Copper mesh grid

Purified NoV VLPs were loaded onto copper mesh grids (Electron Microscopy Sciences, Fort Washington, PA) and stained with 1.5% Uranyl Acetate. The samples were imaged using a F20 FEI Technai 200 kV field transmission electron microscope at the Cornell Center for Materials Research. Images were taken with a Gatan Orius dual-scan CCD camera at 1–3 s exposure time.

Uranyl acetate was used as negative stain, which allows the formation of a uniform, consistent, and high contrast staining. The sample was prepared on carbon film 400 copper mesh grids purchased from Electron Microscopy Sciences (Hatfield, USA). The specimen grids were exposed to glow-discharge treatment under air plasma for 20 s (2.0 × 10⁻¹ atm. and 35 mA) using a MED 020 modular high vacuum coating system (BAL-TEC AG, Balzers, Liechtenstein). Negatively charged carbon grids were used within 5 min after treatment to ensure hydrophilicity. The on-grid negative staining was performed using a slightly modified single-droplet negative-staining procedure. 1.5 μL sample droplet of NP concentration ranging from 6 to 15 nM followed by three 2.5 μL droplets of 0.25% weight/volume (w/v) uranyl acetate aqueous solution were placed on a clean Parafilm piece. The treated grid was incubated on the sample droplet for 1 min and then on the staining droplets for 3, 3, and 60 s, respectively. After each incubation step the excess fluid was nearly fully removed by touching the grid edge with Whatman filter paper. Finally, the sample was fully dried for 20 min at 2.0 × 10⁻¹ atm.

Exosome isolates were fixed in 2% paraformaldehyde, mounted on copper‐mesh grids (Electron Microscopy Sciences), and uranyl acetate at 2% was used for negative staining. JEOL 1230 transmission electron microscope (JEOL) was used for imaging. Image analysis was performed using a software package bundled with the microscope.