NetNotes
Edited by Thomas E. Phillips University of Missouri
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from March 1, 2015 to April 30, 2015. Complete listings and subscription information can be obtained at
http://www.microscopy.com . Postings may have been edited to conserve space or for clarity.
Specimen Preparation: critical point drying of hydrogel
I have a thin membrane that contains a hydrogel that has pores estimated to be 100-200 nm. I would like to visualize the pore structure on SEM and I heard that critical point drying is a suitable method for preparing my sample. I wondered if my sample prep can be as easy as exchanging the water in my hydrogel with ethanol, placing the sample directly with ethanol in the critical point dryer, and dry the sample. If I want a cross-sectional view of the membrane, is it suffi cient to cut the membrane while it is soaked in ethanol? Or are there other precau- tions I should take into account? Also, is there any recommendation for how much gold I can sputter on the dried sample? 50 nm of gold would already infl uence my pore size signifi cantly and I don’t know if gold gets deposited uniformly everywhere in the gel. Daan Witters dwitters@
caltech.edu Tue Apr 28
Others can probably comment better than I can about the entire plan, but there are a couple items I would address immediately. First, I would be careful about cutting the membrane to reveal a cross section. I have clients cut samples to see the structure of a 300 nm layer on the surface. Even when using a fresh razor blade, they are surprised at the amount of damage leſt behind. It is ugly at 15k×. Your situation could be the same. However, if you are not looking at the surface but at a rather thick core area, simple cutting might work for you. We used to apply 15 nm of gold with our old coater. T at is terribly thick by today’s standards. We now oſt en coat with as little as 2 nm of iridium. I would worry if you would still have charging due to the porous nature of your material. You probably want small volumes with something nearby to conduct away the charge. You might also want to look into variable pressure SEM to help mitigate charging. Warren Straszheim
wesaia@iastate.edu Tue Apr 28
You might have to resort to cryo-SEM of uncoated, properly frozen samples, if you really want to get the real answer. Tricky, though, for the unexperienced. You need to have the right cryo-SEM and experience. Reinhard Rachel
reinhard.rachel@biologie.uni-regensburg.de Tue Apr 28 CryoSEM is a good idea. With 200-300 nm pore size, you should be able to freeze the hydrogel onto a stub with little or no ice crystal formation within the membrane itself, then place on cryostage in the SEM and carefully etch away the ice. We’ve done this quite a bit with plant material, and plant cell walls are essentially hydrogels though with much smaller pores. You’d need to be able to do well-controlled coating in the cryo-transfer unit, which the modern instruments should be capable of. Soaking the hydrogel in something conductive before freezing would help too and may allow you to image the membrane uncoated. Rosemary White
rosemary.white@csiro.au Tue Apr 28 T e question really is: do you need an analysis of the structure of the pore or do you just need to determine their size accurately? If you “only” need the size, I would recommend other techniques than
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EM, because hydration is probably critical as it was already mentioned. Perhaps you could consider for example atomic force microscopy (AFM), which can be performed in liquid? Stephane Nizets nizets2@
yahoo.com Wed Apr 29
Specimen Preparation: osmium problem
I just had a big problem with the osmium fi xative I used (2% in 0.1 M cacodylate buff er) turning a purplish black aſt er 3 hours of fi xation. T e tissue was slimy and basically ruined. Any ideas of what could have gone wrong? T e osmium was perfectly clear and slightly yellow as always when I made the solution. I also used the same type vials and buff er that I used before. What kind of solutions could cause this reaction? JoAnn Buchanan
buchsmith@gmail.com Wed Apr 29 T e most common causes of osmium turning color are because there is still glutaraldehyde in the sample or at the top of the tube. It mixes with the osmium and oxidation occurs. I buff er wash 3 times 20 minutes and make sure that I rinse the whole vial and inside of the cap then I wipe the top of the vial to dry it. T e extended wash time is used to stop “peppering” of mitochondria and other dense structures. T e osmium fi xative is in cacodylate buff er, as you use, but at 1%. Several microscopists that I know do use 2% osmium. I have never gone much over an hour and do not understand the need for 3 hours as you mention. I had learned that osmium penetrates into a sample about 0.5 mm in an hour then it starts to block itself from going deeper into a tissue sample. T at is why we keep the size of all samples to 1 mm cubic or smaller. If a larger sample is cut in two aſt er an hour one can see that it is white in the center and hence the osmium did not reach that point to do its work as a fi xative. I had a sample back in 2007 or 2008 that had a huge amount of fat in it. I saw that the osmium did start to turn within the hour. I assume that the glutaraldehyde was not washed out completely, for back then I buff er washed only a half hour. Pat Connelly
connellyps@nhlbi.nih.gov Wed Apr 29
Specimen Preparation: hair samples
We are trying to preserve newborn hair samples by HPF/AFS for TEM analysis. We are having the problem that we cannot section the hair samples because the sample keeps popping out of the Epon blocks. I would appreciate learning how you did your infi ltration, what resin you used and if you have any tricks to overcoming the problem of small samples popping out of blocks. Erin Tranfi eld etranfi
eld@igc.gulbenkian.pt T u Mar 12 Hair and other keratin fi bers are not easy tissues and must be
treated diff erently than normal tissue samples. However, what method you need depends on if you are looking at the hair above the skin only, or if you are also looking at the hair follicle. For the moment I’ll assume you are interested in only the hair above the skin. Hair is already fi xed by nature. It is also very dense. T e water content is very low, and the cells are dead. It is the opposite of normal living cells in terms of the
doi: 10.1017/S1551929515000528
www.microscopy-today.com • 2015 July
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