The use of backscattered electron imaging, X-ray microanalysis and X-ray microscopy in demonstrating physiological cell death.

The cytochemical localization of enzymatic activity by means of backscattered electron imaging (BEI) is reviewed and the application of BEI to changes in acid phosphatase and ATPase distribution during physiological (programmed) cell death in Heliothis midgut is explored. Programmed cell death entails the release of nascent free acid phosphatase as extracisternal hydrolase. This shift can readily be detected by means of the atomic number contrast imparted by BEI of the lead phosphatase reaction product, thus enabling the distribution of dying cells to be mapped. BEI is particularly useful in this context as it allows the examination of bulk specimens at low magnification. Death of cells is also accompanied by a collapse in ATPase activity which shows up as cytochemically negative areas in the X-ray microscope and by means of BEI. Acid phosphatase in normal cells is localized in the apical microvilli and lysosomes. Senescent or dying cells, however, clearly show a basally situated free hydrolase which migrates throughout the cell. Parallel TEM results confirm that this enzyme is ribosomal and extracisternal rather than lysosomal in origin. ATPase activity is largely limited to the apical microvilli, although there is some activity associated with the basal plasma membranes. The apical ATPase, however is partially resistant to ouabain. Young and mature cells are positive although in the latter case some microvilli may be lost as the cells acquire a negative cap or dome. Inhibition by bromotetramizole indicates that apical activity is not to any significant extent contributed to by alkaline phosphatase. Degenerate or dead cells are negative and can be seen as a mozaic of "black patches" among normal cells when imaged by means of BEI or X-ray microscopy.