Crossbeam 550 fib sem microscope

Cryo-FIB milling of the specimen was performed as described previously (10 (link)) using a Zeiss Crossbeam 550 FIB-SEM microscope and generated 200-nm thick lamellae. Subsequently, the EM grids were transferred to a Titan Krios transmission electron microscope operating at 300 kV, equipped with a Falcon 3 direct electron detector (Thermo Fisher Scientific) for cryo-ET data collection. Tilt series images were acquired bidirectionally using the SerialEM software (50 (link)) at 18,000× or 22,500× magnification (pixel sizes 4.6 Å or 3.7 Å, respectively) with a defocus of −4 μm, ±60° oscillation, 1° increments with a total final dose of 100 e⁻/Ų.

Cryo-fluorescence microscopy samples were transferred to the cryo-FIB-SEM microscope in liquid nitrogen using a Leica EM-VCM500 machine and a cryo-holder suitable for c-clipped grids (Leica Microsystems, Vienna, Austria). Prior to sample loading into the FIB-SEM microscope, grids were metalized by platinum sputtering (4 nm) using a Leica ACE600 cryo-sputter coater equipped with a cryo-stage (Leica Microsystems). Metallized samples were then transferred to a pre-cooled Crossbeam 550 FIB-SEM microscope (Carl Zeiss) equipped with a cryo-stage (Leica Microsystems) using an EM-VCT100 shuttle (Leica Microsystems). The cryo-stage was positioned at a working distance of 5.1 mm and tilted to 11° and further protected by a layer of cold-deposited organic-platinum (3 depositions in cycles of 30 s not heating the source at a working distance of 8 mm). Serial sectioning of the sample was followed to generate the FIB-SEM volumes using SmartFIB software (Carl Zeiss). Imaging conditions were 1.8 kV acceleration voltage and a probe current of 36 pA using the InLens detector and at a final pixel size between 4 and 9 nm. FIB milling was done at 30 kV and 300 pA with a sectioning step between 20 and 25 nm. Four different cells from each condition were imaged.