Are your drosophila still alive when you start fixation? If not, it is possible that air drying (and collapsing) of specimens already took place. I never worked with drosophila, but I would try to place a piece of wet filter paper on top of floating specimens. By the way, for insects proper dehydration could be even more important than fixation. Slow dehydration (like 24 hours for each step) may help. You may want to try HMDS instead of CPD.

I too have not prepped flies. See https://einsteinmed.org/labs/bakerlab/labmanual/22.html (searched SEM prep of drosphila) recommending CPD.

Dear Pranjali Priya many thanks for asking this very interesting technical question. In the case that your drosophila samples keep floating on the surface of the fixative, you can add a few drops of a non-ionic surfactant such as Triton X-100 or related to get the flies submerged. Good luck and best wishes!

Fixatives, as you have described, are generally thought of as fairly shallow penetrators into biological tissues, perhaps 1/2 - 1 mm maximum. Additionally, the exo-skeleton of the fly may be a formidable barrier to fixation chemicals. My personal experience with related biologicals indicates only the outer surfaces of the fly experience fixation when attempting whole body fixations. Once the “fixed” specimen is placed under the vacuum of the SEM, body collapse is likely to occur because the fly‘s internal structure is not fixed and contains air spaces.

Low vacuum or environmental-type SEM’s may be a better option for SEM level viewing, which are much less harsh on this type of specimen.

Dear Var St. Jeor , for described task only outer surfaces are of interest, so depth of fixation is not important here. Moreover, for many insects just air drying is sufficient (no fixation, no dehydration with alcohol/acetone). As I understand drosophila eyes are not tough enough and require fixation/dehydration, but again only surface layer is of importance.

Vladimir, Priya is concerned with body damage occurring with his current methods, there fore surface fixation is not doing the job for him. ESEM or low vac. SEM would likely remedy that, or in alternative, whole body fixation (inside and out) and a slow dehydration regime may be appropriate. Thanks for your invites, Vladimir.

I don't work with insects but have come across in my experience also samples not being submerged. If the sample is well covered it should get fixed with the accumulated vapor around. But as pointed out earlier insects are hard to penetrate with fixative. If possible you can try to place the sample upside down meaning the intended part of the sample will be in contact with the fixative.

As last resort, you can use Osmium(OsO4) as a fixative but it's not a good stuff to work with and must follow proper precautions while handling Os. Os will fix very fast though. Furthermore some of the artifacts could be avoided by chemical drying with HMDS rather than air drying. Good luck!