If you want to perform imaging I would use refractive objectives. The imaging quality is far superior with refractive optics.
We use Leica objectives: 5X, 0.10 NA, 50 mm WD and 50X, 0.45 NA, 20.6 mm WD.
We operate at elevated temperatures (} 200 oC) and jacket the objective in a water cooled housing. According to Leica the optical cements fail above 110 degrees.
Message-Id: {MAILQUEUE-101.931129104500.288-at-parmly1.parmly.luc.edu} To: Microscopy-at-anlemc.msd.anl.gov
} We are doing microscopic image processing to access physical properties } (and their change) of lipid membranes and polymers in different setups. } In the near future we will face the problem to escape the limits of standard } video frequency (30/25 Hz). Therefore I want to ask whether anybody is } listening, who has experience with higher frame-rate cameras and other } devices and who would be willing to give me some advice in this field. Question deleted... } I am quite unsure whether the answers to this query might be of general } interest or not, so please feel free to email me directly at } mschindl-at-physik.tu-muenchen.de. I think this is of interest...I would certainly like to know the answers to these questions. Phil Oshel
Before I start let me admit that I have not tried this. However, I have heard other people doing it and it works. You can completely dissolve the scotch tape with acetone. I mean you can dissolve both the adhesive and the backing in aceotne leaving no residues. I have heard of acetone evaporaotrs where a little amount of acetone is heated and the vapors condense on the sample. This acetone dissolves any soluble substance and drips down into the bath to be reevaporated. The solubility of both adhesive and backing is higer at near melting point of acetone and hence this process substantially speeds up the removal. Hope this helps.
Naresh Shah University of Kentucky 233 Mining & Mineral Bldg. Lexington, KY 40506-0107 (606)257-4045 naresh-at-funky.mm.uky.edu
International Microscopy and Image Analysis Conference and Exhibition
12 - 15 September 1994 Earls Court Park Inn, Lillie Road, London
Organized by the Royal Microscopical Society in association with Microscopy and Analysis
Second Circular
Dates
Conference: Monday 12 September - 2.00 pm - 4.15 pm Tuesday 13 September - 10.00 am - 4.15 pm Wednesday 14 September - 10.00 am - 4.15 pm Thursday 15 September - 10.00 am - 4.15 pm
Exhibition: Monday 12 September - 2.00 pm - 7.00 pm Tuesday 13 September - 9.30 am - 6.00 pm Wednesday 14 September - 9.30 am - 6.00 pm Thursday 15 September - 9.30 am - 4.30 pm
On the Monday evening there will be a wine reception at 5.30pm, followed by the AGM and Presidental Lecture at 7.00pm.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
CONFERENCE
Scientific Programme The Conference will be run in seven half-day sessions. The Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) and The Physiological Society are each sponsoring separate lectures within the Conference.
The Programme will consist of tutorial lectures and posters and will feature the following topics:-
Monday 12 September (pm) - Materials I þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ EM in the assessment of semiconductor epitaxial growth Reactions to the surface of implanted bioceramics X-Ray microanalysis in biomaterials Optically active nanostructured materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (am) - Materials II (including EMAG) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Auger electron spectroscopy Imaging time of flight SIMS Formation of strained layer superlattices by phase separation Electron microscopy of weakly ordered III-V semiconductor materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (pm) Scanning Probe Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Applications of atomic force microscopy to thin film research and technology Scanning probe microscopy: near field imaging of surfaces using electrons, forces and photons SPM of living biological systems Environmental SEM þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (am) - Image Processing and Analysis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Sampling in 3D for quantitative microscopy Digital image processing techniques Image analysis of multicoloured biological specimens In vivo microscopy by video imaging þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (pm) - 3D Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Supercomputing in confocal microscopy 3D atomic-scale microanalysis of materials Spatial distribution of fibres in composite materials Confocal polarised-light microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (am) - Flow Cytometry and Proliferation Markers þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Cell cycle control Proliferation-related proteins Proliferation in human tumours Apoptosis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (pm) - Living Cell Cytochemistry (including The Physiological Society) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Living cell cytochemistry: Ratio Imaging Living cell cytochemistry: Confocal scanning laser microscopy
Thursday 15 September (pm) - Proteases in (patho) physiological processes
Use of selective protease inhibitors in the study of collagen breakdown Role of proteases in invasion and metastasis of cancer cells, arthritis and rheumatism and infections þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
Experts in the fields listed above have been invited to give lectures. Each speaker will provide a review of the particular topic in question and ample time for discussion will be provided.
In addition to the above, a special session will be held on the afternoon of Monday 12 September on how to use the light microscope.
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
INVITED SPEAKERS
It is hoped that the following people will be presenting papers at the MICRO 94 Conference:-
Dr Paul D. Brown (University of Cambridge) Professor Peter Marquis (University of Birmingham) Dr Henk Koerten (University of Leiden, The Netherlands) Dr Peter Dobson (University of Oxford) Dr R. K. Wild (University of Bristol) Dr Paul Denison (University of Sheffield) Dr Andrew Norman (Imperial College, London) Dr Caroline Baxter (University of Cambridge) Dr Alan Pidduck (RSRE, Malvern) Dr M. Miles (HH Wills Physics Laboratory, Bristol) Dr H Ho”rber(European Molecular Biology Laboratory, Heidelberg, Germany) Mr Chris Gilpin (Manchester Biological EM Centre) Dr Vyvyan Howard (Royal Liverpool Children's Hospital) Dr Dominique Jeulin (Ecole Nationale Superiere des Mines, Fontainebleau, France) Dr Hans Tanke (University of Leiden, The Netherlands) Dr Andreas Kriete (Der Justus Liebig Universitat, Germany) Dr Alfred Cerezo (University of Oxford) Dr A. R. Clarke (University of Leeds) Dr Alan Entwistle (Ludwig Institute for Cancer Research, London) Dr A Bagg (TNO Rijswijk, The Netherlands) Dr Michael Ormerod (Sutton, Surrey) Dr Peter van Mier (Washington University School of Medicine, USA) Professor P. A. McNaughton (King's College London) Dr R. Jacob (King's College London) Dr Vincent Everts (University of Amsterdam, The Netherlands) Dr Ron Van Noorden (University of Amsterdam, The Netherlands)
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
CONTRIBUTED PAPERS In addition to invited papers, contributions are invited on all aspects of microscopy and related techniques.
All contributed papers will appear in the poster sessions of the Conference. Time will be allowed in the Programme for the viewing of posters, and posters will be on display for the maximum time possible. At certain times authors will be in attendance by their posters to discuss their work.
