EV Immobilization and SEM Imaging

A drop of 0.01 % (w/v) of poly-L-lysine (Thermo Fisher Scientific, Waltham, MA) was spread over the surface of a coverslip and incubated for 30 min. The functionalized coverslip was subsequently washed with deionized water and air-dried. The EVs collected from the suspended ^EVμBRs were purified via TFF^{43 (link)} and were electrostatically immobilized onto the surface for 30 min. The EVs were fixed in 2 % (v/v) glutaraldehyde (Sigma-Aldrich, St. Louis, MO) and 0.1 M sodium cacodylate (Electron Microscopy Sciences, Hatfield, PA) for 3 h. The EVs were then incubated in 1 % (w/v) osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) and 0.1 M sodium cacodylate for 2 h. The sample was dehydrated by incubating the immobilized EVs in varying concentrations of ethanol, including 50 % (v/v), 70 % (v/v), 85 % (v/v), 95 % (v/v), and 100 % (v/v) for 30 min each with intermediate washes of 0.1 M sodium cacodylate. The sample was critical-point dried with a CO₂ critical point dryer (Tousimis, Rockville, MD). The fixed and dehydrated samples were coated with a 2-nm gold coating using a sputtering machine (Leica EM ACE 600, Buffalo Grove, IL) and were subsequently imaged by SEM (Apreo ii, FEI, Thermo Fisher Scientific, Waltham, MA).