Time-resolved surface-sensitive waveguide scattering microscopy of single extracellular vesicles reveals content and biomarker heterogeneity

Deciphering the extracellular vesicle (EV) heterogeneity is essential for understanding their biological functions and enhancing their utility in drug development and medical diagnostics. Here, we demonstrate the efficacy of label-free surface-based waveguide scattering microscopy, coupled with multiplexed fluorescence readout, for quantifying size, inner cargo concentration, and surface protein markers at the individual EV level. By modulating the optical contrast between EVs and the surrounding medium using membrane-permeable and -impermeable solutes, we identify subpopulations of low-cargo EVs and cell-specific markers for EVs derived from platelets and red blood cells. Time-resolved label-free antibody binding to single blood-derived EVs reveals variations in binding rates and facilitates the quantification of surface protein marker density. Additionally, we observe that the presence of tetraspanins, well-known EV markers, correlates with biomolecular content but not size. These findings highlight the crucial role of time-resolved surface-based quantitative EV analysis in complementing current methodologies, offering unparalleled insights into EV characterization.