An ultrastructural study of fixation artifacts in lens epithelium

The conditions providing for optimal preservation of the ultrastructure of the lens epithelium of embryonic and young chicks were sought, especially with regard to avoiding fixation artifacts and to enhancing the lens cytoskeleton. The optimal fixative/buffer solution for these purposes was found to consist of 2% glutaraldehyde in 0.05M phosphate buffer containing 0.2% tannic acid and 0.002% CaCl2, pH 7.2, whose total osmolarity is about 340 mOsm, and postfixation in 1% osmium. As the osmolarity was increased by use of 0.075M or 0.1M phosphate buffer, intercellular spaces and myelin-like figures appeared along the cell membranes of the epithelial cells and superficial cortical fibers. As the osmolarity was decreased, using 0.02M phosphate buffer, plasma membranes became flaccid and interrupted, nuclei underwent severe shape changes, and the cytoplasm became electron-lucent. Delays of 30 minutes or one hour before fixation caused swelling of cytoplasmic organelles and the nuclear envelope. Primary fixation in osmium resulted in interrupted cell membranes and in swollen organelles. Many of these artifacts seen in the superficial chick lens, produced merely by manipulating the tonicity or the time of fixation after death, have been previously attributed in the literature to cataractous or aging changes. Based on the present findings caution in interpreting electron micrographs of pathologic changes in lens is urged.