FIGURE 1.

Procedure of single‐vesicle imaging and co‐localization analysis. A simple fluidic channel made of DDS functionalized cover and slide glasses was incubated with biotinylated BSA, then passivated with Tween‐20. Avidins were then introduced to the surface to immobilize biotinylated EVs. Unbound molecules were washed out after each step to prevent unwanted interactions among the molecules. Immobilized EVs were labelled with probes, and the EVs were imaged with multiple excitation lasers and a multi‐colour simultaneous fluorescence imaging device equipped with EMCCD camera. Acquired signals were analyzed for signal counts and co‐localizations for investigating EV heterogeneity and subpopulation