NetNotes
Specimen Preparation: chitin
Can someone lead me to a good set of protocols for fi xing chitin? My biology friends tell me that conventional methods for fi xing chitin oſt en fail to adhere the plastic to the fi bers. I am doing an analytical study of the inorganic components in the chitin so I don’t want to use any staining and I want to preserve the integrity of the Ca phases present. T anks from a materials oriented microscopist! Ken Livi
klivi@jhu.edu Wed Sep 16
I work on cell walls of plants not fungi. But I believe the issue is related. From my reading of the literature (and this is far from complete), classical chemical crosslinking fi xatives, like formaldehyde and glutaraldehyde, react to only a limited extent with cell wall polymers. I have always used fi xatives when I want to keep the tissue or cytoplasm in good shape. But the wall itself I think does not need fi xation (except perhaps to inactivate cell wall degrading enzymes that live out there). Cell walls are tough and can withstand typical dehydration / embedding schedules without too much trouble. You wrote about plastic adhering to the chitin. T is seems like more of an infi ltrating/embedding issue. An old trick that works to embed certain tricky plant samples is to start infi ltrating with really low concentra- tions of your plastic, like 1% then 2%, 5%, 10% and then as normal. Tobias Baskin
baskin@bio.umass.edu Wed Sep 16
Any chitin in particular? Arthropod cuticle, fungus, or somebody
else? T is is a ubiquitous polysaccharide, but not necessarily identical. Especially for minerals, like Ca. And: Your question implies you’re wanting to do TEM, but perhaps SEM would be better? If you’re not interested in the ultrastructure of biological components (like cells), you could let the critter dry, then cryofracture in liquid nitrogen. T is would expose the internal structure of the chitin and contained mineralized phases. If you need a polished surface for x-ray spectroscopy or EBSD, then this wouldn’t work. Phil Oshel
oshel1pe@cmich.edu Wed Sep 16 I think that (at least some kinds of) chitin have a waxy coating. If that is true of your chitin, Ken, and if the wax structure is part of what you’re interested in—it may contain some Ca—any additive that could dissolve the wax will be problematic. Perhaps Phil’s suggestion of SEM to characterize the wax (if any), then prep for TEM would be best. Can chitin be placed in the SEM without treatment and/or coating? If so, then I’d defi nitely try it. Bill Tivol wtivol@sbcglobal. net Wed Sep 16
Waxy epicuticles are common in insects and terrestrial arthropods, but that’s about it. Not part of the chitin per se. And any wax has an unfortunate tendency to melt under the beam. Cryomethods are needed to study it - and the wax on plant leaves. T at said, waxy coats on critters and plants are very poorly studied and I’m sure have more uses than just slowing or stopping dessication. Phil Oshel oshel1pe@
cmich.edu T u Sep 17
Specimen Preparation: SEM of leather
I’m looking for suggestions on how to prepare leather for SEM viewing; tanned & untanned, tanned is worse for charging. Currently I slice a cross section by hand as thin as possible. Mount with a small dab of Duco cement, and silver paint the edges. (Tried mounting with a dab of silver paint- not much improvement). T e corium takes gold coating reasonably well but the grain seems to be resistant to all but long sputter coating. I get charging at higher magnifi cations. Would a diff erent metal coating do better? Perfuse some conductive material into the sample? Although that might disturb structure. Joe Uknalis
joseph.uknalis@
ars.usda.gov Mon Oct 5
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My fi rst question would be what accelerating voltage are you using, and what spot size when compared with your normal operating conditions? With this information we may move forward. Steve Chapman
protrain@emcourses.com Tue Oct 6 T e Duco cement isn’t helping you—you’re mounting with an insulator. I would use a conductive carbon tab/tape and silver paint around the edges. T en sputter coat, using 60/40 gold/palladium. Cheaper than pure gold and it gives a better (fi ner grain coat). More importantly, what kV and spot size are you using? Reducing both will go a long way to reducing or eliminating charging. T is may be all you need to do. You could also try adding osmium vapor “fi xation”. Place the sample in a petri dish and a crystal of OsO 4 , or a drop of 2-4% OsO 4 in water in a smaller dish, so the OsO 4 doesn’t contact the sample. Don’t cover the same dish with the OsO 4 but cover and Parafi lm sealed the big dish. Let sit at room temperature overnight to 24 hrs. T e OsO 4 doesn’t bind that well to proteins, but it will still bind. Phil Oshel
oshel1pe@cmich.edu Tue Oct 6 I was using 10 kV spot 3 on a FEI 200 FEG I dropped to 5 kV but the charging got worse? Unfortunately I don’t have a carbon coating option. Low vacuum/ESEM has poor resolution at 50,000×. Will try the osmium vapor. Gold/palladium is more $$ than gold alone, may give that a try. Will try diff erent spot sizes. Joe Uknalis
joseph.uknalis@
ars.usda.gov Tue Oct 6
I would start at about 800 V and work my way up. I try to get to the point where the electrons in (beam) are close to the electrons out (SEI, BSE, etc.), and I have the best resolution I can, with minimal charging. With the FEG, I think you will be able to get the mag you want, though I am not familiar with your SEM. I start at low kV and work my way up. If you start high and go down, you will never see that area as best as you could have. Darrell Miles
milesd@us.ibm. com Tue Oct 6
Sounds as if you are trying, but if the charge is worse with dropping
to 5 kV that tells me you have lost the BSE contribution, this will be a big problem when using a through the lens (TTL) system. T e converted BSE provide SE, but because they are derived from BSE they do not carry so much charge information T e approach to a problem specimen should be as follows - 1. Use the smallest possible piece of material, fi xed to the stub with a good quality conducting media. 2. Start at the lowest kV that you are able to work with, and a small spot size; all aimed at not damaging or charging the specimen. If you use a TTL system go to the standard detector in the lower chamber, with about 7 mm working distance. You need a contribution of converted BSE (less charge) which you are unlikely to have with TTL detection. If the signal is too low, slightly increase the spot size. 3. If the specimen is not giving you a problem other than resolution, increase the magnifi cation using a fast scan if possible, to reduce the intensity of beam on a particular area. 4. Having increased the magnifi cation, as resolution is the problem, increase the kV slightly (e.g. if you were using 1 kV go to 1.5 kV). 5. As long as there is no charge continue stepping up the kV until you are happy with the result or the specimen charges. If you charge the specimen take it to air to discharge it, and go back to an earlier kV. Slight charge may be reduced by reducing the spot size slightly. 6. If resolution is still the problem, try moving the specimen closer to the fi nal lens by a few mm, not too close as you will lose the very valuable converted BSE. 7. Still a resolution problem, if you have a TTL system try 10 mm working distance (WD) then in steps down to 5 mm WD with this system, and see if you are able to obtain a satisfactory result (with many TTL systems between 7mm and 5mm works). 8. All of the time you are looking for a mix of kV, spot size, and WD that provides the best mix of signal and therefore performance. Steve Chapman
protrain@emcourses.com Wed Oct 7
www.microscopy-today.com • 2016 January
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