An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 33 IFCM to discriminate single-size populations of fluorescent sub-micron beads by measuring two commercially available mixtures of FITC-fluorescent polystyrene (PS) beads of known sizes (Megamix-Plus FSC – 900, 500, 300 and 100 nm, and Megamix-Plus SSC – 500, 240, 200, 160 nm). Within the Megamix-Plus FSC mix, we acquired a 300/500 nm bead ratio of 2.2, which is within the manufacturers internal reference qualification range (1.7 – 2.7 ratio). Next, we mixed both bead sets in a 1:1 ratio (‘Gigamix’) and performed acquisition. Figure 1a shows that IFCM is able to discern all seven fluorescent bead populations, as well as the 1 µm-sized Speed Beads (SB), via the FITC (Ch02) and side scatter (SSC - Ch06) intensities. Calibration of scatter intensities through Mie theory The output of IFCM signal intensities are presented in arbitrary units (a.u.), which hinders data comparability (and reproducibility) with different flow cytometers. Since light scattering of spherical objects is dependent on particle size and refractive index, Mie theory can be used to relate the scatter intensity of events to their size given their refractive index 32. Generally, Mie theory is applied to calibrate the scatter channels of a FC (forward- and/or sideward-scattered light - FSC or SSC, respectively); however, IFCM utilizes a brightfield detection channel (BF, Ch04) as opposed to FSC. Mie theory was applied on both scatter detection channels (BF and SSC). As a first step, we extracted the BF and SSC median scatter intensities of each identified size population of PS beads (Figure 1b). Coefficient of variation (CV) analysis for each single PS bead population showed scores ≥8% for the BF detector irrespective of bead size, whereas CV scores for the SSC detection channel were observed to increase with decreasing bead sizes – indicating that the detection of smaller particles is close to the detection limit of the SSC detector in our setup. Next, 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 (Figure 1c). Thus, signals from sub-micron PS beads measured with the BF detector do not provide quantitative information. The SSC detector, on the other hand, can be readily calibrated. For the SSC detector, the theoretical model indicates a plateau for EVs with a diameter between ~400 to ~800 nm, which translates into a low resolution when determining EV sizes based on SSC intensities within this region. To ensure inclusion of sub-micron EVs, a gate was set at SSC below the scattering intensity corresponding to the plateau, namely 400 nm EVs, corresponding to a value of 900 a.u. SSC intensity.
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