Thin frozen‐dried cryosections and biological X‐ray microanalysis

Abstract

The application of X-ray microanalysis to problems of cell physiology [Calliphora salivary gland and rat exocrine pancreas and liver] required the development of methods to retain diffusible substances within the subcellular compartments that they occupied in vitro. Several methods of rapidly freezing small samples in ways that minimize mechanical trauma and ice crystal formation have been developed. 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 (< 0.5 .mu.m) cryosections that are apparently free of diffusion can be routinely performed when temperature parameters are kept < 173.degree. K. Efficient cryosectioning requires several modifications to commercially available machines to improve the ease and reliability with which various manipulations can be carried out. Initial attempts to localize Ca 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 mitochondia were Ca-rich organelles. When tissue freezing and cryosectioning were performed under optimized conditions, mitochondrial Ca was so low as to be quantifiable only with difficulty. Available microanalytical results show that ER[endoplasmic reticulum]-rich cytoplasm and terminal cisternae of the sarcoplasmic reticulum seem to contain higher levels of Ca than mitochondria. Nuclei and secretory granules also contain more Ca than mitochondria.