Chapter 3 58 the log of these values against the log of the known ERF values. The resulting linear function was used to relate the log of BV421/CFSE/APC fluorescent intensities to the log of ERF values. Light scatter theory and Mie calculations for IFCM Light scattering signals of bead populations from Gigamix were fitted with Mie theory using a previously described model 32. The BF detector was modelled as a forward scattered light detector collecting light using a lens with a numerical aperture (NA) of 0.9, which corresponds to the NA of the 60x objective. The center wavelength of brightfield detection was 618.5 nm. The SSC detector was modelled as a detector that is placed perpendicular to the propagation direction of the laser beam. The NA of the collection lens was 0.9 and the wavelength was 785.0 nm. PS beads 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 were modelled as core-shell particles with a core refractive index of 1.38, shell refractive index of 1.48 and a shell thickness of 6 nm for both wavelengths as the dispersion relation for the core and shell of EVs is unknown. Beads were measured in water, and EVs in PBS. Therefore, the refractive indices of PBS and water were assumed to be 1.3345 and 1.3325, respectively, at a wavelength of 618.5 nm (BF) and 1.3309 and 1.3289, respectively, at a wavelength of 785.0 nm (SSC). Effective scattering cross sections of the calibration beads were calculated by integrating the amplitude scattering matrix elements over 576 collection angles 32. Data and theory were log10-transformed to scale the data onto the theory using a least-square-fit. Data acquisition (Step VII) All samples were analyzed on an ImageStreamX MKII instrument (ISx; Luminex, Texas, USA) equipped with 4 lasers set to the following powers: 405 nm: 120 mW, 488 nm: 200 mW, 642 nm: 150 mW, and 775 nm (SSC): 1.25 mW. The instrument calibration tool ASSIST® was used upon each startup to optimize performance and consistency. The ISx was equipped with three objectives (20x/40x/60x) and 1 CCD camera. Settings previously established by Görgens et al. 21 were used in our experiments. All data were acquired using the 60x objective (numerical aperture of 0.9 – wherein 1 pixel images an area of 0.1 µm2) with fluidics settings set to “low speed/high sensitivity” – resulting in a flow speed of 43.59 ± 0.07 mm/sec (mean ± standard deviation).
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