The chemical is hexamethyldisilazane, also known as HMDS and several other names. There's a Wikipedia article, of course: https://en.wikipedia.org/wiki/Bis(trimethylsilyl)amine
I first encountered the chemical in a paper titled "An in-depth description of head morphology and mouthparts in larvae of the black soldier fly Hermetia illucens", Daniele Bruno et.al, Arthropod Structure & Development 58 (2020) 100969, currently available at https://www.entomofago.eu/wp-content/up ... hparts.pdf . The relevant paragraph there says:
For our purposes here, the most important thing about this paragraph is what it does not mention: critical point drying.2.4. Scanning electron microscopy (SEM)
For SEM analyses, all the samples were fixed with 4% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) for 1 h at room temperature. After 5 washes in Na-cacodylate buffer, they were postfixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 1 h in the dark. After 5 washes in Na-cacodylate buffer, samples were dehydrated in an increasing series of ethanol and washed twice (8 min each) with hexamethyldisilazane, promoting the drying of tissues. Dried specimens were mounted on stubs, gold-coated with a Sputter K250 coater, and then observed with a SEM-FEG XL-30 microscope (Philips, Amsterdam, The Netherlands).
Let me digress for a moment. The standard method for preparing highest quality SEM specimens is critical point drying, or CPD. CPD works because of an odd quirk of physics. We are all familiar with solid, liquid, vapor phases and the transitions between those: solid to liquid is melting, solid to vapor is sublimation, and liquid to vapor is evaporation. The odd quirk is that at temperatures and pressures above a "critical point", the liquid and gas phases merge into a single regime called "supercritical fluid". A supercritical fluid can have any density between liquid and vapor, and it will never separate into high- and low-density phases with a discrete boundary between them. Instead, a supercritical fluid simply expands to uniformly fill whatever space is available to it. So, by following a temperature/pressure trajectory that loops around the high side of the critical point, you can go from a high density liquid to a low density vapor without ever forming a liquid/vapor surface. With no surface, there is also no surface tension, and that's the key aspect. With critical point drying, you can go from a specimen that is totally wet and immersed in liquid, to one that is totally dry and surrounded by vapor, without ever subjecting it to the surface tension forces that would occur with normal evaporation. The result is similar to freeze drying, with the additional advantage that the specimen is not exposed to damage by crystal growth during freezing.
The one downside to critical point drying is expense. A significant piece of equipment, initially costing $10K and up, is needed to control the fluid flow and pressures involved. Even using carbon dioxide, which goes supercritical at a fairly low point, the dryer has to safely handle pressures over 1070 psi, about 73 atmospheres, in a chamber that can be opened for specimen insertion and removal.
Because of equipment cost for critical point drying, some alternative method is often desired. Freeze drying can work, but requires significant vacuum to work quickly, and the freezing process can damage specimens. For a while, a fluorocarbon tradenamed "Peldri" was used as a sublimation agent (heat to liquid, saturate subject, cool to solid, allow to sublimate), but that apparently became unavailable a couple of decades ago. The remaining alternative is some liquid that can manage to evaporate without tugging hard enough on the subject to cause problems.
Hexamethyldisilazane is one of those liquids. It does not work well for all subjects, but it has been tested favorably against CPD for a wide range of animal tissues and some plant tissues.
A freely available reference is https://phorid.net/hmds.php , (Brown, B.V. 1993. A further chemical alternative to critical-point-drying for preparing small (or large) flies. Fly Times. 11: 10). It concludes that "Specimens come out exactly like CPD-prepared specimens, ready for SEM or for general mounting for the collection." The images in the paper look great -- I would be hard pressed to tell that his specimen dried with HMDS is not fresh.
