Elsa cryo holder

Specimens were prepared by
plunge freezing suspensions (at the original concentration) on copper
grids (300 mesh) containing lacey carbon film. Prior to use, the grids
were glow discharged using a Quorum Technologies GloQube instrument
at a current of 25 mA for 60 sec. The sample (3 μL) was pipetted
onto a TEM grid, blotted for 3 sec at blot force -5 using dedicated
filter paper, and immediately plunged into liquid ethane using a Vitrobot
Mark IV. The Vitrobot chamber was set to 4 °C and 95% humidity.
Specimens after vitrification were kept under liquid nitrogen until
they were inserted into a Gatan Elsa cryo holder and imaged in the
TEM at −178 °C. Images were collected using a Thermo Scientific
(FEI) Talos F200X G2 microscope at 200 kV at low dose using a Ceta
16M CMOS camera.

Cryo-TEM images were obtained using a Thermo Scientific (FEI Company) Talos F200X G2 microscope operating at 200 kV. Images were recorded at low dose using a Ceta 4k × 4k CMOS camera and Velox software. Specimens were vitrified by plunge freezing an aqueous suspension of bacteria (with salinity of 0.01% KCl) onto copper grids (300 mesh) with lacey carbon film. The sample was transferred onto the TEM grid, blotted for 3 s at blot force −10 using dedicated filter paper, and immediately frozen by plunging into liquid ethane using a fully automated and environmental controlled blotting device, Vitrobot Mark IV. The vitrobot chamber was set to 4°C and 95% humidity. After vitrification the specimens were kept under liquid nitrogen until they were inserted into a Gatan Elsa cryo holder and imaged in the TEM at −178°C.