DEOXYRIBONUCLEIC ACID CYTOPHOTOMETRY OF STAINED HUMAN LEUKOCYTES II. THE MECHANICAL SCANNER OF CYDAC, THE THEORY OF SCANNING PHOTOMETRY AND THE MAGNITUDE OF RESIDUAL ERRORS

Abstract

The mechanical scanner of CYDAC is a dual beam cytophotometer with a Nipkow scanning disc, analogue photometric circuits, digital conversion and accumulation of the photometric values and digital control of the scanning procedure. A measuring sequence involves object and clear field scans, and the final readout is virtually independent of fluctuations and heterogeneities in illumination and of variations in the size of the scanning apertures. Optical sources of error are analyzed both theoretically and experimentally as they apply to scanning cytophotometers. The effects of conical and polychromatic illumination, stray light and heterogeneous distribution of chromophore lead to negative averaging errors that are a function of optical density. The role of scan aperture size and optical resolution on distributional error is discussed and calculated in terms of three possible models. Non-absorptive phenomena, such as scattering and diffraction, lead to positive errors that are complementary, in a qualitative sense, to distributional error. In attempting to isolate these different errors experimentally, we show that measurements of the relative deoxyribonucleic acid stain content of human leukocytes are independent, within wide limits, of condenser and objective numerical aperture and of changes in absorptivity secondary to changes in wavelength. Where an adequate theory exists, these experimental results agree well with the theoretical predictions and, in any case, they indicate that for stained leukocytes the net effect of all optical errors must be less than 2%. Random measuring errors include both photon shot noise and errors associated with the geometry of the scanning disc. These errors contribute to an error of 1.2% among replicate measurements of leukocytes made under standard conditions. The previously reported differences in stain content among leukocyte types and among individual cells within types cannot be ascribed to either optical or instrumentation errors. Thus, the measured differences must reflect actual differences either in the amount or in the absorptivity of the stain.