VALIDATION OF QUANTITATIVE ENERGY-DISPERSIVE ELECTRON-PROBE X-RAY MICROANALYSIS OF ELECTROLYTES IN THIN CRYOSECTIONS OF ERYTHROCYTES.

Biological materials consist of extracellular and intracellular compartments containing diffusable ions which are in some cases maintained at vastly different concentrations. For example, in both man and mouse the erythrocytes suspended in the blood plasma are known to have dramatic intracellular-to-extracellular concentration gradients with the concentration of K**30 within the erythrocyte about 10 times that in the blood plasma, whereas the concentration of Na** plus is about 10 times lower and the concentration of Cl** minus is also somewhat lower. To prevent the possible diffusion of such ions along their concentration gradients during preparation for microprobe analysis, the biological specimens are usually rapidly frozen and thin frozen sections of the specimen are cut and freeze-dried at low temperatures. We were interested in an assessment of what temperatures are needed to prevent diffusion along the known intracellular-extracellular ion gradients during specimen-preparation procedures. In this regard, packed and unpacked erythrocytes were prepared identically except that the cryosectioning and cryosorption were done at minus 40 degree C in one case and at minus 100 degree C in the other. Our overall objective was to validate our electron-probe x-ray microanalysis methods by obtaining quantitative agreement with known and accepted wet chemical measurements on erythrocytes.