Can someone please suggest stereology methods (assessing number of nuclei) at the light microscope level involving electronic imaging and appropriate software? We have concerns over randomization of fields...grid overlay?..x-y stage coordinates? Any good books on the subject?
Yes it does matter what you pay for the diamond knife. There are various qualities of diamonds which can be used for knives. Also the company's garantee(sp) will matter. Go with the higher price knives and little trouble will happen.
Weibel's book on stereology is still my favorite. It allows an intuitive understanding of the subject of stereology for those who only need this information or who are just beginning the subject, then presents the mathematics later for those who need or want it. As long as the subject is up for discussion, I will state a bias, related to automated (electronic) systems. The subject is peripherally related to the question under consideration. It also applies to both stereology and statistics where new automated or semi-automated systems are proliferating. 1. It is often easier and more efficient to carry out some of the analyses and tests using hand calculators, overlay sheets, and other low-tech means than using the fancy programs.
2. Both statistics and stereology require a lot on the user to know which is the best method, most useful, or most efficient means of carrying out data collections or analysis.
It follows from these then that a program alone is only a tool. The investigator must first invest in acquiring the knowledge of how to use the tool. i.e. Always buy the book and learn the techniques before buying the program.
For many ,this would appear to be very obvious. I state this "obvious" fact because of the number of papers I have reviewed lately where statistical methods were used incorrectly. In most cases the methods stated that such-and-such a program was used for the statistics. This suggests to me that far too many people are using these programs to generate numbers they do not understand because they failed to invest the time in understanding the discipline. Thats MHO, anyway.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Fri, 1 Jul 1994, BOBROWW wrote:
} Can someone please suggest stereology methods (assessing number of nuclei) at } the light microscope level involving electronic imaging and appropriate } software? We have concerns over randomization of fields...grid overlay?..x-y } stage coordinates? Any good books on the subject? } } Walt Bobrowski } bobroww-at-aa.wl.com } } }
In my experience over the years, diamond knives seem to differ mainly in 2 factors: price and quality control. Without mentioning any specific manufacturers, the most expensive ones are made by a unique process which does give them some desireable features...ie many resharpenings, better edge quality, greater durability, etc. All the others are made by essentially the same process and differ as to type of mounting, boat, etc. Most companies will give you several weeks following purchase to try it out and determine if the quality meets your standards. I myself have bought the cheapest (from a single manufacturer) for many years, and have only had to return a single knife. I also tend to buy the largest size available (4 mm or larger) as these tend to last longer, have better quality control, and seem to give you the most for your money.
consider a package called "Stereology" by Kinetic Imaging in Liverpool UK. It's a very useful windows based package, developed by a team led by V. Howard. It's very userfriendly and kind cheap. Give me a mail if you want specifics.
} Can someone please suggest stereology methods (assessing number of nuclei) at } the light microscope level involving electronic imaging and appropriate } software? We have concerns over randomization of fields...grid overlay?..x-y } stage coordinates? Any good books on the subject? } } Walt Bobrowski } bobroww-at-aa.wl.com } } }
While we are on the subject of diamond knives, does any one have a trick to get the knife edge to be a little more hydrophilic? my knife edge appears very clean but it is getting harder to keep the edge wet with out greatly increasing the water level in the boat. any thoughts on this would be appreciated.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Stereology Walt Bobrowski asks - Can someone please suggest stereology methods (assessing number of nuclei) at the light microscope level involving electronic imaging and appropriate software? We have concerns over randomization of fields...grid overlay?..x-y stage coordinates? Any good books on the subject?
I agree with Jay Jerome in recommending the book by Weibel (1979, Academic Press); Practical Methods for Biological Morphometry. vol 1 Sterological Methods. A more recent update can be found in Gundersen 1986 J. Microsc. 143:3-45. Look out also for other reviews by Cruz-Orive and by Gundersen. When using machines it is not easy to avoid bais. For example many counting frames will only score objects that are totally in the frame. This is OK for small particles but large objects may be under sampled. There are many alternative modern techniques which are simple to apply and give efficient and unbiased results. These techniques do not require the purchase of expensive machines or complicated software. The best way to find out about these techniques is to take a short course, one of which we are offering at Yale this August. Paul Webster, Yale School of Medicine.
Re} Diamond knife wetting The trick from the past is to run some siliva over the edge with an eyelash! A more modern way is to rinse out the boat, and the knife surface, with water containing a small amount of detergent. Try very low concentrations of Photoflo (Kodak).
Reply to: RE} Stereology Walt Bobrowski asks - Can someone please suggest stereology methods (assessing number of nuclei) at the light microscope level involving electronic imaging and appropriate software? We have concerns over randomization of fields...grid overlay?..x-y stage coordinates? Any good books on the subject?
I agree with Jay Jerome in recommending the book by Weibel (1979, Academic Press); Practical Methods for Biological Morphometry. vol 1 Sterological Methods. A more recent update can be found in Gundersen 1986 J. Microsc. 143:3-45. Look out also for other reviews by Cruz-Orive and by Gundersen. When using machines it is not easy to avoid bais. For example many counting frames will only score objects that are totally in the frame. This is OK for small particles but large objects may be under sampled. There are many alternative modern techniques which are simple to apply and give efficient and unbiased results. These techniques do not require the purchase of expensive machines or complicated software. The best way to find out about these techniques is to take a short course, one of which we are offering at Yale this August. You may also like to contact the International Society for Stereology in Freiburg, Germany for details of other courses. The secretary is Heimo Kurtz, tel 011 49 761 203 5092 and fax 5054. Good Luck Paul Webster, Yale School of Medicine.
For the very basics, Weibel Er. Stereologic principles for morphometry in electron microscopic cytology. International Review of Cytology, 1969, 26:235-302 has a nice presentation. Of course this predates the dissector and its offspring, so there is a lot more one can do than you could back in '69. Taking a course on stereology is an efficient way to learn, especially the pitfalls. The jump from 2-D sampling to 3-D numbers is not always intuitively obvious to the beginner. The number of times I have seen counts of the number of objects found in micrographs directly transposed to number of objects in cell is becomming legion. For those that don't realize it- You can't do that.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
Subject: Time:3:15 PM OFFICE MEMO RE:LM book Date:7/1/94
If I were looking for a book on light microscopy, I'd check with the McCrone Associates in Chicago (Ph. 708-887-7100; Fx: 708-887-7764). They are experts in all kinds of LM methods, and for years have offered courses on all phases of the subject. I believe they have written books for these courses, and perhaps you could adopt one of them for your course.
I use my saliva to wet the edge of the knife. Wet your fingers with saliva, run fingers over your eyelash "pushstick", then wet the knife edge. Works everytime. Best of luck.
XEI Scientific (Ph: 415-369-1133; Fx: 415-363-1659) market a complete nitrogen-purge system (SEM-Clean) designed to remove oil contamination from specimen chambers of SEMs. They can unquestionably give you details of how the system is optimally designed and operated, and would undoubtedly be willing to design a system specifically to meet your needs. Carl Henderson, manager of the Microprobe/EM lab in our Geology Department here has an XEI system on an SEM and has been very pleased with it. I don't know his email address, but his FAX is 313-763-4690. If you get in touch with him I am sure he will be able to give you considerable information on how this kind of a system performs and operates. Danielson Associates (Ph: 708-960-0086; Fx: 708-960-0546) market a device (Phototron) that produces ultraviolet light with a wavelength distribution optimal for desorbing molecules from surfaces inside vacuum systems. This might be of use to you, either alone or in conjunction with a nitrogen purge system, as discussed on page 201 of my recently published book on Vacuum Methods in Electron Microscopy (available from Ashgate Pub. Co., Fx: 802-276-3837)
Message-ID: {MAILQUEUE-101.940701154911.256-at-eye1.eye.ufl.edu} To: microscopy-at-anlemc.msd.anl.gov
We have the same problems with wetting of diamond knives, and have looked at an old one using SEM.. the amount of plastic adhering to the edge was unbelievable. I believe that this layer contributes both to the "dulling" effect after long use, i.e. that the diamond crystalline structure is not so much dull, just coated, and to the hydrophobic quality. We are all using what are really only partially polymerized epoxies, and the unpolymerized component ends up on our knives. Our best success has been to carefully cure the epoxies, and to soak tghe knives overnight in weak detergent, rinse, and run a styrofoam stick wedge across the edge as recommeded by one of the companies. This doesn't have to be done very often. Also in using photoflo as the detergent, make sure it is very dilute, we soaked off all of the finish coating on the outside of one knife boat! Evelyn Clausnitzer U of Florida Ophthalmolgy
Message-Id: {9407012222.AA07599-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: N2 Vacuum Cleaning Orig-Author: {Alan Wilson {wilsona-at-blackjack.cis.dsto.gov.au} }:ddn:wpafb ----------------------------------------------------------- Does anyone have experience with cleaning (FE)SEM vacuum chambers using a N2 gas bleed at around 100-150Torr. 1. How much gas is used in this process (1 G size bottle = 6.4m3 of gas)? 2. Presumably the cleaning depends on the quantity of gas passed through the system. Thus a lower speed rotary pump would take longer than a higher speed rotary pump? 3. Would a higher speed pump be better due to the greater gas flow speeds giving a greater "scouring" action?
Alan Wilson *:-{)} wilsona-at-blackjack.cis.dsto.gov.au Airframes and Engines Division DSTO-Aeronautical and Maritime Reseach Laboratory 506 Lorimer St Fishermens' Bend 3207 Victoria AUSTRALIA
-- John F. Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, 2455 Hayward St., Ann Arbor, MI 48109-2143 Phone: (313)-936-3352 FAX: (313)-936-3352 jfmjfm-at-umich.edu
We soak dirty/difficult to wet diamonds overnight in diluted triton X-100, .1% or less. Then run alcohol-0washed styrofoam cleaning stick alon g the edge.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
I am currently doing TEM on freshwater fish. The fish I am looking at is the "brown bullhead". Does anybody have a suggestion as to the fixative that would be best for this type of fish? The osmolarity and pH is important to the fixation of the fish. The fish can tolerate salinities from 0-8ppt. Should a different fix with different osmolarities and pH be used for fish taken from higher salinities or can the same fix be used for the fish taken in both 0 and 8ppt salinity? Thanks, Phil
I forgot to mention that I am also doing an exposure study at different salinities (5,10,15,25,35ppt) using both a Euryhaline and Anadromous species of fish in the larval and mature life stage. Is it necessary to adjust the pH and osmolarity for the differnt salinities given these criteria? i.e. habitat and life stage. This is for TEM studies also. Thanks again, Phil
Phil, Regarding the issue of osmolarity and TEM fixation: I have no experience with fish but I do a lot of fixation of platyhelminths (flatworms) from a variety of environments (including fish parasitic species). I use boiled, filtered water from the environment of the animal to make up the buffers for fixations and washing, and usually a pH of around 7.4 for marine and 7.2 for freshwater species. Good luck
Aquatic organisms tend to be a problem, and often considerable experimentation is necessary. In general though, here's what you have to consider with freshwater beasts: Invertebrates tend to be osmoconformers...they are about isotonic to their environment. The blood of some freshwater mussels I've worked with measures about 30 mOsm, which roughly conforms to their pondwater. Most buffers are ineffectual at this concentration, so my best results seemed to be from fixing in a glutaraldehyde-pondwater mixture. Freshwater vertebrates are hypertonic to their environment. I would suspect that under different salinities, their kidneys will keep their blood osmolarity rather constant. You should formulate your fixative based on blood osmolarity (as determined by freezing point depression), keeping in mind that tissues look better by TEM when fixed in a slightly hypertonic fixative. Keep in mind when formulating your fixative, the EOP (effective osmotic pressure) of your fixative. Formaldehyde is so membrane-permeable that you don't have to consider the osmotic pressure contributed by it. Glutaraldehyde is not so permeable and only contributes about half of its calculated osmotic pressure. I hope this will be of some help and at least get you started in the right direction. W.L. Steffens University of Georgia
Phil, Regarding pH and osmolarity for TEM of fish tissues: I used to work with lamprey in both salt and fresh water and we used the same buffer for both with no problems-Millonig's phosphate pH7.2-7.4 with 7% sucrose I believe. We made it up using distilled water from the lab.
Hi I'm the one that ask if there is a future in EM. Most of the responses I received thought that there is. (Although, these were from people who have a reason to hope there is.) I am, however, branching out. I have also been put incharge of the department's confocal microscope. I will take a class with Biorad in Sept. I heard on this news group that there was a confocal news group. Could someone send me the address. I have been unable to find it in my list of news groups. Also, is there anyone else who reads this group that will be taking the Biorad class in Sept. in New York? I would be interested in hearing from you.
I'm looking for additional ideas for a specimen which would be useful for training SEM novices. The idea is to have something which could be distributed to students with training materials (instructions and illustrations) keyed to this specimen. Qualities of importance include: 1. robust - capable of taking careless handling without major change in properties. 2. can be easily cleaned or rejuvenated with minimal equipment. 3. inexpensive. 4. reasonably consistent copies can be produced or obtained in quantity. 5. interesting structure from low magnifications to 10,000X or so to encourage exploration. 6. suitable structures to practice skills in focusing and stigmating. 7. produces interesting micrographs. 8. ideally -- interesting contrast in both SE and BSE modes.
I stress the word "novice". Assume that this specimen could be handed to a high-school student and would both survive and stimulate interest.
Also, I would appreciate any suggestions for a basic EDS training specimen to be used in the same way. Here one would like to have a segregation of elements to encourage "what's this ?" kind of questions and produce interesting dot-mapped images -- the goal here is strictly qualitative -- suitability for quant semi-quant analysis would be a bonus. Of course, it would be ideal if this could be the same specimen as the first!
I am familiar with some specimens which have been used for this purpose, but hope someone might have some imaginative suggestions.
I would like to make contact with anyone using MPM 201 micicrophotometer from Zeiss with MSP 21 processor. We do single cell Calcium measurements but have a number of specific problems concerning the procesors signals to the fast shutter. Specifically I would like to be able to vary the time between measurements in dual wavelength mode. Thanks in advance Gary Jamieson Haematology Austin Hospital Heidelberg VIC 3084 AUSTRALIA Internet gapja-at-austin.unimelb.edu.au Phone 613 496 5518 Fax 613 459 1674
Here's what I've used for the past 20 years: find an old windup mechanical watch, preferably a ladies model. On disassembly, you will have numerous very small screws, gears, bearings, etc. Many are excellent for learning mounting techniques, focussing, stigmation, demonstrating depth-of-field, secondary vs backscatter imaging, etc. And of course they are durable and recyclable.
Received: From MCONET(MAILER) by ANLNRH(ANJE6.10) for MICROSCO-at-ANLEMC; Thu, 7 Jul 94 08:28 Received: from opus.mco.edu by cutter.mco.edu (PMDF V4.2-14 #5375) id {01HEF4OFW0F4000NG8-at-cutter.mco.edu} ; Thu, 7 Jul 1994 09:26:38 EDT Received: from opus.mco.edu by opus.mco.edu (PMDF V4.2-14 #5375) id {01HEF4HT56U8001NKT-at-opus.mco.edu} ; Thu, 7 Jul 1994 09:21:19 EDT
Fred,
My recommendation for SEM training material would be, hair. It is always handy and in large quanties. If sample is taken at the end of a day, all sorts of things can be found on it, such as pollen, which is good material for high mag. and low mag work. I do not do much EDS work but any rusty bolt should work fine for this. Good Luck.
I need microscopy-oriented symbols to use when composing flyers, brochures for MSEM and for EM public relations work at Chicago State University. I use Harvard Graphics for Windows, but the symbol library is oriented more toward business use rather than science. Can anyone help out with this? I can use anything from atoms to a drawing of a JEOL TEMSCAN. A simple light microscope would be nice. Thanks. Joyce Craig Biology Department Chicago State University Chicago, IL 60628 Internet bafpjec-at-uxa.ecn.bgu.edu Phone 312 995 3800 FAX 312 995 3759bafpjec-at-uxa.ecn.bgu.edu
Mosquitoes and fruit flies make very useful specimens. While they are not robust, they have wonderful detail up to magnifications of 20,000 and provide excellent opportunites to demonstrate the effects of foreshortening, charging, background interference, and composition.
Rod Kuehn University of Minnesota
On 6 Jul 1994, Fred Schamber wrote:
} I'm looking for additional ideas for a specimen which would be useful for } training SEM novices. The idea is to have something which could be distributed } to students with training materials (instructions and illustrations) keyed to } this specimen. Qualities of importance include:
Well, may not be too imaginative, but may be samples worth considering: (ie: they are common and long-lasting, and fairly easy to prepare)
- a carbon steel with nice pearlitic and cementite structures etched w/ either Picral and/or Nital acids (~4%); (ex/ 1080, 52100);
- a cast iron, etched as above, revealing graphite nodules surrounded by ferrite in a matrix of pearlite (ex/ grade 80-55-06 ductile iron, or a class 30 gray iron that has graphite flakes in matrix of pearlite);
- may also desire to collect a few -rusty- bolts, nuts, etc. and prepare cross-sections of 'em to reveal and compare the corrosion products vs the remaining steel/iron (may be interesting to you/your "students" to have different amounts of corrosion to study on different items of the same grade of steel/iron);
- various aluminum alloys can be very interesting (ex/ samples of brazed joint areas, alloys of the 3xx series which have quite a few various precipates, and if can find 'em, a very-slow cooled Al alloy revealing many, large dendrites);
- other metals that could be very interesting and informative: tin alloys (Sn-Pb, Sn-Zn-Cu), lead alloys (Pb-Sn-Sb-Cu), and copper (brass);
- and lastly, some geological samples may be worth while, although harder to an extent to keep in good condition, and may be harder for you to acquire and prepare. Examples: ore samples, metamorphic & igneous rocks of various grades, and possibly sedimentary rocks with various mineral stainings.
