Size and refractive index dependence of simple forward angle scattering measurements in a flow system using sharply-focused illumination.

Abstract

We have investigated the properties of three forward angle light scattering signals in a flow cytophotometer (Bio/Physics FC-200) using a line focused illuminating beam of about 5 ,z half-intensity width: (a) extinction of the illuminating beam; (b) scattering in the direction parallel to the focal line, integrated between 2 and 20#{176}; (c) scattering perpendicular to the focal line, integrated between 5 and 25#{176}. Signals were compared qualitatively in terms of pulse shape, and quantitatively in terms of total intensity (time integral) and pulse width (integral rise time) measurements, with respect to variations in particle diameter and refractive index at two wavelengths (488 or 633 nm). Test particles were transparent, spherical dextran gel (Sephadex) beads, having diameters continuously distributed between about 10 and 40 �z of several distinct refractive indices in the range 1.01-1.04, relative to water- comparable to fixed (hydrated) and living cells. Parallel and perpendicular scatter signals exhibit different, characteristic pulse shapes which remain distinctive over the whole range of diameter, refractive index and wavelength studied. These shapes evidently depend on geometrical factors which are different for the two scattering directions. Parallel scatter pulses rise and fall rapidly at the edges, but their amplitudes change relatively little while the center of the particle traverses the beam. Perpendicular scattering arises primarily from the edges of the particle, each of which produces a sharp pulse as it passes through the beam. Leading and trailing edge pulses are detected unequally and mirror-symmetrically on opposite sides of the beam. Perpendicular scatter pulsewidth (i.e. , the separation in time of these two pulses) is accurately proportional to particle diameter (at constant flow velocity) and independent of refractive index and wavelength within experimental error. Integrated extinction intensity is comparable to extinction under uniform, parallel illumination; its dependence on diameter, refractive index and wavelength is in good agreement with the predictions of anomalous diffraction theory. This permits a simple absolute calibration ofthe pulsewidth scale. Perpendicular scatter intensity increases smoothly with bead diameter, and also depends strongly on refractive index in the range studied. Parallel scatter intensity shows a more complex dependence on these parameters, but is relatively much less sensitive to refractive index. These results are discussed in terms of an approximate separation of refractive from diffractive effects due to the undirectionally focused illumination. We conclude that unambiguous and fairly precise estimates of both size and refractive index for cell-like particles can be obtained from a single (perpendicular) scatter signal in a flow system using line-focused illumination.