NetNotes
calcium as black precipitate, but concentrated in mitochondria and not diffused. But I don’t remember the paper, nor the author, and I am not sure how accurate is that identification. Josif Mircheski
jmircheski@us.es Tu May 12 Te dreaded black dot plague! I’ve struggled with this precipitate
here in Tampa for years. It seems that our groundwater in Florida is rich in phosphates, and that the Millipore and similar filtration units don’t always take all of the phosphates out of the water when the systems filter the water. Tis results in phosphate microcrystal precipitates in tissue processed for TEM if I use filtered water. When I switched to true distilled water, this issue resolved itself. I now use true distilled water to make my reagents in, and for my rinses. I work with phosphate buffered fixatives, which preserve the cytosol of mammalian tissue better. Aſter glutaraldehyde fixation, I do a half hour buffer rinse, 3 × 10 minutes each, minimum, before osmication, and even longer if I am processing tissue such as myelinated nerve, in which case I rinse overnight in buffer. Aſter osmication, I rinse in distilled water, again 3 × 10 minutes each, minimum, and longer if the tissue is thick or dense, to remove both the osmium and the phosphate salts. Tis has cured the precipitate problems for me. Some advice I gave another E.M. technician was to rinse in Tris buffer aſter glutaraldehyde and osmicate in Tris. Te two buffer systems are compatible. You could consider this, also. Let me know what you find out. If you switch to cacodylate buffer, you should not get this precipitate, if phosphate salts are the culprit. I don’t know if this is a possibility for you. I don’t like the look of my tissue when working with cacodylate buffer, and the arsenic in the buffer is something I try to avoid. Ed Haller
ehaller@health.usf. edu Tu May 12 Electron microscopy and electron probe analysis of
mitochondrial cation accumulation in smooth muscle. Somlyo et al.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2109306/pdf/723. pdf. Perhaps this? Frederick C. Monson
fmonson@wcupa.edu Tu May 12
Specimen Preparation: microtoming material specimens I have a student who wants to microtome some materials samples.
Te samples are GaSb and Si wafers. Te length and the width are less than 1 cm each. Te thickness, which is the most important, is less than 0.5 mm for GaSb and 1 mm for Si. Te structures are on top of the support surfaces (same material). Te GaSb samples are really not very hard. My concern is that these are students who have never touched a microtome. Tey need advise as to type of microtome knife to purchase and any other hints you have for success with these samples. Will it be necessary to embed them in resin (which one?) to stabilize the samples in the chuck? Most likely the resin will peel away from the cut sections but that is not a problem. I will teach them using a resin block and glass knives so that they get the idea but that is a long way from success with their samples. Tey are hoping for thicknesses of 100–300 nm and will be imaging them with a 300 kV FEI Titan TEM. FIB is not an option with these samples as the ion beam destroys the detail of interest. Debby Sherman dsherman@
purdue.edu Mon Jun 13 It has been done with semiconductors, but the results give you
a bunch of cleaved stuff that doesn’t look great. If they are looking for a relatively straightforward process for making TEM samples that doesn’t require very expensive equipment, I highly recommend the MicroCleave(TM) technique, also known as the Small Angle Cleavage Technique. Tis technique is fairly easy to learn and it will make absolutely fantastic samples with these types of materials. Here
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are several links to application notes on our website. We also have a video on how to do the technique that we can send out. http://
southbaytech.com/appnotes/62%20The%20Small%20Angle%20 Cleavage%20Technique%20An%20Update.pdf. http://southbaytech. com/appnotes/61%20SACT%20Prepared%20MBE%20QWIP%20 Structure.pdf.
http://southbaytech.com/appnotes/59%20EELS%20of %20PLD%20DLC.pdf.
http://southbaytech.com/appnotes/55%20GaN Sapphire%20Prepared%20by%20SACT.pdf.
http://southbaytech.com/ appnotes/60%20PreThinning%20for%20FIB%20TEM%20 Sample%20Preparation%20Using%20the%20Small%20Angle%20 Cleavage%20Technique.pdf. Te first of these files have references for the technique. If you like, have your students give me a call and I will talk to them about the technique. It is a much better approach for these materials than microtoming. For about the cost of a knife (or less), they could buy the equipment and supplies to do it or they could purchase our MicroCleaveTM kit. Disclaimer: South Bay Technology manufactures and sells the MicroCleaveTM Kit Model 520. Scott D. Walck
swalck@southbaytech.com Mon Jun 13
SEM: cross section study I am a relative newcomer to the microscopy field and need some
advice for the sample preparation for cross section study of fiber material. Tis kind of film material (polyethylene, polypropylene etc.) has pores structure and orientation. Te thickness is around 30–50 microns. It is too tough to break in liquid nitrogen. We had even tried to cut it in liquid nitrogen by a blade. But the pores structure is some distorted. I found cryostat microtomy doesn’t work for our case. Is there any trick for this? I got some information from this website before. I still have some questions about it. “(1). Insert the material in a small diameter tube (thin drinking straws are ideal). Cut the straw down to about 3 cm tall. Block one end with wax, modeling clay or similar material.” Is wax, modeling clay or similar material only used to block end? Is it also used to fill in the big gap between straw and film materials? “(2) Using a syringe, force water into the straw and block the end as above.” Is water filled in the big gap between straw and film? “(3) Drop the straw into liquid nitrogen then follow method A part 2 above.” Should we cut the sample by blade or break it by tweezers? Yun Peng
yun.peng@
ge.com Tue Jun 7 I wrote the paper that you discuss so let me try to help you.
Te basic idea is a media stiffens the fiber and forces it to fracture. Firstly to answer your questions: (1) Te wax is simply to block one end. (2) Water may be used to provide a solid interface that will fracture cleanly. (3) Use strong tweezers or grippers (in the UK we would say pliers) to bend the straw unit until fracture occurs. Or (A) Infiltrate the fibers within the drinking with straw a water soluble carbon solution. (B) Once fully infiltrated block one end with wax as above; allow to dry fully (hours). (C) Place in liquid nitrogen until it stops bubbling. (D) Remove and with two strong grippers (UK we would say pliers) bend the straw unit to fracture. We have many times conducted investigation of fiber internal structure by using this method. Steve Chapman
protrain@emcourses.com Tue Jun 7
I don’t have any comments about the methods you list below,
but I would be inclined to try a typical metallurgical cross-section. Mount the sample in a material with similar hardness (epoxy may work fine), and grind and polish to achieve your cross-section. Tere should be plenty of facilities and people at GE-GRC that are familiar with metallurgical sectioning. Diane Ciaburri
diane.ciaburri@gd-ais. com Tue Jun 7
www.microscopy-today.com • 2011 November
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