Chapter 3 34 These data show that 1) IFCM is able to readily discern sub-micron sized EVs based on their emitted fluorescence and SSC intensities, and 2) SSC – but not BF – light scattering intensities can be used to approximate particle sizes (following Mie calculations). The standardization of SSC signal intensities followed by the setting of a sub-micron gate provides a tool to selectively analyze all fluorescent EVs in complex samples such as plasma, as long as these particles emit detectable fluorescent intensities. Figure 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.
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