Cells were grown on gridded sapphire discs coated with poly-L-lysine for 2 h. Samples were immediately frozen on a Leica EM HPM100 high-pressure freezing machine. Freeze substitution was done as recently described (Müller et al., 2021 (link)) with some modifications. Briefly, samples were substituted in acetone containing 2% osmium tetroxide, 1% uranyl acetate, 0.5% glutaraldehyde, 5% water, and 1% methanol at −90°C for 46 h. Afterward, the temperature was raised to 0°C over 15 h. At this temperature, the substitution medium was changed to pure acetone and the temperature was increased to 22°C in four 15-min steps. Between steps, the acetone was exchanged for the new one at the same temperature. Afterward, samples were incubated in 0.2% thiocarbohydrazide in 80% methanol at RT for 1 h and washed four times for 15 min in acetone, followed by 2% osmium tetroxide for 1 h, brief washing in acetone, 1% uranyl acetate in 10% methanol for another hour, and washing for 4 × 15 min in acetone. Then the samples were embedded into resin with increasing concentrations (Araldite 502/Embed 812; 25, 50, 75, and 100%), 1 h each step, and left overnight in the freshly made 100% resin. After 48 h of polymerization at 60°C, the sapphire discs were removed and samples were coated with 25 nm of the platinum and imaged by FIB-SEM (FEI Helios NanoLab 660 G3 UC).