Abstract Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high‐speed atomic force microscopy (HS‐AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high‐temperature, high‐pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real‐time observation by HS‐AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS‐AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS‐AFM for structural characterization and assessment of nanoparticles.
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