Light scatter applications

Abstract

Forward scatter Forward light scatter (also referred to as ‘FALS’, forward angle light scatter) is very rarely specified adequately and almost invariably is equated directly with cell size. Forward scatter is proportional to particle size at narrow forward angles. The operative word here is narrow , and the relationship is strictly only valid over collection angles between about 0.5 and 1.5°. At wider angles the correlations tend to break down. This is illustrated in figure 10.1 by the work of Mullaney et al . (1976) where total light scatter intensity at 632.8 nm (helium-neon) is plotted on the ordinate versus diameter for particles with two different refractive indices. The latter were 1.533 and 1.373, which correspond approximately to fixed and unfixed cells respectively, and the collecting angle was between 3 and 5°. It will be noted that the scatter intensity between these particular angles is very insensitive for diameters of 7 to 13 μm which is the range of most interest in biology. Another problem encountered with all scatter measurements, irrespective of the collection angle, is the dependence on cellular orientation. A spherical cell with a centrally placed spherical nucleus constitutes no problem as the light scatter pattern will be orientation independent. However, cells which are not radially uniform in all orientations, e.g. nucleated disks such as chicken red blood cells (CRBCs) or elongated fibroblasts, can give rise to artefactual light scatter distributions. Loken, Parks and Herzenberg (1977) found a bimodal distribution with light scatter measurements from fixed CRBCs.