2.8. Cryogenic Transmission Electron Microscopy

Cryogenic transmission electron microscopy (CRYO TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hilsboro, OR, USA) equipped with field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Eagle 4k HS camera (FEI Company, Hilsboro, OR, USA) and processed with TIA software (FEI Company, USA). Specimen preparation was carried out by vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Grosslobichau, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Ebhausen, Germany). Cryo samples were prepared by applying a droplet (3 µL) of the solution to the grid, blotting with filter paper and rapid freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, Hilsboro, Ore, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, CA, USA) and analyzed in the TEM at −178 °C.