Leo 912 ab omega tem

2-D TEM was carried out in a LEO 912 AB Omega TEM (Zeiss, Oberkochen, Germany) at 80 kV. The images were filtered at zero energy loss and recorded with a TRS 2k Slow-Scan CCD camera (Tröndle Restlicht Verstärker Systeme, Moorenweis, Germany).
The “slice and view” technique was carried out at a Zeiss Auriga 40 crossbeam workstation (Carl Zeiss Microscopy, Oberkochen, Germany) to obtain tomographic datasets. FIB milling was performed with 2−5 nA milling current of the Ga-emitter. The slice thickness was chosen between 10−16 nm. SEM micrographs of the block faces were taken with an aperture of 60 µm in high-current mode at +0.5 kV of the in-lens EsB detector. The alignment (semi-automatically) of the FIB/SEM image series and the segmentation (manually) was done with Amira™ (Thermo Fisher Scientific).

2-D TEM was performed either in a LEO 912 AB Omega TEM or in a Libra 120 TEM (both Zeiss, Oberkochen, Germany) operated at 80 kV. 3-D TEM tomography was done in a LEO 912 AB Omega TEM at 120 kV. Images were always filtered at zero energy loss and were captured with a TRS (Tröndle Restlicht Verstärker Systeme, Moorenweis, Germany) 2k Slow-Scan CCD camera at both TEMs employed.
For TEM tomography, Formvar coated grids with approximately 200 nm sections were incubated in 2 μl of 15 nm colloidal gold particles (BBI Solutions, Cardiff, United Kingdom) on both grid sides for subsequent alignments. During the tomographic process, samples were automatically tilted between −70° and +70° with an increment of 1°. Acquisition and calibration was performed by iTEM Software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). The tilting series of the TEM tomography were first stack aligned with iTEM several times. For the actual alignment with IMOD (University of Colorado, Denver, CO, United States), a conversion with ImageJ (Schneider et al., 2012 (link)) was done. Segmentation was carried out manually with Amira Software (Visualized Sciences Group, Hilsboro, OR, United States).