Photographic Densitometry with the Two - Wave-length Method

Abstract

THE appreciation of distributional error in the measurement of complex microscopical objects has led in recent years to the development of scanning methods of photometry and the two – wave-length method of microspectrophotometry. The two – wave-length method has been used successfully in a number of laboratories since its inception by Ornstein¹ and its elaboration by Patau². It is based on an empirical correction for distributional error derived from transmission readings of the entire object taken with two wave-lengths which have been pre-determined to give true absorbances in the ratio of 1 : 2 for the chromophore being measured. The successful application of the method depends on the spectral characteristics of the chromophore, and on the presence in the object of suitable areas in which the chromophore is randomly distributed and can be measured for the selection of appropriate wavelengths. Three types of material will not meet these requirements. They are objects without homogeneous portions, objects in which more than one independent chromophore is present, and objects in which the chromophore has too flat an absorption spectrum to supply the necessary ratio.