Quantification of Light Scattering Detection Efficiency and Background in Flow Cytometry

Knowledge of the sensitivity of a flow cytometer is essential for data interpretation and comparison. However, light scatter sensitivity currently cannot be quantified due to a lack of a standardized unit. Analogous to the QB approach for fluorescence, we show that it is possible to derive the detection efficiency Q and background B for light scatter detectors, using the scattering cross section (σs) in nm2 as a standardized unit. Side scatter of a mixture of polystyrene beads with known diameter and refractive index was measured at different illumination powers on a customized BD FACSCanto. Poisson statistics was used to derive the number of statistical photoelectrons from the measured coefficient of variation of the bead populations. The resulting light scatter sensitivity was expressed as Q in photoelectrons/nm2, B in nm2, and a resolution limit (R) in nm2. As expected, Q scales linearly with the illumination power (R2 = 0.999), and B is constant over the illumination power (difference < 15%). The resulting R decreased from 1.75·105 nm2 at 20 mW, to 4.03·104 nm2 at 200 mW, which resembles the light scattering of a 450 nm and a 314 nm polystyrene bead, respectively. Comparison of R with the side scatter histograms confirmed that R describes the lower limit at which light scatter signals can be fully discriminated from the background noise. In conclusion, as a proof of principle, we derived Q, B, and R for a scatter detector of a flow cytometer where photon noise associated with the light scattering signal originating from the particle is the dominant source of the measured variation. To quantify the sensitivity of scatter detectors with nanoparticle (<100 nm) sensitivity, our approach requires monodisperse (variation coefficient < 2%) nanoparticles that scatter light isotropically. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.