Image analysis and X-ray microanalysis in cytochemistry

When cytochemical reaction products are homogeneously distributed within an organelle, point analyses suffice for the quantitative approach. However, quantitative analysis becomes tedious, when the elements in the reaction product are inhomogeneously distributed. Problems arise when elements from two reaction products have to be related to each other, or to endogenous cytological products (ferritin, haemosiderin, calcium, electron dense markers), either topographically or in concentration. When analyzing inhomogeneous/heteromorphical reaction product-containing organelles special attention has to be paid to measure and relate both volume and concentration. In this paper a relative simple structure (eosinophil granules) is chosen to demonstrate that the acquisition of the requested morphometrical plus chemical information and their integration is possible. The following points will be covered to acquire the morphometrical and chemical information: a). How to estimate the total cell cross-sectioned area. b). How to estimate the total cross-sectioned area of all reaction product-containing particles inside that cell. The ratio of these two areas will provide the requested information about the particle volume fraction. By using the X-ray detector in addition: c). How to acquire the chemical information at the requested resolution, within a reasonable total acquisition time d). How to integrate the morphometrical and chemical data per organelle, by matrix analysis in a reduced scan area. e). How to acquire quantitative chemical information, by the use of cross-sectioned standards. f). How to make this acquisition method independent from changes in the instrumental conditions during the acquisition