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No, that title is not a swear word,nor (heaven forbid) an abbrevia-tion. In fact it is the name of a

chemical I have been using quite a lotrecently.When a specimen is imaged in the SEM itneeds to be thoroughly dry, at least for thehigh vacuum mode of operation, or anyresidual water vapour will interfere withthe electron beam. It is common to displaceany water during preparation with a vola-tile organic solvent such as acetone oralcohol, but when that solvent evaporatesfrom the specimen the resulting surfacetension can significantly deform its shape.There are two ways round this problem.One is to use a “Critical Point Dryer”(CPD) as described in the first issue ofSEM Diaries. This involves displacing theorganic solvent with liquid CO2 in a pres-sure vessel, and raising its pressure and

temperature to the “critical point” whichis where the properties of liquid andgaseous CO2 are identical (around 1050p.s.i. and 31.5 C). A simpler alternative fornon-critical applications is to use anorganic solvent that has a relatively lowsurface tension. This is where hexameth-yldisilazane (henceforth referred to asHMDS) comes in.The advantage of using HMDS is that it isrelatively quick and simple. Among itsdisadvantages are its cost, at about £300per litre, and its smell, which is not pleas-ant!My protocol is to pass the specimenthrough a number of baths of iso-propylalcohol and then finally through a bath ofHMDS, before removing the specimenfrom the bath and leaving the HMDS toevaporate at room temperature. In theoryit is best to use increasing concentrations

SEM Diaries - 16HEXAMETHYLDISILAZANE

Jeremy Poole

Fig. 1: Small container of Hexamethyldisilazane and stainless steel mesh “saucer”

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of alcohol through the bath sequence, butsince my specimens are quite robust I justuse 100% in each of my four baths. Obvi-ously each bath becomes slightly dilutedas it removes water from the specimen. (Imentioned my process in passing to DavidSpears when I last saw him, and I wassurprised but gratified to learn that hehimself likes to use HMDS in preferenceto his CPD when he can.)So, why all this activity with HMDS? Well,since I advised readers of the launch of mynew “spider” website in the last issue ofSEM Diaries I have contributed an articleon it to the British Arachnological Society.I am keen to get electron micrographs ofas many species as I can before the nextissue of their Newsletter hits the door-mats. Although it has not exactly been thebest season for collecting spiders I havemanaged to raid my preserved specimensand added a few more species to my collec-tion of micrographs, and by the time youread this, hopefully to my website as well.In preparing my specimens for mountingon stubs, I have two choices. I can eitherdesiccate the whole spider and then dissectout the desired parts, or I can carry out thedissection under water or dilute alcoholand desiccate the resulting anatomicalfeatures. These days I normally use thislatter order since it reduces the chance ofparts flying off when they are cut, as hasbeen described in SEM Diaries - 7.Each spider should yield up about 13 partsof interest. These are: 8 legs, 2 pedipalps(male or female), spinnerets, head withchelicerae (or alternatively just the cheli-creae) and epigyne (females only). Theseare dissected in the alcohol in which theywere stored and then placed in the firstbath of neat alcohol.Since I use five baths (including theHMDS) the idea of transferring each ofthese fragile parts individually betweenbaths using tweezers is not very appealing,not least because of the high likelihoodthat some part or other might be damaged

by the tweezers at some stage during theroutine, or even mislaid.Taking a cue from the late great EricMarson, of Northern Biological Suppliesfame, I decided that what was needed wasa container with a perforated bottom thatcould be moved between baths withouthaving to handle the specimens at all.While Eric used glass tubes with stockingmaterial lashed to the bottom of them, Imanaged to find some fine stainless steelmesh material which could be cut up andfolded to make saucer-like containers(grids) that fitted the small (5ml) plasticpots that I use for my baths. A grid,together with a pot containing anothergrid, are illustrated on the frontispiece(Figure 1).Now, one of my favourite spiders is theLinythiid Labulla thoracica (Figure 2). Ilike it for no other reason than that thesexual organs of the male are so totally outof proportion to the rest of the spider. Thebusiness end of its pedipalp is around 1.5mm across its widest part, and this is fora spider whose overall length (head tospinnerets) is about 5 mm. One only hasto view that in a human context to realisehow impressive that is (if you areimpressed by that sort of thing!).The structure of the pedipalps resemblesa flat disc, with a “hose pipe” coiled aroundit. Ironically, it took me quite a while tofind this in the Field Guide [1], as the view

Fig. 2: The impressive pedipalps on the maleLabulla thoracica (arrowed)

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in that volume is edge on, and thereforeless impressive. As part of my recentcapturing of images of this creature, I tookmicrographs from two different angles,which are reproduced in Figure 3. For mywebsite I like to black out any distractingbackground. I am not looking forward todoing this (if I actually attempt it at all)on account of the myriad hairs around thesubject in both views.

