Preparation of small and soft specimen for scanning electron microscopy: Investigation of mouse optic nerve

We have previously reported details of the dimensional changes taking place during the processing of soft tissue specimens for scanning electron microscopy. Mouse embryo limbs were used for many of these measurements and the present paper deals with the associated morphological findings. Effects of fixation, dehydration and drying are considered. Freeze drying and critical point drying of glutaraldehyde and glutaraldehyde and osmium fixed samples give perfectly acceptable results for scanning electron microscopy. The best volume retention with freeze dried material is matched by the best morphological appearance of the specimen surface, except when ice crystal damage occurs due to a failure to freeze the tissue rapidly enough. For CPD tissues, perforation of the plasmalemma may occur in glutaraldehyde-only fixed tissue, this being prevented by post-osmication if the glutaraldehyde fixation is not unduly prolonged. This perforation may be due to the extraction of some plasmalemma component during dehydration or further solvent substitution on critical point drying. Solvent evaporation drying usually causes recognizable distortion due to shrinkage: this is minimal in the case of solvent evaporation drying in a nearly saturated atmosphere of the same solvent if this is a very volatile solvent. The examples of Freon 113 and diethyl ether are given here. Swelling during early stages of ethanol dehydration can be prevented by using 70% or 100% ethanol as the first step, with marginal reduction in the post CPD volume and no apparent differences in the SEM. The severe swelling causing sample disruption which can occur with GA + OsO4 fixed tissue can also be prevented by treating the sample with divalent cations, such as Ca++ or Cu++, at any stage.