Basic Science Imaging Platform

Electron tomography (ET) allows the 3D-visualization of subcellular structures. It is a powerful alternative to the 3D-visualization obtained from ultrathin serial sections. ET is performed with semithin sections with a thickness of 200-300 nm. These sections can be obtained from tissues or cultured cells embedded in resin.


Brief technical description

In our department, ET is performed using a Tecnai-20 transmission electron microscope (FEI, Eindhoven, The Netherlands) containing a eucentric goniometer. For single axis ET, a single tilt holder (FEI), for dual axis ET, a rotation holder (Gatan, Inc., Pleasanton, USA) is used in order to orientate the sections. Series of tilted images (maximal range: -70° to +70°) are acquired with a tilt increment of 1-2° by the help of the Explore 3D software (FEI), which performs a holder calibration procedure to compensate dislocations of recorded tilt images and allows an automatic adjustment of eucentric height and autofocus before acquisition. The volume of the semithin sections is reconstructed into serial slices using the software packages Inspect 3D (FEI) or IMOD (Boulder Laboratory, University of Colorado) implicating alignment and filter tools as well as a reconstruction module. Reconstructions are performed by weighted back projection (WPB) and/or by the simultaneous itinerative reconstruction technique (SIRT). 3D models are created by tracing the structures of interest in every slice with colored contours that are merged in the Z axis using the Amira 4.3 software (Mercury Computer Systems, Merignac Cedex, France). (J Neumüller/M Pavelka, March 2012)