Lacey carbon grid

Three microliters of the EV sample were placed on a Lacey carbon grid with a mesh size of 200 (Electron Microscopy Sciences, Hatfield, PA). After a 30-second waiting period, excess liquid was blotted, and the grid was submerged in liquid ethane for 4 seconds. Subsequently, the grid was transferred to liquid nitrogen and mounted on a Gatan 4022 cryo-holder to maintain a temperature below -180°C during cryo-TEM. The TEM images were captured using a Gatan Rio 16 camera.

Base and Etop -MNPs were prepared for scanning transmission electron microscopy (S/TEM) and energy dispersive x-ray spectroscopy (EDS) analysis with 10 washes of OmniTrace® Ultra picopure water (EMD Millipore Corperation, Billerica, MA) to remove contaminants. MNPs were drop cast onto Lacey Carbon Grids (Electron Microscopy Sciences, Hatfield, PA) and dried sterilely for 24 h. MNPs were visualized using a JEOL-ARM200CF electron microscope (JEOL USA, Inc., Glen Ellyn, IL) operated at 200 keV. EDS line scan data was collected 0–10 keV with 10 eV per channel and dwell time of 15 μs.
Samples for scanning electron microscopy (SEM) were dehydrated with ethanol followed by hexamethyldisilazane. Glass coverslips were adhered to aluminum mounts, then sputter-coated with 6.0 nm of Pt/Pd in a low pressure argon atmosphere. Surface morphology was examined using a Hitachi S-3000N Variable Pressure SEM (Hitachi America, Ltd., Santa Clara, CA) using secondary and backscatter detectors. MNPs have a high electron density and therefore appear bright on the images. For transmission electron microscopy (TEM), cells were fixed in 2.5% glutaraldehyde, dehydrated by an ethanol series, and embedded in epoxy resin. Ultra-thin sections (70 nm) were collected and stained with uranyl acetate and lead citrate. Specimens were examined using a JEOL JEM-1220 transmission electron microscope at 80 kV.

Lacey carbon grids (200 mesh, Electron Microscopy Sciences, Inc.) were glow-discharged for 20 s in a Pelco easiGlow glow-discharger (15 mA, 0.24 mBar). Each sample (4 uL) was pipetted onto a grid and plunge-frozen into liquid ethane using an FEI Vitrobot Mark IV cryo plunge freezing robot with a blotting time of 5 or 5.5 seconds and blotting pressure of 1. Frozen grids were stored in liquid nitrogen and then loaded into a Gatan 626.5 cryo transfer holder cooled down to −180 °C prior to image within a Hitachi HD2300 STEM at 200kV. Frozen samples were previewed with the phase contrast TE and HAADF detectors to verify sample preparation. With the sample still in the microscope, the cryo holder was slowly warmed up to −95 °C over the course of 30 minutes to sublime away ice within the vacuum. Imaging in the microscope continued using an SE detector in the STEM to image the surface of our particles. Image data was collected with Gatan Digital Micrograph and a Digiscan system. Any further image processing conducted on the aligned frames was completed in ImageJ.