Camera-ready sheets and instructions for the submission of short abstracts can be obtained from the Royal Microscopical Society office. The deadline for submission is 4 May 1994. These abstracts will appear in the Conference Programme, which will be published in a special MICRO 94 issue of the Proceedings of the Royal Microscopical Society.
Authors will be notified regarding acceptance of their papers by the end of June 1994.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
EXHIBITION An Exhibition of the latest microscopes and ancillary instrumentation and equipment will be held at the Earls Court Park Inn, adjacent to the Lecture Theatre. Admission to the Exhibition is free, by conference badge, or by exhibition only badge which will be obtainable at the registration desk.
By 1 November 1993, the following firms had reserved exhibition space:-
Agar Scientific Ltd Alrad Instruments Ltd Bemax (UK) Ltd Bio-Rad Laboratories Ltd British BioCell International Burleigh Instruments (UK) Ltd Cambridge Scanning Co Ltd Confocal Technologies Ltd Cryophysics Ltd Data Cell Ltd Drukker International Edwards High Vacuum International Emitech Ltd Finlay Microvision Co Ltd Fisons Instruments Foster Findlay Associates Ltd Hamamatsu Photonics UK Ltd Hitachi Scientific Instruments ISS Imaging Associates Ltd J K Instruments Ltd JEOL UK Ltd K E Developments Ltd Lasertec Corporation Leica Cambridge Limited Leica UK Limited Microfield Scientific Ltd Microscopy and Analysis Newport Ltd Nikon UK Limited Olympus Optical Co (UK) Ltd Oxford Instruments Microanalysis Group Oxford Instruments Philips Electron Optics Photonic Science Polaroid (UK) Ltd Princeton Gamma-Tech (UK) Ltd Pyser (Holdings) plc Synoptics Ltd Taab Laboratories Equipment Ltd Tracor Europa Carl Zeiss (Oberkochen) Ltd þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
RECEPTION AND ASSOCIATED EVENTS On Monday 12 September there will be a wine reception in the Exhibition between 5.30 pm and 7.00 pm. The Annual General Meeting of the Royal Microscopical Society, and the Presidential Address will also take place during the evening.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ACCOMMODATION Academic Accommodation A limited amount of academic accommodation has been booked for the use of delegates at Imperial College. Rooms have been booked there on a bed and breakfast basis at a cost of œ2.00 pounds6 per night. This academic/student accommodation will be filled on a 'first-come first-served' basis. From the nearby Underground Station at South Kensington, Earls Court is two stops along the District or Piccadilly Line.
Hotel Accommodation There are some rooms available in the Earls Court Park Inn at the special MICRO 94 rate of œ65.00pounds per night. If you would like to reserve accommodation at these special rates, please contact the Earls Court Park Inn directly, quoting that you are a MICRO 94 visitor. To be sure of booking rooms at these rates, it is advisable that you book well in advance. Telephone: 071 385 1255 Telex: 917728 Fax: 071 381 4450.
Other Hotel Accommodation Delegates who wish to make their own accommodation arrangements may wish to use the services of Expotel Executive Travel - Europe's leading hotel booking agent, who have been appointed the official hotel agency for MICRO 94. The hotel of your choice or a similar alternative can be booked through Expotel often at discounted rates. By making one telephone call to Expotel on 071 735 0060 stating the event code 'MICRO 94', your reservation will be confirmed verbally followed by confirmation in writing. This free booking service is available to anyone attending MICRO 94. Telephone: 071 735 0060 Telex: 8811951 EXPOTL G Fax: 071 735 2839.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
REGISTRATION AND PAYMENT
Registration Registration will take place at the Earls Court Park Inn from 1.00 pm on Monday 12 September 1994, and from 9.00 am on subsequent mornings.
Payment Payment may be made by sterling cheque payable to the Royal Microscopical Society (please add œ12.00pounds to cover exchange and bank charges if the cheque is not a UK Bank Cheque) or by credit card (Visa/Barclaycard or Access/Eurocard/Mastercard).
Cancellation and Refunds Cancellations received before 12 July 1994 will be subject to a full refund. No refunds will be made if cancellation is made after this date. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
HOW TO GET THERE
The Conference, Exhibition, Posters, and Refreshments will all take place at the Earls Court Park Inn, Lillie Road, London SW6.
MICRO 94 is organized by the Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, UK, in association with Microscopy and Analysis. Telephone: 0865 248768, Fax: 0865 791237, Email: RMS-at-UK.AC.OX.VAX.
I am now in the process of designing a new EM suite dedicated to biological EM. I would like input from those of you who work in what they feel is an ideal or close to ideal physical arrangement. I would also like to hear from those who have envisioned an ideal EM suite but have never seen it built. The restrictions are that a space of 2200 sq ft should house 2 TEMs and 2 SEMs as well as a prep lab, darkrooms ancillary equipment and 2 offices. The overall design is of more interest than actual use of the stated space. So I would like to receive copies of plans you might have or rough * Director, ICBR EMCL * Any and all contributions would be greatly appreciated. ***************************** * Greg Erdos * * ICBR EM Lab * * 218 Carr Hall * * University of Florida * * Gainesville, FL 32611 * * gwerdos-at-gnv.ifas.ufl.edu * * 904-392-1295 * *****************************
Here's yet another addition to the EMail server. Periodic updates of upcoming Journal articles. Both the Journal of Microscopy and the Microscopy Society of America (MSA) Bulletin have agreed in principle to forward this (abstract) information to me and I will post it to the subscribers. In many cases the information distributed here may preceed the actual publication of the full article by a few weeks.
Hope you find it informative! I certainly did.
Nestor Z. ANL EM Center =============================================================
JOURNAL OF MICROSCOPY - ABSTRACTS OCTOBER 1993 - DECEMBER 1993
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 3--12. Quantitative acoustic microscopy of individual living human cells by G. A. D. BRIGGS,* J. WANG* and R. GUNDLE, *Department of Materials, University of Oxford, Oxford OX1 3PH, U.K. Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford OX3 7LD, U.K.
