Cell physiological and pathophysiological studies often require information about
the elemental composition of intracellular organelles in situ. Electron probe X-ray
microanalysis (EPXMA) is one of the few methods by which intracellular elemental content
and distribution can be measured simultaneously. While several cryofixation techniques
for EPXMA have been utilized on isolated cells, few have been applied successfully
to whole tissue in vivo or in situ. A recently developed, commercial, portable, metal-mirror
device was used for preserving kidney in situ to determine the intracellular element
distribution in proximal tubule cells. Kidneys of male rats were exposed, cryofixed,
and analyzed for organelle elemental contents by EPXMA imaging. In addition, some
portions of the frozen tissue were prepared for conventional transmission electron
microscopy. Proximal tubules were preserved with intact brush borders and open lumens.
The quality of preservation of tubule cell organelles varied inversely as a function
of depth from the point of first contact with the mirror surface; the best preservation
was within 15 microns, while the poorest preservation was deeper than 30 microns.
Analysis of EPXMA images from the best-preserved regions revealed that proximal tubule
cell cytoplasmic K/Na was approximately 6, cytoplasmic Cl was low relative to other
subcellular compartments, and mitochondrial 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
include acquisition of organelle elemental content data in vivo, ease of use, reproducibility,
portability, applicability to other tissues, and suitability for pathophysiological
studies.