The localization of histochemical and autoradiographic products in the scanning electron microscope by means of atomic number contrast

Abstract

The role of atomic number contrast and backscattered imaging in the localization of histochemical products and reagents is reviewed. Particular attention is given to the localization of enzymatically released products. A backscattered imaging method is presented which permits the simultaneous demonstration of two label elements in a cryosection examined in the scanning electron microscope. An azo-dye method for the localization of acid phosphatase is combined with an autoradiographic technique for demonstrating [3H]thymidine incorporation. Atomic number contrast, produced by backscattered electrons, permits the detection of a bromine-labelled azo dye, denoting sites of acid phosphatase activity within a cryosection and the simultaneous display of the sites of the silver deposited in an overlying photographic emulsion. The former reveals acid phosphatase to be predominantly associated with thymocyte death and macrophage activity in mouse thymus. The latter pin-points the thymocytes as the main sites of [3H]thymidine incorporation. Atomic number contrast has a useful potential for differentiating between spatially separated histochemical products of differing atomic number in a structural context and also allows a flexible degree of subsurface localization.