Summary The elastic properties of cells can be measured with microscopic resolution by acoustic microscopy. By measuring the waveform of very short pulses, the thickness, and the acoustic velocity, impedance and attenuation can be determined from the two separate signals reflected from the top and the bottom of the cell.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 13--21. Random models for morphological analysis of powders by D. JEULIN, Centre de Geostatistique, E.N.S.M.P., 35 rue St-Honore, 77305 Fontainebleau, France
Summary Models to estimate powder size distributions or fractions of components in a mixture of powders were developed and tested for various specimens. The models derived from the Boolean model and from the dead leaves models can be implemented on rough secondary electron scanning electron microscope images, obtained after minimal sample preparation. With the dead leaves tessellation, the size distributions of spherical particles or short fibres can be estimated either from the size distribution of intact grains, or from the area fraction measurement after binary erosions. With the dead leaves random function, there is no need for image segmentation. It provides data on the size and shape of a population of particles from an estimation of the distribution of grey-level images after erosions by convex structuring elements of increasing size. A version of these models for long fibres is developed for estimating their diameter distribution. Allowing for superposition of particles, the proposed methods enable an unbiased estimation of the size distribution and characterization of the shape of complex particles. The approach is illustrated by applications to spherical particles obtained by simulations and from scanning electron microscope micrographs.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 23--29. Spatial distribution of curve length: concept and estimation by N. ROBERTS and L. M. CRUZ-ORIVE*, Magnetic Resonance Research Centre, PO Box 147, University of Liverpool, Liverpool L69 3BX, U.K. *Stereology Unit, Department of Anatomy, University of Berne, Postfach 139, CH-3000 Berne 9, Switzerland
Summary The length of a curvilinear feature, such as a dendrite tree of a neuron, can, in principle, be estimated by the recent, non-invasive method of total vertical projections (TVPs). Curve length is a measure of size, but it reports nothing about curve shape. The shape of a tree-like structure can be described to some extent by the distribution of branch length in properly defined regions of three-dimensional (3-D) space. A definition of curve length distribution in three dimensions is proposed and implemented here on a human neuron. The relevant 3-D regions overlap after projection, and therefore the TVPs method cannot be used directly to estimate the corresponding feature lengths. However, using the ANALYZE software system running on a SUNSPARC workstation, dendrite subsets sitting in predefined regions of space were rendered in different colours and measured separately by the TVPs method using a cycloid test system. In combination with non-invasive image acquisition and processing techniques, the length distribution concept is likely to be useful in the metrical analysis of either microscopic or macroscopic arborizations in a wide variety of contexts, including living cells and organisms.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 31--39. Image sharpness and contrast transfer in coherent confocal microscopy by R.OLDENBOURG,* H. TERADA,~~ R. TIBERIO## and S. INOUE*, *Marine Biological Laboratory, Woods Hole, MA, U.S.A. Martin Fisher School of Physics, Brandeis University, Waltham, MA, U.S.A. ~~Hamamatsu Photonics Kabushiki Kaisha, Hamamatsu City, Japan. ##National Nanofabrication Facility, Cornell University, Ithaca, NY, U.S.A.
Summary Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non-confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non-confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non-confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non-confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 41--48. A light-emitting diode light standard for photo- and videomicroscopy by J. M. BEACH* and B. R. DULING, Departments of *Biomedical Engineering and Physiology, University of Virginia, Charlottesville, VA, U.S.A. Summary A light calibration system consisting of a compact light-emitting diode (LED) source with feedback control of intensity is described. The source is positioned in the focal plane of the microscope objective and produces flat-field illumination of up to 31microW. The source can be easily used to determine the performance of microscope optics and camera response. It can also be used as a standard light source for calibration of experimental systems. Selectable light intensities are produced by controlling the LED input power via a feedback circuit consisting of a photodiode that detects output light intensity. Spectral coverage extends between 550 and 670nm using green, yellow and red LEDs mounted side by side, which are selected individually. The LED chips are encapsulated in plastic diffusers which homogenize the light, and a flat field of illumination is obtained through a thin 1-mm-diameter aperture positioned directly over each chip. Provision is made for insertion of Ronchi rulings over the aperture to enable measurements of contrast modulation in a uniform field. The light may be pulse-modulated to assess camera response times and the device can be synchronized with video frames. Narrow bandpass interference filters can be placed between the objective lens and the LED source to produce monochromatic light without affecting the spacing of controlled light intensities since emission spectra do not shift appreciably over the range of LED powers chosen in this design. Results of tests using controlled light intensity and uniform illumination are presented.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 49--54. Video camera calibration for optical densitometry by R. A. Baldock and I. Poole, MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, U.K.
Summary An efficient technique for calibrating video cameras to record optical density (OD) from microscopic images is described. The method corrects for variation over the field of the brightfield and darkfield intensities, does not assume a linear response of the camera to the incident intensity and requires a single calibration filter.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 55--61. Vitrification depth can be increased more than 10-fold by high-pressure freezing by N. SARTORI, K. RICHTER* and J. DUBOCHET, Laboratoire d'Analyse Ultrastructurale, Batiment de Biologie, Universite de Lausanne, CH-1015 Lausanne, Switzerland
Summary Biological specimens prepared for cryoelectron microscopy seem to suffer less damage when they are frozen under 2kbar pressure rather than under normal conditions. The volume that can be well preserved is larger. This fact has been illustrated in a number of publications on a number of different samples. However, there is a lack of quantitative data concerning the depth of this good specimen preservation. Catalase crystals in various sugar solutions have been used as test objects and vitrification, as determined by electron diffraction, has been used as the criterion for good freezing. Keeping all other conditions equal, the depth of vitrification is approximately 10 times larger with freezing at high, rather than normal, pressure. The high-pressure vitrification depth in a 15--20% sugar solution averages 200micrometre. Fully vitrified specimens up to 700micrometre in thickness are obtained. When crystalline water is observed it is frequently in the form of high-density ice II, III or IX. These results are probably also relevant for typical biological specimens. The advantage of high-pressure freezing must be balanced by the possible consequences of a considerably increased cooling time and by the damage that may be induced by the pressure.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 63--69. A portable cryo-storage system for low-temperature scanning electron microscopy, suitable for international transport of cryo-specimens by C. E. Jeffree* and P. R. van Gardingen, *University of Edinburgh, Science Faculty Electron Microscope Facility, Daniel Rutherford Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JH, U.K.
