Nanoscopic Profiling of Small Extracellular Vesicles via High‐Speed Atomic Force Microscopy (HS‐AFM) Videography

Small extracellular vesicles (sEVs), which carry lipids, proteins and RNAs from their parent cells, serve as biomarkers for specific cell types and biological states. These vesicles, including exosomes and microvesicles, facilitate intercellular communication by transferring cellular components between cells. Current methods, such as ultracentrifugation and Tim-4 affinity method, yield high-purity sEVs. However, despite their small size, purified sEVs remain heterogeneous due to their varied intracellular origins. In this technical note, we used high-speed atomic force microscopy (HS-AFM) in conjunction with exosome markers (IgG^CD63 and IgG^CD81) to explore the intracellular origins of sEVs at single-sEV resolution. Our results first revealed the nanotopology of HEK293T-derived sEVs under physiological conditions. Larger sEVs (diameter > 100 nm) exhibited greater height fluctuations compared to smaller sEVs (diameter ≤ 100 nm). Next, we found that mouse-origin IgG^CD63, and rabbit-origin IgG^control and IgG^CD81, exhibited the iconic 'Y' conformation, and similar structural dynamics properties. Last, exosome marker antibodies predominantly co-localised with sEV_{d ≤ 100 nm} but not with sEV_{d > 100 nm}, demonstrating the CD63-CD81-enriched sEV and CD63-CD81-depleted sEV subpopulations. In summary, we demonstrate that nanoscopic profiling of surface exosome markers on sEVs using HS-AFM is feasible for characterising distinct sEV subpopulations in a heterogeneous sEV mixture.