An application of rapid freezing, freeze substitution–fixation for scanning electron microscopy

A combined technique of the rapid freezing, freeze substitution–fixation method and the osmium–DMSO‐osmium method was devised. By this combined method we clearly observed the architecture of intracellular components in three dimensions. Morphological characteristics were generally similar to those of tissue prepared by the osmium–DMSO‐osmium method but different in some respects. Mucigen droplets in intestinal goblet cells, for example, appeared as separated spheres, while in specimens prepared by chemical fixation they were observed as a mass of fused droplets. In the Golgi complex, all cisternae were extremely flat, although they usually dilated on the cis side after chemical fixation. Particles on the mitochondrial tubules of liver cells were well distinguished. They were mushroom shaped, as are those observed by negative staining. The combined method, that is, the rapid freezing, osmium–DMSO‐osmium method, is thought to be effective for studying the true structure of intracellular components by scanning electron microscopy.