Cytochemical applications of X-ray microanalysis.

X-ray microanalysis (XRMA) has been applied to a wide variety of cytochemical problems, but the most valuable applications have been to the validation of cytochemical methods (by the qualitative or quantitative analysis of reaction products), and to the simultaneous localization of more than one substance, which cannot easily be achieved by using alternative methods. The latter applications involve stoichiometric studies (the quantitative relationships between reaction products and substrates), and distribution studies. Ultrastructural cytochemistry with XRMA is limited by the need to use high-brightness electron sources. Apart from the limited availability of such sources, they may cause unacceptable damage to the specimen. Preparation methods for cytochemistry using XRMA are reviewed; in principle these do not differ from those used for other cytochemical applications, but it is important not to introduce extraneous elements (from fixative, buffer, or embedding medium) into the specimen, where the additional X-ray peaks may interfere with the analysis. Quantification in XRMA of cytochemical preparations poses special problems, because the addition of the reaction product to the specimen alters the yield of continuum X rays, used for assessing the mass of the specimen, and also dilutes endogenous elements. However, measurement of ratios between characteristic elemental peaks is a useful method in X-ray microanalytical cytochemistry, and it is concluded that one of the most important attributes of XRMA for cytochemical purposes is the ease with which the substances of interest can be measured.