An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 73 Framework Criteria What to report Please complete each criterion 4.4 Light Scatter Calibration. State whether and how light scatter calibration was implemented. Light scatter parameters may be reported in standardized units of nm2, along with information required to reproduce the model. Light scattering signals were fitted with Mie theory using a previously described model. 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 (n) 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. Data and theory were log10-transformed to scale the data onto the theory using a least-square-fit. 5.1 EV diameter/ surface area/ volume approximation. State whether and how EV diameter, surface area, and/or volume has been calculated using FC measurements. BF and SSC data of the PS beads were scaled onto Mie theory, resulting in a scaling factor (F) of 1.3518 and a coefficient of determination (R2) of 0.00 for the BF detector and a scaling factor of 8.405 and an R2 of 0.91 for the SSC detector. For the SSC detector, the theoretical model indicated a plateau between ~400 to ~800 nm, which translates into a low resolution when determining EV sizes based on SSC intensities within this region. The highest dynamic range was observed up to 400 nm - corresponding to a value of 900 a.u. SSC intensity. 5.2 EV refractive index approximation. State whether the EV refractive index has been approximated and how this was done. EV refractive index has not been approximated in this work - for Mie theory application, 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. 5.3 EV epitope number approximation. State whether EV epitope number has been approximated, and if so, how it was approximated. Other than conversion of fluorescent intensities into standardized units (ERF), no EV epitope numbers have been approximated in this work.
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