Shrinkage in preparatory steps for SEM. A study on rabbit corneal endothelium

Since specular microscopy of the cornea offers the opportunity to observe and measure cells in vivo without any outside interference this method forms an unrivalled basis for estimation of tissue shrinkage during various preparatory methods. Therefore a study was performed with the purpose of evaluating the degree of artifacts in each preparatory step from the living tissue "in vivo" to the final SEM specimen. The study was performed on rabbit corneas, the endothelium serving as measuring target. The in vivo state was recorded by specular microscopy. Unfixed corneas were studied by light microscopy unstained and stained by alizarin red S or silver nitrate. Fixation was performed intracamerally with 1.5% glutaraldehyde (Gla) by a pH, osmolarity, viscosity and intraocular pressure identical with the physiological values of rabbit eyes. Fixation was completed by immersion in 2.5% Gla for 1/2 h. Gla-fixed corneas were evaluated as above before osmification. Dehydration was performed either by graded acetone, by acetone in a gradient-free system, both followed by critical point drying (CPD). At all steps cells were counted using the same reference frame. The number of cells/mm2 was estimated and statistical analysis showed a shrinkage of 22 per cent (area) in unfixed tissue, 26 per cent (area) in normally dehydrated tissue and 37 per cent (area) in gradient free dehydrated tissue processed for SEM.