Enjoy, -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /\v/\ | | Metallographic Lab. | Missouri Speleological Survey /\v/\ | | Rolla Research Center | Bat Conservation International /\v/\ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /\v/\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
John- try Diatome they are a proven product with great support. the size and thickness of your sections will determine the knife size, angle, and grade of diamond -Mike
On Thu, 30 Jun 1994, HARDY, JOHN wrote:
} } With such a wide range of prices - through various suppliers - for a } specific size of diamond knife (for thin sectioning) does the saying } "you get what you pay for" apply here, or is a diamond knife a diamond } knife? Thanks for any experienced comments. } } jhardy-at-smtplink.coh.org }
Tom- wetting the edge is easy if you add some sort of surfactant, saliva, dilute photo-flow, triton x-100, tween-80, etc. saliva on the tip of your (dog eye-lash) brush tool works best, just pull the water up with the spit laden brush tip. -Mike
On Fri, 1 Jul 1994, Tom Phillips wrote:
} While we are on the subject of diamond knives, does any one have a trick to } get the knife edge to be a little more hydrophilic? my knife edge appears } very clean but it is getting harder to keep the edge wet with out greatly } increasing the water level in the boat. any thoughts on this would be } appreciated. } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax) } } }
The most effective and flexible method of producing black backgrounds seems to be by blocking collection of background electrons. This can be accomplished on a regular stub by tilting the sample 90 degrees so the bottom of the chamber becomes the background. The chamber floor is coated with carbon paint to reduce secondary electron generation. On the side of the chamber floor, just to the collector side of the viewing area, tape a carbon-coated barrier. Ours is made of cardboard but in humid weather it absorbs water and requires extensive pump-down periods. Its dimensions are roughly 2 cm high x 3 cm long. For other samples, such as hair, we use a custom-designed stub made from a hollowed brass cylinder. The bottom is solid. The top is square with a square opening of about 1 cm. The cylinder is about 20 mm deep and is coated with carbon paint. The sample, of course, lies across the opening. No experimentation has been done with other sizes or shapes so it is likely that more compact designs would work as well. Both methods produce uniform jet-black backgrounds.
Also, higher kv expands the contrast range at the extremes. Light objects become white, dark objects turn black. Therefore, increase kilovoltage as much as the sample allows. Sorry for the tardy reply.
Rod Kuehn University of Minnesota
On Tue, 7 Jun 1994, Jim Romanow wrote:
} } I have been doing low magnification SEM (50 to 150x) on insects } using a 16 year old Coates and Welter HPS-50B FESEM (TV rate only). } Some of my clients desire a "black background" on the photographic } images (analog 4200 line photo monitor/ Polaroid Type 52) and I } find this is not possible in most cases due to imaging of the mounting } device and stage area around the sample. I have tried mounting on } pins which will eliminate holder interference but I still see gray } shapes in the background from several centimeters away. } } Is a black background the norm with some SEM designs, a } darkroom technique that can be applied to modify photographs that } originally looked like mine or achievable if I change my SEM } techniques? } } Jim Romanow } The University of Connecticut } Physiology and Neurobiology Dept. } Storrs, CT } bsgphy3-at-uconnvm.uconn.edu
You can try using a butterfly or moth wing for the SEM training. That's what we show students when they come in to see a demonstration of our SEM. As long as the student doesn't squash the wing with their finger, the wing should be a pretty good specimen to look at. You definitely can go from low to very high magnifications to see alot of different things and it can be used to practice focusing and stigmating. There are plenty of "holes" and microsculpture. I'm not sure if you can see interesting contrast in both SE and BSE modes. I've never tried it. Another thing is that most if not all people should be familiar with "the powder" that comes off the wing when a person touches it. Once they know what they are viewing, they can in a way, identify with it.
One thing that comes to mind that can be used for EDS work is flakes of paint. I know that paint is composed of various elements and the students can look at different colors or different shades of the same color. They should be able to find out for example, what makes one shade of red different from another shade of red. The paint samples would need to be carbon coated.
Just afew ideas.
Good luck! Peling Melville
On July 6, 1994 Fred Schamber writes:
} I'm looking for additional ideas for a specimen which would be useful for } training SEM novices. The idea is to have something which could be distributed } to students with training materials (instructions and illustrations) keyed to } this specimen. Qualities of importance include: } 1. robust - capable of taking careless handling without major change in } properties. } 2. can be easily cleaned or rejuvenated with minimal equipment. } 3. inexpensive. } 4. reasonably consistent copies can be produced or obtained in quantity. } 5. interesting structure from low magnifications to 10,000X or so to encourage } exploration. } 6. suitable structures to practice skills in focusing and stigmating. } 7. produces interesting micrographs. } 8. ideally -- interesting contrast in both SE and BSE modes. } } I stress the word "novice". Assume that this specimen could be handed to a } high-school student and would both survive and stimulate interest. } } Also, I would appreciate any suggestions for a basic EDS training specimen to } be } used in the same way. Here one would like to have a segregation of elements to } encourage "what's this ?" kind of questions and produce interesting dot-mapped } images -- the goal here is strictly qualitative -- suitability for quant } semi-quant analysis would be a bonus. Of course, it would be ideal if this } could be the same specimen as the first! } } I am familiar with some specimens which have been used for this purpose, but } hope someone might have some imaginative suggestions. } } Fred Schamber } RJ Lee Group, Instruments Division
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
As you suggested, a simple light microscope will indeed be very suitable, because more people are able to relate to a light microscope than an electron microscope. You have an excellent one already in Chicago - the pin designed by McCrone Research Institute for the Illinois State Microscopy Society. Call MRI at 312-842-7100 to see if they can send you a copy of the design or a pin itself.
Shu-Chun Su Hercules Inc. Research Center Wilmington, DE 19808 Phone: 302-995-3498 Fax: 302-995-4135
On Thu, 7 Jul 1994, Joyce Craig wrote:
} I need microscopy-oriented symbols to use when composing flyers, } brochures for MSEM and for EM public relations work at Chicago State } University. I use Harvard Graphics for Windows, but the symbol library } is oriented more toward business use rather than science. Can anyone } help out with this? I can use anything from atoms to a drawing of a JEOL } TEMSCAN. A simple light microscope would be nice. Thanks. } Joyce Craig } Biology Department } Chicago State University } Chicago, IL 60628 } Internet bafpjec-at-uxa.ecn.bgu.edu } Phone 312 995 3800 } FAX 312 995 3759bafpjec-at-uxa.ecn.bgu.edu }
I recieved a call this morning from Clint Mills. He works in the EM lab at a VA hospital in Alabama. It seems that his water chiller has been a source of chronic problems over the past two years. He was calling to inquire what brand/make of chiller we use. Since we are on a house chilled water system, I wasn't much help. If anyone would like to call him with comments and suggestions, I am sure he would appreciate it. His phone number is (205)-933-8101 ext. 6719. He has a Hitachi H-600, which came with a variety of specimen exchange rods. He would also like to sell the bulk specimen holder and the rotational holder. ________________________________________________________________
Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
Message-Id: {9407081619.AA06260-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: SEM Training Specimen Orig-Author: {Fred Schamber {73751.3677-at-compuserve.com} }:ddn:wpafb ----------------------------------------------------------- I'm looking for additional ideas for a specimen which would be useful for training SEM novices. The idea is to have something which could be distributed to students with training materials (instructions and illustrations) keyed to this specimen. Qualities of importance include: 1. robust - capable of taking careless handling without major change in properties. 2. can be easily cleaned or rejuvenated with minimal equipment. 3. inexpensive. 4. reasonably consistent copies can be produced or obtained in quantity. 5. interesting structure from low magnifications to 10,000X or so to encourage exploration. 6. suitable structures to practice skills in focusing and stigmating. 7. produces interesting micrographs. 8. ideally -- interesting contrast in both SE and BSE modes.
I stress the word "novice". Assume that this specimen could be handed to a high-school student and would both survive and stimulate interest.
Also, I would appreciate any suggestions for a basic EDS training specimen to be used in the same way. Here one would like to have a segregation of elements to encourage "what's this ?" kind of questions and produce interesting dot-mapped images -- the goal here is strictly qualitative -- suitability for quant semi-quant analysis would be a bonus. Of course, it would be ideal if this could be the same specimen as the first!
I am familiar with some specimens which have been used for this purpose, but hope someone might have some imaginative suggestions.
I am looking for a US contact that is affiliated with (BASF A. G., Ludwigshafen, Germany) or a source for tetcyclasis.
Any help will be most appreciated.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
On Fri, 8 Jul 1994, Francisco Javier H Blazquez wrote:
We are studying the absorption of macromolecules (proteins) by the intestinal cells of a freshwater fish (Prochilodus scrofa). We are using ferritin as an eletron microscopic and optical microscope marker and it can be observede in the enterocytes of the fish. When we tried to isolate the intestine and added ferritin to Eagle's medium where a isolated segment was incubated for 5 hours we fail to observe ferritin absorption by the enterocytes. The literature reports some insucess like this one. The time employed permits observations when this ferritin is given by oral route. Why isolated intestine don't phagocytes macromolecules? I think that problably some hormones are needed. May someone helps us?
============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
JOHN CHANDLER ASKED ABOUT THE UNCURED EPOXY ON THE EDGE OF A DIAMOND KNIFE-- I AM TALKING ABOUT CURING THE EPOXY AS WELL AS POSSIBLE BEFORE SECTIONING. THIS ALL REFERS BACK TO SOME READING I DID A COUPLE OF YEARS AGO IN SOME BOOKS ABOUT THE CHEMISTRY OF PLASTICS (I'LL HAVE TO LOOK UP THE REF.) THE EPOXY PLASTICS THAT WE USE ARE ALSO USED IN INDUSTRIAL APPLICATIONS, AND THE POLYMERIZATION THAT THEY USE IS ON THE ORDER OF 200 DEGREES OR MORE FOR DAYS! WE WOULD NEVER BE ABLE TO SECTION STUFF LIKE THAT; WHAT WE USE IS BARELY STARTING TO CURE. IT SEEMS THAT ALL THE UNPOLYMERIZED MONOMERS IN OUR PLASTICS ARE WHAT COLLECT ON THE KNIFE EDGE, AND SLOWLY HARDEN. Evelyn Clausnitzer U of Florida Ophthalmolgy
My lab is beginning the hunt for the perfect FEG SEM. We're a service lab in a sizeable chemical company so we see all manner of samples - but the bulk of our work is with polymeric samples. Good low kV operation is an obvious must but the choosen machine had better perform well doing EDS and some high kv, high resolution work (catalytic materials). Ease of operation and reliability are also musts - like many of you, we really depend on SEM info and finicky instrumentation and downtime is not something we want to spend all that money on. So what's the experience out there? Any horror stories? Any very happy users (especially those working with polymers)?
Re: Discussion concerning the remains of uncured epoxy on the back of a diamond knife: Industrial epoxies are indeed cured at temperatures very much higher than those we use, but this is due to the absence of a catalyst in the industrial formulations. The ca. 1% catalyst that we use makes it possible to cure our plastics at 50 - 60 C within a reasonable time - BUT - it is quite true that our formulations are not fully cured after 12h at typically 60C.
When the increase in hardness of a variety of EM embedding epoxies is measured during curing, all of them need approximately 40-48h at 60C for a full cure. This result is true for the generally-used catalysts DMP30, BDMA and DMAE (or S1) at normal concentrations of 0.5 - 1%, used in conjunction with a variety of modified and unmodified epoxy formulations. The choice of anhydride hardener also does not influence the length of the curing times to any great extent.
With the above in mind we have, in this lab, been polymerizing our epoxy embeddings for 48h at 60C. The embedded material does not seem to be influenced by the increased time, but we get: more reproducible sectioning qualities, cleaner knives, increased beam stability and possibly less contamination in the EM. The blocks are slightly harder than with the short cure times, but still section well with a diamond knife. (All this courtesy of Chris van der Merwe in this lab.)
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Microscopy Forum {microscopy-at-anlemc.msd.anl.gov}
To microscopist everywhere:
My lab is beginning the hunt for the perfect FEG SEM. We're a service lab in a sizeable chemical company so we see all manner of samples - but the bulk of our work is with polymeric samples. Good low kV operation is an obvious must but the choosen machine had better perform well doing EDS and some high kv, high resolution work (catalytic materials). Ease of operation and reliability are also musts - like many of you, we really depend on SEM info and finicky instrumentation and downtime is not something we want to spend all that money on. So what's the experience out there? Any horror stories? Any very happy users (especially those working with polymers)?
I would like to get hold of (pay for) a late model freeze fracture device in good working order with all the gizmos and widgets etc.
If you, or anyone you know is looking to sell an instrument (Balzers, Cressington etc) which is, in the "single owner, well serviced, less that 20,000 miles group, preferably with A/C and electric windows" please could you drop me a line
Thanks
Simon C. Watkins Director SBIC University of Pittsburgh Pittsburgh PA 412-648-3051
I publish a monthly newsletter on analytical instrumentation, and I encourage subscribers to make use of Internet and other information sources on line. Could you send me information on your forum, so I can pass it on to my readers?
Our monthly survey for July 1994 covered SEMs and x-ray detection systems.
Jo Rita Jordan Analytical Consumer jjordan-at-world.std.com
In response to Dave Calvert's questions about FEG SEM instrumentation, I can't say much about the bad or the ugly, but we have had a very good experience here at NRL with a Hitachi S-800 that was purchased several years ago. It has proven to be very reliable over its years of service, and its operation is actually much simpler than most of the more conventional SEM's that we have scattered around. It has been used by a large number of researchers, ranging from high school students to senior PhD's, and most users are able to get good results the first day they are on the machine, assuming they need fairly standard operating conditions. The setup we have includes a thin-window EDS system and a Robinson backscattered electron detector.
Altough our machine is an older model, I think that two of the features that have contributed to its reliability are worth considering when evaluating a newer instrument. First, it has a fairly robust vacuum system with an easy-to-use specimen airlock and good isolation and differential pumping of the specimen chamber and the rest of the column. This is essential for polymer specimens and for cases that we occasionally see, when an over-anxious operator neglects to let the silver paint dry long enough. Second, the S-800 is arranged so that its more advanced features, such as gamma control and special conditions for low-voltage operation, do not get in the way of its basic operation.
I hope these thoughts are useful.
James Sprague Surface Modification Branch Code 6670 Naval Research Laboratory Washington, DC 20375
We have been using aminopropyltriethoxysilane coated slides for LM immunohistochemistry and in situ hybridization. We were using xylene as clearing agents at first but because of the health problems, etc with xylene we decided to try the substitutes, mainly Hemo-De and Histoclear. We seem to be having a problem with these however, and it has gotten worse in the last few months. We seem to be getting alot of water in our clearing agent, even after well dehydrating. Could this be an artifact of the silane coating?? Why not the problem with xylene?? Has anybody come across the same problem?? Thanks for any suggestions.
Mark Elliott, PhD UBC-pulmonary Research Lab St. Paul's Hospital Vancouver, BC Canada
The following recipes are reasonable guesses from Bernie Kestel at Argonne National Laboratory. They are guesses since he has not thinned cast aluminum. The results are from polishing done with a South Bay 550-B single vertical jet polisher. It supplies the higher voltage needed by some electrolytes, in-situ process viewing, and allows for rapid foil rinsing. Use only ethyl alcohol to rinse ploished aluminum (also copper) to avoid methanol's corrosive attack of the finished surfaces.
Suggestions:
1. 150 ml nitric acid, 350 ml methanol, 40 ml butyl cellosolve (2-butoxy ethanol). Try -40C to -45C (a colder solution leaves a textured surface), 80 volts, 50-75 mA, medium flow rate.
2. 5.3 g lithium chloride, 11.16 g magnesium perchlorate, 500 ml methanol, 100 ml butyl cellosolve. To produce this electrolyte safely, first mix the liquid components together, then slowly add the dry granular salts to the stirred liquid mixture. Do not mix the dry components together! Continue stirring until the solids dissolve completely. Use at -50C, 100-150 volts, 12 mA, and a slow flow rate. Different concentrations of the salts may also work in special cases, i.e. more dilute or more concentrated solutions may help.
3. The following change was made to recipe #2 to thin Al-6wt%Ge, and it retained precipitates which were electron-transparent. Add 200 ml (or so) of acetic acid to the cooled solution. Use at -25C, 100 volts, and 15 mA with a slow to medium flow rate.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
In message Mon, 11 Jul 1994 00:16:38 -0400, Dave Calvert {73363.1014-at-compuserve.com} writes:
} To microscopist everywhere: } } My lab is beginning the hunt for the perfect FEG SEM. We're a } service lab in a sizeable chemical company so we see all manner of } samples - but the bulk of our work is with polymeric samples. Good low } kV operation is an obvious must but the choosen machine had better } perform well doing EDS and some high kv, high resolution work (catalytic } materials). Ease of operation and reliability are also musts - like many } of you, we really depend on SEM info and finicky instrumentation and } downtime is not something we want to spend all that money on. } So what's the experience out there? Any horror stories? Any very } happy users (especially those working with polymers)? } } Thanks in advance for the information. } } } Dave Calvert - Eastman Chemical Company } ================ We have been very happy with the low kV operation of our HITACHI S4500 FESEM (turbo pump). We have successfully imaged many uncoated material including polymers, diatoms (silica shells) and frozen material at kVs ranging from 0.5 to 2.0. We have had the instrument for about 15 months and it has been very reliable and has not had any down time. Since ours is a multiuser facility as well as a teaching facility, the instrument has tolerated some mishandling. We have not yet had a great demand for X-ray analyses so I cannot as yet comment much about it. M.V. Parthasarathy Section of Plant Biology Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734 Fax: 607-255-5407
Message-Id: {9407111755.AA00390-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM specimen prep. of powder Fe ? Orig-Author: {dakshs-at-rpi.edu}:ddn:wpafb -----------------------------------------------------------
Greetings, I need to study the microstructure of Fe powders (ranging from 5 - 50 microns in size) under the TEM. What is the specimen prep. procedure for such powder materials? Also, is there a quick and dirty method for a first look? Any help would be appreciated. TIA, yours, Sharath dakshs-at-rpi.edu
If you can use a microtome such as the Reichert Ultracut E, very thin samples can be made using a diamond knife. I've used Araldite 502 to embed Ti-Al powder and microtomed the blocks at 0.2-0.5 mm/sec to produce 60nm-thick sections.
If you are interested in the details, call or drop me an e-mail note.
Dear Microscopists, We have been told to stop using molecular sieves to "dry" our solvents, ie ethanol, acetone and methanol. The reason is because molecular sieves have been implicated in damaging diamond knives if fine "grit" from the sieves ends up in the final resin block. When the block is cut the grit damages the knife edge.
The recommmendation was to use copper sulphate however I am not convinced that this won't present a similar problem. I am interested to hear from others how they keep their absolute solvents absolute.
For Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
I'm looking for a company that carries 0 or 00 coverslips. I've looked in all the usual sources (Fisher, VWR, Polysciences) and nobody has them. If anyone knows an address of a company that does please let me know.
