Three-dimensional high voltage electron microscopy of thick biological specimens

Abstract

The procedures recently developed in our laboratory to observe three-dimensional structures of cell organelles in thick biological specimens by means of high voltage electron microscopy are reviewed. Thick biological specimens such as whole mount cultured cells seeded and grown on grid meshes in culture vessels or thick sections cut from embedded tissues and stained by histochemical reactions can be readily observed three-dimensionally by high voltage transmission electron microscopy at 400–1000kV. Cultured cells used were both primary cultures from animal tissues and established cell lines maintained in our laboratory. The livers of adult Wistar rats were isolated by collagenase perfusion, and hepatocytes were suspended in a Leibovitz medium and seeded on formval coated gold grid meshes in Petri dishes, incubated in a CO2 incubator in a humidified atmosphere containing 5% CO2 in air at 37°C for a few days. Established cell lines, CHO-K1 cells, were cultured in Ham's F12 medium, while HeLa cells were cultured in Eagle's MEM under the same condition. Some of the cells were cultured under experimental conditions such as hepatocyte culture in the medium containing peroxisome proliferating agents such as clofibrate or bezafibrate and some of them were labeled with 3H-thymidine, 3H-uridine, 3H-labeled precursors and 14C-bezafibrate. Also some cells were incubated in medium containing HRP to induce pinocytosis. All the whole mount cultured cells on grid meshes were prefixed in buffered 2.5% glutaraldehyde, stained with various histochemical reactions and postfixed in 1% osmium tetroxide. The histochemical reactions used were glucose-6-phosphatase (G-6-Pase), thiamine pyrophosphatase (TPPase), cytochrome oxidase, acid phosphatase (AcPase), DAB, ZIO, PA–TCH–SP reactions and radioautography was performed after labeling with radiolabeled compounds. The whole mount cultured cells were dried in a critical point dryer and were observed with JEOL JEM-4000EX or Hitachi H-1250M high voltage electron microscopes at 400–1000kV. By tilting the specimens' stereo-pair micrographs were recorded and they were observed with stereoscopes. Rat liver, mouse intestine and pancreas tissues, fixed and stained as above, were embedded in Epoxy resin, thick sectioned at 1–2μm and were observed as for the whole mount cultured cells at 1000kV. Stereo-pairs were further analyzed with an image analyzer JEOL JIM-5000 (JEOL, Tokyo, Japan), producing two contour lines plotted from the micrographs at a thickness of 0.2μm and were observed with anaglyph type glasses, demonstrating the depth or heights of respective cell organelles. The results show that whole mount cultured cells and thick sections stained with histochemical reactions reveal cell organelles corresponding to marker enzymes, such as G-6-Pase in endoplasmic reticulum, TPPase and ZIO in Golgi apparatus, cytochrome oxidase in mitochondria, AcPase in lysosomes, DAB in peroxisomes and pinocytotic vesicles, PA–TCH–SP in secretory granules, 3H-thymidine and 3H-uridine in nuclei, 3H-animo acids in endoplasmic reticulum and secretory granules, 14C-bezafibrate around ER and peroxisomes. The ultrastructure of these cell organelles as well as the structural relationship between them can be demonstrated three-dimensionally with stereo-pair images. Overall, these procedures are useful for analyzing stereologically the ultrastructure of cell organelles in cells and tissues.