No advice on gelatin slides (never tried them), but you may consider HMDS as an alternative to CPD. In many cases it works just as good with much less physical disturbances to specimen.

By the way, gelatin could be pretty fragile under an electron beam. I wonder if treating it with OsO4 can help.

I never tried to CPD gelatine but would assume to convert it into powder by removing all the water which makes it sticky. Why not precipitating the sludge on some epoxy resin or methacrylate glue prior to CPD?

Paavo, I have seen the comment in Romeis'  "Mikroskopische Technik" [p.149] on "fixing" small specimens (like protozoans) to coated cover slips or object slides....

There is a hint to the technique of making the film of Gelatine in Chapter 10.5.2 (p.180, which only says: "....Die Beschichtung mit Chromalaungelatine ist ebenfalls eine lange gebräuchliche Methode...."(transl.: "....Coating [i.e. of object slides/cover slips for ] with chrome-alum-gelatine is a method used for long ....... Admitting that I haven't used the C-A-G (CAG)-coating method for SEM-specimen preparation but for better adhering of semithin resin sections prior to classical histochemical staining (with harsh removal of resin) I only can speculate and tell that there wasn't any problem with the coating itself during and after staining [i.e. for example stained remnants). I understood the coating to be very very thin (it is only 0.5% gelatine and therefore it might be that the gelatine, in combination / together with the chrome provided acts only as +/- a molecular layer changing the surface properties of glass) so I don't think you might contaminate your CPD-apparatus / chamber and/or the needle valve anyway.... 

BTW: a recipe for CAG-coating (subbing object-slides) besides a possible mentioning in Romeis:   cf.:  http://openwetware.org/wiki/Kubke_Lab:Protocols/Gelatin_Subbing#Acid_alcohol_solution.

About a possible 'reaction mechanism' I only can cite from an MSA-Listserver thread between  09.05.2007 until 10.05.2007   ["Adhesion of Epoxy-embedded tissue (Semithin sections) to Gelatin subbed Slides and alternatives to Chrome-alum Technique" ]:  citation:

Reply by: [email protected]  (2007-05-10)

I got my recipe for chrome-alum from an old copy of Berlin and Miksche's "Botanical Microtechnique and Cytochemistry".  In the chapter covering microtomy, in the section under adhesives, there is a subsection for gelatin adhesives.  The first recipe is for "Haupt's adhesive".  This is prepared by dissolving 1g of gelatine in 100 cc water, smearing a thin film of this onto slides, and then flooding with 4% formalin.  The second recipe is for the chrome-alum that we all know and love.  The paragraph which introduces the chrome-alum adhesive technique begins: "A gelatin adhesive that does not use formalin may be prepared and used as follows...".

I know I'm telling this poorly, but the point is that I think the chrome-alum is used to "fix" the gelatine in place in a manner analogous to what the formaldehyde would do in the Haupt's solution.  This is the purpose (I believe) in using chrome in the tanning industry.

Furthermore, the treatment of the gelatine layer with formaldehyde or chrome would then leave free aldehydes/uncomplexed chrome atoms embedded in the gelatine which might then react with your sections, adhering them to the slide.  Using glutaraldehyde instead of formaldehyde might even provide a stronger bond, if that is what you decide to go with.

So here is a possible reason for the chrome-alum and a possible (although old-school) alternative that still uses gelatine as the adhesive but does not contain chromium. [NB:  = Haupt's adhesive as mentioned above]

Andy Bowling, Plant Physiologist; USDA-ARS-SWSRU.    END of CITATION 

I would try the CAG-method on cover-slips or object slides (perhaps also using either FA and / or GA as additional fixative),  best of luck and best wishes!

Dear colleagues,

Thank you very much for your valuable suggestions. My initial idea was to precipitate the sample from an aquous solution in order to get a convenient sample for further processing. I came up with the chromalaun-gelatine because I was using it routinely for sticking semithin sectionings to glass slides with good success, only I am not sure if very fine particles will stick as good as sectionings, and if it behaves well in CPD. Thank you Wolfgang Muss for pointing me to the possibility of fixing the gelatine with aldehydes instead of /in addition to chrome salts. I may try if aldehyde-fixed gelatine stays stable during CPD. Then it might be possible to adhere the particles to hydrated gelatine and  fix everything together, followed by osmium postfixation. Thank you Vladimir Dusevich for suggesting to stabilize the gelatine against the electron beam. Instead of fragile gelatine, hydrophilic methacrylate surfaces might do the trick, thank you Heinz -R. Köhler for that suggestion. I´ll let you know how it turned out.

Cheers, Paavo

Hello all,

here is how the first try played out: I used raw gelatine, and fixed it with glutardialdehyde together wih the sample after sedimentation, followed by postfixation with osmium tetroxide.

Biological components stuck to the surface sufficiently. Anorganic matter did not stick particularly well to the gelatine at first, so I lost some of the material during the washing steps prior to dehydration, but nevertheless satisfactory results could be obtained, and the evenly scattered particles even facilitated representative imaging.

structural preservation was good, and the osmificated gelatine layer was stable under beam conditions and resulted in a smooth and even background. After sputter-coating (6nm Pt), surface charge was not a problem, and clear pictures could obtained. Sample handling was very convenient, compared to other approached I tried (filter membranes, powder). At least for qualitative imaging, the protocol seems appropriate.  For quantification, i´d need to check whether the particles stuck to the surface represent a random sample, or if some sorting/bias is taking place compared to the powder preparation.

Thank you all very much for your valuable and much appreciated input.

Cheers, Paavo