The use of off-peak measurements in Feulgen cytophotometry is reported, using intact diploid (x) and tetraploid (y) nuclei of the human anterior pituitary gland as the experimental model studied. Results indicate that the ratio y/x is similar at the 14 wavelengths examined over the range 450 m# to 650 m#. The value of this ratio was 1.95, falling slightly under the theoretical ratio of 2.0. It was concluded that off-peak absorption measurements are of value in Feulgen cytophotometry. A discussion of the possible justification of off-peak absorption measurements was presented for the case in which the Beer-Lambert relationship was experimentally determined to be followed at a peak wavelength, for all concentrations under discussion, and the curves preceding and/or following the peaks were straight lines coming from a common point. If the curve best fitting the data is a straight line, it follows that the rate of change of absorbance with respect to a linear wavelength scale is constant. This means that for a given increase, or decrease, in wavelength, the same change in absorbance is obtained. From this it follows that absorbance readings at any wavelength in such a region will be equally valid to those taken at the peak. While the finding of such a linear relationship at several concentrations does not guarantee that it will occur at all other concentrations, it is suggestive. The closer the spectra approximate straight lines, the more valid does the use of off-peak measurements become.The literature records considerable discussion about the appropriateness of off-peak measurements, or relative extinctions, in cytophotometry (4,6,8,12). It is the purpose of this report to present data on the consistency of the results obtained experimentally when using off-peak compared with maximal absorption measurements for Feulgen-stained human pituitary nuclei measured in the Barr and Stroud integrating microdensitometer (2), and a discussion of the possible justification of such measurements.
MATERIALS AND METHODSTISSSUE PREPARATION: Imprints from the cut surface of fresh, human, anterior lobe were made directly after receiving pituitary glands from postmortem examination. The preparations, on long coverslips, were snap frozen in isopentane over dry ice and then freeze-substltuted with ethanol (14). When the substitution was complete, they were transferred to alcoholic formalin for fixation prior to Feulgen staining. In our hands the optimal time of hydrochloric acid hydrolysis for these preparations was 20 minutes, after which the aldehydes formed from the deoxyribonucleic acid were demonstrated by Lillie's cold Schiff reagent (7) at pH 2. After staining, the imprints were covered with a mylar strip over immersion oil of refractive index 1.5150.INSTRUMENTATION: For determining the absorption of the Feulgen-stained nuclei, we used the Barr and Stroud, Ltd. integrating microdensitometer,