The Contribution of Lower Oxides of Osmium to the Density of Biological Specimens in Electron Microscopy

Centrifugally stratified eggs of the sand-dollar, Dendraster excentricus, have cytoplasmic structures segregated into distinct layers. Fat droplets, yolk granules, and mitochondria are separated by "hyaline" layers of protoplasm. Aggregations of particles of 150 to 200 A diameter ("heavy bodies") are found near the mitochondrial layer and a concentration gradient of free 150 to 200 A particles corresponds to a similar gradient of basophilia in thick sections. Eggs fixed in buffered osmium-tetroxide at pH 7.4 and embedded in methacrylate were sectioned and floated on a "bleaching" solution of acidified hydrogen peroxide. "Bleached" sections showed a considerable loss in general density and especially a loss in the sharp images of cellular membranes. It was shown that such loss is not due to sublimation of structure in the electron beam. Assuming that the "bleach" acts principally to reoxidize lower oxides of osmium, it was concluded that reduced and bound lower oxides of osmium play a major role in creation of the electron micrograph image, especially in the delineation of phospholipide components of cellular membranes. Particles of 150 to 200 A diameter showed little or no loss in density, but rather a high intrinsic electron density. Refractometric data were presented to substantiate the tentative conclusions.