A procedure for minimizing cellular shrinkage in electron microscope preparation: a quantitative study on frog gall bladder

SUMMARY Nomarski differential interference contrast microscopy of whole‐mount preparations of frog gall bladders has been used for light microscopic measurement of epithelial cell luminal area. Measurements were performed: in buffer after fixation, and in cured Epon after step‐by‐step and after continuous dehydration and infiltration procedures. Sections cut perpendicularly to the luminal surface were used for the measurement of epithelial cell height. From these measurements epithelial cell mean volumes were calculated. The comparative measurements showed that a step procedure results in significantly smaller cellular dimensions than does a procedure of slow and continuous change of concentration of the preparative media, the difference in epithelial cell mean volume being about 40%. Electron microscopy of thin sections of continuously processed specimens showed superior ultrastructural preservation of cytoplasmic density and microplical arrangement compared to the step processed counterparts. The principle was successfully applied in processing for scanning electron microscopy of osmotically extremely fragile preparations of erythrocyte ghosts; similarly, formation of the luminal cobblestone appearance of gall bladder epithelium was prevented. The present studies support the view that at least part of the volume shrinkage through the procedures of embedding is caused by osmotic gradients set up in the tissue.