Summary A cryo-specimen storage system for low-temperature scanning electron microscopy (LTSEM) specimens is described, which: liberates multi-specimen experiments from sampling restrictions imposed by the rate at which LTSEM specimens can be examined in the SEM; provides security against experiment loss resulting from breakdown of the SEM or cryo-system; enables collection of specimens in the field or in laboratories remote from the SEM laboratory; and facilitates international air transport of LTSEM specimens. The com-ponents of the system, which has a capacity of 98 stub-mounted specimens, are readily made in a laboratory workshop. The details of the design may be altered to suit particular specimen types or experimental approaches.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 71--79. Empirically determined freezing time for quick-freezing with a liquid-nitrogen-cooled copper block by P. H. W. W. BAATSEN, Departement de Physiologie generale, FYMU, Universite Catholique de Louvain, Ave. Hippocrate 55, 1200-Brussels, Belgium
Summary A method is presented to determine freezing time empirically. The method is based on determining the amount of stretch of a skinned muscle fibre while it is being frozen. Freezing time, as determined with this method, lies in between 0 and 1ms.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 81--88. An application of scanned focused ion beam milling to studies on the internal morphology of small arthropods by R. J. YOUNG,* T. DINGLE,* K. ROBINSON and P. J. A. PUGH, *FEI Europe Ltd, Brookfield Business Centre, Cottenham, Cambridge CB4 4PS, U.K. British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, U.K.
Summary For the first time a scanned focused ion beam of approximately 50nm diameter has been used to prepare biological material. Small defined areas of the surface were removed by ion etching to allow examination of the underlying structures with a scanning electron microscope. Different milling procedures were carried out on two anatomical features in mites of the genus Halarachne (Halarachnidae: Mesostigmata). In the first, square holes were milled into the surface of the peritrematal plate to reveal the structure of the underlying respiratory peritrematal groove. In the second, transverse cuts were made across the shafts of the sensory sensilli which make up the sensory Haller's organ on tarsus I. This latter procedure revealed detail both within the core and walls of sensilli. Details of specimen preparation and milling procedures, as well as suitability and interpretation of results, are presented.
Journal of Microscopy, Vol. 172, Pt. 1, October 1993, pp. 89--92. Variable-depth electropolishing of TEM samples by S. W. LEONARD, Department of Physics, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Summary A variable-depth electropolishing technique has been developed for transmission electron microscopy samples using copper as sample material. This was required for an experiment concerning the measurement of the variation of dislocation density with depth for ion-implanted materials. The polishing technique was achieved by a two-step process, involving the measurement and use of the polishing rate to polish to a specific depth and the application of a transparent cover to one side of the sample for back-thinning. With this technique, any sample depth can be made accessible for observation with a transmission electron microscope and the method should be applicable to many different materials and electropolishers.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 97--107. Computer simulation of a mirror STEM by A. V. CREWE, Department of Physics and the Enrico Fermi Institute, The University of Chicago, 5640 S. Ellis, Chicago, IL 60464, U.S.A.
Summary The results of a computer simulation indicate that it is possible to design and build an STEM that is free from spherical aberration and should therefore have a very high resolution. The computer program was written in APL. The calculations include the effects of apertures and, consequently, mimic a realistic situation.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 109--119. The effect of soft X-radiation on myofibrils by P. M. BENNETT,* G. F. FOSTER, C. J. BUCKLEY and R. E. BURGE, *MRC Muscle and Cell Motility Unit, The Randall Institute, King's College London, 26/29 Drury Lane, London WC2B 5RL, U.K. Wheatstone Physics Laboratory, King's College London, The Strand, London WC2R 2LS, U.K.
Summary Myofibrils, the contractile organelles from striated muscles, have been examined in the X-ray microscope to determine the effect of radiation on their function and structure. Using X-rays of energy 350--385eV in the water window we find that after an exposure to 7.5 x (10 to the fifth power) photons per square micrometre (calculated to give an absorbed dose of 20 000 Gy) the myofibrils will no longer contract. The use of the free radical scavenging agent, DMSO, gives some protection to the fibrils. It has also been found that after this much irradiation the fibrils lose up to 20% of their mass. Further substantial mass loss occurs on subsequent irradiation. After 25 times the loss-of-function exposure only 30% of the mass remains. Analysis of a series of images of the same myofibril covering this range of exposures shows that the mass is preferentially lost in some areas of the structure and consequently significant structural changes occur.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 121--129. Scanning luminescence X-ray microscopy: imaging fluorescence dyes at suboptical resolution by C. JACOBSEN, S. LINDAAS, S. WILLIAMS and X. ZHANG, Department of Physics, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A.
Summary Scanning luminescence X-ray microscopy is based on the use of the very small focused probe of a scanning X-ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X-ray source and a Fresnel zone plate to produce a focused X-ray probe, images of P31 phosphor grains with a resolution of 50--75nm have been obtained, and luminescence from polystyrene spheres loaded with 50--100micromol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X-ray probe (the microscope has imaged features at 36-nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye-tagged sites of biochemical activity at the resolution of the X-ray microscope in wet, unsectioned, and unfixed cells, especially with soft X-ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye-tagged features should be 2--22 times lower than it is in transmission X-ray microscopy. A possible extension of the technique for three-dimensional imaging at the transverse resolution of the X-ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 131--136. High-spatial-resolution maps of sulphur from human hair sections: an EELS study by P. HALLEGOT and P. CORCUFF, Laboratoires de Recherche Avancee, L'Oreal, Departement de Biophysique, 1 avenue Eugene-Schueller, 93 600 Aulnay sous Bois, France
Summary High-resolution sulphur maps have been acquired from human hair using a Zeiss CEM 902A transmission electron microscope equipped with an energy filter. Analysis by electron energy-loss spectroscopy (EELS) was performed on ultrathin sections of hair shafts embedded in three different types of resin: Nanoplast (water-soluble), Spurr (epoxy) and Lowicryl (low-temperature resin). Good-quality energy-loss images have been obtained with the three resins, although it was found that Nanoplast gave the best image contrast. For the first time, the results obtained for the detection of sulphur by silver staining of hair sections, which until now has been the only way to map sulphur at the electron microscopic level, have been confirmed. The results are compared with local sulphur concentrations from bulk analysis.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 137--151. Improvements in the technique of vascular perfusion-fixation employing a fluorocarbon-containing perfusate and a peristaltic pump controlled by pressure feedback by J. Rostgaard, K. Qvortrup and S. S. Poulsen, Institute of Medical Anatomy Department B, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N, Denmark
