Extracellular vesicles (EVs) are fundamental intercellular signaling organelles; these lipid-bilayer-enclosed particles are secreted by all cell types and carry a cargo of proteins, lipids, and nucleic acids to facilitate such cell communication and transfer. Advances in the field of single EV analyses, including profiling techniques, capture and detection strategies, digital technologies, multiplexing capability, machine learning, and resolution of molecular analysis have led new insights into EV biology, crucial for detecting subtle variations and rare EV populations, determine the origin and molecular features of EVs, and the absolute quantification of biomarker expression levels. This issue focuses on how single EV analysis through omics and imaging technology is advancing the characterization and refinement of EVs. These studies highlight how single-EV approaches reveal multimodal distributions, challenging assumptions of discrete EV classes toward often shared physical and molecular properties (including markers across EV types) and extending this toward other EV types, refining technology pipelines, and integrated analytical strategies.
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