Microwave Fixation and Localization of Calcium in Synaptic Terminals and Muscular Cells by Electron Probe X-Ray Microanalysis and Electron Energy-Loss Spectroscopy Imaging.

Abstract

The distribution of calcium ions in the rat brain synaptic terminals, in their synaptic vesicles, membranes and mitochondria; and in the sarcoplasmic reticula, mitochondria and their endosacs of skeletal or cardiac muscle cells, and in the caveolae of endothelial cells of blood capillaries was investigated with a two-step inorganic chemical precipitation method using potassium oxalate followed by potassium antimonate.During the process, calcium ions were precipitated with potassium oxalate containing aldehyde fixative as an insoluble precipitate of calcium oxalate under simultaneous computerized microwave irradiation fixation. Then the precipitates were chemically changed to calcium antimonate during the postfixation procedure with an osmium tetroxide fixative containing potassium antimonate. The chemical natures and elemental binding ratios of this precipitate of calcium and antimony in the tube test and in sections were investigated by computerized electron probe X-ray microanalysis using an energy dispersive type X-ray detector (EDX) and electron energy-loss spectroscopy (EELS) imaging. The EDX analyzed chemical nature and calculated values of precipitates, in both atomic binding ratios (AT, %) and molecular weight ratios (WT, %), were exactly the same as those of the theoretical calculated values based upon the chemical formula of calcium antimonate. The data indicated that calcium ions exsisted in the site of precipitate.Electron energy-loss spectroscopy imaging showed the distribution of digital net calcium images at electron microscopic resolution. Calcium exsisted in the nerve terminals, synaptic vesicles, mitochondria and synaptic membranes; in the skeletal and cardiac muscular endosacs of sarcoplasmic reticula, mitochondria, and in the endocytotic vesicles of cardiac sarcoplasmic membranes, or in the caveolae of blood vessel capillary endothelial cells.High magnification O-loss images and EELS spectra confirmed differences in the fine precipitates in a section, as most of the precipitates were Ca-antimonate showing a clot of fine needle-like crystals, but in some cases we found antimonic acid showing an electron opaque foamy spot, or just contamination with an irregularly shaped homogeneous opacity. They were found mixed together, and it was not easy to differentiate them under EDX analysis.Summarized data were reported in the 10th International Congress on Histochemistry and Cytochemistry, Kyoto, Japan, 1996 [24] and XXlth International Congress & International Academy of Pathology, and 12th World Congress on Academy of Enviromental Pathology, Budapest, Hungary, 1996 [26].