An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 51 background produced by samples. We established the background level of the IFCM with respect to sub-micron particle quantification at ~E5 objects/mL (after sample dilution correction). Previously published work by Görgens et al. showed that IFCM is able to accurately quantify single EV (cell culture-derived) up to concentrations of ~E8 objects/mL 21. Together, these data suggest that single EV quantification with IFCM is optimal for samples between E5 – E8 objects/mL (as demonstrated in this work). To identify single EVs present in the PPP samples, we designed a gating strategy based on the imaging capabilities of IFCM. Several key features or advantages that contribute to IFCM being a more powerful platform for EV analysis compared to conventional FC include the slower flow rate, CCD-camera based detection (enabling higher quantum efficiency compared to conventional photon multiplier tubes), and integration of detected signals over time using TDI 21. Additionally, IFCM allows automatic triggering on all channels during acquisition, and thus EVs devoid of SSC signals may still be detected based on their fluorescent probes. Conversion of scatter intensities from arbitrary units into standardized units (using light scatter theory and Mie calculations 32) enhances reproducibility across different FC platforms. By performing these calculations for the BF and SSC detector channels, we demonstrated that measured PS bead signal intensities in the BF channel did not correlate with the theorized model. Thus, although the BF channel has its merits for cell-based research, it should not be used for EV-based research. The high degree of correlation between predicted and measured scatter intensities (R2 = 0.91) for the SSC detection channel underlines the utility of the SSC channel to relate scatter signals to standard units. Both size and fluorescence calibrations are key in the validation of sub-micron sized particle detection and reproducibility of the generated data, respectively 18. In line with previously published literature, we have shown that IFCM is able to discriminate PS particles down to 100 nm on the basis of their emitted fluorescent intensities 21. Regarding fluorescent calibration, we standardized the generated fluorescent intensities into ERF values using Rainbow Calibration Particles (RCP). It should be noted that ERF assignments to RCP are derived from a reference instrument, and comparisons across instruments are expected to vary with filter and laser configuration, variations that can be measured and accounted for by cross-calibration against MESF or antibody capture beads 33.
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