Summary A new, improved technique for whole-body perfusion-fixation of rats and other small animals is described. The driving force is a peristaltic pump which is feedback regulated by a pressure transducer that monitors the blood/perfusion pressure in the left ventricle of the heart. The primary perfusate-fixative is composed of a blood substitute---13.3% oxygenated fluorocarbon FC-75---in 0.05 M cacodylate buffer (pH 7.4) with 2% glutaraldehyde. The secondary perfusate-fixative is composed of 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) with 20 mM CaCl2 A double-barrelled, self-holding cannula is used to cannulate the heart; the outer and inner barrels of the cannula are connected to the peristaltic pump and to the pressure transducer, respectively. The tissue oxygen tension in the rat is monitored by a sub-cutaneous oxygen electrode. Measurements showed that tissue hypoxia/anoxia did not develop before or during the perfusion-fixation. Thus, the technique permits study of specimens which do not exhibit fixation gradients and do not contain cells fixed in a state of asphyxia. This is substantiated by electron micrographs of cells from different organs, revealing new fine structural elements. By adding oxygenated fluorocarbon to glutaraldehyde perfusate-fixatives, enough oxygen is made accessible for cellular respiration as well as for the oxygen-consuming chemical reactions of glutaraldehyde with the tissue. Data on anaesthesia, operative manoeuvres, mechanical components of the system, preparation of fixatives and flow of the perfusate-fixatives are furnished and discussed.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 153--156. A device for picking up ultrathin serial sections by E. P. Meyer and V. J. Domanico, Department of Zoology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
Summary Reconstruction from serial sections is often required to understand the architecture of biological tissue. The sampling of serial sections calls for an enormous amount of patience and skill. Sections are cut with a diamond knife, sampled in a trough and must then be collected on grids for examination with the electron microscope (EM). This last step, in which the section ribbons have to be aligned with the EM grid, is especially difficult as both hands have to work in perfect co-ordination. A simple manually operated or motorized mechanical device has been designed which facilitates the collection of EM section ribbons.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 157--162. An unbiased and efficient procedure for 3-D connectivity measurement as applied to porous media by H. Q. ZHAO and I. F. MACDONALD, Porous Media Research Institute, Department of Chemical Engineering, University of Waterloo, Ontario, Canada N2L 3G1
Summary A new stereological technique to measure the mean genus (connectivity) per unit volume of a porous medium is described and applied to a real sandstone sample. The technique is based on the `net volume tangent counts' performed on disector samples, i.e. pairs of consecutive sections of an interconnected structure. It consists of a set of simple counting rules applied to the features on the sections of the structure and can be easily implemented manually. The applicability and efficiency of the procedure is evaluated by applying it to a Berea sandstone sample which has been studied previously using a network analysis approach by interactive three-dimensional computer reconstruction. It is shown that the procedure yields results in good agreement with the network analysis result, but has the advantages that it is much easier to implement, is more flexible in how the data are collected, is more efficient, and is known to provide an unbiased estimate of the mean genus per unit volume of the whole structure.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 163--176. Remapping disparate images for coincidence by W. Galbraith* and D. L. Farkas, *Department of Laboratory Medicine, Allegheny Singer Research Institute, 320 East North Ave., Pittsburgh, PA 15212, U.S.A. Center for Light Microscope Imaging and Biotechnology, Carnegie-Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, U.S.A.
Summary With the development of complex multimode computerized microscope systems, it is possible and necessary to obtain images of the same area of the microscopical preparation by several methods of microscopy, such as differential interference contrast, reflection interference microscopy, several wavelengths of fluorescence microscopy, laser scanning and confocal modes. Thus, varied information may be obtained about a single field, in the form of a set of images, taken at different ports of the microscope, using different digitizing cameras, each appropriate to certain tasks. For comparative purposes, the images should be superimposable, pixel by pixel, but in general they are not --- they differ in image shape and size, magnification, distortion, centration and orientation. This paper shows how the problem may be approached, using an extension of the remapping procedures described in a previous paper, in which images of a separate grid reference slide are used to detect, quantify and correct the image errors. Affine remapping, without the use of grid images, is also described.
Journal of Microscopy, Vol. 172, Pt. 2, November 1993, pp. 177--180. Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope by O. GUNTINAS-LICHIUS,* J. MOCKENHAUPT,* E.’STENNERT and W. F. NEISS*, *Department of Anatomy and Department of Oto-Rhino-Laryngology, University of Cologne, Lindenthal, D-50924 Koln, Germany
Summary A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6micrometre thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 189--194. A compact Schwarzschild soft X-ray microscope with a laser-produced plasma source by Y. HORIKAWA, K. NAGAI, S. MOCHIMARU and Y. IKETAKI, T. Morokuma Research Laboratory, Olympus Optical Co., Ltd, 2--3 Kuboyama-cho, Hachioji-shi, Tokyo 192, Japan
Summary A compact Schwarzschild soft X-ray microscope using a laser-produced plasma soft X-ray source has been developed. The laser-produced plasma source, which is small but of high brilliance, has made it possible to use the soft X-ray microscope in a small laboratory. The microscope is composed of a Schwarzschild objective and a grazing incidence mirror condenser. Image contrast for biological specimens in soft X-ray regions is investigated briefly. It is possible to observe the fine structures of a thin specimen at a wavelength of 15nm; at this wavelength high-contrast images of biological specimens have been obtained with a single laser shot of pulse width of 8ns at a resolution of 0.3micrometre. The resolution of the system is limited by the detector.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 195--203. Liquid substitution: a versatile procedure for SEM specimen preparation of biological materials without drying or coating by H. J. Ensikat and W. Barthlott, Botanisches Institut der Universitat, Meckenheimer Allee 170, 53115 Bonn, Germany
Summary Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo-preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 205--214. Conformational characterization of nucleosomes by principal component analysis of their electron micrographs by M. M. Z. ZABAL*, G. J. CZARNOTA*, D. P. BAZETT-JONES and F. P. OTTENSMEYER*, *Department of Medical Biophysics, The University of Toronto and Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9
Summary Optimized fixation conditions were determined for protein--protein and protein--DNA crosslinking within calf-thymus nucleosomes in low monovalent salt concentrations. Nucleosomes were examined without heavy-atom staining by darkfield electron microscopy. The dimensions of these macromolecular complexes and those of HeLa core particles optimally fixed in divalent salt were analysed using principal component analysis. According to this analysis the structure of the calf-thymus nucleosomes was best presented by a prolate ellipsoid. Particle images had average major and minor axis lengths of 14.1 and 10.5nm, respectively. In contrast, the HeLa nucleosomes were best modelled by an oblate ellipsoid from the analysis of their images, which had average major and minor axes of 13.3 and 11.5nm. The applicability of this multivariate statistical analysis to the interpretation of macromolecular images is illustrated and discussed.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 215--221. An investigation of substrate and sample preparation effects on scanning tunnelling microscopy studies on xanthan gum by M. J. WILKINS, M. C. DAVIES, D. E. JACKSON, C. J. ROBERTS and S. J. B. TENDLER, The Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
