Principles of cytophotometry in enzyme histochemistry and validity of the reactions

Essential conditions for valid quantification of enzyme histochemical reactions by means of cytophotometry are discussed. It is shown that a scanning and integrating microdensitometer or an image analysing system is needed to avoid distributional errors. Other possible errors on the instrumentation side are reviewed as well, such as the out-of-range error, and errors due to glare, out-of-focus effects or random signal variation. Furthermore, conditions of enzyme histochemical reactions are discussed because validity of cytophotometrical analysis depends largely on these conditions. Finally, it is concluded that fundamental enzyme histochemistry should especially aim now for the development of valid methods for functionally important enzymes such as flux-generating enzymes.