EM-UKI was able to attend “EM-UK” at WarwickUniversity in early January 2019. This isan annual event, organised by the RoyalMicroscopical Society, which bringstogether members of the electron micros-copy community in the UK (both academ-ics and suppliers) to discuss matters ofcommon interest.I was possibly a bit out of place at ameeting aimed at academics, although itmade me nostalgic for the days when Icarried out university research! It did,however, give me the opportunity to catchup with two of my suppliers, including thevendor of the “dodgy” filaments that Idescribed in SEM Diaries - 14.

While many of the lectures were of generalinterest to me, two stood out to grab myattention. The first was entitled “UnusualSample Preparation for TEM”. In this thelecturer described an exercise he carriedout with two 17 year old work experiencestudents. The object of the exercise was tocollect water samples, stain them (withuranium salts) and image them on atransmission electron microscope. A widevariety of viruses were discovered, even inthe drip tray from the water cooler in theJunior Common Room. This exercise wasused as the basis for a “Citizen Science”project, called “Virus Hunters”, whichinvited schoolchildren to send in watersamples and to help analyse the imagesobtained from a transmission electronmicroscope [2].The second lecture was on the status oftechnicians at universities and an initia-tive being undertaken to enhance therespect in which they are held. Whilst myrecollections from school days was that thelab technician was the person who toppedup the reagent bottles in the chemistry laband washed up the beakers and pipettes,the role of a technician in a university or

Fig. 3: Two views of male pedipalp of Labulla thoracica. On the left is the left pedipalp viewedfrom the side, as is shown in the references. The right hand micrograph is of the right pedipalp

viewed from underneath. Scale bar 500µm.

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research laboratory (or even schools) thesedays is much more skilled.The problem is that the original stereotypestill exists, and is reflected in salaries andjob progression opportunities. In reality,someone in charge of an electron micros-copy laboratory, who might well be classedas a technician, will almost certainly be agraduate or of graduate level, and knowmuch more about the instruments in hisor her charge than the people they reportto. Similarly, they would be perfectlycapable of carrying out research to PhDlevel and possibly beyond.An initiative called “The Technician Com-mittment” [3] has been set up to tacklethese issues in four key areas:● Visibility - within their organisation● Recognition - professional registra-

tion● Career Development - through a

defined pathway● Sustainability - to ensure a flow of

new recruits to provide requiredskills

Nearer HomeIn SEM Diaries - 13 I mentioned that Inow use numbered laser-engraved stubson which to mount my specimens, to aidtraceability and identification. I also men-tioned that on delivery my 1000 stubsrequired to be sorted manually into

numerical order. Well, in January 2019 Iplaced one of my compartmentalised boxeson my office chair in the laboratory, havingextracted a stub of some sort. A while laterI heard a crash.The open box had fallen off the seat andspread numbered stubs all over the labfloor and under my desk. Thus I had toboth clean and then sort approximately500 stubs once more, before they could beput away in their correct locations.It had been about 15 months since myservice engineer, Don, had visited when Iheard a grinding noise from the mecha-nism that rotates my stage. This noisesoon went away and I had the dilemma ofwhether to call in Don (who might not findanything when he did visit) or to wait untilthe noise persisted and Don would havesomething to get his teeth into (so to speak).Well, since running my SEM Lab is some-thing of a lonely occupation I decided onthe former approach, and Don dulyarrived. He soon located the source of theproblem and carried out the necessaryadjustment, so we spent much of the restof the day carrying out routine mainte-nance including changing the oil in myrotary pump and topping up the distilledwater bottle for my low vacuum mode.Don was able to confirm that my instru-ment was working well, and I even learnta few more features of the computer inter-face that I did not even know existed! Allin all this was a very worthwhile visit, buthopefully I shall not need another for sometime yet.

References1. Roberts, M.J. Spiders of Britain and

Northern Europe. Collins FieldGuide, Harper Collins, London 1996

2. https://warwick.ac.uk/fac/sci/lifesci/outreach/virushunters/

3. http://technicians.org.uk/techniciancommitment/