Correcting cell density measurements for tissue hydration changes in scanning electron microscopy--application to the rabbit corneal endothelium.

Chemical fixation with glutaraldehyde (followed by osmium tetroxide treatment, methanol dehydration and critical point drying) of biological tissue can result in a reduction in tissue size. As a result, cell density estimates can be much higher than in the original tissue. For rabbit corneal endothelium (cell density of 3300 cells/mm2); preparation of fresh tissue (73% hydrated) results in a net 2-fold increase in apparent endothelial cell density unless the dimensions of the tissue before and after processing for scanning electron microscopy (SEM) are taken into consideration. The increase in endothelial cell density (ECD) is the same regardless of whether the fresh tissue is subjected to primary fixation for 1-14 days or if the fixed (and osmium tetroxide treated) tissue is stored in methanol for up to 80 weeks. However, if the tissue hydration is acutely altered during ex vivo perfusion techniques, the cell density, as assessed by SEM, is found to increase proportionately to the tissue hydration (as measured by corneal thickness); the increase was 600 cells/mm2 for each 100 microns increase in corneal thickness. The latter phenomenon does not appear to be the result of a differential reduction of tissue size associated with either primary fixation or secondary processing. For some tissues, therefore, a change in the tissue hydration level prior to fixation, may produce secondary effects in measures of cell densities.