Femto plasma cleaner

Cryogenic transmission electron microscopy (cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, OR, USA) equipped with a field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Gatan Rio 16 CMOS 4k camera (Gatan Inc., Pleasanton, CA, USA) and processed with Gatan Microscopy Suite (GMS) software (Gatan Inc., Pleasanton, CA, USA). The specimen preparation was done by vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, 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 suspension to the grid, blotting with filter paper, and immediately freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, 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.

The organic functional groups present on the exterior surface of the mesoporous silica films were removed by applying a CO₂ plasma treatment using a Diener Femto plasma cleaner (Diener electronic, Ebhausen, Germany) at a pressure of 0.4 mbar and power of 20% for 12 s according to a previously published protocol by Babu et al. [34 (link)].

The AAF solution with a protein concentration of 1 mg mL⁻¹ was placed in an ultrasonic chamber (Pol-Sonic, Poland) for 10 min at 40°. Next, the preparation of the AAF specimen consisted in vitrification of an aqueous suspension on the TEM grid with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using the Femto plasma cleaner (Diener Electronic, Ebhausen, Germany). The samples of AAF were vitrified by applying a droplet (3 μL) of the suspension to the grid, blotting with filter paper, and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, Hillsboro, Oregon, USA). After preparation, the vitrified specimens were kept in liquid nitrogen until they were inserted into the Cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, USA) providing a sufficiently low temperature (− 178 °C) during the transfer of the samples to the microscope and during the TEM analyses^{25 (link)}. Cryogenic Transmission Electron Microscopy (Cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, Oregon, USA) equipped with a field emission gun (FEG) operating at the acceleration voltage of 200 kV. Images were recorded with an Eagle 4k HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA).

Cryogenic Transmission Electron Microscopy (cryo-TEM) images were collected with a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, OR, USA) equipped with a field emission gun operating at an acceleration voltage of 200 kV. Images were recorded on the Gatan Rio 16 CMOS 4 k camera and processed with Gatan Microscopy Suite (GMS) software (Gatan Inc., Pleasanton, CA, USA). Specimen preparation was done by the vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Prior to use, the grids were treated 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 suspension to the grid, blotting with filter paper, and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). The vitrified specimens were kept under liquid nitrogen prior the insertion into a cryo-TEMholder Gatan 626 (Gatan Inc., Pleasanton, CA, USA) and analyzed at −178 °C.