Summary Scanning tunnelling microscopy is developing as an important biophysical tool for the molecular imaging of biological material. In this study, the effect of sample deposition technique and substrate employed on the resultant images of a microbial polysaccharide is investigated. Scanning tunnelling microscopy topographs of xanthan gum, pipette deposited and spray deposited onto highly orientated pyrolytic graphite and mica substrates are presented. The use of pipette deposition of the aqueous sample solution is shown to result in a xanthan network. In contrast, images of isolated xanthan molecules are obtained when spray deposition with glycerol is employed. The effect of these deposition techniques on the macroscopic distribution of sample material across substrates is shown using confocal fluorescence microscopy.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 223--232. Fractal characterization by frequency analysis. I. Surfaces by E. ANGUIANO, M. PANCORBO and M. AGUILAR, Instituto de Ciencia de Materiales, Sede B (CSIC), Universidad Auto¨noma de Madrid (C-III), 28049-Madrid, Spain
Summary A study of the quality and accuracy of the methods based on frequency analysis for the fractal characterization of surfaces as measured by scanning tunnelling microscopy (or profilometry) is made. The study is based on computer simulation of images of fractal surfaces. A discussion of the mathematical algorithms used for computer generation of fractal surfaces then follows. The main conclusion is that studies of fractal characterization by frequency analysis reported in previous papers in the STM field, as well as conclusions about the performance of the various methods, are doubtful. New methods for frequency analysis that in some cases produce more reliable results are proposed.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 233--238. Fractal characterization by frequency analysis. II. A new method by M. AGUILAR, E. ANGUIANO and M. PANCORBO, Instituto de Ciencia de Materiales, Sede B (CSIC), Universidad AutÆnoma de Madrid (C-III), 28049-Madrid, Spain
Summary A new frequency analysis method, fractal analysis by circular average (FACA), and an image replication procedure are proposed that together produce accurate measurements of the fractal dimension of surfaces and profiles, eliminating Fourier transform artefacts which arise from the lack of periodic continuity in real surfaces and profiles.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 239--248. Effects of noise and anisotropy on the determination of fractal dimensions by J. C. RUSS, Materials Science and Engineering Department, North Carolina State University, Raleigh, NC 27695-7907, U.S.A.
Summary Measurement of the fractal dimension of surfaces imaged by the scanning tunnelling microscope, atomic force microscope or similar instruments can be performed using several different algorithms. Dimensional analysis---plots of log (perimeter) vs. log (area)---are compared to Korcak and slit-island methods, which also employ a horizontal plane section, and to Fourier analysis of the two-dimensional array of elevation values. The effects of surface anisotropy and instrument noise on each of these measurement techniques is investigated.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 249--256. Evaluation of the precision of systematic sampling: nugget effect and covariogram modelling by J. THIOULOUSE,* J. P. ROYET, H. PLOYE* and F. HOULLIER~~, *Laboratoire de Biometrie, Genetique et Biologie des Populations, (U.R.A.CNRS 243), Universite Claude Bernard-Lyon 1, F-69622 Villeurbanne Cedex, France. Laboratoire de Physiologie Neurosensorielle, (U.R.A. CNRS 180), Universite Claude Bernard-Lyon 1, F-69622 Villeurbanne Cedex, France. ~~Ecole Nationale du Genie Rural, des Eaux et Forets, Laboratoire de Recherches en Sciences forestieres, 14, rue Girardet, F-54042 Nancy Cedex, France
Summary Systematic sampling designs are widely used in stereology. When an estimator of the total amount, Q, of the sampled variable is evaluated by such a procedure, the coefficient of error can be predicted by applying Matheron's theory of regionalized variables. To evaluate the accuracy of the estimate of Q, it is necessary to study the behaviour of the regionalized variable and to model its covariogram. Histological data with a low short-range variability and agronomic data with a pronounced nugget effect provided the biological material for extreme case studies. Results show that the short-range variability, if present, cannot be detected when only small samples are available. An underestimation of the coefficient of error is then to be expected. We propose several models of the covariogram, which can be used to test for the presence of a nugget effect. If a nugget effect is present, these models will provide better estimates of the coefficient of error. If there is no nugget effect a simplified method can be used and will provide reliable estimates of the coefficient of error.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 257--261. Directional analysis of planar fibre networks: application to cardboard microstructure by I. MOLCHANOV,* D. STOYAN* and K.’FYODOROV, *Bergakademie Freiberg, FB Mathematik, Bernhard-v.-Cotta-Str. 2, D-09596 Freiberg, Germany. Kiev Technological Institute of the Food Industry, Vladimirskaya, 68, 252017 Kiev, Ukraine
Summary This paper discusses the problem of determining suitable roses of intersections for systems of planar thick fibres which cross and overlap. A planar Boolean model with long rectangular (or similar) grains is suggested as an appropriate mathematical model. It leads to a statistical estimator for the rose of intersections of the system of fibre spines. The method is used to analyse the microstructure of the outer layer of two samples of cardboard.
Journal of Microscopy, Vol. 172, Pt. 3, December 1993, pp. 263--266. Edge detection in petrographic images by J. STARKEY and A. K. SAMANTARAY, Department of Earth Sciences, The University of Western Ontario, London, Ontario, Canada N6A 3B7
Summary The automatic detection of mineral grain boundaries in images obtained from a polarized-light microscope requires special techniques. Observations in both plane- and cross-polarized light may be necessry and the section must be rotated relative to the plane of polarization of the microscope to see all the grain boundaries. In computer-based microscopy this can be accomplished by the sequential accumulation of individual images captured from one microscope field of view with different polarizer orientations. ƒFor real-time implementation the sequential images are segmented individually by applying Canny's algorithm. A separable Gaussian mask is used for smoothing and a 3 x 3 convolution mask is used to generate 1-pixel-wide boundaries, which are located at the zero-crossing of the second-order derivative of the intensity gradient. The boundaries are extracted and accumulated in a composite image. The resulting composite image is a synoptic grain-boundary image of the rock.
Message-Id: {9312010147.AA22920-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM:Celli Tape Orig-Author: {"Nestor J. Zaluzec (708)-252-5075, -4964" {ZALUZEC-at-anlemc.msd.anl.gov} }:ddn:wpafb ----------------------------------------------------------- Re: TEM specimen preparation of Graphite:
Here's a question for those of you that used to use the old Celli tape popular in England about 10+ years ago.
In the past I had some of this tape which I used to use to prepare specimens of graphite for TEM. Here I would use the old trick of just continually touching the surface of the graphite with fresh tape to delaminate the graphite gradually until I made a nice thin & optically transparent section, which stuck to the tape and was usually perfect for TEM. The nice thing about this old type of scotch tape was that the adhesive used would disolve beautifully in Acetone and the backing for that tape would just float away as it was not soluablein the acetone. Unfortuantely, the current type of "SCOTCH BRAND" transparent tape completely dissolves in Acetone and most other solvents I tried.
