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FLUORESCENCE SPECTROSCOPIC AND FADING BEHAVIOR OF EHRLICH'S HYPERDIPLOID MOUSE ASCITES TUMOR CELLS SUPRAVITALLY STAINED WITH ACRIDINE ORANGE

Corrected fluorescence emission spectra and measurements of fluorescence intensity and fluorescence fading were recorded from Ehrlich's hyperdiploid mouse ascites (EHD) tumor cells supravitally stained with acridine orange (AO). After equilibration of the staining reaction, some EHD tumor cells with dye content greater than 5 x 10–15 mole AO/cell spectroscopically exhibit both red and green fluorescence while the rest fluoresce only green. This behavior contrasts with that previously observed for normal mouse leukocytes. Below 5 x 10–15 mole AO/cell, all EHD tumor cells fluoresce green, but the intensity varies considerably from cell to cell. The fading rate of the green fluorescence appears to be a linear function of fluorescence-exciting light intensity. Absolute light intensity measurements, a necessity for interexperiment comparisons and automation, were based upon a phosphor particle standard. The results presented are indicative of the possibilities for automated cytopathology that can be uncovered by rapid recording, corrected spectrum microspectrofluorophotometry and related biophysical cytochemical techniques.