Three to four microliters of the EV solution (1:20 dilution) was added to Lacey carbon grids (300-mesh; Ted Pella, Inc., Redding, CA, USA) that were negatively glow-discharged for 80 s at 30 mA. Excess sample was removed by blotting once for 3 s with Vitrobot filter paper (Ted Pella, Inc, Redding, CA, USA) and then the grid was plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot plunge-freezer (ThermoFisher Scientific, Hillsboro, OR, USA).
The vitrified vesicle samples were imaged using a Talos Arctica 200 kV transmission electron microscope (ThermoFisher Scientific, Hillsboro, OR, USA) equipped with a Gatan K3 camera (Gatan, Inc., Pleasanton, CA, USA) The SerialEM software was used to collect images under low-dose conditions at 36,000 × magnification corresponding to a pixel size of 1.14 Å/pixel. For each image, 50 frames were recorded over 2.5 s exposure time at a dose rate of 35 electrons/pixel/s. The movie frames were aligned using MotionCorr2 (2) under Relion (Zivanov et al. 2018 (link)).
The vitrified vesicle samples were imaged using a Talos Arctica 200 kV transmission electron microscope (ThermoFisher Scientific, Hillsboro, OR, USA) equipped with a Gatan K3 camera (Gatan, Inc., Pleasanton, CA, USA) The SerialEM software was used to collect images under low-dose conditions at 36,000 × magnification corresponding to a pixel size of 1.14 Å/pixel. For each image, 50 frames were recorded over 2.5 s exposure time at a dose rate of 35 electrons/pixel/s. The movie frames were aligned using MotionCorr2 (2) under Relion (Zivanov et al. 2018 (link)).