I'm back to trying to make more graphite samples as my old ones have finally bit the dust so to speak. Since the new scotch tape failed, I tried extraction replica tape which is available from most EM supply vendors, however it does not have the adhearance of either the old Celli tape or the newer scotch tapes. (BTW the new scotch tape very nicely delaminated the graphite and gives beautifully thin sections if I could ever remove them from the adhesive). Does anyone know of a solvent which works on the newer scotch brand tapes or have another/similar idea/procedure for layered structures which are not strongly bound?.
I'd prefer to avoid ion milling the graphite although I may have to resort to this in the end.
International Microscopy and Image Analysis Conference and Exhibition
12 - 15 September 1994 Earls Court Park Inn, Lillie Road, London
Organized by the Royal Microscopical Society in association with Microscopy and Analysis
Second Circular
Dates
Conference: Monday 12 September - 2.00 pm - 4.15 pm Tuesday 13 September - 10.00 am - 4.15 pm Wednesday 14 September - 10.00 am - 4.15 pm Thursday 15 September - 10.00 am - 4.15 pm
Exhibition: Monday 12 September - 2.00 pm - 7.00 pm Tuesday 13 September - 9.30 am - 6.00 pm Wednesday 14 September - 9.30 am - 6.00 pm Thursday 15 September - 9.30 am - 4.30 pm
On the Monday evening there will be a wine reception at 5.30pm, followed by the AGM and Presidental Lecture at 7.00pm.
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CONFERENCE
Scientific Programme The Conference will be run in seven half-day sessions. The Electron Microscopy and Analysis Group of the Institute of Physics (EMAG) and The Physiological Society are each sponsoring separate lectures within the Conference.
The Programme will consist of tutorial lectures and posters and will feature the following topics:-
Monday 12 September (pm) - Materials I þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ EM in the assessment of semiconductor epitaxial growth Reactions to the surface of implanted bioceramics X-Ray microanalysis in biomaterials Optically active nanostructured materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (am) - Materials II (including EMAG) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Auger electron spectroscopy Imaging time of flight SIMS Formation of strained layer superlattices by phase separation Electron microscopy of weakly ordered III-V semiconductor materials þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Tuesday 13 September (pm) Scanning Probe Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Applications of atomic force microscopy to thin film research and technology Scanning probe microscopy: near field imaging of surfaces using electrons, forces and photons SPM of living biological systems Environmental SEM þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (am) - Image Processing and Analysis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Sampling in 3D for quantitative microscopy Digital image processing techniques Image analysis of multicoloured biological specimens In vivo microscopy by video imaging þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Wednesday 14 September (pm) - 3D Microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Supercomputing in confocal microscopy 3D atomic-scale microanalysis of materials Spatial distribution of fibres in composite materials Confocal polarised-light microscopy þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (am) - Flow Cytometry and Proliferation Markers þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Cell cycle control Proliferation-related proteins Proliferation in human tumours Apoptosis þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Thursday 15 September (pm) - Living Cell Cytochemistry (including The Physiological Society) þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ Living cell cytochemistry: Ratio Imaging Living cell cytochemistry: Confocal scanning laser microscopy
Thursday 15 September (pm) - Proteases in (patho) physiological processes
Use of selective protease inhibitors in the study of collagen breakdown Role of proteases in invasion and metastasis of cancer cells, arthritis and rheumatism and infections þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
Experts in the fields listed above have been invited to give lectures. Each speaker will provide a review of the particular topic in question and ample time for discussion will be provided.
In addition to the above, a special session will be held on the afternoon of Monday 12 September on how to use the light microscope.
Technical Lectures will be organized by Exhibitors to act as a bridge between the specialized review lectures and the equipment being exhibited. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
INVITED SPEAKERS
It is hoped that the following people will be presenting papers at the MICRO 94 Conference:-
Dr Paul D. Brown (University of Cambridge) Professor Peter Marquis (University of Birmingham) Dr Henk Koerten (University of Leiden, The Netherlands) Dr Peter Dobson (University of Oxford) Dr R. K. Wild (University of Bristol) Dr Paul Denison (University of Sheffield) Dr Andrew Norman (Imperial College, London) Dr Caroline Baxter (University of Cambridge) Dr Alan Pidduck (RSRE, Malvern) Dr M. Miles (HH Wills Physics Laboratory, Bristol) Dr H Ho”rber(European Molecular Biology Laboratory, Heidelberg, Germany) Mr Chris Gilpin (Manchester Biological EM Centre) Dr Vyvyan Howard (Royal Liverpool Children's Hospital) Dr Dominique Jeulin (Ecole Nationale Superiere des Mines, Fontainebleau, France) Dr Hans Tanke (University of Leiden, The Netherlands) Dr Andreas Kriete (Der Justus Liebig Universitat, Germany) Dr Alfred Cerezo (University of Oxford) Dr A. R. Clarke (University of Leeds) Dr Alan Entwistle (Ludwig Institute for Cancer Research, London) Dr A Bagg (TNO Rijswijk, The Netherlands) Dr Michael Ormerod (Sutton, Surrey) Dr Peter van Mier (Washington University School of Medicine, USA) Professor P. A. McNaughton (King's College London) Dr R. Jacob (King's College London) Dr Vincent Everts (University of Amsterdam, The Netherlands) Dr Ron Van Noorden (University of Amsterdam, The Netherlands)
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CONTRIBUTED PAPERS In addition to invited papers, contributions are invited on all aspects of microscopy and related techniques.
All contributed papers will appear in the poster sessions of the Conference. Time will be allowed in the Programme for the viewing of posters, and posters will be on display for the maximum time possible. At certain times authors will be in attendance by their posters to discuss their work.
Camera-ready sheets and instructions for the submission of short abstracts can be obtained from the Royal Microscopical Society office. The deadline for submission is 4 May 1994. These abstracts will appear in the Conference Programme, which will be published in a special MICRO 94 issue of the Proceedings of the Royal Microscopical Society.
Authors will be notified regarding acceptance of their papers by the end of June 1994.
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EXHIBITION An Exhibition of the latest microscopes and ancillary instrumentation and equipment will be held at the Earls Court Park Inn, adjacent to the Lecture Theatre. Admission to the Exhibition is free, by conference badge, or by exhibition only badge which will be obtainable at the registration desk.