Dear microscopists, Wanted your views/opinions on black and white automatic photographic printers. The features we need are: variable speed, variable temperture, automatic replenishing, an efficient washer and a dryer or dryer attachment.
for Deborah MacLead
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
} ...We have been told to stop using molecular sieves to "dry" our solvents, ie } ethanol, acetone and methanol. The reason is because molecular sieves have } been implicated in damaging diamond knives if fine "grit" from the sieves } ends up in the final resin block. When the block is cut the grit damages } the knife edge. }
I have for years been using four angstrom molecular sieves, activated by heating to 205-315 degC for two hours, to dry solvents and keep them dry for freeze substitution. I have had not problems with premature dulling of diamond knives, but I have always (a) let the "fines" settle for a day or two minimum before using the solvent, and (b) removed any remaining suspended matter by centrifugation or filtration before use. I and others have found that reagent grade acetone or tetrahydrofuran from a freshly opened bottle generally does not need to be further dried for use in freeze substitution.
We regularly use molecular sieves but we extract the solvent ONLY with a pipette to avoid stirring up dust. We have recognized no problems from this practice. However, I am also not convinced that the solvent needs to be this dry. Has anyone experimented with eliminating dried acetone/ethanol?
Rod Kuehn University of Minnesota
On Tue, 12 Jul 1994, Richard Easingwood wrote:
} Dear Microscopists, } We have been told to stop using molecular sieves to "dry" our solvents, ie } ethanol, acetone and methanol. The reason is because molecular sieves have } been implicated in damaging diamond knives if fine "grit" from the sieves } ends up in the final resin block. When the block is cut the grit damages } the knife edge. } } The recommmendation was to use copper sulphate however I am not convinced } that this won't present a similar problem. I am interested to hear from } others how they keep their absolute solvents absolute. } } For Allan Mitchell } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 } }
Message-Id: {MAILQUEUE-101.940712073223.320-at-vanlab.paprican.ca} To: microscopy-at-anlemc.msd.anl.gov
There was an article in "The Microscope" Vol 42 No.1, 1994 on the McArthur microscope and some other portable microscopes. According to the author, the McArthur microscope is available from Prior Scientific Inc. (80 Washington St., Bldg. 0-54, Norwell, MA 02061, U.S.A. (617) 878-8442. I hope this helps.
James Drummond Pulp and Paper Research Institute of Canada Vancouver, B.C.
We have been using the Ilford 2150 RC for the past several months. It fulfills all the reqyuirements except variable speed. Frankly we don't know how we ever managed with out it. The results are equivalent to tray development and you have a finished, dry print in 59 seconds. Standardizing our negatives exposure and development has been important so taht any given batch of negatives can usually be done with out readjusting for exposure. This makes it a real time saver when you can run them through without thinking about it ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
=From: TOWER::GWERDOS "Greg Erdos ICBR EM Core Lab Univers" =To: IN%"richard.easingwood-at-stonebow.otago.ac.nz" =CC: GWERDOS =Subj: Re: Solvent drying = =} From: IN%"richard.easingwood-at-stonebow.otago.ac.nz" =} Subj: Solvent drying =} =} Return-path: {richard.easingwood-at-stonebow.otago.ac.nz} =} Received: from anlemc.msd.anl.gov by gnv.ifas.ufl.edu (PMDF V4.2-11 #3240) id =} {01HELE2ULG5S8WX95Z-at-gnv.ifas.ufl.edu} ; Mon, 11 Jul 1994 21:00:20 EST =} Date: Tue, 12 Jul 1994 11:42:29 +1200 =} From: richard.easingwood-at-stonebow.otago.ac.nz (Richard Easingwood) =} Subject: Solvent drying =} To: microscopy-at-anlemc.msd.anl.gov =} Message-id: {199407112342.AA19751-at-arwen.otago.ac.nz} =} MIME-version: 1.0 =} Content-type: text/plain; charset="us-ascii" =} Content-transfer-encoding: 7BIT =} X-Sender: st004718-at-brandywine.otago.ac.nz =} =} Dear Microscopists, =} We have been told to stop using molecular sieves to "dry" our solvents, ie =} ethanol, acetone and methanol. The reason is because molecular sieves have =} been implicated in damaging diamond knives if fine "grit" from the sieves =} ends up in the final resin block. When the block is cut the grit damages =} the knife edge. =} =} The recommmendation was to use copper sulphate however I am not convinced =} that this won't present a similar problem. I am interested to hear from =} others how they keep their absolute solvents absolute. =} =} For Allan Mitchell =} =} Richard Easingwood =} Department of Anatomy and Structural Biology, =} P.O. Box 913 =} University of Otago, =} Dunedin, New Zealand. =} Fax:64-3-479 7254 =} Telephone:64-3-479 7301 =} =} =############################################################# = I put the molecular sieve inside a dialysis bag, tightly tied at =both ends. It is made from 1 inch tubing and looks like a sausage at the =bottom of the vessel. Lets water in but won't let the grit out fd=********************************************************** =* Greg Erdos ** * =* Director, ICBR EMCL ** Phone 904-392-1295 * =* 218 Carr Hall ** FAX 904-392-8598 * =* University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * =* Gainesville, FL 32611 ** * =********************************************************** ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
Thomas Scientific(Arthur THomas Co.) lists cover slips of 0 thickness of at= least two different brands. They have various 800 numbers, depending where= you are: Far West region is (800) 345-2102. Their standard for that= thickness, by the way, is 0.085-0.13mm. Good luck.
Does anyone out there know exactly how the WPE of a resin is determined?? = Please, no answers telling me what it is--I want to know what is involved,= physically, with the determination of its value..... Incidentally, my= apologies to Nestor-I accidentally sent this to him, instead of the= bulletin board.....Thanks, to anyone who can help. Grace Kennedy/UCSD
To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM sample prep for Fe powder Orig-Author: {COOK-at-anlemc.msd.anl.gov}:ddn:wpafb ----------------------------------------------------------- If you can use a microtome such as the Reichert Ultracut E, very thin samples can be made using a diamond knife. I've used Araldite 502 to embed Ti-Al powder and microtomed the blocks at 0.2-0.5 mm/sec to produce 60nm-thick sections.
If you are interested in the details, call or drop me an e-mail not
I stopped using molecular sieves about 10 years ago for all applications, and haven't seen any noticeable change. For critical point drying however, I do always use a freshly opened bottle of absolute ethanol. A chemist advised me some years ago that ethanol is far too hygroscopic for molecular sieves to keep it absolute. This requires a special distillation step in the manufacturing process. They will keep acetone dry, however I too question just how dry it needs to be for resin embedding.
} Wanted your views/opinions on black and white automatic photographic } printers. The features we need are: variable speed, variable temperture, } automatic replenishing, an efficient washer and a dryer or dryer } attachment.
In December, 1993, I asked a similar question about B&W print processors. I got 23 excellent responses to the query which I edited into a compact format. I will forward it to anyone who is interested. Just contact me by e-mail.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
We use molecular seives for drying purposes. I also do have some problems with knife marks on sections. I returned a knife once and the manufacture said there were no chips out of the edge by it was faulty. Due to molecular seives?? Could be, I have never heard of this. When we change the seive material we do rinse it very well with solvents. Is there breakdown of the seive material during use? Will have to look into another dehydrant.
Malis and Steele have listed a reference for microtomy of Fe: L. Reimer, Z. Metallkunde, 50 (1959) 37-41. There are many references to the microtomy of materials in T. F. Malis and D. Steele, "Ultramicrotomy for Materials Science", in "Specimen Preparation for Transmission Electron Microscopy of Materials II", ed. Ron Anderson, Materials Research Society Synposium Proceedings, vol. 199, Materials Research Society, 1990.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
I can report only one negative incident with using molecular sieve. A client sucked up acetone from within the pellets during dehydration for critical point drying prior to SEM viewing. The plant leaf surfaces were litered with particulates. Subsequent comparative EDS analysis showed that the particles were sieve.
Since then, whenever I set up new sieve, or prior to heat-drying sieve, I always rinse it 3-4 times with the solvent to wash out as much dust as possible. Use relatively large bottles to put sieve and solvent into and replenish with pipet (dribbling solvent down inside wall of bottle) when 2/3 has been used, leaving 1/3 solvent as a buffer against turbulent mixing with the solvent near the sieve pellets. Handle the bottles gently to prevent stirring up the sieve. And NEVER take solvent from right above or within the sieve. Let settle overnight after adding new solvent to the bottle.
Now, I'm glad this subject came up as I've been wondering if there is any quick , cheap and reliable method to measure water in these solvents in the range .01 to 2.0% by volume, just to see if the sieve is really doing what we think it is. It seems that gravimetric methods based on specific gravity or weighing precise amounts on an analytical balance would not be easy to do or accurate. Is there some absorption method available?
How often do you heat-dry the sieve, how many bottle volumes do you run through before drying the sieve?
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Message-Id: {9407121551.AA05059-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM specimen prep. of powder Fe ? Orig-Author: {dakshs-at-rpi.edu}:ddn:wpafb -----------------------------------------------------------
Greetings, I need to study the microstructure of Fe powders (ranging from 5 - 50 microns in size) under the TEM. What is the specimen prep. procedure for such powder materials? Also, is there a quick and dirty method for a first look? Any help would be appreciated. TIA, yours, Sharath dakshs-at-rpi.edu
---------------------- Replied Message Body ---------------------- To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: TEM specimen prep. of powder Fe ? Orig-Author: {dakshs-at-rpi.edu}:ddn:wpafb -----------------------------------------------------------
Greetings, I need to study the microstructure of Fe powders (ranging from 5 - 50 microns in size) under the TEM. What is the specimen prep. procedure for such powder materials? Also, is there a quick and dirty method for a first look? Any help would be appreciated. TIA, yours, Sharath dakshs-at-rpi.edu
Try Thomas Scientific (800) 345-2100, they have 0 cover slips in a wide range of sizes.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Mon, 11 Jul 1994, Mike Folsom wrote:
} } Folks - } } I'm looking for a company that carries 0 or 00 coverslips. I've } looked in all the usual sources (Fisher, VWR, Polysciences) and } nobody has them. If anyone knows an address of a company that } does please let me know. } } My thanks in advance - } } Mike Folsom } } } _______________________________________________________________________________ } M.W.Folsom/Biology/UNM/Albuquerque,NM~87131/505.277.4277/mwfolsom-at-hydra.unm.edu } }
Why do you dry solvents? Is this to be able tyo keep large quantities in opened containers? For EM and SEM I've always gotten by purchasing dehydrants in pint containers and relegating the last inch of an old opened bottle to less than anhydrous uses.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
} Does anyone out there know exactly how the WPE of a resin is determined?? Please, no answers telling me what it is--I want to know what is involved, physically, with the determination of its value..... Incidentally, my apologies to Nestor-I accidental ly sent this to him, instead of the bulletin board.....Thanks, to anyone who can help. Grace Kennedy/UCSD } } } Grace: If you can give me a fax number I will fax a copy of how to determine the WPE. I will also include a table for mixing. The table will be for epon 812. The system may work for other resins. You can get the WPE straight from the manufacturer if you can find a telephone number for the company. We used to do this when we used Epon 812. I started using Araldite 502 15 years ago and I like it better than the epon or the equivalents of epon. I can get beautiful serial sections and the staining of LM sections is very good. It's a very simple resin to make up and you can freeze it in batches like you can do with epon. Here's the receipe just incase you want to give it a try.
Araldite 502..............12.5g DDSA....bring to..........22.5g DMP-30.....................0.3ml (I use tuberculin syringe) Mix well. I use applicator sticks to mix.
I mix this up in the morning and by that afternoon when I get ready to embed, the air bubbles are out of the resin. Hope this helps. Phil 8-{)
Like Greg Erdos, we too have an Ilford 2150 print processor. Ours is 4 years old.
Yes it can give you dry prints in about 60sec. but if you want the prints to last for years you will probibly need to refix and rewash them. Some of ours began to turn brown after about a year.
Yes the prints are consistent. When you get the exposure right for one publication or grant photo you can print the next 8 as fast as you can change paper and expose it.
We have had a number of things that have had to be replaced in the last 4 years.
One heating element
one gear for one of the roler
the water selinoid switch
twice I replaced the nut on the heat sensor (this made a big mess both times with developer all over the counter) (I was most displeased.)
The contacts for the drier heaters kept corroding until I replaced them with the coated ones that now come with the instrument. I could not talk Ilford into giving me a set, so I had to buy them.
i also know that I will have to replace the drier rolers soon.
This processor is not used a great deal. We average about 300 to 400 prints a month.
It is nice to be able to print one print if you want and not waste chemical. We can print 16" X 20" prints, if we want. (That is fun.) We can print 8" X 10" EM prints as fast as we can change negs.
It costs us about 40 cents a print for 8 X 10s in chemicals. This is not to bad for multi uses darkroom.
This is all I know. I hope it is helpful to some one.
Larry Hawkey Neurobiology Duke Hawkey-at-neuro.duke.edu
I've done critical point drying for years using abolute alcohol from the bottle, not freshly opened but discarded if 3/4 empty or been sitting too long ie month or more. I have not observed any difference. Note that some cylindars of CO2 are contaiminated with water... this hows up by freezing in the output line of CO2 escaping to atmoshere, usually as start & stop flow, sort of snorting and popping. Test with pure CO2 (ie no specimen in chamber) and if its still snorts, repl;ace the CO2 (about 1 cylindar in 10 or more has had this.
I find that pollen grains are very good specimens with which to train students on SEM. You can fix them, but if you have a mature plant with a stamen, just shake it onto a stub with adhesive of some sort. Set it to dry in a box with drierite for 24 hours....and then sputtercoat and observe. Diatom shells are really good too. Nina Allen
We have had a Kodak Dektomatic print processor for the past five years. This unit has all the variables requested in the original posting.
Over the years this processor has been nearly unstoppable except for routine cleanings and one broken bearing. At various times during the year a variety of users will produce as many as 1000 prints in a month.
The recommended processing speed produces a completely dry and archive level print in 93 seconds. No further finishing has ever been required. melsen-at-MICROBIO.emory.edu
I am tempted to try benzyl dimethyl amine (BDMA) in place of DMP-30 but don't have the original reference. How much does one use? Anyone have the original citation? I usually use: 20 g EmBed812 + 10 g DDSA + 10 g NMA + 0.6 g DMP-30.
Thanks.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
We have used black and white print processers for the last 12 years. Our original Durst Variospeed finally died last year and we now use a Durst Printo model. This is a modular system for the advanced amateur and performs very well. It can be set up with all the functions you want apart from continuously variable speed (you can set up 4 fixed speeds for differing chemistries). We found with the variospeed that we hardly ever altered the speed .
We use a very simple and inexpensive version without an attached drier and this is excellent for all our printing needs.
We've been having an intermitten problem over the last 2 years that we have been unable to solve and hoped someone out their might have had the same problem and found a solution.[I am forever the optimist]. The problem is with 4489 film. Occasionally we process a batch which comes out with abnormally shaped densities in the emulsion. It is not on the surface of the film, but rather in the emulsion and is not related to exposure or silver grain distribution. These areas print light because of the extra density. It is intermittent enough that it is hard to track down. It seems to be related to certain batches of film, since changing lot numbers gets rid of the problem. We investigated every aspect of our handling and processing and can't figure determine anything we are doing that could damage the emulsion. We've even tried brutalizing the film in various ways to cause the problem. The problem comes in spurts and usually limits itself to our most important images- the one where the sample can't be reproduced. Its bloody maddening and has us stumped. Any suggestions for things to try would be helpful.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I am not much of a fan of molecular sieves. A long time ago when I was doing some freeze-substitution work, I put some radioactive H20 into various organic solvents with molecular sieves. what I found was that the sieves and water reached some consistent equilibrium dependent on the solvent. For instance, ethanol is commercially available with 0.008% water but I found molecular sieves were ineffective in removing trace amounts of added radiolabeled water. This is why 100% ethanol is so much more expensive than 95%; a simple solution like sieves isn't good enough. removing the last 5% is not as simple as using molecular sieves. acetone is commercially available with {0.5% water and sieves removed 76% of the water. THF comes at 0.005% water and sieves removed 95% of the water. for routine TEM, we simply use fairly recently opened pint bottles and have no problem. for freeze-substitution, we always use a freshly opened pint bottle.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Do any EM users know of a substitute petri-dish for the old Lux Permanox tissue culture dish for processing of cell cultures directly for resin embedding. I have tried several possible substitutes that the manufacturers assured me were the equivalent but as soon as propylene oxide was added to them they quickly melted into a plastic mess.
Terry Robertson
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
} We've been having an intermitten problem over the last 2 years that we } have been unable to solve and hoped someone out their might have had the } same problem and found a solution.[I am forever the optimist]. The } problem is with 4489 film. Occasionally we process a batch which comes } out with abnormally shaped densities in the emulsion. It is not on the } surface of the film, but rather in the emulsion and is not related to } exposure or silver grain distribution. These areas print light because of } the extra density. It is intermittent enough that it is hard to track } down. It seems to be related to certain batches of film, since changing } lot numbers gets rid of the problem. We investigated every aspect of our } handling and processing and can't figure determine anything we are doing } that could damage the emulsion. We've even tried brutalizing the film in } various ways to cause the problem. The problem comes in spurts and } usually limits itself to our most important images- the one where the } sample can't be reproduced. Its bloody maddening and has us stumped. Any } suggestions for things to try would be helpful. } } Jay Jerome } ************************************************************** } * aka: W. Gray Jerome * } * Pathology * } * Bowman Gray School of Medicine of Wake Forest University * } * 910-716-4972 * } * jjerome-at-isnet.is.wfu.edu * } ************************************************************** } } } }
Dr Terry A. Robertson Telephone: 61-9-3462935 Department of Pathology Fax: 61-9-3462891 University of Western Australia Nedlands WA 6009 Australia Email: troberts-at-eosin.path.uwa.edu.au
My suggestion is to not use proplene oxide. Use ethanol for final dehydration and infiltration mixture. Works well. Might take a little longer but does not eat up petri dishes. Are you using Epon (or subitutes)? Spurr's Resins will eat up normal perti dishes. Good Luck.
Please send us details of your full name and address (The Royal Microscopical Society, 37/38 St Clements Oxford, OX4 1AJ (Tel: (01865) 248768/Fax: (01865) 791237)), indicating your requirements, if you would like further information on any of the RMS' events listed below:
1994 1 - MICRO 94 Conference and Exhibition (London) 12-15 September 2 - Immunocytochemistry Course (Oxford) 5-9 September 3 - Flow Cytometry Course (Cambridge) 19-23 September 4 - Microscopy and Catalysis (London) 27 October 5 - Ultrastructural Immunocytochemistry Course (Sutton) 14-18 November
1995 A - Electron Microscopy Course (Manchester) 9-13 January B - Microscopy in Geology (London) 8 March C - Annual Immunocytochemistry Meeting (London) 23 March D - Botanical Microscopy (Oxford) 27-31 March E - Microscopy of Magnetic Materials (Oxford) 3-5 April F - Annual Light Microscopy Meeting (London) 11 April G - Microscopy of Biomaterials (Oxford) 19 April H - Scanning Probe Microscopy Meeting (Nottingham) 24-25 April I - CYTO 95 Conference Cell Signalling (Southampton) 3-6 July J - Summer School in Light Microscopy (Leeds) 17-21 July K - Immunocytochemistry Course (Oxford) 4-8 September L - Immunophenotyping Meeting (London) September M - Cryotechniques Course (Glasgow) 11-15 September N - Flow Cytometry Course (Cambridge) 18-22 September O - Computers in Microscopy Course (Cambridge) 18-21 September P - Ultrastructural Immunocytochemistry Course (Sutton) 13-17 November
To N.L. Desmond: My attempt to send you the print processor summary keeps getting returned, "unrecognized host" message attached. Please contact me and send your e-mail address so I may try again. Thanks.
