Autotem 5

We used an FIB/SEM (Helios 5 UX, Thermo Fisher Scientific) with the latest automation software (AutoTEM 5, Thermo Fisher Scientific). A motorized manipulator (EasyLift, Thermo Fisher Scientific) is integrated with the FIB/SEM and controlled via AutoTEM 5 software. Detail workflow of AutoTEM 5 is explained in the main text and Supplementary Methods.

Vitrified cells were further processed by cryo-FIB milling for the preparation of lamellae. A dual-beam microscope FIB/SEM Aquilos 2 (Thermo Fisher Scientific) equipped with a cryo-transfer system (Thermo Fisher Scientific) and rotatable cryo-stage cooled at −191 °C by an open nitrogen circuit was used to carry out the thinning. Prior to the milling, the grids were mounted on the shuttle and transferred onto the cryo-stage, followed by the coating with an organometallic Platinum layer using the GIS system (Thermo Fisher Scientific) for 5–6 s. Then, cells positioned approximately in the centres of grid squares were selected for thinning. The thinning was conducted by the automated milling software AutoTEM 5 (Thermo Fisher Scientific) in a stepwise manner from current 0.5 μA to 30 pA at 30 kV, and the final thickness of lamellae was set to 120 nm.

TEM lamellae were prepared by FIB (Helios 5 UX, Thermo Fischer Scientific) using AutoTEM 5 (Thermo Fischer Scientific). Carbon deposition was used to protect the surface. The chunk milling and lamellae thinning were done at 30 kV with an FIB current from 20 nA to 90 pA. Then the lamellae were polished at 5 kV and finished at 2 kV. TEM characterisation was performed on a TEM (Talos F200X, Thermo Fischer Scientific) operating at 200 kV. EDS mapping was acquired by using quadrant EDS detectors (Super-X, Thermos Fisher Scientific, the Netherlands) in STEM mode.