Further studies of specimen volume changes during processing for SEM: including some plant tissue

The dimensions of specimens undergoing preparation for examination in the SEM were measured throughout the preparative sequence or at various important stages. The tissues studied included 15-day mouse embryo limbs (MEL), small blocks of adult mouse liver and brain, and potato tuber. The animal tissues were fixed in 3% glutaraldehyde (GA) in 0.15M cacodylate buffer, and the potatoe tissue in 2% GA in water. The effects of various secondary fixation and other treatments were investigated. The results show that lithium salts cause a reduction in the shrinkage of MEL in 100% ethanol but this effect was not significant in the other tissues investigated, and did not persist in specimens stored after critical point drying (CPD). All CPD specimens were shrunken. However postosmication and treatment with uranyl acetate (UAc) and cetyl pyridinium chloride (CPC) all reduced specimen shrinkage in 100% ethanol and after critical point drying. The volume gains with Os + UAc and Os + CPC are both very significant, but it was found that these larger specimens shrank more on storage. Thus rapid examination in the SEM is recommended. Ethanol and Freon 113 were compared as intermediate fluids and it was found that ethanol-CO2 critical point dried specimens shrank more before and after CPD than Freon 113-CO2 specimens. The latter technique is, therefore, to be recommended. Potato tissue shrinks in 30% ethanol, whereas animal tissues all swell in this concentration. The potato tissue also shrank very litte on critical point drying in contrast to the animal tissue specimens.