One of the seminal papers in this area seems to be https://analyticalsciencejournals.onlin ... 1070260603 (Comparison of hexamethyldisilazane (HMDS), Peldri II, and critical-point drying methods for scanning electron microscopy of biological specimens", D. F. Bray,J. Bagu,P. Koegler, First published: 15 December 1993 https://doi.org/10.1002/jemt.1070260603 ). The freely available abstract says:
I paid for short-term access to the full paper. That mostly led me to conclude that the abstract is a great summary of the full paper.Abstract
Three different drying methods, critical-point drying (CPD), Peldri II, and hexamethyldisilazane (HMDS), were compared using representative animal (rat kidney, trachea, duodenum, lung, and red blood cells) and plant (leaves from ten species of monocotyledons and dicotyledons) specimens. All three drying methods produced identical results with animal specimens. Plant specimens showed signs of shrinkage regardless of which drying method was employed. The order of preservation quality from best to worst for leaves was CPD > Peldri II > HMDS, with the CPD method providing substantially better results in all but one case. Postfixation of leaves with osmium tetroxide resulted in poorer preservation in all instances. Peldri II caused complete extraction of leaf cuticular wax, while both both CPD and HMDS showed minimal extraction compared with samples air dried directly from acetone. These results indicate that HMDS provides a time-saving and inexpensive alternative to CPD for animal specimens. Plant specimens, particularly those containing cells with large central vacuoles, are adequately preserved only with the CPD method. In addition, postfixation with osmium should be avoided when processing plant specimens for scanning electron microscopy. © 1993 Wiley-Liss, Inc.
However, there was one additional snippet that definitely caught my attention:
It's also worth noting that Bray et.al. report that for their rat samples, "specimens dried directly from acetone showed severe shrinkage, which was visible to the naked eye as well as under the microscope". But in contrast, my black soldier fly larvae mouthparts came out quite nicely from just acetone, even skipping all the fixation processes described by Bruno et.al.The mechanism of action of HMDS is not known. It is a reagent commonly used in gas chromatography for making silyl ethers of compounds with one or more reactive compounds such as sugars, amino acids, and alcohols (Nation, 1983). The combined properties of low surface tension and cross-linking potential are likely to be important factors in the suitability of HMDS as a drying agent for animal specimens.
So, at this point it's totally unclear to me when the HMDS really is and is not required. Since I now have a bottle of the stuff, I'll probably use it often. On the other hand, at a total cost of $115.44 for 250 ml (from https://www.polysciences.com, including tax and shipping), I'm not inclined to tell other people "This stuff is magic, you gotta try it!"
Adding some visual description of what HMDS does, here is a series of three photos showing what happens when a thoroughly dry split body of Noctua pronuba is saturated with acetone alone for one half, HMDS alone for the other half, and just allowed to air dry:
It's very clear that when wet, both fluids cause the long scales to collapse down tight against the body. In other words, "low surface tension" is not nearly low enough to keep the scales from moving. On the other hand, after both fluids have evaporated, the scales treated with HMDS recover much more of their original loft. I do not know why this is, but I repeated the experiment for a couple of other moth specimens, and got the same results.
So, there is something about HMDS that causes it to work nicely as a drying agent for both rat samples (fixed proteins and lipids) and for moth scales (bare chitin on the surface), but not for plant samples of the type tested by Bray et.al.
Meanwhile, on yet another hand, there are other reports that HMDS also works nicely for pollen. From https://www.tandfonline.com/doi/abs/10. ... 9409106286
Hexamethyldisilazane as a Drying Agent for Pollen Scanning Electron Microscopy
William F. Chissoe,Edward L. Vezey &John J. Skvarla
Pages 192-198 | Published online: 12 Jul 2009
https://doi.org/10.3109/10520299409106286
Abstract
Use of hexamethyldisilazane (HMDS) as a final dehydrating solution provides robust, undistorted secondary electron images of a variety of angiosperm and gymnosperm pollen grains, including those considered to be susceptible to collapse in the scanning electron microscope. Ease of handling, low cost, lack of specialized equipment, minimal expenditure of time, and high rate of success are factors that favor HMDS over other drying agents for preparing pollen grains for scanning' electron microscopy.
I wish I could be more definitive, but it seems that this is a case of "your mileage may vary!"
If anybody has other references or experiences, please share.
--Rik
