IN-SITU CRYOFIXATION OF KIDNEY FOR ELECTRON-PROBE X-RAY-MICROANALYSIS

Cell physiological and pathophysiological studies often require inform ation about the elemental composition of intracellular organelles in s itu. Electron probe X-ray microanalysis (EPXMA) is one of the few meth ods by which intracellular elemental content and distribution can be m easured simultaneously. While several cryofixation techniques for EPXM A have been utilized on isolated cells, few have been applied successf ully to whole tissue in vivo or in, situ. A recently developed, commer cial, portable, metal-mirror device was used for preserving kidney in situ to determine the intracellular element distribution in proximal t ubule cells. Kidneys of male rats were exposed, cryofixed, and analyze d for organelle elemental contents by EPXMA imaging. In addition, some portions of the frozen tissue were prepared for conventional transmis sion electron microscopy. Proximal tubules were preserved with intact brush borders and open lumens. The quality of preservation of tubule c ell organelles varied inversely as a function of depth from the point of first contact with the mirror surface; the best preservation was wi thin 15 mu m, while the poorest preservation was deeper than 30 mu m. Analysis of EPXMA images from the best-preserved regions revealed that proximal tubule cell cytoplasmic K/Na was similar to 6, cytoplasmic C l was low relative to other subcellular compartments, and mitochondria l Ca levels were 1.8 nmole/mg dry weight; these observations indicate that the cells were physiologically viable at the time of cryofixation . The advantages of in situ cryofixation by this metal-mirror method i nclude acquisition of organelle elemental content data in vivo, ease o f use, reproducibility, portability, applicability to other tissues, a nd suitability for pathophysiological studies. (C) 1994 Academic Press , Inc.