By 1 November 1993, the following firms had reserved exhibition space:-
Agar Scientific Ltd Alrad Instruments Ltd Bemax (UK) Ltd Bio-Rad Laboratories Ltd British BioCell International Burleigh Instruments (UK) Ltd Cambridge Scanning Co Ltd Confocal Technologies Ltd Cryophysics Ltd Data Cell Ltd Drukker International Edwards High Vacuum International Emitech Ltd Finlay Microvision Co Ltd Fisons Instruments Foster Findlay Associates Ltd Hamamatsu Photonics UK Ltd Hitachi Scientific Instruments ISS Imaging Associates Ltd J K Instruments Ltd JEOL UK Ltd K E Developments Ltd Lasertec Corporation Leica Cambridge Limited Leica UK Limited Microfield Scientific Ltd Microscopy and Analysis Newport Ltd Nikon UK Limited Olympus Optical Co (UK) Ltd Oxford Instruments Microanalysis Group Oxford Instruments Philips Electron Optics Photonic Science Polaroid (UK) Ltd Princeton Gamma-Tech (UK) Ltd Pyser (Holdings) plc Synoptics Ltd Taab Laboratories Equipment Ltd Tracor Europa Carl Zeiss (Oberkochen) Ltd þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
RECEPTION AND ASSOCIATED EVENTS On Monday 12 September there will be a wine reception in the Exhibition between 5.30 pm and 7.00 pm. The Annual General Meeting of the Royal Microscopical Society, and the Presidential Address will also take place during the evening.
þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ACCOMMODATION Academic Accommodation A limited amount of academic accommodation has been booked for the use of delegates at Imperial College. Rooms have been booked there on a bed and breakfast basis at a cost of œ2.00 pounds6 per night. This academic/student accommodation will be filled on a 'first-come first-served' basis. From the nearby Underground Station at South Kensington, Earls Court is two stops along the District or Piccadilly Line.
Hotel Accommodation There are some rooms available in the Earls Court Park Inn at the special MICRO 94 rate of œ65.00pounds per night. If you would like to reserve accommodation at these special rates, please contact the Earls Court Park Inn directly, quoting that you are a MICRO 94 visitor. To be sure of booking rooms at these rates, it is advisable that you book well in advance. Telephone: 071 385 1255 Telex: 917728 Fax: 071 381 4450.
Other Hotel Accommodation Delegates who wish to make their own accommodation arrangements may wish to use the services of Expotel Executive Travel - Europe's leading hotel booking agent, who have been appointed the official hotel agency for MICRO 94. The hotel of your choice or a similar alternative can be booked through Expotel often at discounted rates. By making one telephone call to Expotel on 071 735 0060 stating the event code 'MICRO 94', your reservation will be confirmed verbally followed by confirmation in writing. This free booking service is available to anyone attending MICRO 94. Telephone: 071 735 0060 Telex: 8811951 EXPOTL G Fax: 071 735 2839.
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REGISTRATION AND PAYMENT
Registration Registration will take place at the Earls Court Park Inn from 1.00 pm on Monday 12 September 1994, and from 9.00 am on subsequent mornings.
Payment Payment may be made by sterling cheque payable to the Royal Microscopical Society (please add œ12.00pounds to cover exchange and bank charges if the cheque is not a UK Bank Cheque) or by credit card (Visa/Barclaycard or Access/Eurocard/Mastercard).
Cancellation and Refunds Cancellations received before 12 July 1994 will be subject to a full refund. No refunds will be made if cancellation is made after this date. þÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ
HOW TO GET THERE
The Conference, Exhibition, Posters, and Refreshments will all take place at the Earls Court Park Inn, Lillie Road, London SW6.
MICRO 94 is organized by the Royal Microscopical Society, 37/38 St Clements, Oxford OX4 1AJ, UK, in association with Microscopy and Analysis. Telephone: 0865 248768, Fax: 0865 791237, Email: RMS-at-UK.AC.OX.VAX.
Summary of the procedure for cleaving graphite into TEM specimens using 3M brand Scotch (Magic) tape.
Begin using with the Scotch Brand Magic Tape using it to cleave the graphite flakes several times to get a "clean" i.e. fresh surfaces. Then begin with a fresh piece of tape. Continue to delaminate until you see a number (~10+) of optically transparent "flakes" stuck onto the tape (many more will not be optically transparent) . During the delamination /peeling process try not to press the tape pieces together with alot of force this only serves to make complications later. Use only sufficient force to cause the graphite flakes to stick & peel off. Now trim off excess tape where there is no potential graphite specimens attached. Immerse the remaining tape in Toluene at room temp , this starts to seperate/loosen the backing from the glue, after about 5 minutes slowly add Methanol until you get to a ~50/50 mixture Let this sit a few more minutes then gently loosen the glue mass (looks like a wrinkled gelatinous mass and acts like it too) from the backing . To remove the plastic backing, you may need to use a pair of tweezers to entice the seperation where the glue has been forced down into good contact with the plastic backing at the sites of the graphite flakes. Toss the plastic backing in the trash.
Now begin to add acetone which dissolves the glue. As the acetone is added use a pair of tweezers to agitate the glue/graphite mass. This allows the solvent to get inbetween the graphite flakes and glue mass. Remove as much of the glue mass as possible before it completely dissolves in the solvent mixture. This is a real sticky "goo" at this point and it will only serve to contaminate your solution further. Be realistic here you will not get all of the graphite off of the tape. I estimate that there were about 2 dozen specimens on the tape, as the graphite released from the glue it fractured into maybe a hundred smaller flakes which were just floating in the solvent. At some point it nolonger is worth while trying to release the remaining graphite flakes from the glue mass, too much agitation only causes folds which trap the flakes and makes it nearly impossible to release all the graphite. Waiting to long only causes the glue mass to dissolve into your solvent and possibly contaminate your specimens.
Decant off excess solvent and add fresh acetone to continue dissolution of any remaining glue on the flakes. Now just pickup the flakes on grids by floating them over grids and lifting out of the solvent. (i.e. play chase the flake with the grid. As I recall the life science microscopists have a special tool for this but I just went fishing with a pair of tweezers and a grid under a low power steroe microscope). If they (the graphite flakes) are optically transparent (very light gray to clear) you've got a reasonably good specimen to work with at 100 kV.
Via TEM I see the usual basal plane dislocations, and little surface contamination there is some but it is obvious and can be easily avoided. The flakes show the usual bending that I would expect from the peeling procedure, but are reasonably uniform in thickness over wide areas (~sq microns). No thickness measurements yet, I'll report those when the data comes out of the experiments.
Thanks to all who made suggestions:
Nestor Z. ANL EM Center
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