'pologies to all other netters for this intrusion.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
A recent article by EA Stanley in Microscope 41: 15-27 (1994) lists a source for the McArthur (portable) microscope as Prior Scientific Inc., 80 Washington Street, Bldg. 0-54, Norwell, MA 02061, Telephone (617) 878-8442.
Dear Jay, 4489 is the usual film in use here; we've shot over 10**5, and I'd think we've seen everything, but I don't recognize the problem from your de- scription. By "abnormally shaped densities in the emulsion" do you refer to grains, clusters of grains, or larger-scale phenomena? Does the problem occur for several sheets of film from one pack (& pk/100 or pk/250?), or is it less predictable. Have you looked at a film in bright safelight to see if there are any visible surface characteristics? If so, and if the film is not uniformly OD 6 after this, do the lighter areas referred to correlate with anything noted on the surface? What voltage are your electrons, or, rather is V { { 100 kV? Our HVEM (V = 10**6 V) may penetrate the abnormalities leaving little to dis- tinguish them by.
Jay Jerome posted the message concerning problems with Kodak 4489 films. From my experience this type of problem causes a lot of pain and frustration, especially when it occurs to your most important electron images. Unfortunately you do not notice this until you developed the films.
Perhaps the best way to solve the problem is to stop using film to record images. If you can afford a commercial CCD camera, your pain and frustration are gone forever ! With such a device, what-you-see-is-what-you-get. You never miss any action ! Plus you got all the image processing capabilities either on-line, or off-line. We have had very pleseant experience with a commercial CCD camera. Our sample is radiation sensitive material.
Just my own opinion.
Ming Pan Center for Solid State Science Arizona State University
To all who responded to the announcement on the microscopy bulletin board:
Thank you for your interest! Analytical Consumer is available by subscription only; we do not accept advertisements. We sell all back issues, including the SEM one in July 1994. That issue is $40 in the USA (and territories) and Canada, and $45 elsewhere. Subscriptions are $260 in North America and $310 elsewhere (US funds).
We survey a different technology for analytical chemistry each month, asking labs what equipment (manufacturers) they own, why they bought it, and their opinion of the instrument and its service. The result is a customer satisfaction analysis for that equipment. In the four years of publication, we have covered a wide variety of instruments, supplies, and software, from gas chromatography to robotics or FTIR spectrophotometers to LIMS.
All those who gave me a mailing address will receive information, including a list of back issues, about Analytical Consumer in the mail. If you need something faster, please call me at (508) 369-9079 or e mail to jjordan-at-world.std.com.
Thank you for your interest, Jo Rita Jordan, PhD Analytical Consumer (508) 369-9079 jjordan-at-world.std.com CompuServe 76150,2171
In reply to my 7/6 posting requesting ideas for SEM training specimens there have been a number of responses -- all useful and appreciated. I intend to contact a number of individuals for more specifics. I would also invite any with an ongoing interest in this subject to contact me directly for further sharing of ideas.
In reponse to my posting, John Chandler of Colo State replied:
} } OK, spill the beans! Which specimens have you thought about? I'd be interested in knowing. This is a really good question. { {
Herewith my reply to John and others who might be interested -- things I had thought about:
(1) Prickly gold grids -- Pro: easy to get from supply houses, coarse and fine structures, good depth of field and focus/stigmation specimen. Con - somewhat expensive to buy, can't be cleaned, no useful BSE or EDS, doesn't have any everyday associations to interest students, BORING!
(2) Semiconductor circuit dies -- Pro: consistent and inexpensive, good macro structure, interesting and recognizable features, robust, easy to mount and clean, strong BSE and X-ray/mapping contrast. Con - little fine structure, limited contrast, rectilinear patterns are not very good for focus and stigmation practice, gets boring after a while.
(3) Burnt-out bulb filaments -- Pro: can make them oneself, can get really spectacular 3Dstructures with macro and high mag detail, very sharp edges and high contrast -- great pictures! Con -- haven't figured out how to make them consistently, a pain to mount, easy to destroy, can't be cleaned, not useful for BSED or EDS.
(4) Fracture surfaces -- Pro: not hard to obtain, interesting detail, very durable. Con - no major/obvious problems here -- if one could find the right material (displaying elemental segregation) and develop a cheap procedure for preparing and mounting, this may be a good bet.
(5) Insects -- Pro: great to look at and cheap to obtain. Con - have to be mounted carefully. Easily destroyed. Not useful for EDS.
(6) Aluminum dendrite structures -- Pro: durable, cleanable, lots of fine structure, and produce great pictures. Con: have to spend a lot of time finding the structures. Boring EDS and BSE.
For me a big issue is to be able to stamp out a whole lot of nearly identical specimens. I intend to use the specimen for printed or video-taped training materials which a student could take from ground-zero (turning on the SEM) through more advanced topics, and so it is important to be able to predict what the student will see. Of the above, the semiconductor sample and the fracture surface are probably the best candidates I knew of for my purposes. (However, I intend to look at hard at some of the new suggestions received.)
I spent 10 years trying to get TEM blocks that would thin section perfectly every time, and I succeeded by using Quetol. That formula is published in my book "Current Trends In Morphological Techniques, Volume I", 1981, CRC Press, Boca Raton, page 243. It is as follows: Quetol 651 - 30 gm, NSA - 50 gm, NMA 8 gm, and DMP-30 - 2 gm. I tested nearly 100 epoxy formulas to finally arrive at this one, which has high contrast, and sections easily. However, the problem I never solved, although I spent several years and dozens of tests, was the presence of precipitates in the tissues, apparently as a result of the reaction of phosphate and cacodylate buffers with uranyl acetate en bloc treatments. The technique I finally settled on was to get to the 100% alcohol stage for the treatment of the blocks of tissue with uranyl acetate. However, the results with this are less satisfactory in terms of unbroken membranes than when the tissue is stained en bloc before dehydration. My question is: has anyone run similar tests, and found a method of en bloc staining with uranyl acetate before dehydration without getting the pepper-like precipitates in the tissues (they are not caused by staining of the thin sections, but are present in the tissue, as they can be seen even on unstained thin sections)? Organic buffers such as HEPES and PIPES improve the situation, as they do not react with uranyl acetate, but the contrast is much lower. This is a significant problem, and I have seen the precipitates in published micrographs of various authors on numerous occasions. We have all probably had them occur from time to time, and it seems worse with dense tissues such as brain, and not so bad with tissues that are less dense.
John E. Johnson, Jr. Editor, Microscopy Research and Technique
Hi, This sounds too coincidental, but just before checking my mail I was reading the chapter by H.H. Mollenhauer in "Artifacts in Biological Electron Microscopy", edited by R.F.E. Crang and K.L. Klomparens. Mollenhauer's chapter _Dehydration and Embedding Artifacts in TEM_, pg. 43-64, is quite interesting, particularly his discussion of two types of "pepper", which he defines as section contamination of a dense particulate matter having a general appearance of black pepper. Of the two types, embedding pepper seems to be the one that might be contributing to your problem. Mollenhauer states that this type of pepper is influenced by the resin formulation, occurs when sections are stained with lead citrate, is much worse with Spurr resin than Epon or Araldite (unfortunately he doesn't discuss Quetol), and is "exacerbated" when en bloc UA is used before dehydration and embedding. He suggests that pepper may be formed in sections when stains become trapped within the section or react with one of the resin components. To remedy the problem, he recommends pretreatment (I assume before the lead citrate step) with either 1% EDTA or O.5% HCl. Both pretreatments will apparently eliminate the embedding pepper. The other type he calls fixation pepper. This type may be removed by pretreatment with 1-2% periodic acid prior to staining with UA or PbCit. BTW, are you using your resin mixture with animal tissues, plants or both? I work with plant tissue, primarily leaves, and have been using Spurr resin and Mollenhauer's Epon/Araldite formulation. How well does Quetol work with plant material? Hope this info helps. Dwight
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
There has lately been some discussion on the relative merits of various epoxies for embedding samples. It is of course impossible to formulate one resin that will satisfy all applications, but if a resin is formulated to enhance the desirable properties of epoxies and hardeners and to minimize the undesirable characteristics, a useful improvement in qualities can be obtained. Attributes that are important in formulation include: choice of epoxide (influences wettability, crosslinking, beam stability, contrast) anhydride to epoxide ratios (influences elongation after the yield point, which is a measure of sectioning quality) anhydride characteristics (influences hardness) choice of plasticiser (influences hardness and beam stability)
Use of micro-hardness testing to check the relationship between formulation and hardness, and tensile testing to relate Young's modulus, plastic deformation and toughness to sectioning qualities makes it possible to formulate towards a goal.
We have now been using the result of such a formulation (with Quetol 651) for a number of years on all types of material, for EM and for 0.5 - 1 micron sections for LM. It adheres exceptionally well to most sample surfaces, including such difficult samples as plant leaf cuticles, it sections well, has very little own structure, contrasts well and is stable in the beam. Specimens in this also stain well for LM with buffered Toluidin Blue. We like it and use it for more than 90 percent of our biological samples. It is hard - use a diamond knife for best results. It is also not the worst epoxy to use if you would like to do structure and immunolabeling on (in?) the same section. It contains: Quetol 651 19.4g MNA 22.3g DDSA 8.3g Araldite RD2 1.0g S1 (DMAE) 0.5g
We do not use U-acetate during the dehydration, so do not know if this resin formulation also shows pepper-like precipitates. We have occasionally seen pepper deposits after fixing in Glut. in 0.1M (or higher) phosphate buffer, but none if the phosphate conc. is lower. A reference to specimen pepper: Hendriks & Eestermans (1982), Electron dense granules and the role of buffers: artefacts from fixation with glutaraldehyde and osmium tetroxide. J. Microscopy, 126: 161-168.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Are you sure that the PO is necessary (or even useful)? We routinely use acetone for Embed 812 embedding and seldom have problems. On the other hand, PO adds extra steps, extra cost, and is reportedly carcinogenic.
Rod Kuehn University of Minnesota
On Fri, 8 Jul 1994, Terry Robertson wrote:
} Do any EM users know of a substitute petri-dish for the old Lux Permanox } tissue culture dish for processing of cell cultures directly for resin } embedding. I have tried several possible substitutes that the manufacturers } assured me were the equivalent but as soon as propylene oxide was added to } them they quickly melted into a plastic mess. } } Terry Robertson } } } } Dr Terry A. Robertson Telephone: 61-9-3462935 } Department of Pathology Fax: 61-9-3462891 } University of Western Australia } Nedlands WA 6009 } Australia Email: } troberts-at-eosin.path.uwa.edu.au }
John: As to your problem with "pepper", are you washing enough after your fix? If you don't wash the glut out of the specimen enough, especially doing enbloc staining, the osmium will bind to the glut left in the sample and form a pepper like precipitate. If someone needs enbloc staining, I sometimes wash my tissue overnight. For normal processing with post staining I never have a problem with precipitate in the sections. The best thing to do, in my opinion, is to WASH,WASH,WASH, after fix. Or post stain with uranyl acetate. For my post stains I use:
uranyl acetate..........5g 50% ETOH..............100ml stain for 4 minutes (6-700A sections) for 2-400A sections I stain 6 minutes
Hope this helps. If you have any questions you can reach me at: voice: 410-455-3582 fax: 410-455-3875 email: prutle1-at-gl.umbc.edu
John H. Johnson recently asked about "pepper" in ultrathin sections:
..."the problem I never solved, although I spent several years and dozens of tests, was the presence of precipitates in the tissues, apparently as a result of the reaction of phosphate and cacodylate buffers with uranyl acetate en bloc treatments."
In December, 1993, I asked a similar question of the Microscopy network and compiled 11 responses detailing experiences and solutions to this problem. If you would like a copy of this summary, contact me be e-mail and I will send it to you.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Soem time ago, I asked if anyone else was using Quetol 651, and almost no= one replied. Why not?? I see several responses to the "pepper" problem= containing recipes for its use. I'd like to know the source and lot= numbers of this Quetol that you're currently using, plus the same info for= the NSA, if you use that: I've had a persistent, unresponsive problem for= the past year with Quetol system, and would like to solve it once and for= all. Thanks. Grace Kennedy, UCSD
Richard, For sea urchin gametes, we got very good results using a Karnovsky-type fix (glut-formaldehyde) at about 2%-2% in filtered seawater. Seawater itself is a good buffer, though something in it seems to react w/ UA if you use it en bloc. Since this mixture is about isotonic with the urchin cells, we ended up adding a bit of sucrose to make it slightly hypertonic so the mitochondria stayed nice. I don't recall right offhand how much sucrose we did add, but it was only a few grams per liter. Hope this helps.
Re: Suggestion to wash the tissue blocks for extended periods.
Yes, I have tried the washing and washing and washing procedure in a vain attempt to eliminate the pepper. I have tried leaving the blocks in buffer for up to a week before moving on to the en bloc staining with uranyl acetate (the specimens have been glutaraldehyde fixed, washed in buffer for up to 2 days, then osmicated, then washed in buffer for up to 7 days). No matter how long the blocks are washed in buffer after osmication and before en bloc staining, the precipitates are there in the thin sections, even with no lead or uranyl staining of the sections. It does not occur quite so badly on tissues which contain cells not so tightly packed together or in poorly fixed tissues (which would have more broken membranes), but I fix brain tissue very carefully, and it is typically very tightly packed together, which may result in retention of phosphate or cacodylate regardless of washing. The whole thing is more of an irritation than anything else, because it does not really interfere with scientific interpretation, but is rather unaesthetic. As I mentioned, the problem is eleminated by using organic buffers or staining en bloc at the 100% alcohol stage, before propylene oxide and infiltration with epoxy. However, organic buffers react very quickly with osmium tetroxide, and the resulting contrast is much lower than with phosphate buffer or cacodylate. Also, waiting until the alcohol stage results in lower quality of preservation than if en bloc stained before dehydration. Believe me when I say that I have tried EVERYTHING, with no luck in finding a reliable method of en bloc staining with uranyl acetate at the post osmium stage, before dehydration, which is the best place to do it, as uranium helps preserve membranes against the damage caused by dehydration. At present, I am tracing down a possible new technique with borohydride reduction suggested to me by a very brilliant biochemist. I will post the results when I have them.
Re: Referral to H. Mollenhauer's chapter in the 1988 Crang and Klomparens textbook.
Hilton also published on this topic in Microscopy Research and Technique, 1993, volume 26, number 6, pp. 496-512. The situation as I see it is to prevent the precipitates from forming in the first place rather than removing them after they have occurred. Still, the EDTA/HCl method can eliminate their visibility, and may be one of the best remedies so far. Hilton Mollenhauer spent decades dealing with such problems, and I would trust his findings and solutions implicitly.
Re: Quetol 651 problems.
If your difficulty with this epoxy is represented by holes in the thin sections (incomplete infiltration), I went around in circles over that one too. The problem is solved by leaving the tissue blocks in the 50/50 epoxy/propylene oxide overnight rather than just for a short time.
John E. Johnson, Jr. Editor, Microscopy Research and Technique
Message-Id: {9407141741.AA15674-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: OPTICAL MICROSCOPY - MICROSTRUCTURES IN STEELS . Orig-Author: {Griffiths Michael MJ {griffmj-at-msmailipx.bhpese.oz.au} }:ddn:wpafb -----------------------------------------------------------
Do any Materials Scientists / Metallurgists out there know of a reliable,reproducible etching technique to delineate bainite / tempered martensite structures in plain low carbon steel , [0.1% C] , which will yield nice coloured light microscope photomicrographs ?
We have an honors student here who despite trying a plethora of etchants : LePera's Reagent , Klemms Reagent , BASP , Cadmiun Sulphide Reagent , Beraha colour etchant , has had no luck.
Thank you
MIichael Griffiths B.H.P. Steel Newcastle Works Australia.
If this topic is a bit too esoteric for the general Bulletin Board maybe any responses can be E Mailed directly to me .
My Internet address is : griffmj-at-msmailipx.bhpese.oz.au
Greetings! While we are discussing resins, I'd like to get some feedback on the suggestion to replace proplyene oxide with acetonitrile (as mentioned to me by one of the resource people at Polysciences). She said that I could simply introduce acetonitrile in place of propylene oxide in the same ratios with the resin. Anyone tried this? I often use acetone as a dehydrant and so go right into resin from 100%, but I've also used EtOH and done the same thing without the transitional solvent (epoxy). Again, I work only with plant material, primarily leaves, so your mileage may vary.
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
John Johnson says: "...As I mentioned, the problem is eleminated by using organic buffers or staining en bloc at the 100% alcohol stage, before propylene oxide and infiltration with epoxy. However, organic buffers react very quickly with osmium tetroxide, and the resulting contrast is much lower than with phosphate buffer or cacodylate. ..."
does everybody use a buffer for their osmium step? I use HEPES without much problem but remember someone saying they did it in pure water since the osmium permeabilized the membranes and osmolarity no longer counted. In regards to John Johnson's comment, how about a short fixation in osmium/HEPES to ensure stability and loss of osmo-sensitivity then a second fix in osmium/water? is this crazy?
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
It is a coincidence that someone should ask about the use of acetonitrile as a substitute solvent in EM specimens. We just received an article on this subject, and if it is accepted, I will post the author's name and address so you can request a preprint.
John E. Johnson, Jr. Editor, Microscopy Research and Technique
On Thu, 14 Jul 1994, Griffiths Michael MJ wrote: } } Do any Materials Scientists / Metallurgists out there know of a } reliable,reproducible etching technique to delineate bainite / tempered } martensite structures in plain low carbon steel , [0.1% C] , which will } yield nice coloured light microscope photomicrographs ? } } We have an honors student here who despite trying a plethora of etchants : } LePera's Reagent , Klemms Reagent , BASP , Cadmiun Sulphide Reagent , Beraha } colour etchant , has had no luck.
