Gatan 626 cryo tem holder

Venetin-1 at a concentration of 1 mg mL⁻¹ was ultrasonicated for 10 min at 40 °C in an ultrasonic chamber (Pol-Sonic, Poland). Then, the sample was vitrificated in a water solution on the TEM grid covered with Quantifoil R 2/2 carbon film (Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Before observations, the grids were activated with oxygen plasma for 15 s in a Femto plasma cleaner (Diener Electronic, Germany). Next, 3 μL of the Venetin-1 suspension was transferred onto the grid, blotted with filter paper and immersed in liquid ethane for instant freezing by Vitrobot Mark IV (FEI Company, USA). Before observations, the samples were stored in liquid nitrogen. To transfer the specimens to the TEM microscope, they were loaded into the Gatan 626 Cryo-TEM holder (Gatan Inc., USA)^{59 (link)}. The samples were observed using a Tecnai F20 X TWIN microscope (FEI Company, USA) with 200-kV acceleration voltage of the emission gun. An Eagle 4k HS camera (FEI Company, USA) was used to record the images.

Cryogenic
transmission electron microscopy (Cryo-TEM) was performed using a
Tecnai F20× TWIN microscope (FEI Company, USA) equipped with
a field emission gun operating at a 200 kV acceleration voltage. Images
were recorded with an Eagle 4k HS camera (FEI Company, USA) and processed
using the TIA software (FEI Company, USA). Prior to use, the grids
with a holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools
GmbH, Germany) were activated for 15 s in oxygen plasma using a Femto
plasma cleaner (Diener Electronic, Germany). 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 Vitrobot
Mark IV (FEI Company, USA). After preparation, the vitrified specimens
were kept under liquid nitrogen until they were inserted into a Gatan
626 cryo-TEM-holder (Gatan Inc., USA) and analyzed under the microscope
at −178 °C.

Cryo-TEM images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, OR, USA) equipped with field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Gatan Rio 16 CMOS camera (Gatan Inc., Pleasanton, CA, USA) and processed with Gatan Microscopy Suite (GMS) software (Gatan Inc., Pleasanton, CA, USA). 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). Before use, the grids were activated for 15 seconds 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 Vitrobot Mark IV blotting device (Thermo Fisher Scientific, Waltham, MA, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a Gatan 626 cryoTEM holder (Gatan Inc., Pleasanton, CA, USA) and analyzed in the TEM at −178 °C.