Temcam 224a

Ultrathin sections of ~ 70 nm thickness were recorded on a Philips CM 120 BioTwin transmission electron microscope (Philips Inc., Eindhoven, the Netherlands) without counterstaining. Sections were taken in parallel to the growth surface of the cells. Usually, 2D images of at least 100 mitochondria from at least 10 different cells were randomly taken for each sample, using a TemCam 224A slow scan CCD camera (TVIPS, Gauting, Germany).
For ET, tilt series from 210‐nm‐thick sections from Agar100 embedded cells were recorded on a Talos L120C transmission microscope (Thermo Fischer Scientific/FEI company, Hilsboro, Oregon, USA) at 17,500× magnification using a Ceta 4k × 4k CMOS camera in unbinning mode. Orthogonal series were recorded from −64.5° to 64.5° using 3° saxton angular increase. The series were calculated using Etomo (David Mastronade, http://bio3d.colorado.edu/). Tomograms were processed using the nonlinear anisotropic diffusion (NAD) filter in IMOD (David Mastronade, http://bio3d.colorado.edu/imod/). Recordings of thin sections were processed in Fiji using the median filter.

For 2D analysis, images of ultrathin sections of ∼70 nm thickness were recorded on a Philips CM120 BioTwin transmission electron microscope (Philips Inc.). Usually, 2D images of at least 20 different cells were randomly recorded for each sample at 8,600× original magnification using a TemCam 224A slow-scan CCD camera (TVIPS).
For electron tomography, tilt-series of thin sections of ∼230 nm that were in addition decorated with 10-nm gold beads on both sides were recorded on a Talos L120C transmission electron microscope (Thermo Fisher Scientific/FEI Company) at 17,500× original magnification using a Ceta 4k × 4k CMOS camera in an unbinning mode. Series were recorded from −65.0° to 65.0° with a 2° dose-symmetric angular increase. The series were calculated using Etomo (David Mastronade; http://bio3d.colorado.edu/).

A solution containing protein was applied to glow-discharged carbon coated grids and stained with 1 % uranyl acetate (Sigma). Images were taken in a Philips CM120 electron microscope (Philips, Amsterdam, The Netherlands) at a defocus of 2.3 μm using a TemCam 224A slow scan CCD camera (TVIPS, Gauting, Germany).