Might try: [hint: I highly recommend #2 ;)]
1. Beaujard & Tordeux's: 21-28% aqueous NaHSO3; immerse 10-25 sec.
2. Villela's: 5 ml hydrochloric acid + 1 gr. picric acid + 100 ml Ethanol; immerse -few- seconds to minutes; -GREAT- STUFF!!! Works very good for CVD iron also; use polarized light, of course...; be -very- careful not to disturb the final "film" on the polished surface.
3. You say that Beraha's has been tried... which version(s)? There is: a) 1 gram Na2.MoO4 + 100 ml H2.O + 0.1 gram HH4.HF2; immerse 20-30 sec.; b) 3 gram K2.S2.O5 + 10 gram Na2.S2.O3 + 100 ml H2.O; immerse 1-15 min. c) many more...
4. and, tho' not too colourful, Picral [4 gr. picric acid + 100 ml H2.O, and sometimes + few drops (~4) 17% zephiran chloride (wetting agent)] -does- work... (may also need a few drops of HCl to enhance the action)
Good luck, -Rob ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, | Rob Tayloe | MSM Spelunkers Club /\v/\ | | Metallographic Lab. | Missouri Speleological Survey /\v/\ | | Rolla Research Center | Bat Conservation International /\v/\ | | U.S. Bureau of Mines | Missouri Cave & Karst Conservancy | | tayloe-at-rorc.usbm.gov | National Speleological Society #32993 /\v/\ | | (314) 364-3169 x247 | American Cave Conservation Association | ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
Netters, We recently tried acetonitrile as a substitute for ethanol in a dehydration series. The application is immunocytochemistry at the light microscope level and the resin we embed in is a mix of butyl and methyl methacrylate. The tissue is a plant root. In our trial, the acetonitrile was inferior to the ethanol, leading to greater distortion of the tissue (cell separtation and wrinkling), and higher background. We did not "trouble shoot" this, but on the basis of this trial, we were not encouraged to pursue the matter further.
Bonjour! In reference to the posting by Tobias Baskin, my interest is not in _substituting_ acetonitrile for a dehydrant like ethanol, but in using it in place of propylene oxide. My guess would be that like ethanol and the lower percentages of acetone, considerable tissue shrinkage would occur if the acetonitrile is used as a straight dehydrant, as Tobias experienced. Yet another resin question: How do most people store the resin components? I have seen them kept in a large glass desiccator, at room temperature on the shelf, but are there particular tricks that will yield a longer shelf life? Do the different components have different lifetimes? I work with the Spurr formula and the Epon/Araldite mixture of Mollenhauer. I'm also considering trying the Quetol 651 formulation suggested to me by Jan Coetzee (quetol, araldite RD-2 [1,4-butanediol diglycidyl ether], MNA, DDSA, DMAE). Thanks
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
A trick for Mollenhauer's Epon/Araldite, that I got out of an old Harvard Anatomy Department set of protocols, is to put your customary proportions of Araldite, Epon (Poly/Bed 812 or other), and DDSA into a glass bottle, cap it loosely, put it in a 60 degree oven for about an hour, then tighten the cap and mix thoroughly by vigorous agitation (they mix very effectively, because the heat has made them less viscous). Then put the bottle in the refrigerator, where the stock will be stable for six months or more.
When you need catalyzed resin, take the stock out of the refrigerator and let it come to room temperature. Then take out the volume you want, put it in a suitable tricorn plastic disposable beaker, and return the stock to the refrigerator. Add DMP-30 (with a Pipetman) proportionately to 2% (in other words, if you took 10 ml out of the stock bottle, add 0.2 ml DMP-30 to it). You can mix with a metal weighing spatula. You then have the desired volume of catalyzed resin.
I have used this and it works very well. The thorough mixing of the stock components is a considerable advantage (they often don't get mixed properly because they are so viscous). I like being able to prepare only the amount of catalyzed resin that I actually need, often only 10 ml. It is very convenient. The instructions that come with the Polysciences Epon/Araldite kit (cat. #02595) confirm that "In the absence of DMP-30 the mixtures are stable for six months at 4 degrees C, and for several days at room temperature".
} Bonjour! } In reference to the posting by Tobias Baskin, my interest is not } in _substituting_ acetonitrile for a dehydrant like ethanol, but in using } it in place of propylene oxide. My guess would be that like ethanol and } the lower percentages of acetone, considerable tissue shrinkage would } occur if the acetonitrile is used as a straight dehydrant, as Tobias } experienced. } Yet another resin question: How do most people store the resin } components? I have seen them kept in a large glass desiccator, at room } temperature on the shelf, but are there particular tricks that will yield } a longer shelf life? Do the different components have different lifetimes? } I work with the Spurr formula and the Epon/Araldite mixture of } Mollenhauer. I'm also considering trying the Quetol 651 formulation } suggested to me by Jan Coetzee (quetol, araldite RD-2 [1,4-butanediol } diglycidyl ether], MNA, DDSA, DMAE). } Thanks } } Dwight U. Beebe } Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca } Universite de Montreal Voice:514-872-4563 } 4101, rue Sherbrooke est FAX:514-872-9406 } Montreal, PQ H1X 2B2 Canada } } } }
Received: From ANLVM(MAILER) by ANLNRH(ANJE6.10) for MICROSCO-at-ANLEMC; Sun, 17 Jul 94 21:29 Received: from ANLVM by ANLVM (Mailer R2.07B) with BSMTP id 5997; Sun, 17 Jul 94 21:28:09 CDT Received: from earwax.pd.uwa.edu.au by ANLVM.CTD.ANL.GOV (IBM VM SMTP R1.2.2ANL-MX) with TCP; Sun, 17 Jul 94 21:28:00 CDT Received: from [130.95.124.120] (awsj [130.95.124.120]) by earwax.pd.uwa.edu.au (8.1C/8.1) with SMTP id KAA08688; Mon, 18 Jul 1994 10:26:44 +0800
We currently considering setting up a facility to analyse high resolution TEM micrographs using Fraunhofer diffraction. The set up we are considering goes something like this:
He-Ne laser-} collimator-} diaphragm-} the film-} lens-} mirror-} camera or -} lens-} CCD-} image processing
Are there any suggestions, pitfalls etc, that we should avoid or consider. Are there any commercial apparatus available or is it a pick and mix from an optics catalogue?
All suggestions eagerly awaited.
Keith Moulding
Hong Kong University of Science and Technology Materials Characterisation and Preparation Centre.
We store all resin components (for Spurr's, Embed 812, Araldite and Quetol) except DMP-30 and DMAE at room temp. We store the catalysts at 4 degrees and discard them after a year or two. I've seen no hint of a limiting shelf life for the other components.
The complete Quetol resin is made up a gallon-at-a-time, aliquotted into 12 ml plastic vials and stored at -40C. The gallon lasts for about 6 months and is used long before it starts to set.
Rod Kuehn University of Minnesota
On Fri, 15 Jul 1994, Dwight Beebe wrote:
} Bonjour! } In reference to the posting by Tobias Baskin, my interest is not } in _substituting_ acetonitrile for a dehydrant like ethanol, but in using } it in place of propylene oxide. My guess would be that like ethanol and } the lower percentages of acetone, considerable tissue shrinkage would } occur if the acetonitrile is used as a straight dehydrant, as Tobias } experienced. } Yet another resin question: How do most people store the resin } components? I have seen them kept in a large glass desiccator, at room } temperature on the shelf, but are there particular tricks that will yield } a longer shelf life? Do the different components have different lifetimes? } I work with the Spurr formula and the Epon/Araldite mixture of } Mollenhauer. I'm also considering trying the Quetol 651 formulation } suggested to me by Jan Coetzee (quetol, araldite RD-2 [1,4-butanediol } diglycidyl ether], MNA, DDSA, DMAE). } Thanks } } Dwight U. Beebe } Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca } Universite de Montreal Voice:514-872-4563 } 4101, rue Sherbrooke est FAX:514-872-9406 } Montreal, PQ H1X 2B2 Canada } } }
Someone in our department is doing in situ hybridization at the EM level and is having some problems with contamination on the sections.
She is processing thin sections of Lowicryl-embedded material by floating on the hybridization solution. This is done at 42 deg C in a sealed container for 22 hours. After hybridization, grids are washed 5X10 min in PBS at room temperature to remove hybridization solution. This is followed by a histochemical stain.
In the EM, there is an amorphous "sludge" over the section, along with some electron-dense precipitate. Processing for the histochemistry alone, including potassium permanganate, UA and lead citrate stains, eliminates the sludge/ppt problem, so it looks like the in situ step is the culprit.
Does anyone have suggestions for eliminating this contamination? I can get more details, if that would help.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
I've had the best results with picral or nital. Sometimes a few drops of zephyrin chloride to the picral helps.
Let me know how this pans out or if you have tried this already. You can reach me via:
Sue Smith smiths-at-mlc.lib.mi.us
On Thu, 14 Jul 1994, Griffiths Michael MJ wrote:
} } Do any Materials Scientists / Metallurgists out there know of a } reliable,reproducible etching technique to delineate bainite / tempered } martensite structures in plain low carbon steel , [0.1% C] , which will } yield nice coloured light microscope photomicrographs ? } } We have an honors student here who despite trying a plethora of etchants : } LePera's Reagent , Klemms Reagent , BASP , Cadmiun Sulphide Reagent , Beraha } colour etchant , has had no luck. } } Thank you } } } MIichael Griffiths } B.H.P. Steel } Newcastle Works } Australia. } } If this topic is a bit too esoteric for the general Bulletin Board maybe } any responses } can be E Mailed directly to me . } } My Internet address is : griffmj-at-msmailipx.bhpese.oz.au } } } } } } }
In response to the several comments regarding embedding media, I would like to mention some experiences regarding embedding and the ease of sectioning as well as the quality of the sections themselves. I used EPON and Araldite for years, and then, in 1976, switched completely to Quetol 651. However, even though the sections cut easily and the contrast was high, there were often small holes in the sections. I thought there might be water in the media (I believe Quetol 651 is water soluble), or perhaps in the absolute alcohol, propylene oxide, acetone, epoxy plasticizers (DDSA, NMA) or polymerizer (DMP-30). So, I had a local chemical analysis company in Baltimore (this was when I was with NIH, and Johns Hopkins University) test the various reagents for the presence of water, including some bottles that I left open overnight. The results indicated that there was no significant amount of water in any of the reagents, including the ones that I left open overnight (and Baltimore is a humid city). So, I experimented with leaving the samples in various stages of dehydration and infiltration for different lengths of time. It turned out that the 50/50 stage (acetone/embedding media) was the critical step. By leaving the tissue in the 50/50 stage overnight, or longer, the holes were no longer present, and the technician said that the blocks cut better than anything he had ever seen. With EPON, the holes did not occur as often, but the blocks cut less evenly when standard infiltration times were used. I believe that we have been leaving our specimens (depending on the density of the tissue) in the dehydration and infiltration stages for much too short a period of time, and that the holes are caused by incomplete dehydration and infiltration due to this improper procedure. With Quetol, because of its water solubility, I theorize that water left over from incomplete dehydration, rather than any water in the absolute alcohol or media, cannot be displaced. EPON, on the other hand, is not water soluble, and probably displaces residual water. However, extending the dehydration and infiltration times will improve the sectioning quality of any embedding media in my experience. On the other hand, regarding my recent correspondence with all of you on the TEM embedding pepper problem, no amount of rinsing or extended dehydration has eliminated the pepper when the specimens are stained en bloc before dehydration. Because we all use different types of specimens, I would be interested in your comments regarding the above theory, as well as experiences with embedding pepper using different types of specimens (I use brain which is very tightly packed tissue; it does not occur in my lab when I use monolayer cell cultures).
John E. Johnson, Jr. Editor, Microscopy Research and Technique
Your proposed setup--L-} C-} D-} F-} L'-} M-} Cam--looks OK; it's what we put together from components. I don't know of a commercially available kit, but I'm sure anyone who sells one will let us know. If you go the CCD-} Im. proc route, there is no need to do optical diffraction; you can get the same information from FFT of the image. One great convenience is to have a right-angle viewer on the camera--this prevents painful contortions when checking that the pattern is in the right position to show up on the film. Good luck.
I have seen a reference for using acetonitrile as a dehydrating agent as well as substituting it for the PO in epoxy resins - I only tried it once and was not very impressed with the results
*** CAUTION *** Acetonitrile combined with water releases hydrogen cyanide gas !!!
while it is touted as being considerably less toxic than PO users should be aware of the above reaction if it is being used as a dehydrant
I have an Agfa B&W print processor and I have a most annoying problem with it and would appreciate some help from anyone who has had & solved this problem.....
If the processor sits unused for more thatn a few days, I get this icky black mold(?) that grows in the baths - I can keep it somewhat under control by adding a small amount of bleach to the wash water, but I worry about what this is doing to the prints and their longetivity - it seems to originate in the activator- but I can't be certain - also it stains the trays to the extent that they cannot be completely cleaned
Message-Id: {1994Jul19.090536.319743799-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy listserver)
Microscopy Training Trends-Your Input Solicited
Hi fellow microscopists, * We are having a special symposium at MSA in New Orleans (Aug 1-6, 1994) on training trends in microscopy. * I solicit your input on the subject that I will collate and make available at the meeting and summarize and make available to the microscopy list when complete. * Specific examples are encouraged. * Feel free to answer any of the questions or just make some general comments, although specific examples will be extremely helpful. * Send your answers to me directly at my e-mail address: murphy-at-ms.sjdccd.cc.ca.us. * If you have "reply" on your mail option, just click it, write your message, and send it. It should then be sent directly to my e mail address without bottling up the network. * Thank you in advance for your help. * Judy Murphy, PhD, Dept. of Microscopy, San Joaquin Delta College, 5151 Pacific Ave., Stockton, CA 95207, Phone 209/474-5284; FAX 209/474-5649
I am looking for information on the following: 1. Formal courses taught at your institution in electron microscopy (lecture, lab or lecture and lab) and # of students taking the courses (present vs past). Indicate if other types of microscopy courses. 2. Microscopy courses that have decreased in units or been completely eliminated because of lack of enrollment. 3. Informal training in microscopy (indicate type) and # of students taking the training (present vs past). 4. Service microscopy done at your institution A. for graduate students (#) B. for researchers (#) C. other 5. Funding for microscopy labs A. Has this decreased or increased for your lab? ballpark percentage or remained unchanged? B. Specifically, how has this affected your operation? 6. What are the general trends you are aware of in training for microscopists with respect to numbers, material covered, amount of lab, etc. 7. What implications do you think this will have in the job market, at the workplace, etc.? 8. Any other comments?
For those that send information, thank you sincerely.
We have tried unsuccessfully to collect dust mites for an SEM project. Does anyone have suggestions on how to collect and prepare such a sample? So far what we have done is attach double stick tape to stubs and either blot areas (floor corners, bed linens, various anatomical locations) or sweep and area and then press collection onto double stick tape. We then give a brief gold-palladium coat.
I'm having some problems with the Microscopy Listserver system. Please expect some "Old Mail" and "Duplicate" messages traveling across the net for the next day or so. We've had several crashes in the last few days and I've discovered a large queue of messages which may not have been delivered, some of which are several weeks old. I apologize in advance for the hassel of seeing a message twice but rather than check everyone against the archive I've decided the easiest thing to do is just to resubmit the lot. I'll do it gradually over the next day or so.
Once again excuse the test message. I'm still trying to correct the nameserver problems and this is the only way I can test all 1300+ subscribers/sites....
Once again. This is a Listserver test by your friendly neighborhood SysOp. I'm still trying to get the nameserver test to complete. Sorry for the traffic. Just delete this.
We're looking for a high speed, high resolution flatbed scanner that can be used to scan both prints and negatives. The scanner should have a Mac driver (It'll be a super plus if the scanner can be interfaced to a unix system). The price range is below $5000. Any suggestions, comments, or stories are very much appreciated. Thank you in advance.
Best regards,
Xiao Zhang GE Corporate R&D (518) 387-6709 internet: ZHANG-at-CRD.GE.COM
Zhang, I recently bought a Nikon "Coolscan" slide scanner (~$2,000). It scans 2x2 slides, 35mm negatives, in color or monochrome. Resolution is better than 2K dpi. It will interface to anything with a SCSI port and comes with software for MS-Windows and Mac. It serves my needs because just about everything I have ends up as slides or negatives. Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
Although I haven't tried it myself, it seems that the best place to look for dust mites would be in a vacuum cleaner bag (after several vacuum runs across a normal carpet).
We have just completed our latest survey called "BUYING A NEW MICROSCOPE/THE AGONY AND THE ECSTASY". We've had so many calls about the work that it might be helpful to send along this information. The article includes a quantitative system for a selection that is appropriate for any microscope. The survey encompasses 59 scanning electron microscopes. In every case, users were contacted directly. We assessed for Customer Satisfaction, Applications, Microanalytical Accessories, and Service History.
This survey, Volume 6, Numbers 6 and 7 is available to new and existing subscribers of our newsletter. If you are interested in subscriber information, we have an 800 number. 800-440-0311.
This study is a joint effort of MICROSCOPE TECHNOLOGY & NEWS and ANALYTICAL CONSUMER. Additionally, we are working on a similar study for Transmission Electron Microscopes. If you have a TEM and wouldlike to be considered for the survey, please call us.
Our next issue, published in early August, will feature a review of the MSA/MAS meeting in New Orleans. If you can't attend, call us, tell us what you would like to have reviewed, and we will try to accommodate you.
Regards to all, Ellie Solit, Formerly Scientific Marketing Manager for Polaroid
Many years ago (10) we purchased an entire optical diffractometer from Polaron. We still use it occasionally, although lately we have been doing FFT's of images captured by Gatan's slow scan camera. The last addresses I had for Polaron are:
Polaron Equipment Ltd. 53-63 Greenhill Crescent Watford Business Park Watford Hertfordshire WD1 8QS U. K.
Polaron Instruments, Inc. 2293 Amber Drive Line Lexington Industrial Park Hatfield, Pennsylvania 19440 U. S. A. Telephone: 215-822-2665
P.S. Sorry about that previous, garbled message.
Russell E. Cook Electron Microscopy Center for Materials Research Materials Science Division Argonne National Laboratory Argonne, Illinois 60439 USA
Does anyone know any technicques we can use prepare Dioscorea (yam) or other angiosperm material? We are interested in using EM to see polymorphism in chromosomes. We have been using root tips. Thanks. Joyce.
Please excuse this plea for help.I need to access WorldWideWeb on internet (for images) from my home Mac thru a remote workstation account running XWindows and Mosaic as the browser program .Is there a Mac program that will emulate XWindows for this purpose? Is there another way around this? My internet connection is only thru my workstation account. Are there other browser programs for Mac/WWW access via XWindows? Thanks for any help you can provide.
