Scanning Electron Microscopy


Although X-ray microanalysis represents a useful tool for identifying electron dense histochemical end-products, quantitative microanalytic measurements are seriously hampered in the case of the activities of certain membrane-bound enzymes. For example, the electron histochemical methods revealing K+-dependent pNPPase activity result in a very fine, granular reaction product of lead phosphate. Therefore microanalytic, densitometric or similar evaluations of the reaction, even in semiquantitative terms are not practical by the usual procedures.

This paper describes a method of X-ray microanalysis of thick sections (0.5 μm) processed for K+-pNPPase, where a sufficient amount of lead is present for X-ray microanalytic determination. The analysis is performed in the line scan mode on transversely cut membrane profiles by means of the line scan ratemeter of an EDAX System F. This yields quantitative data on the relative lead concentrations in the vicinity of the cell membrane. A method is proposed for calculation of relative enzyme activities based on the Pb-signal of the ratemeter curve and the average "noise"-level of the cytoplasm, containing also non-specifically bound lead. This method avoids the necessity of measuring the section thickness; it may be useful for a variety of purposes in the electron microscopic histochemistry of membrane-bound enzymes.

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