Quantitative histochemistry in pathology and toxicology. An evaluation of the original two-wave length method of...

A quantitative histochemical procedure for use in general pathologic and toxicologic studies, based on the original two-wave length microdensitometric method of Ornstein, has been developed. Basic mathematical formulas and necessary parameters have been derived from Ornstein's calculations. A computer simulation of the behavior of these equations in analyzing a photometric field demonstrated the importance of careful choice of working wave lengths for both reference wave length (lambda ref) and correction wave length (lambda cor), the importance of minimizing "empty space" in the photometric field, and the importance of accuracy and stability of transmission readings. An easy procedure for choosing lambda ref and lambda cor is described. The effects of mounting media, varying chromophores analyzed, internal glare, and measuring diaphragms and monochromator in the photometric apparatus are assessed. This method is compared with a scanning technique, and the formulas employed are related to alternative calculations done by Patau. The application of this two-wave length method is well suited to accurately determine extinction values from tissues and cytologic specimens at varying powers of magnification utilizing a variety of histochemical chromophores, which makes the method ideally applicable to general purpose use in pathologic and toxicologic studies.