Marc C. Brande, M.S. Live Brain Cell Functioning in 3D Culture San Diego 3D Imaging Group 3840 Camino Lindo San Diego, CA 92122 Email: BRANDE-at-SDSC.EDU Voice: (619) 587-4830 SD3D Email Discussion List: All aspects of 3D Imaging To subscribe/unsubscribe,send request to: sd3d-request-at-sdsc.edu To post a message, send message to: sd3d-at-mailserver.sdsc.edu
} } Please excuse this plea for help.I need to access WorldWideWeb on internet } (for images) from my home Mac thru a remote workstation account running } XWindows and Mosaic as the browser program .Is there a Mac program that will } emulate XWindows for this purpose? Is there another way around this? My } internet connection is only thru my workstation account. Are there other } browser programs for Mac/WWW access via XWindows? } Thanks for any help you can provide. } } NCSA provides WWW Browsers for X-Win, MS-Win, and MAC's. So you don't need to setup an X-Server only to browse the WWW (though X might be a nice addition even to MAC's ;). For more information check the NCSA Mosaic Home Page at :
} Sorry for repeating the same subject, but I just couldn't let anything } dangling for too long. To wrap up the discussion on the necessity of using } dry nitrogen when venting the TEM camera chamber, I measured the time it } took for the camera chamber of our JEM-2000FX TEM to pump down to ~0.5 mPa } (at which point the air-lock opens automatically and the green light for } the filament comes on). The results are as follows: } } using dry N2: 6'2" } using room air: 6'15" } } I did not repeat this experiment to determine whether the difference is } within fluctuation, but given the 13 s difference, I do not think it is } worthwhile to do so.
Gary, what was the humidity level in your lab at the time? With relative humidities of 65 - 85% in my lab, I find a remarkable difference in pumpdown time with honest-to-gosh dry nitrogen! If the camera has been open for more than about 60 sec on a humid day (like today, with a hurricane lurking out there) I give the camera a few second burst of nitrogen just prior to pumpdown, as well. It makes a huge difference! So I guess the bottom line is - whatever works. But it pays to check out your own individual situation.
Aloha, Tina Weatherby Carvalho Biological EM Facility University of Hawaii
Applications are invited for the above position in the Department of Histochemistry, Royal Postgraduate Medical School.
The successful candidate will be expected to oversee and expand the existing molecular biology unit within the Department. The unit is mainly engaged in investigating the roles played by specific regulatory factors (hormones, neurotransmitters, enzymes, receptors, free radicals) in the pathogenesis of human diseases, namely vascular remodelling, asthma, osteoporosis and gut dysfunctions. Applicants should have experience in probe preparation, in-situ hybridization and PCR techniques.
Ambitious and well motivated, the successful candidate will be encouraged to develop their own research interests within the scope of the Department's work and take full advantage of academic career opportunities within this prestigious establishment
The salary level range for a this Senior Research Officer 1A would be between GPB 19,326 and GPB 20,953 plus GPB 2,134 London Allowance according to age and experience.
Application forms and further details are available from the Personnel Department, RPMS, Du Cane Road, London W12 0NN, tel 081 740 3204, reference AJAY1.
OUTLINE JOB DESCRIPTION
The Department of Histochemistry is an extremely active research based Department which uses an integrated, thematic approach to its investigation of regulatory factors (hormones, neurotranmsitters, enzymes etc) and their contribution to normal bodily function and disease processes. Research is centred on the study of growth and remodelling, blood flow, inflammation and neuromuscular interaction. The mainstream technology is based on microscopy. The successful candidate will have the opportunity of taking over the existing molecular biology unit and expanding it. He/she will take responsibility for the day to day management of the facility and will be able to develop their own research interests within the scope of the Department's work. As a post-doctoral researcher, they will be expected to contribute fully to the academic as well as practical aspects of the Department, participating in inter- and intra-departmental seminars and helping to maintain the Department's position as an internationally renowned research centre through the production of scientific publications.
Hands on experience of probe preparation, in-situ hybridization and PCR methods will be required along with experience of supervising the work of others.
This post will suit an ambitious individual who is sufficiently motivated to take full advantage of this opportunity to develop a career within this prestigious academic institution.
Here in the Department of Anatomy and Cell Biology in the University of Michigan Medical School, I offered an EM course ("Biological Electron Microscopy") for four years (1988-91). It was a lecture/laboratory course, a rather intensive "hands on" type of course for graduate students. It was limited to 8 students. It seemed to go well, but was very labor intensive, and very expensive (each student received the equivalent of about $500 worth of supplies and recharge time on instruments in our department central central research facility (called the "Cell Biology Laboratories"). It was discontinued because of the expense and the large amount of labor.
For the last two years, I have offered a broader course for graduate students, entitled "Morphology for Molecular Biologists". It is a lecture and demonstration course (students have some "hands on" experience in the course, but less than the previous course). As the name indicates, there is an emphasis on immunocytochemistry, in situ hybridization, localization of reporter gene expression, and confocal microscopy, but there is also a rather extensive introduction to light and electron microscopy (both TEM and SEM), including both instrumentation and specimen preparation. Since many students in molecular biology these days do not have much experience in interpreting light and electron micrographs, the course includes a 2-hour "mini-course" in histology, and a 2-hour introduction to EM of organelles. The course is limited to 16 students, and they seem quite enthusiastic and well motivated, because of the obvious importance of morphological insight for some approaches in contemporary molecular biology.
Our department has a central research facility, the "Cell Biology Laboratories", containing equipment including TEM (Philips CM10), SEM (ISI=Topcon DS-130), confocal facility (BioRad MR600, Meridian, extensive Unix-based image processing and analysis), Balzer freeze fracture, Leica-Reichert ultramicrotomes, photographic darkroom, etc. The CBL was set up in 1979, and is primarily a hardware facility, offering the use of equipment to anyone in the University (on a recharge basis). A CBL Manager keeps the equipment in excellent shape, and gives training and help to users. There is some service (again on a recharge basis), although this has not been a major emphasis. As is often the case for such facilities, it is difficult to generate enough recharge money to operate completely in the black, covering very high annual service contracts (for example, about $12K for the Philips TEM), Manager's salary and other expenses. However, it has generally done quite well.
My own bias is that EM in the future may see some of the same renaissance that LM has seen in molecular biology over the last few years. I think that the questions and localizations will be more and more intracellular, where the resolution of EM will be needed to provide answers.
Kent
A. Kent Christensen Department of Anatomy and Cell Biology University of Michigan Medical School Ann Arbor, MI akc-at-umich.edu
Our laboratory will be introducing a new technology during the Poster Session at the MSA meeting for relatively rapid 3-D reconstruction from a series of tilted images. Simpler than a complete computed tomography reconstruction, the software can be run on a Macintosh or DOS computer and is highly adaptable to extracting quantitative information. If you want to see it in action, We will have several examples for demonstration. The poster is PQ# 435 THREE-DIMENSIONAL RECONSTRUCTION OF ATHEROSCLEROTIC FOAM CELLS USING TOMOSYNTHESIS.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I need some advice from someone who has had success preparing TEM samples of metallic film / ceramic substrate systems. I have been trying to make samples perpendicular to the interface of the film/substrate and have been unable to keep the metal layer from debonding from the substrate during either cross sectioning or mechanical thinning. Any thoughts?
Eric Stach Grad Student Department of Materials Science and Engineering University of Washington email: stach-at-u.washington.edu
Help! On attempting to section blocks from a recent fixation, I encountered a frustrating series of problems with compression, wrinkling, and related faults. After checking my sectioning methods and not finding any noticable mistakes, I went back to my notebook and realized that I had added twice the accelerator (DMP-30) to my Epon/Araldite mixture. A look at some of the available texts yielded info about the necessity for correct catalyst amounts, but specific info about what to do wasn't there. Is this excess catalyst the cause of the sectioning problems (the blocks seem too soft)? Or is something else the culprit? I had thought that too much accelerator would have caused the blocks to become very brittle, but the opposite seems to be the case. The mixture was: 7.5 gm EMBed 812 12.5 gm Araldite 502 27.5 gm DDSA 1.4 gm DMP-30 (should be 0.7gm) Polymerization was at 70 C for 43 hr.
Thanks in advance,
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
I am about to start a new project doing TEM of inactivated (I hope!) HIV. I'll be doing straight morphology and immuno-gold labelling of viral surface glycoproteins. I haven't hit the library yet and was wondering if anybody out there had a good fix for straight TEM and/or IMC of HIV. Citations are also welcomed.
Thanks in advance.
John Aghajanian Worcester Foundation for Experimental Biology
jjerome: Try a small, low powere vacuum-cleaner like a dust-buster, or a whisk broom. Gather a buch of dust and run through a Berlese funnel. Also: have you tried plucking eyebrows and eyelashes for Demodex? Should be able to get several from any healthy person. Soft opisthosomas, so they are a good drying-test. Phil Oshel poshel-at-luc.edu
For the last few days I have been receiving multiple "error messages" and "returned mail" from the Post Office at ecf.toronto.edu which have the text of postings to the microscopy list from other individuals. i had previously received these postings. Why do we get all these error messages? Am i the only one?
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
} For the last few days I have been receiving multiple "error messages" and } "returned mail" from the Post Office at ecf.toronto.edu which have the text } of postings to the microscopy list from other individuals. i had } previously received these postings. Why do we get all these error } messages? Am i the only one? } } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax)
Nope! Me, too.
Mike Wilson
------------------------------------------------------------------------ | Mike Wilson | | | Otolaryngology Dept. | "Don't take life so serious, son -- | | Univ. of Texas Health Sci. Ctr.| it ain't nohow permanent!" | | San Antonio, TX 78284-7777 | -- Pogo (Walt Kelly) | | Phone (210)567-6507; FAX -3617 | | ------------------------------------------------------------------------
I hope that this doesn't sound corny but, I just wanted to say that I am glad that this list exists. It is nice to be able to have a place to write to if you are having problems with a fixation process or if you have questions on a particular type of microscope, etc.
Thanks to everyone for being helpful in answering various questions and of course, thanks Nestor for taking up the task of maintaining and monitoring this list.
This is to everyone attending the MSA meetings in New Orleans, see you there!!
-------------------------------------------------------------- Peling Melville peling-at-amnh.org Interdepartmental Laboratories American Museum of Natural History
John- We've done immunogold labeling of HIV gp120 and found that it was sensitive to all aldehydes and ethanol fixatives. We do now do immunostaining on unfixed tissue and then fix. For immunochemistry inside of cells we permeabilize with BRIJ 57 (Triton seems to extract everything). I hope this is helpful. I am anxious to hear the other answers you get since they may solve some of our problems too.
Best-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Fri, 22 Jul 1994 JOHNA-at-SCI.WFEB.EDU wrote:
} Greetings folks, } } I am about to start a new project doing TEM of inactivated (I hope!) HIV. } I'll be doing straight morphology and immuno-gold labelling of viral } surface glycoproteins. I haven't hit the library yet and was wondering if } anybody out there had a good fix for straight TEM and/or IMC of HIV. } Citations are also welcomed. } } Thanks in advance. } } John Aghajanian } Worcester Foundation for Experimental Biology } } JOHNA-at-sci.wfeb.edu } }
I use to work with HIV alot in my old job. I would fix viral suspensions with 1-2% glut. in 0.1M PIPES buffer. The problem with this is that the fixation would make the particles clump together, thus for your case hindering labeling sites. If you are using cell culture, fix with a 1% of less of glut. I would use 1% ammonium molybdate as the negative of choice. Some references to check out are by S. Hearn and by G. Herrera. There are many references out there on viral immunocytochemistry.
} } I am tempted to try benzyl dimethyl amine (BDMA) in place of DMP-30 but } don't have the original reference. How much does one use? Anyone have the original citation? I usually use: } 20 g EmBed812 + } 10 g DDSA + } 10 g NMA + } 0.6 g DMP-30. } } Thanks. } } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax)
I recently tried using BDMA as a catylst using the EMS formulation EMBed 812 16.2 mls DDSA 10 mls NMA 8.9 mls BDMA 1-1.4 mls (EM Sciences recommends using 2X the volume of DMP-30 when substituting BDMA for DMP-30)
The blocks shared similar characteristics of cutting etc as epon batches made with DMP-30. One significant difference was in the shelf life. I store mixed batches of epon plus accelerator in aliquots at -20 C. The batches with BDMA were significantly more viscous after 1 month at -20. Furthermore, leaving 100% BDMA-epon for 24 hours on the rotor produced a taffylike material that made final embedding more difficult. By comparison, the DMP-30 batches retained characteristics similar to freshly mixed batches.
Regarding the original citation, give Electron Microscopy Sciences a call at 800-523-5874.
steve ---------------------------------------------------------- Dr. Steven Barlow EM Facility/Biology Dept. San Diego State University San Diego CA 92182-0057 phone: (619) 594-4523 fax: (619) 594-5676 email to sbarlow-at-sunstroke.sdsu.edu
} For the last few days I have been receiving multiple "error messages" and } "returned mail" from the Post Office at ecf.toronto.edu which have the } text of postings to the microscopy list from other individuals. i had } previously received these postings. Why do we get all these error } messages? Am i the only one? } } } Thomas E. Phillips, Ph.D. } Associate Professor of Biological Sciences } Director, Molecular Cytology Core Facility } 3 Tucker Hall } University of Missouri } Columbia, MO 65211 } (314)-882-4712 (voice) } (314)-882-0123 (fax) }
Resent-Date: Fri, 22 Jul 1994 13:58:31 -0500 (CDT) Resent-From: microscopy-at-ANLEMC.MSD.ANL.GOV Resent-To: MICROSCOPYLIST-at-ANLEMC.MSD.ANL.GOV To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Metal Film / Ceramic Substrate TEM Prep Orig-Author: {microscopy-at-ANLEMC.MSD.ANL.GOV}:ddn:wpafb -----------------------------------------------------------
I need some advice from someone who has had success preparing TEM samples of metallic film / ceramic substrate systems. I have been trying to make samples perpendicular to the interface of the film/substrate and have been unable to keep the metal layer from debonding from the substrate during either cross sectioning or mechanical thinning. Any thoughts?
Eric Stach Grad Student Department of Materials Science and Engineering University of Washington email: stach-at-u.washington.edu
I have received a number of requests for information regarding permeabilization mentioned in my reply about HIV immunostaining and so I am passing on brief comments to the users group.
The method we use is described in a paper by one of our post docs: Landers et al, 1993. Am J Pathol. 142:1668.
it is a modification of a method first described by Schliwa. Schliwa et al, 1981. PNAS, USA 78:4329.
We use Brij 58 in a concentration range of 1-3 percent to vary the harshness of permeabilization. Addition of PEG 20000 can also soften the harshness. We use PHEM buffer for the solutions. Concentration of PEG is 0-5%. Not all cells will be effectively permeabilized and a few cells will be completely trashed. However, the cells which are permeabilized show very good structural preservaion. The timing of permeabilization should be brief but will be determined by the cells you use. I.E we hit or miss each time out. Why do you think they call it magic? ;-)
I hope this is helpful-
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
In message {199407122042.QAA15951-at-ahab.rutgers.edu} Alan Pooley writes: }
...... Note that some } cylindars of CO2 are contaiminated with water... this shows up by freezing in the output line of CO2 escaping to atmoshere, usually as start & stop flow, } sort of snorting and popping. Test with pure CO2 (ie no specimen in chamber) } and if its still snorts, replace the CO2 (about 1 cylindar in 10 or more } has had this.
Note: above discussion in context of using ethanol as transition fluid .
Alan,(sorry for late response)
I may be wrong, but I always thought that the snorting and popping of ices out the exhaust tube was either CO2 freezing up (dry ice) in the needle valve and exhaust line as it expands, cools, and is released to atmospheric pressure (the ol' Joule-Thompson effect), or an ethanol slush forming in the cooled CO2. Usually, after 4-5 successive rinse cycles to flush ethanol out of samples, the ices dissapear, so I've assumed it is ethanol freezing up. Question is, does the ethanol really cool enough during expansion out the valve to cool to its freezing point, which is about -117.3 C, (from CRC Handbook)?. In fact, my Ladd CPD unit has heaters surrounding the valves to prevent such ice-up (but I seldom use them because they tend to heat up the champer during the CO2 flushing procedures). So my theory is that due to the high cooling rate of the liquid CO2 due to its conversion to gas as it expands through the exhaust or drain valve from a pressure of 500 to 1000 lbs/in2 in the CPD chamber to atmospheric pressure some CO2 cools enough to form dry ice, or cools the mixed in ethanol enough to freeze out.
To test for water in the CO2, do a blank run with no sample in the chamber. Introduce liquid CO2 into the chamber at a typical working temperature of 5-10 degrees C. Vent it out the chamber's drain valve and trap or collect the "ices" formed in the exhaust. If it is dry ice, it should sublimate quickly leaving no liquid behind. If it is water ice, it should melt into liquid water which will not readily evaporate because it is cold after just melting (artefact possible during humid summer months: the cooled CO2 vapor will cool the lab bench, filter paper, or whatever you collect the exhaust ices and vapors on, and moisture can condense out of the air, misinterpreted as water in the CO2. Thus each new tank of CO2 can be tested to see if it contains any significant water. When I do this test, I usually get no ices out nor accumulating; just a cool spot on the hood wall, with sometimes a bare trace of moisture which I usually attribute to condensation out of the air on the cool spot.(or...water in my CO2 tank???)
Then do a test with some ethanol in the cooled chamber (3 squirts from a pipet, again no actual sample) , fill with liquid CO2, vent out and see what you get out. When I do this test, I get little icy patches, which when they melt, sure smell like ethanol.
My Ladd CPD unit uses CO2 expansion into the chamber (with vents wide open to create quick flow-through to set up a large pressure drop into the chamber) for cooling and I usually see liquid CO2 bouncing around in the chamber when I cool it in this manner, but not ices.
