Quantitative analysis of electrolytes in frozen dried sections

During recent years our group has employed the technique of electron microprobe analysis to determine the electrolyte concentrations in various epithelial tissues. The specimen preparation is characterized by shock-freezing of small tissue pieces in liquid propane/isopentane mixtures at 77 K, cryosectioning of 1 micrometer thick serial sections at 170 K and subsequent freeze-drying at 190 K and 10-4 Pa. The analysis of the frozen dried cryosections is performed in a scanning electron microscopy which is equipped with an energy dispersive X-ray detector. The measuring conditions selected are 17-20 kV acceleration voltage and 0.1-0.5 nA probe current. For quantification, the cellular X-ray spectra are compared with those of an internal albumin standard layer. The evaluation of the characteristic X-ray intensities is performed using a computer program. Some critical points of this technique will be discussed.