Abstract
The procedures recently developed in our laboratory to observe three-dimensional structures of cell organelles in thick histochemical and cytochemical specimens by means of high voltage electron microscopy are reviewed. Various tissues obtained from the organs of experimental animals in vivo such as rat and mouse liver, intestine and pancreas; or cultured cells, either primary cultures from animal tissues or established cell lines maintained in tissue culture laboratory such as Wistar rat hepatocytes or CHO-K1 cells cultured under normal or experimental conditions, were prefixed in buffered 2.5% glutaraldehyde, stained with various histochemical and cytochemical reactions and postfixed in 1% osmium tetroxide. The reactions used were acid phosphatase, cytochrome oxidase, glucose-6-phosphatase, thiamine pyrophosphatase, DAB, ZIO, PA-TCH-SP reactions, silver impregnation, immunostaining and radioautography. After the reactions the tissues in vivo were embedded in epoxy resin and thick sectioned at 0.5-2.0 μm, while the whole mount cultured cells in vitro were dried in a critical point dryer. All the specimens were observed in JEOL JEM-4000EX or Hitachi H-1250M high voltage electron microscopes at 400-1000 kV. By tilting the specimens, stereo-pair micrographs were recorded and they were observed with stereoscopes or further analyzed with an image analyzer and observed with anaglyph type glasses. The results showed that the stereo-pair images obtained from thick specimens revealed 3-D ultrastructures and relationships. The procedure is useful to analyze 3-D and 4-D ultrastructures of cells and tissues.
