High-throughput single-vesicle imaging platform for direct extracellular vesicle profiling of human plasma.

Extracellular vesicles (EVs) are nanoscale particles secreted by cells that carry diverse biomolecules reflecting their cell of origin. Single-EV imaging approaches have enabled precise characterization of heterogeneous EV populations; however, their broader application is limited by low-throughput workflows and cumbersome EV isolation procedures. Here, we introduce a streamlined, high-throughput imaging platform capable of analyzing protein expression of individual intact EVs directly from unprocessed biological samples at the single-vesicle level. Our approach employs a functionalized glass surface optimized for high-throughput single-EV imaging, facilitating specific capture of EVs and enabling integration with existing automation technologies. We evaluate the platform's analytical capabilities by characterizing various recombinant EV samples and demonstrate its clinical utility by analyzing EVs in a total of 191 human plasma samples with high-throughput efficiency. This technology will offer a pathway for high-precision and large-scale characterization of EVs in clinical samples.