Quantitative electron probe X-ray microanalysis of electrolyte elements within epithelial tissue compartments

The electron probe X-ray microanalysis of 1-micron thick frozen-hydrated sections provides a method to measure local concentrations of electrolyte elements (and H2O) in and around the cells in situ with an analytical spatial resolution of better than 0.2 micron and a sensitivity limit better than 10 mM with a standard error of less than 10%. Our microprobe studies on several epithelia transporting isotonic fluids have provided strong evidence that the electrolyte concentration in the interspaces suspected to be the sites of solute-solvent coupling may, on average, be as much as 35% more than in the bathing fluid. There also appear to be distinct concentration gradients in interspaces, the profiles of which differ according to the tissue geometry but particularly according to the location of the leaky cell junctions, suggesting that the osmotic equilibration of the transported fluid may require substantial mass flow through the junctions. In addition, there is evidence a) that the major electrolytes in the cytoplasm of the epithelial cells are not uniformly distributed but may have distinct axial and radial concentration gradients that depend on active solute transport (e.g., that abolished by ouabain); b) that in some epithelia (e.g., rabbit ileum) there is a peripheral cytoplasmic zone that may support fast convective flow for transcellular fluxes; c) that the extracellular structures like the glycocalyx and basement membrane preferentially sequester potassium (and calcium) and could have a non-zero reflection coefficient; and d) that in normal cells, nuclei have ionic composition similar to that of the circumnuclear cytoplasm.