Estimation of cell size from pulse shape in flow cytofluorometry.

Abstract

The fluorescence pulse widths (pulse duration) generated by fluorochromed cells in a flow-through cytofluorometer provide useful information regarding cell (or nuclear) size and possibly other morphologic features. Simple fixed thresholds just above background noise can be used to identify these pulses, but measurements are then strongly affected by random noise and will vary as a result of both pulse amplitude and pulse shape. In this paper, we propose two alternative, amplitude-independent estimates of pulse width. The first is based on a threshold at some fraction of pulse height, or on a pair of thresholds scaled to some fixed central fraction of the total integrated intensity. The second is based on the ratio of pulse area to peak height. The quantitative properties of these width estimators is studied with simulated fluorescence pulses and with experimental specimens of fluorchromed polystyrene spheres, pollen and spores of known different diameters. The results indicated that absolute particle diameters can be measured within a precision of approximately 1 mu using instruments for flow cytofluorometry.