Fig. 1. Calibration of scatter intensities through Mie theory.

a Gigamix polystyrene (PS) bead populations with sizes from 900 nm down to 100 nm were identified on the basis of SSC and FITC fluorescent intensities. b Counts and median scatter intensities of each PS bead population as detected by the brightfield (BF) and side scatter (SSC) detectors (Ch04 and Ch06, respectively). c Diameter vs Scattering cross section graphs. PS beads (green lines) were modelled as solid spheres with a refractive index of 1.5885 for a wavelength of 618.5 nm (brightfield) and 1.5783 for a wavelength of 785.0 nm (SSC). EVs (orange lines) were modelled as core-shell particles, with a core refractive index of 1.38 and a shell refractive index of 1.48 and a shell thickness of 6 nm for both wavelengths. The obtained scatter intensities of the PS beads as described in b were overlayed and a least-square-fit was performed to correlate theory and practice. Based on these correlations, SSC signal intensities were found to be indicative of particle size and a SSC cut-off of 900 a.u – corresponding to particles of 400 nm – was used in the rest in this work. F: scaling factor between scattering intensity and scattering cross section; n: refractive index.