Microdensitometry

Microdensitometry, or microspectrophotometry, is the measurement of the concentration or mass of a chromophore in microscopically defined regions, and is governed by well-established laws of physics. Initially it proved of value in Feulgen cytophotometry of the relative amounts of DNA in individual nuclei of isolated cells. It has now achieved wide applicability to the measurement of cellular biochemical activity by means of stoichiometric chromogenic reactions. The validity of some of these measurements has been confirmed by comparative biochemical and microdensitometric assays. Thus microdensitometry, even of heterogeneously distributed chromophores, can be precise, provided that the technique is operated with due regard to its limitations within the laws of physics. The potential errors include: variation in thickness of tissue sections (path-length); scatter; glare; diffraction; occlusion of light by optically dense particles; and the inhomogeneity error. However, under correct conditions for the cytochemical reactions and for operating the microdensitometer, these potential errors become small or negligible. Thus the highly sensitive cytochemical bioassay of thyrotropin exemplifies the precision that can be achieved by controlled use of microdensitometry.