The Utility Value of High Voltage Electron Microscopy for X-Ray Microanalysis

Abstract

X-ray microanalysis is a useful technique to qualify and quantify basic elements in biological specimens. This article reviews the principles and techniques for applying intermediate high voltage electron microscopy to X-ray microanalysis of various elements in biological specimens developed in our laboratory since the late 20th century. We first quantified the endproducts of histochemical reactions such as Ag in radioautographs, Ce in acid phosphatase reaction and Au in colloidal gold immunostaining using semi-thin sections by high voltage electron microscopy at 300-400 kV. We then analyzed various trace elements such as Zn, Ca, and S, which originally existed in the cytoplasmic matrix or cell organelles of various cells in different organs, and some absorbed elements introduced by experimental administration into cells and tissues such as Al, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning, freeze-drying, or freeze-substitution. As a result, we showed that the peak to background (P/B) ratios of all the elements analyzed had high P/B ratios at 300-400 kV. It was concluded that X-ray microanalysis using semi-thin sections by high voltage electron microscopy is of great utility for quantifying trace elements in biological specimens.