Multifluorescence Single Extracellular Vesicle Analysis by Time-Sequential Illumination and Tracking.

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.