Label-Free and Three-Dimensional Visualization Reveals the Dynamics of Plasma Membrane-Derived Extracellular Vesicles

Plasma membrane-derived extracellular vesicles (PEVs) are carriers of biological molecules that perform special cell-cell communications. Nevertheless, the characterization of complicated PEV biology is hampered by the failure of current methods, mainly due to lack of specific labels and insufficient resolution. Here, we employed atomic force microscopy and scanning ion conductance microscopy, both capable of three-dimensional nanoscale resolution, for the label-free visualization of the PEV morphology, release, and uptake at the single-vesicle level. Except for classical microvesicles, we observed a cluster-like PEVs subtype in tumor cells. Moreover, both PEV subtype release times positively correlated with size. Through three-dimensional nanoscale imaging, we visualized the multiform PEV-cell interaction behaviors of individual vesicles, which was challenged in conventional PEV imaging. Finally, we developed single-cell manipulation strategies to induce micrometer-sized PEV generation. Collectively, these results revealed the heterogeneous morphology and dynamics of PEVs at the single vesicle level, which provided new insight into the PEV biology.