PHYSICAL METHODS IN QUANTITATIVE HISTO- AND CYTOCHEMISTRY

Some of the older members of this society will recall both our first symposium in 1953, which was entitled “The Structure and Biochemistry of Mitochondria,” and the address given by the late R. R. Bensley of the University of Chicago (1). At the age of 85, Dr. Bensley was the most forward-looking participant of that symposium, and, if the younger members of this society have not read his presentation, I would strongly recommend that they do so because his statements are as appropriate today as they were in 1953. Dr. Bensley had made a most fundamental contribution to cytochemistry in his isolation and characterization of mitochondria. Despite his personal commitment to mitochondria, Dr. Bensley urged his listeners to broaden their research horizons by looking beyond the mitochondria, for he was convinced that the functional interrelationships of mitochondria with the other subcellular organelles were extremely important. The developments in cell biology over the past decade have proven fully the wisdom of his advanced thinking. In selecting “Physical Methods in Quantitative Histoand Cytochemistry” as the theme of this symposium, I used my prerogative as President of this society to emphasize the more quantitative aspects of histochemistry. I believe that increased use of quantitative methods in histochemistry will contribute greatly to the development of biology and medicine during the next decade. Unfortunately, the time limitations imposed by a single half-day session prevented the inclusion of many important quantitative physical methods such as polarization optics, phase microspectroscopy, mass determination through use of interference microscopy and elemental analysis using long x-ray diffraction. The papers by Drs. Kvistberg (“Infared Spectrophotometry”) and Glick (“The Laser Microprobe”) were chosen because these methods are relatively new and because their potential application to histochemistry and cytochemistry is not generally appreciated. The papers by Drs. Carpenter (“Scanning Methods”) and Swift (“Microspectrophotometry”) were chosen because they are of special interest in our laboratory at Minnesota and because we believe that the combination of these two techniques into a single histochemical method offers much promise. Casperson (2), using an integrating microspectrophotometer, has estimated the total deoxyribonucleic acid content in the nucleus by integrating the point-by-pointabsorbance over the nucleus as it was scanned. Our approach’ is to quantitate the amount of histochemical reaction product for each different cell type present in a complex tissue section and to express the results as relative absorbance per unit volume of cell. A television-scanning system is used with our instrument assembly to project the entire field of scan; this permits visualization and identification of the component cell type while the scanning and absorbance measurements are being recorded. The point-by-point absorbance is measured and recorded during a series of successive linear scans and each absorbance value is referenced to the appropriate cell type. All original data, i.e., the component scan intercepts for each cell type, and corresponding point-by-point absorbance values (as measured at two different wave lengths) are directly recorded on paper-punched (or magnetic) tape and this information is transferred to the computer. Through use of appropriate computer programs, each absorbance value is corrected by subtracting the corresponding blank values and, when necessary, correction for heterogeneity in distribution of absorbing materials is made using the two-wave length method (3, 4). The program then integrates the corrected absorbance values for each different cell type in the tissue section and this integrated value is divided by the corresponding volume for each component as determined by the linear scan method. Thus, the volumes for each cell type are expressed in rel-