Thin frozen-dried cryosections and biological x-ray microanalysis.

The application of X-ray microanalysis to problems of cell physiology required the development of methods to retain diffusible substances within the subcellular compartments that they occupied in vivo. Several groups have developed methods of rapidly freezing small samples in ways that minimize mechanical traumae and ice crystal formation. This provides a narrow zone from which cryosections, believed to be representative of the in vivo distribution of electrolytes, can be cut. The production of thin (less than 0.5 micrometers) cryosections that are apparently free of diffusion can be routinely performed when temperature parameters are kept below 173 K. Efficient cryosectioning requires several modifications to commercially available machines, in order to improve the ease and reliability with which various manipulations can be carried out. Initial attempts to localize calcium at the subcellular level were disturbed by the use of mechanically damaged specimens and by insufficiently cold conditions in the cryochamber. Such sections indicated that mitochondria were calcium-rich organelles. When tissue freezing and cryosectioning were performed under optimized conditions, mitochondrial calcium was so low as to be quantifiable only with difficulty. Available microanalytical results show that ER-rich cytoplasm and terminal cisternae of the sarcoplasmic reticulum seem to contain higher levels of calcium than mitochondria. Nuclei and secretory granules also contain more calcium than mitochondria.