See the Unseen: Rapid, Multiparametric Single‐Particle Analysis of Extracellular Vesicles With Nano‐Flow Cytometry

Extracellular vesicles (EVs) are heterogeneous lipid membrane-bound nanoparticles that carry diverse biomolecular cargos, including proteins, nucleic acids, lipids, and metabolites. Serving as fundamental mediators of intercellular communication in both physiological and pathological contexts, EVs have become a central focus in basic and translational research. Their participation in diverse biological processes-from immune regulation and tissue homeostasis to cancer progression and neurodegenerative diseases-highlights their dual functional roles. However, the intrinsic heterogeneity of EVs poses significant challenges for accurate characterization using conventional ensemble-averaging methods. Nano-flow cytometry (nFCM) represents a transformative technological advancement for single-particle EV analysis, offering high sensitivity, throughput, and multiparametric detection capabilities. This review first examines recent progress in nFCM instrumentation that improves sensitivity and resolution. We then summarize key applications of nFCM in uncovering previously inaccessible biophysical and biochemical characteristics of EVs at the single-particle level. Finally, we discuss current limitations hindering broader implementation and outline future directions to advance the field.