Nanoscale Fluorescent Characterizations and Origin Tracing of Extracellular Vesicles through Standard Preparation...

Extracellular vesicles (EVs) facilitate intercellular communication and have emerged as valuable diagnostic and prognostic biomarkers, therapeutic tools, and delivery systems. Due to their heterogeneity, precise profiling of EVs requires sub-EV resolution beyond standard EV biomarkers. Additionally, there is a growing need to trace the biogenesis of individual EVs. While super-resolution microscopy enables EV profiling at the nanoscale with biochemical specificity, the lack of standardized workflows for EV purification, immobilization, and labeling continues to limit reproducibility and biological interpretation. Here, we demonstrate the use of two standard purification methods to prepare EVs isolated from two cellular sources for nanoscale characterizations of CD81 using direct stochastic optical reconstruction microscopy (dSTORM). Using polymer-based precipitation and size-exclusion chromatography, we isolated EVs from cultured cells and immobilized them on poly-l-lysine-coated glass surfaces for immunofluorescence staining. dSTORM imaging revealed broad size distributions of EVs and variations in the nanoscale spatial organization of CD81. In both Jurkat T cells and hepatocellular carcinoma cells (HCC), we identified a subpopulation of EVs below the optical diffraction limit with asymmetrical CD81 distributions and developed quantification metrics to characterize this feature. In addition, we demonstrated the successful distinction between ectosomes and exosomes using a pan-membrane-protein labeling strategy. Together, these findings establish EV isolation methods coupled with dSTORM as a robust strategy for high-resolution sub-EV analysis, enabling the precise characterization of EV heterogeneity and biomarker discovery.