Light-Scattering Sizing of Single Submicron Particles by High-Sensitivity Flow Cytometry.

Abstract

Rapid and reliable size measurement of single submicron particles (100-1000 nm) is important for quality control of particulate matter, biomedical research, environmental study, and drug delivery system development. Though direct measurement of the elastically scattered light from individual submicron particles represents the simplest method for particle size measurement, the inadequate instrument sensitivity and complicated relationship between scattering intensity and particle size render it a great challenge. Combining the superior sensitivity of a laboratory-built high-sensitivity flow cytometer (HSFCM) in the side scattering (SSC) detection of single nanoparticles and the great efforts in synthesizing 38 highly monodisperse silica spheres ranging from 180 to 880 nm with small size intervals, here we report the first comprehensive comparison between the experimentally measured and Mie theory calculated intensities of light scattered by single submicron particles. Good agreements were observed for both the silica spheres and polystyrene beads at both the perpendicular and the parallel polarizations of the incident laser beam. Compared with perpendicular polarization, parallel polarization can resolve differently sized beads better due to the continuously increased scattering intensity with particle size. The predictive capability of the simple numerical model constructed in present work can be exploited to allow us to foresee scattering behavior on flow cytometers. More importantly, the linear correlation between the measured and the calculated scattering intensities enables us to develop a method that can measure the particle size of submicron particles with the precision and accuracy of Mie theory rather than a calibration curve fitted by several sparsely separated size reference standards. Comparable sizing resolution and accuracy to those of electron microscopy were demonstrated for Gram-positive bacteria Staphylococcus aureus. The as-developed method shows great potential in guiding the accurate size measurement of submicron particles.