A Quantitative Method for Enzyme Cytochemistry applied to Alkaline Phosphatase

Abstract

IN the Gomori–Takamatsu technique for localizing alkaline phosphatase, tissue sections are incubated in a solution containing a phosphate ester and calcium ions. The phosphate ions liberated as a result of enzyme activity are precipitated in the section as calcium phosphate, which is made visible by converting it to cobalt sulphide. The cobalt sulphide is dark brown and gives only a rough indication of the degree of enzyme activity. A quantitative method¹ has recently been developed for measuring the mass of substances (other than water; that is, dry mass) in cells. It has been shown that the dry mass can be determined from measurements of optical path difference which can be made with an interferometer microscope. This note describes measurements made with this technique of the amounts of calcium phosphate deposited as a result of phosphatase activity. We have found that the interferometer-microscope method can be used to study the magnitude of possible artefacts due to diffusion of enzyme and diffusion of the calcium phosphate. We have also found that interferometry provides a most promising approach to the quantitative determination of enzyme activity at sites in cells.