Fluorescence Characterization of Extracellular Vesicles Using Single‐Molecule Confocal Microscopy

Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the extracellular environment. They play a pivotal role in cell communication and have recently gained prominence as biomarkers. However, their low abundance and high heterogeneity challenge their accurate characterization using conventional approaches. To enable the specific detection of individual EVs, EV-specific antibodies labeled with two different fluorophores are coupled with fast-flow microfluidics and single-molecule confocal microscopy. This allows for the determine the concentration of EVs down to sub-femtomolar levels (≈10⁷ EVs mL^-1), and the approach demonstrates the capacity to detect EVs even in the presence of other lipid vesicles. The ability to quantify EVs in serum and plasma samples, without the need for purification is highlighted. Furthermore, the yield of EVs extracted from both serum and plasma is compared using ultracentrifugation and various size exclusion chromatography approaches. Overall, the method offers a highly specific, sensitive and easy-to-use solution for characterizing EVs from different sources.