Determination of backscattering cross section of individual particles from cytometric measurements: a new methodology.

Abstract

A methodology is developed to derive the backscattering cross section of individual particles as measured with the CytoSense (CytoBuoy b.v., NL). This in situ flow cytometer detects light scatter in forward and sideward directions and fluorescence in various spectral bands for a wide range of particles. First, the weighting functions are determined for the forward and sideward detectors to take into account their instrumental response as a function of the scattering angle. The CytoSense values are converted into forward and sideward scattering cross sections. The CytoSense estimates of uniform polystyrene microspheres from 1 to 90 μm are compared with Mie computations. The mean absolute relative differences ΔE are around 33.7% and 23.9% for forward and sideward scattering, respectively. Then, a theoretical relationship is developed to convert sideward scattering into backscattering cross section, from a synthetic database of 495,900 simulations including homogeneous and multi-layered spheres. The relationship follows a power law with a coefficient of determination of 0.95. To test the methodology, a laboratory experiment is carried out on a suspension of silica beads to compare backscattering cross section as measured by the WET Labs ECO-BB9 and derived from CytoSense. Relative differences are between 35% and 60%. They are of the same order of magnitude as the instrumental variability. Differences can be partly explained by the fact that the two instruments do not measure exactly the same parameter: the cross section of individual particles for the CytoSense and the bulk cross section for the ECO-BB9.