Automatic sizing and separation of particles by ratios of light scattering intensities.

Abstract

Up to three different sizes of polystyrene beads (0.48-2 � in diameter) were examined at various angles. The ratio function resolved the populations in the form of well-separated peaks even when the intensity distributions showed severe overlap. The distributions of the ratio function were also more symmetric and narrower than those derived from the signals directly. Separations were carried out on binary mixtures of beads 0.794 and 1.001 � in diameter, with the deflection categories gated to each peak of the measured bimodal ratio function. The analysis of the separated fractions indicated a purity of > 95(7 and recoveries of > 90� . The experimentally derived ratios from a variety of combinations of bead sizes and observation angles agreed very well with theoretical prediction. The laser beam in the apparatus is focused achromatically with mirrors and the light scatter signals are detected with a pair of fiber optic assemblies which can be positioned at any desired observation angle 0. The signal peaks and integrals are digitized and transferred to a minicomputer which performs further analysis and implements the separation logic. Both the original signals and the software-generated ratio are displayed as pulse-height analyses and can be used in programmed sorting criteria together with Within the last decade, a number of' flow well defined particles is understood in detail systems have been developed for the identifIca- (28, 61), biological structures constitute a level tion and sorting of biological cells or particles on the basis of' spectroscopic parameters. A repre- sentative but by no means comprehensive re- view of such systems in which the light scatter properties of individual cells or particles are exploited is given in Table I. The applications of light scatter in this context have included counting, sizing, discriminating dead from live cells and distinguishing between cells Ofl the