Dual-wavelength angle-resolved light scattering used in the analysis of particles by scanning flow cytometry

Abstract

For the first time, a scanning flow cytometer (SFC) was applied for the analysis of individual particles using angle-resolved light scattering at two wavelengths simultaneously. The SFC was equipped with three lasers with wavelengths of 405, 444 and 660 nm. The performance of dual-wavelength angle-resolved light-scattering (DWARLS) flow cytometry was demonstrated by the analysis of polymer beads, spherized red blood cells (RBCs) and blood platelets. The main advantage of dual-wavelength flow cytometry relates to an increment in the precision of the inverse light-scattering problem solution. The solution allows one to retrieve the characteristics of individual particles by measuring the light-scattering profiles of these particles. DWARLS has allowed us to measure diameters and refractive indices (RIs) of polystyrene beads with record precision: a median error of 12 nm and a few thousandths for diameter and RI, respectively. Analysis of spherized RBCs with DWARLS flow cytometry has provided record precision in the sizing of spherized RBCs, with a median error of 16 nm for diameter, and an adequate value of the specific refraction increment for hemoglobin. By means of DWARLS, we have formed a numerical criterion for shape separation of blood platelets, which can be described by an oblate spheroid model to increase the precision of the distribution parameters of the platelet fractions.