QUANTITATIVE ELECTRON PROBE ANALYSIS OF SOFT BIOLOGIC TISSUE FOR ELECTROLYTES

Abstract

Electron probe analysis of soft biologic tissue requires the use of a tissue preparation that (a) presents a flat surface of the specimen to an electron beam in a vacuum, (b) can withstand the rigors of a vacuum, (c) can withstand damage by an electron beam and (d) causes no redistribution of the elements under study. A tissue preparation which also does not involve the use of aqueous solutions for any step of the preparation has been used for the examples of electron probe analyses presented here. Tissue is prepared by quick freezing at about –196°C, drying in a vacuum at between –60°C and –85°C, fixing with osmium tetroxide vapor and embedding in a low Cl epoxy. Calibration of the electron probe signals is accomplished with prepared solutions of 20% albumin and known quantities of electrolytes. Cell morphology is well preserved to the submicron level, but counting rates and signal to background ratios for the x-ray signals are low. With a 10-kV, 50-na electron beam, the spatial resolution is comparable to that of light microscopy with detection limits for K, Cl and Na of a few milliequivalents per liter. Analysis of frog skeletal muscle fibers yielded concentrations of 122.4 ± 2.8 (s.e.) mEq/liter for K, 10.5 ± 0.7 mEq/liter for Cl and 9.2 ± 1.0 mEq/liter for Na as the averages of nine fibers from one muscle. The Ca content in a discontinuous extracellular layer in frog skin was found to be as high as 4-5 M with a Ca:P ratio of an apatite when referenced to a natural fluoroapatite crystal.