Visualizing Extracellular Vesicles by SEM

For SEM imaging, typically 200–400 μL of EVs were mixed with uranyl acetate (0.05 wt%) or hemin (final 1 mg mL⁻¹ w/v) for 10 min at room temperature to allow labeling. EVs were purified by size-exclusion chromatography (SEC), characterized and loaded into hydrogels as described above. Hydrogels containing labeled EVs, native EVs, or PBS control gels were dehydrated with increasing amounts of methanol (10–100% v/v in water), for 1 h at each step and air dried, snap frozen in liquid nitrogen, and cut with a razor blade. Samples were attached to aluminum stubs with carbon tape, silver paint was spread on the sample sides, and coated with 5 nm carbon (Quorum Technologies Turbo-Pumped Thermal Evaporators model K975X). Gels were imaged by SEM (Zeiss Auriga) operated at 5 kV, equipped to record in-lens and secondary electrons, and in backscatter mode. The density-dependent color SEM analysis was executed as described previously,[17 (link)] briefly the in-lens or secondary electron image was assigned to the green channel while the backscattering signal was assigned to the red channel; images were stacked using ImageJ.