Keep in touch.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
JOURNAL OF MICROSCOPY - VOLUME 175 PART 2, AUGUST 1994
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 93-99
Quantitative analysis of the microstructure of the human cornea and sclera using 2-D Fourier methods
Shahram Vaezy & John I Clark, Department of Biological Structure SM-20, University of Washington, Seattle, WA 98195, USA,
SUMMARY
A two-dimensional (2-D) Fourier analysis was used to characterize the microstructure of the human cornea and sclera. The average centre-to- centre spacing of collagen fibrils was found to be 59nm for the cornea and 285nm for the sclera. These results agreed with those obtained by direct measurement using the electron micrographs, and those reported in the literature. The spatial order in the microstructure of the cornea was much greater when compared with that of the sclera. The results of the 2-D Fourier analysis were consistent with the theory of transparency of the eye. The 2-D Fourier analysis will be useful in quantitative characterization and analysis of the complex microstructure of biological cells and tissue in normal development and abnormal pathogenesis.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 100-107
A high sensitivity CCD system for parallel electron energy loss spectroscopy (CCD for EELS)
Zizhou Tang, Ruoya Ho, Zengli Xu, Zhifeng Shao & Andrew P Somlyo, Molecular Physiology & Biological Physics, University of Virginia, Box 449 Jordan Hall, Charlottesville, Virginia 22908, USA
SUMMARY
A cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy-loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal-to-noise ration and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorous L edge generated from a phosphoprotein, phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required at elemental mapping for a given sensitivity.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 108-120
Kinetics of intracellular Ca2+ concentration changes and cell contraction of electrically stimulated cardiomyocytes as analysed by automated digital-imaging microscopy
Hubert Schneider, Marc Fallert & Ernst Dieter Wachsmuth, K 125.507, CIBA-GEIGY Ltd, CH-4002, Basel, Switzerland,
SUMMARY
Enzymatically disaggregated, electrically stimulated cardiomyocytes from adult rats were examined by television-mediated vital microscopy for intracellular Ca2+concentration and contractile activity. Using an inverted microscope in the epifluorescence mode, the Ca2+ signal was imaged with a low-light-level CCD camera and traced by means of the intracellular concentration of the fluorescent complex of Ca2+ with its indicator Fluo-3. Using the transmitted-light mode, cardiomyocytes that were not loaded were imaged with a conventional CCD camera with automatic gain control and traced by length measurements. Optical images of at least 40 cardiomyocytes per batch of cells from one heart were recorded in up to 20 microscopic fields of observation on videotape within 20min. They were consecutively analysed by a personal computer installed with an image analysis card at a time resolution of 20ms, employing a discrete convolution operation, filtering and threshold setting for fluorescence measurements, and contour description and vectorial analysis for length measurements. Frames of fluorescent images were corrected for the halo effect caused by the increase in the Ca2+- dependent fluorescence signal after electrical stimulation. The cell contraction had to be measured in the transmission mode without Fluo-3 due to the inhibition caused by the intracellular Fluo-3. the following coefficients of variation (V) were determined: Vfluorescence { 0.033 and Vtransmission { 0.003 for the precision of measurement, and Vfluorescence { 0.05 and Vtransmission { 0.04 for the reproducibility. The system was validated with isoprenaline and ouabain as agents to modify the Ca2+-signal and the contraction. The response of the cardiomyocytes of various rats to electrical stimulation, with respect to amplitude and its time point, has a V { 0.08 for both the Ca2+ signal and the contraction.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 121-134
Reduced carrier single-sideband microscopy: a powerful method for the observation of transparent microscopical objects
F Bretschneider & P F M Teunis, Comparative Physiology Neuroethology Gp, University of Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands,
SUMMARY
The theoretical and practical properties of different forms of contrast formation in the microscope based on anaxial illumination are investigated: so-called single-sideband (SSB) techniques. The use of anaxial illumination in transmitted light microscopy is by itself a form of phase contrast (asymmetric illumination contrast, or AIC), but needs enhancement via a video circuit coupled to the microscope. The addition of a partially absorbing mask, known as a carrier attenuation filter (CAF), in a proper, conjugate plane in the microscope, improves contrast substantially. The imaging properties of this reduced-carrier, single-sideband imaging method (RC-SSB) were tested using the transparent parts of a compact disc (CD); the tracks may be treated as small objects with a controllable phase shift. the results were compared both theoretically and experimentally with Zernike's phase contrast and with Nomarski differential interference contrast. The SSB technique has been shown to reveal transparent, submicrometre parts of living unstained tissue, such as the microvilli on sensory receptor cells of the transparent catfish, Kryptopterus. The high resolving power, together with the variable spatial-frequency contrast enhancement, makes it a powerful technique for the imaging of in vivo subcellular details.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 135-142
Particle-surface interaction in thin vitrified films for cryo-electron microscopy
Marek Cyrklaff, Norbert Roos, Heinz Gross & Jacques Dubochet, European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012 Heidelberg, Germany,
SUMMARY
The concentration of particles in thin vitrified films of suspensions is described as a function of various parameters such as the type of particles observed, the time the sample is left on the grid and the effect of different washing procedures. The thin films are prepared for cryo- electron microscopy by the classical single-side blotting method or by blotting both sides of the grid simultaneously. The single-side blotting method results in particles preferentially absorbing to the non-blotted surface. This has the advantage that the concentration of particles in the thin vitrified film is higher than in the original suspension. The energy involved in adhesion of particles to the surface appears to be generally small. In most cases, it does not cause significant deformation of the particles or of the surface of the film. However, there are cases, for example with lipid vesicles, where the particles are broken as a result of absorption. Since particles remain absorbed to the air-liquid interface, it is possible to wash or dialyse the solution directly on the grid with negligible loss of particles. This represents a very rapid and handy method for micro-dialysis. A thin film is then formed by blotting the specimen and vitrified by rapid cooling.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 143-153
Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections
Alice Warley, Katherine P B Cracknell, Helen B Cammish, Charles H C Twort, Jeremy P T Ward & Stuart J Hirst, Sherrington School of Physiology, St Thomas' Hospital Medical School, Lambeth Palace Road, London, SE1 7EH, U.K.
SUMMARY
Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X-ray microanalysis studies are presented. The cells are grown on Pioloform-covered gold grids supported on Thermanox coverslips. This provides a growth-compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze- drying. The effects of different washing media (0.3M sucrose, 0.15M ammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled-water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 154-161
Fluorescence bleach rate imaging
G J Brakenhoff, K Visscher & E J Gijsbers, Electron Microscopy & Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands,
SUMMARY
Bleach rate imaging on a (cooled) CCD can be easily achieved using a confocal microscope with bilateral scanning and detection coupled to a workstation; it is as easy as acquiring regular fluorescence images. Several analysis and display methods for bleach rate imaging are presented such as the bleach map (and its inverse) and a matrix-based decomposition method for multi-labelled specimens based on the bleach rate differences between the dyes used. With these tools, bleach rate based imaging can become a viable alternative to multiple labelling techniques for component identification in fluorescent specimens.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 162-165
Fluorescence saturation in confocal microscopy
K Visscher, G J Brakenhoff & T D Visser, Electron Microscopy & Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands,
SUMMARY
The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approaches that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 166-170
Calcium alginate encapsulation of small specimens for transmission electron microscopy
A M Page, J R Lagnado, T W Ford & G Place, Department of Biology, Royal Holloway, University of London, Egham, Surrey, TW20 0EX
SUMMARY
A technique of encapsulating small objects in calcium alginate for further processing for transmission electron microscopy is described. Five methods are outlined which enable a variety of specimens, including single cells (in suspension and on agar plates), small organisms and monolayers of tissue culture cells to be processed. A method for immunolabelling alginate-entrapped material is also outlined.
Journal of Microscopy, Vol. 175, Pt. 2, August 1994, pp. 171-174
Sequential treatment by phosphotungstic acid and uranyl acetate enhances the adherence of lipid membranes and membrane proteins to hydrophobic EM grids
Alexander N Barnakov, Biochemistry Biophysics Department, Washington State University, Pullman WA 99164, U.S.A.
SUMMARY
A simple procedure for screening by electron microscopic observations of conditions for the reconstitution of membrane proteins into lipid bilayers is described. This procedure consists of a 5-10s treatment of electron microscopic grids, to which the sample has already been applied, with 1% phosphotungstic acid before proceeding with final staining in uranyl acetate. The method substantially enhances the adherence of lipid membranes and membrane protein particles to hydrophobic collodion/carbon grids
ANLEMC is dying..... I've managed to get it back up and running, however, expect that sometime in the next few days that this mail server will have a new home.
Please keep an eye on your mail for the announcement of a new host address. I will have to permanently move the microscopy mailing list so that the problems of the last few days do not repeat themselves.
Sorry for the headaches many of you have had in the last week, it wasn't fun for me either.
Nestor -------------
Nestor J. Zaluzec Argonne National Lab. Materials Science Division
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
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G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Nestor, I am sure you hear about anyone who has problems however I just wanted to send a note to thank you for putting in the time and energy to provide this service to the microscopy field. It is an invaluable network for all of us. Judy M _______________________________________________________________________________
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
A few weeks ago I volunteered to prepare a set of test images for round robin testing of grayscale printers for images. The images are now ready and accessible via ANONYMOUS FTP. But before you all rush off to grab them let me spell out a few details.
1.) Only grab the images now if you intend to print them and bring copies of your output to the computer workshop at the MSA meeting in New Orleans. I will leave the images posted on the site for those of you who wish to download them later, but let those who are definitely intending to participate at MSA have first crack.
2.) Download the README file in addition to the images! It contains important information as well as a form for you to fill out and bring to the workshop.
3.) The images are B&W TIFF files. There are 2 copies one at 100 dpi (~1 Mbyte) and one at 300 dpi (~10Mbytes). These are not small images and you should realize that they will take time to download and requires approximately the disk space I have indicated to store. Both images should be downloaded and printed. They are identical, however, they WILL stress the limit of your printers.
4.) Each of the image files is stored both in BINARY and BINHEX format choose your pleasure.
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Our lab has been making cross sections for TEM analysis utilizing the Anderson tripod polisher technique. The good news is that this technique seems to work, the bad news is I know of only one supplier for the abrassive diamond films (South Bay Technology). Not that there is anything wrong with South Bay, in fact they are always quite helpful when called on. But as the only supplier of these films that I know of, I am limited by their in stock inventory. Does anyone know of other suppliers for the diamond abrasive films?
----Mail status follows---- Have been unable to send your mail to {gillen-at-[129.6.98.22]} , will keep trying for a total of three days. At that time your mail will be returned.
----Transcript of message follows----
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G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Message-Id: {9407271840.AA02306-at-odin.morph.med.umich.edu} Errors-To: {dennis%odin-at-odin.morph.med.umich.edu} To: MICROSCOPY-at-ANLEMC.MSD.ANL.GOV Cc: dennis-at-odin.morph.med.umich.edu
} ...Does anyone know of other suppliers for the diamond abrasive films? } ... } John Phelps } NIST, Boulder
3M used to sell adhesive backed paper with alumina and diamond coatings of various grit sizes. I believe that the product was called 'Imperial Lapping Paper.' Check with your local rep.
} ...Does anyone know of other suppliers for the diamond abrasive films? } ... } John Phelps } NIST, Boulder
} 3M used to sell adhesive backed paper with alumina and diamond coatings } of various grit sizes. I believe that the product was called 'Imperial } Lapping Paper.' Check with your local rep.
If you do buy 3M, I've been told to make sure to get the Type A 3M lapping discs. The quality of other types are not as high (diamonds pull out of disc too easy) and who wants to ruin their samples.
A grad student in my lab needs to do some counting using a light microscope. A grid would be most helpful, but at present we don't have the bucks to shell out for a new occular. Are there other alternatives out there, such as a transparent film with a grid or some such that one can place inside an occular? Thanks for your help.
Gail Celio Botany and Plant Pathology Michigan State University
I hear that 3M company make diamond lapping films (down to 3 micron), large pieces, bulk packs, at low prices. Sorry, I don't have a name or #.
On Wed, 27 Jul 1994 PHELPS-at-ENH.NIST.GOV wrote:
} Hello, } } Our lab has been making cross sections for TEM analysis utilizing the } Anderson tripod polisher technique. The good news is that this technique } seems to work, the bad news is I know of only one supplier for the abrassive } diamond films (South Bay Technology). Not that there is anything wrong with } South Bay, in fact they are always quite helpful when called on. But as the } only supplier of these films that I know of, I am limited by their in stock } inventory. Does anyone know of other suppliers for the diamond abrasive films? } } thanks in advance, } John } } John Phelps } NIST, Boulder }
Try to trim block down as small as possible. Then soak in propylene-oxide (pop) to remove soft plastic, then re-infiltrate stepwise, 1:1 pop:resin, 1:2 pop:resin, 1:3, 100% resin 2changes, embed in a hard resin mix polymerize hot. This may work, but no promises, I've had mixed results. Maybe microwave polymerizing the block you have might work?
On Wed, 27 Jul 1994 dennis%odin-at-odin.morph.med.umich.edu wrote:
} I have embedded some samples of domestic cat spermatozoa in an incorrectly } mixed batch of Poly/Bed 812. The plastic came out excessively soft with no } possibility to be hardened further. } } I have tried one of the re-embedding techniques recommeded in Hayat's 1989 } book on EM technique using 100% EtOH saturated with KOH, but it destroyed the } fragile sample. } } Any recommendations on how these samples can be re-embedded safely? They are } difficult to obtain, and I only have a few with which to work. Any } suggestions are appreciated! } } Thanks, } } Dennis Shubitowski } dennis-at-odin.morph.med.umich.edu }
Gail Celio asks about using a homemade grid occular. I have made some experimental grids with a photocopier and transparency film. Cut to the right size this could easily be used for your purposes. Two notes of caution: you have to have the right kind of eyepiece. Some eyepieces have a lens in front of the image plane, and you can't get access to it. If you take the eyepiece off the microscope and look through it while shoving a cotton swab in the other end you should be able to get the end of the swab in focus before hitting the first lens if you can't you are out of luck. Second, the reticle has to be exactly in focus, meaning that the image from the objective is at focus in the plane of the reticle. This also makes dirt, etc on the reticle in focus.
If you are lucky enough to have an eyepiece that was specifically designed for holding a reticle there will be a nice ledge to push the reticle up against. If not you need to make a spacer which will take some adjustment, which which is what graduate students do well. The reticle can be held against the spacer or ledge with a spring clip. regards, Mark W. Lund MOXTEK, Inc Orem UT
Greetings! Ed Basgall responded to my resin storage question and asked me to forward his note to the group as he's been having problems getting notes posted. In a second note, he wrote
"Joe Mascorro at Tulane Univ, New Orleans, LA (EMSA august 1992) did a comparison of viscosities of variuos resin components. and found that mixtures using BDMA instead of DMP-30 had less than half the viscosity even after 60 minutes post mix. EMSA proceedings 1992, p 746."
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
---------- Forwarded message ----------
Please unsubscribe me from the Microscopy list Thank you Daniel Dagan
I forgot to tell you that I have managed to save all your subscriptions. You will *NOT* have to resubscribe. Just change your local nicknames/aliases....
Can anyone tell me what the status of the electron microscope company Camscan is? It would probably be best to respond to me directly, rather than post to the list.
regards Mark W. Lund MOXTEK, Inc. Orem UT lundm-at-xray.byu.edu
We are thinking of upgrading an existing TEM to allow acquistion of digital images that could then be ported directly to our image processing and analysis software. Does anyone out there have experience with digital cameras for TEM? I am interested in what models people are using and the quality of the image. Are the digitized images suitable only for morphometry or can they be used for publication prints. Thanks
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Gail- If your occulars are either non-focusing, or you are anxious about taking them apart, let me suggest a simple fix. Photograph a grid (like that of a bright-line hemocytometer) using 35mm color film. Use several maginifications to make grid images with different spacings. I like to give the image a color cast, usually light blue to correct for the yellowing of many illuminators at low power. The developed transparancy can be placed in the light path at any point which also has an image of the field-limiting diaphragm. I'd put it right on top of the exit of the diagphram of a Zeiss, for instance. Now focus your condensor so that the image of the grid is focused onto the object plane.You should be able to sector the field of view without much confusion. Hope this helps- let me know if there are any problems or questions Hal Krider Biotechnology Center Image Analysis Facility The University of Connecticut 203-486-4860
Does anyone know off the top of their head the electric field required for room temperature field emission from a standard tungsten gun and what type of force this exerts on the filament?
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: No Need to Resubscribe Orig-Author: {"Nestor J. Zaluzec-Argonne Nat. Lab." {ZALUZEC-at-AAEM.AMC.ANL.GOV} }:ddn:wpafb -----------------------------------------------------------
Sorry Folks...
I forgot to tell you that I have managed to save all your subscriptions. You will *NOT* have to resubscribe. Just change your local nicknames/aliases....
a bit more on resin storage - I follow basically the same procedure as Ed Basgall (mix up a batch, place aliquots in syringes for storage) one way to minimize leaks is to plug the "needle-end" with a toothpich or shaved applicator stick, leaving just enough sticking out to be able to grab it later, then replace the original plastic tip cap and parafilm - I almost never get leakage, but just to be on the safe side I store the syringes in Ziplock freezer bags
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----Transcript of message follows----
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Does anyone have information on the durability of images printed on the Fargo Primera printer, and is it similar in quality to the $7K Alden Electronics 9315CTP? I have had a demo print from the Alden printer near my window on the wall for a few weeks, and it is REALLY brown. Any way to fix thermal paper?
christoffersen-at-snmail.jsc.nasa.gov wrote: } Can someone suggest some entry points (books, papers, EMSA proceedings etc.) } that can update me on the state of the art in cathodoluminescence spectroscopy } applications in SEM/TEM? A book or article on current CL theory would also be } most helpful. Thanks.
Try:
1. AUTHOR: Yacobi, B. G. TITLE: Cathodoluminescence microscopy of inorganic solids / B.G. Yacobi and D.B. Holt. PUBLICATION: New York : Plenum Press, c1990. DESCRIPTION: ix, 292 p. : ill. ; 24 cm.
or
2. AUTHOR: Marshall, Donald J. TITLE: Cathodoluminescence of geological materials : an introduction / D. J. Marshall, with a chapter contributed by Anthony N. Mariano. PUBLICATION: Boston : Allen & Unwin, 1988. DESCRIPTION: xiv, 146 p., {12} p. of plates : ill. ; 29 cm.
Hope this helps Roy
Ed Vicenzi tel (609) 258-1464 office Princeton University tel (609) 258-1406 lab Princeton Materials Inst. fax (609) 258-6878 70 Prospect Ave. Princeton, N.J. 08540-5211 email: vicenzi-at-phoenix.princeton.edu
A student was staining some arthropod antennae with methylene blue for a project. One day she couldn't find the stain, and so arbitrarily picked another off the shelf (which contained a bunch of really antique bottles). It was azocarmine and, fortunately, worked unbelievably well in staining the structures we were looking for (aesthetascs) a nice pink! Unfortunately, we don't know why. I can't find anything about azocarmine itself. It sounds like carmine will stain glycogen and mucous, but that doesn't explain why it did this type of chemoreceptor so well, and did not stain the other types at all. Does anyone have any clues? I'm an electron microscopist, so anyting that happens in color is beyond me!
