Improving Multicolor Colocalization in Single-Vesicle Flow Cytometry with Vesicle Transit Time

Immunophenotyping of vesicles, such as extracellularvesicles (EVs),is essential to understanding their origin and biological role. Wepreviously described a custom-built flow analyzer that utilizes agravity-driven flow, high numerical aperture objective, and micrometer-sizedflow channels to reach the sensitivity needed for fast multidimensionalanalysis of the surface proteins of EVs, even down to the smallestEVs (e.g., & SIM;30-40 nm). It is difficult to flow focussmall EVs, and thus, the transiting EVs exhibit a distribution inparticle velocities due to the laminar flow. This distribution ofvesicle velocities leads to potentially incorrect results when immunophenotypingnanometer-sized vesicles using cross-correlation analysis (Xcorr),as the order of appearance of the vesicles might not be the same atdifferent spatially offset laser excitation regions. Here, we describean alternative cross-correlation analysis strategy (Scorr), whichuses information on particle transit time across the laser excitationbeam width to improve multicolor colocalization in single-vesicleimmunoprofiling. We tested the performance of the algorithm for colocalizationanalysis of multicolor nanobeads and EVs experimentally and via simulationsand found that Scorr improved both the efficiency and accuracy ofcolocalization versus Xcorr. As shown from Monte Carlo simulations,Scorr provided an & SIM;1.2-4.7-fold increase in the numberof colocalized peaks and ensured negligible colocalization of peaks.In silico results were in good agreement with experimental data, whichshowed an increase in colocalized peaks of & SIM;1.3-2.5-foldand & SIM;1.2-2-fold for multicolor beads and EVs, respectively.