Aloha, Tina Weatherby Carvalho Biological EM Facility University of Hawaii
Franklin RJM, Barnett SC, 1991. The electron microscopic appearance of the beta-galactosidase reaction product. Acta Neuropathol 81:686-687.
Engelhardt JF, Allen ED, Wilson JM, 1991. Reconstitution of tracheal grafts with a genetically modified epithelium. Proc Natl Acad Sci USA 88:11192-11196.
Bob Cardell, of the Department of Anatomy and Cell Biology at Univ of Cincinnati Med School has also published on this, but I can't find the reference.
Regards. A. Kent Christensen, Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor {akc-at-umich.edu}
--------------------------------
On Thu, 28 Jul 1994, Nestor J. Zaluzec-Argonne Nat. Lab. wrote:
} From: SMTP%"delannoy-at-welchlink.welch.jhu.edu" 25-JUL-1994 13:13:25.58 } To: MICROSCOPY } CC: } Subj: b-gal react. prod. EM } } Date: Mon, 25 Jul 94 14:13:53 EDT } From: delannoy-at-welchlink.welch.jhu.edu (MICHAEL DELANNOY ) } Message-Id: {9407251813.AA01300-at-welchlink.welch.jhu.edu.bubba} } To: microscopy-at-anlemc.msd.anl.gov } Subject: b-gal react. prod. EM } Cc: delannoy-at-welchlink.welch.jhu.edu } Mime-Version: 1.0 } Content-Type: text/plain; charset=us-ascii } Content-Length: 161 } } Does anyone have any experience detecting the beta galactosidase } reaction product at the EM level? } } Please respond to mwilson-at-welchlink.welch.jhu.edu. Thanks! }
1) It appears that the problems with the LISTSERVER have not been entirely sorted out, as I continue to recieve quite a lot of it at the moment. I don't know if this is a throw-back from the prevoius problems or something new.
2) Does anyone have any experience of stage control on optical microscopes linked to image analysis systems. We intend to purchase or develope a system to analyse cracks in material surfaces over an approximately 10x10mm area on a light microscope. This requires very accurate (preferably sub micron) stage positioning to link sets of video shots together into one large file. We had considered using ether an off-the-shelf system or building one based in NIH-Image. Any commentsor recommendations would be of help.
Thanks.
Dr Doug Arrell Mechanical Performance and Joining Institute for Advanced Materials 1755 ZG Petten Netherlands
As Diamond Lapping film seems to be the hot topic of the day, I thought it might be appropriate for me to give you all an update on the lapping film products from South Bay Technology.
It is important to note that the SBT films are not 3M films and that they are made through a different process. SBT films are considered to be a "closed coat" product whereas the 3M films are considered an "open coat" product. The differences between the products do not make one better or worse than the other - just different. If you have used a South Bay film and switch to a 3M, then you will notice a difference in the performance. Each type of film has a place in tripod polishing and in many other applications. On some materials a closed coat type film may work better than an open type film and vice versa.
To meet the varied needs of our customers, South Bay Technology plans to introduce a new line of "open coat" films that are designed as a direct replacement to 3M films (and at a much more reasonable price). We will continue to offer the "closed coat" film as we have many customers who are very pleased with this product and are reluctant to change. The new "open coat" product is not due out for several months. I normally would not make such a premature announcement, but the volume of diamond film postings here forced my hand. We currently have several beta sites lined up for testing of this film, but we would welcome requests from other people who would be interested in testing out the film as well.
I have seen several postings on this list server referring to the price and availability of these films. These films are expensive and when you need them - you need them. I'm the same as many of you - "if I needed the film tomorrow, I'd order it tomorrow". There are ways to minimize your cost and ensure that the film is available when you need it. At SBT we offer quantity discounts for as few as 25 pieces of film (assorted sizes). You can save money by prchasing a reasonable supply of film at one time rather than buying 2 or 3 pieces every 2 weeks. If it is not possible or desirable for you to stock the film yourelf, you can enter into a blanket or open order agreement.
A blanket order is an order for a specified group of items with a scheduled delivery. For example, you may want a total of 15 discs per month in 3 different sizes. While 15 discs doesn't qualify for a quantity discount, 15 discs x 12 months would qualify for a sizable discount if a blanket order was arranged. A blanket order offers the highest discounts as it helps us to plan our inventory more precisely. Also, with a blanket order, we maintain "reserved stock" just for you. This means that we always have the product available to ship - even if you decide to that you need it 3 weeks earlier than planned.
An open order is an order placed for a specific dollar amount, but without specific products or deliveries. You simply guarantee that you will spend x number of dollars within a specified period of time.
You may also want to consider purchasing all of your sample preparation supplies together. You can combine supplies from your met lab etc. to increase the size of your blanket or open order and therby increase your discount. As we do offer every type of sample prep supply, you can include much more than just your diamond films.
I realize that this has turned into a bit of an advertisement, but I felt that I had to say something to clarify the Diamond Film Debate. The concerns people seemed to have about diamond lapping films are those of price, performance and delivery. I hope I have provided some insight on these topics and made some valid sugestions that will enable you to save money and frustration. We are a small, family owned business that has been around for over 30 years. We've lasted that long by working very hard to offer our customers superior products and service at very reasonable prices. We take great pride in what we do and always strive to meet our goal of absolute customer satisfaction. We are always interested in hearing input from our customers (and those of you we haven't gotten to yet!). If any of you plan on being in New Orleans for the MSA/MAS Meeting, please stop by our booth (No. 534) and say hello. I'll be there myself and will look forward to meeting you. I encourage anyone who has any questions or comments to contact me directly as follows:
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
Mr-Received: by mta PETVAX.MUAS; Relayed; Fri, 29 Jul 1994 07:25:47 -0400 Mr-Received: by mta PETVAX; Relayed; Fri, 29 Jul 1994 07:25:47 -0400 Mr-Received: by mta SRVR01; Relayed; Fri, 29 Jul 1994 07:25:23 -0400 Disclose-Recipients: prohibited
Does anyone have experience embedding cell cultures plated out on your typical plastic multi-well plate (Falcon, Corning) using LR White as an embedding media? A 24 Hr Heat cure at 50C causes the plate to dissolve. We need to avoid catalytic cure for immunocytochemistry.
Walt Bobrowski Parke-Davis Research Ann Arbor, MI 48105
To all microscopists on the listserver: since there have been listserver problems over the last few weeks, I thought it would be useful to re-post this message, in case the original did not get through.
The following is an announcement for the INTERNATIONAL WORKSHOP ON ELECTRON HOLOGRAPHY. A circular for this meeting has recently been mailed to a selected mailing list, but the listserver may reach a number of people who may be interested and who were not included on our original list. I welcome all responses by e-mail (privately, of course). The workshop has an outstanding list of speakers, and, because of large private sponsorshop, will be very inexpensive for participants (in other words, a cheap ticket). Don't hesitate to reserve your spot; the attendance will be limited because of space limitations.
INTERNATIONAL WORKSHOP ON ELECTRON HOLOGRAPHY - Theory, Applications and Future Prospects -
August 29 -31, 1994 Holiday Inn World's Fair Knoxville, Tennessee, USA
Organized by
Exploratory Research for Advanced Technology (ERATO), Research Development Corporation of Japan (JRDC) Oak Ridge National Laboratory (ORNL) University of Bologna University of Tennessee
Purpose: This workshop offers an international forum for experts from all over the world to discuss recent developments in the techniques, theory and application of electron holography in materials and biological science research.
Organizing and Program Committee
A. Tonomura ......................................ERATO, JRDC and Hitachi Advanced Research Laboratory (HARL) L.F. Allard .....................................................Oak Ridge National Laboratory T.A. Nolan ......................................................Oak Ridge National Laboratory G. Pozzi ............................................................................University of Bologna D. C. Joy ......................................................................University of Tennessee Y. A. Ono ..........................................................................ERATO, JRDC and HARL
REGISTRATION: Free of charge (includes extended abstracts and hard-cover proceedings, to be published by Elsevier) ACTIVITY FEE: $100 (partial support for Sunday reception, 3 continental breakfasts, 2 buffet lunches, workshop banquet, box lunch and transportation to ORNL on Wednesday.) PROGRAM: The workshop will be comprised of technical sessions held over two and one-half days, with a tour of microscopy facilities and demonstrations of holography at ORNL scheduled for Wednesday afternoon. The workshop banquet, to be held at the Knoxville Museum of Art, will be highlighted by an after-dinner talk by T. Mulvey on Gabor and the early history of holography. The museum will be available entire evening for the workshop attendees. OFFICIAL LANGUAGE: English
Invited Speakers and Topics:
G. Ade (PTB, Braunschweig) .........................Digital recording and processing L. Allard (ORNL) .................................................................Holography of fullerenes J. Bonevich (NCEM,LBL) ............Observation of vortices in superconductors J. Chen (ERATO, JRDC) ........................................Real-time electron holography A. Datye (Univ. New Mexico)............................................Holography of catalysts V. Dravid (Northwestern Univ.) .......................Holography of electroceramics H. Fink (IBM Zurich) .......................................Electron point source microscopy B. Frost (ORNL)................................................Holography of electrostatic fields R. Herring (ERATO, JRDC) ...............................Diffracted beam interferometry T. Hirayama (ERATO, JRDC) ...........................Holography of magnetic domains K. Ishizuka (ERATO, JRDC) ........................Tilted single side-band holography D. Joy (Univ. Tennessee) ..................................New fields and future prospects G. Lai (ERATO, JRDC) .....................................Holographic computed tomography M. Lehmann (Univ. Tubingen) .................Holographic reconstruction methods H. Lichte (Univ. Tubingen) ........................High resolution electron holography M. Mankos (ASU) ..................................................................................STEM holography G. Matteucci (Univ. Bologna) ..............................Holography of magnetic fields M. McCartney (ASU) ....................................................Holography of p/n junctions T. Mulvey (Univ. Aston) ............................Early history of electron holography M. Op de Beek (Univ. Antwerp)..........................................Through-focus methods Q. Ru (ERATO, JRDC) .............................................Phase-shifting interferometry J. Spence (ASU) ......................................Projection microscopy and holography J. Steeds (Univ. Bristol) ..............................................Coherent beam diffraction T. Tanji (ERATO, JRDC) ...........................Atomic surface potential holography A. Tonomura (ERATO and HARL) ..................................ERATO and HARL research D. van Dyck (Univ. Antwerp) ....................Reconstructed image interpretation E. Volkl (ORNL) .....................................................Digital processing of holograms
Posters(tentative) K. Aoyama (ERATO, JRDC)................Holography of Thin biological filaments A. Carim (Penn State Univ.)...................................Holography of fine particles D. Joy (Univ. Tennessee) ........................Holography of ferroelectric domains T. Matsumoto (ERATO, JRDC).................Frozen-hydrated DNA super-helices Q. Ru (ERATO, JRDC) ..........................................Incoherent electron holography E. Volkl (ORNL)................................................................Extended Fourier analysis
Contributed Poster Papers
We have room for a few additional contributed papers which will be accepted as posters. Contributed papers will also be eligible for publication in the hardcover proceedings. Please e-mail your interest in submitting a poster to allardlfjr-at-ornl.gov and we will fax a circular with instructions to you. Any other queries about the workshop are also welcome.
Hi, I'm sectioning Spurr embedded (hard formulation) leaf tissue on an Ultracut using Diatech diamond knives (two different ones) and I've been encountering what appears to be a static problem. Every other section crumples and wrinkles badly. As the block approaches the knife the water in the boat can actually be seen to bulge or lift upward toward the descending block face. When the block touches the edge of the knife (water bulge?) the water attaction is instantly released, but the section appears almost to be cut almost under the surface. The section has water on top of it. The block face is quite small, { 0.5mm in both height and width; the edges are clean and smooth, the piece is oriented with its long axis perpendicular to the knife edge. I have tried adjusting: the speed of the cutting stroke, the clearance angle, the level of water in the boat, and have used two different knives. The instrument was recently serviced by a competent person and I have no reason to believe that the microtome is malfunctioning. I want to emphasize that this occurs regularly, every other section. I also tried cutting sections of different thickness, but the problem remained. I have tried touching a damp filter paper wedge to the block (not the face) during the return stroke and that seems to have a positive effect, but it is very awkward to do.
Any experience with a phenomenon like this? Any likely solution?
Thanks in advance!
Dwight U. Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
this message repeats an announcement mailed during the recent period of problems with the host computer
OPENING FOR ELECTRON OPTICS FACILITY ENGINEER The Department of Metallurgical and Materials Engineering at Michigan Technological University has an opening for an engineer who will be responsible for operation, maintenance, repair, and supervision of microanalytical instruments in its Electron Optics Facility. This facility includes several scanning electron microscopes, transmission electron microscopes, and an electron microprobe. Successful candidates will have training and experience in operation, maintenance and repair of similar instruments in a research setting. Duties may include collaboration with faculty and graduate students in preparation of research proposals, development of new microanalytical methods and publication of research. Interested applicants should send a resume , including names and addresses of three professional references, to the following address:
Chair, Electron Optics Facility Engineer Search Committee Department of Metallurgical and Materials Engineering Michigan Technological University 1400 Townsend Drive Houghton, MI 49931-1295
Salary range for this position is $34,261-$54,818/yr, starting salary will depend on experience and qualifications. The search committee will begin reviewing applications on August 1, 1994. Applications will be accepted until the position is filled.
Michigan Technological University is an equal opportunity employer/educational institution and welcomes applications from all qualified applicants.
It sounds like you should spit in your boat! Really, the surface tension of the water can be reduced by a tiny bit of saliva. I keep toothpicks in alcohol, and before using them put them in my mouth. That little bit of saliva when you use the stick to push the water to the edge of the knife is enough to make a difference. The only problem this might cause is epithelial cells which will be visible in your thick sections. Also be careful about lipstick, but from your name I would as same that probably shouldn't be a problem. If you don't like that idea, you can use Photoflo, diluted by about 1000 I think-anyway enough so you don't get soapsuds in your boat. Also-don't fill your boat too full. Just enough so you can push the water up to meet the section. Good luck. Joyce.
----Mail status follows---- Have been unable to send your mail to {gillen-at-[129.6.98.22]} for two days, will keep trying for another 24 hours. At that time your mail will be returned.
----Transcript of message follows----
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
} A student was staining some arthropod antennae with methylene blue for a } project. One day she couldn't find the stain, and so arbitrarily picked } another off the shelf (which contained a bunch of really antique } bottles). It was azocarmine and, fortunately, worked unbelievably well in } staining the structures we were looking for (aesthetascs) a nice pink! } Unfortunately, we don't know why. I can't find anything about } azocarmine itself. It sounds like carmine will stain glycogen and } mucous, but that doesn't explain why it did this type of chemoreceptor so } well, and did not stain the other types at all. Does anyone have any } clues? I'm an electron microscopist, so anyting that happens in color is } beyond me! } } Aloha, } Tina Weatherby Carvalho } Biological EM Facility } University of Hawaii
Thank God for ignorant students. There are two types of azocaramine (B & G). Gomori 1939 Anat. Rec. 74:439 & 1941 Am. J. Path. 17:395 used azocarmaine G and orange G to stain alpha, beta, and D cells in Islets of Langerhans. Mollier 1938 Z. Wissen. Mikr. 55:472 (in German) used azocaramine G in a quadruple stain for elastic tissue, muscle, collagen and epithelium. Lille 1965 Histopathologic Technic and Practical Histochemistry (McGraw Hill 3 ed) reports a use of azocaramine B, orange G and aniline blue to stain muscle, glia fibrils, collagen erythroctyes and nuclei. good luck.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Could anyone tell me how to mount grains of sodalite for analysis in an AFM? Or point me to some work in this field?
Also, does anyone know the, or a source of, the atomic structure of H-ZSM5, in coordinates ( For an EMS file.) I have some references with angles and lengths etc but not enough to be able to calculate the coordinates.
Subject: Time:8:38 AM OFFICE MEMO Re} Round -robin Images Date:7/29/94
I was not clear in my earlier message. I know where the Images are posted, but need to know where and when the images will be compared at the meeting in New Orleans. Thanks for your help.
Mike Mike_Schwartz-at-qm.yale.edu Fax 203-785-5263
Michael Schwartz, Ph.D. Section of Neurobiology Yale University School of Medicine 333 Cedar St. New Haven, CT 06510
} Does anyone know off the top of their head the electric field required } for room temperature field emission from a standard tungsten gun and what } type of force this exerts on the filament? } } Daniel L. Callahan } Department of Mechanical Engg. and Materials Science } Rice University } dlc-at-owlnet.rice.edu
Assuming the following 1) a fixed angular intensity of 0.1 mA/sr 2) radius of tip = 0.1microns 3) T = 300K 4) W {310} work fct 4.5eV
The field on the tip is ~5 V/nm
A good reference is: "Point Cathodes for Use in Virtual Electron Optics," Tuggle D.W. Journal of Microscopy, Vol 140, December 1985.
For anyone who is interested, FEI has an extensive list of technical articles relating to thermal and field emission, which is available upon request.
Best regards, Damon L. Heer
FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830
----Reason for mail failure follows---- Sending mail to {gillen-at-[129.6.98.22]} : Could not be delivered for three days.
----Transcript of message follows----
Return-Path: {MICROARCHIVE-at-ANLEMC.MSD.ANL.GOV} Received: from ANLEMC.MSD.ANL.GOV by gapnet.nist.gov with SMTP ; Wed, 27 Jul 94 10:10:28 EST
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
----- Transcript of session follows ----- pop: not running setuid to pop 451 {l.s.smith-at-diana.met.bham.ac.uk} ... Operating system error
----- Unsent message follows ----- Received: from cuchulain by diana.met.bham.ac.uk; Wed, 27 Jul 1994 16:30:22 +0100 Received: from bham.ac.uk by met.bham.ac.uk; Wed, 27 Jul 1994 15:30:26 +0100 Received: from anlemc.msd.anl.gov by bham.ac.uk with SMTP (PP) id {26371-0-at-bham.ac.uk} ; Wed, 27 Jul 1994 15:30:12 +0100
G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
Dear Microscopists, I am helping a student set up a cryofixation (plunge or slam on a KF80) and cryosubstitution project on blades of rye grass. She hopes to immunolabel for CI proteins in the blade. This protein is a Poty virus-induced protein. Our problem is that we can't find much published material suggesting the best substitution medium, best fixative and recommended Lowicryl to use. There doesn't seem to be much botanical cryofixation/crysubstitution literature out there. Can anyone offer some suggestions?
Allan Mitchell Technical Officer South Campus EM Unit Dept of Anantomy and Structural Biology Otago Medical School NZ
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
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