Single high tilt holder

Electron tomography of epoxy resin sections of HP frozen, rapidly freeze-substituted ovisacs of A. franciscana was performed using a Tecnai-20 electron microscope at 200 kV equipped with an eucentric goniometer and a single high-tilt holder (FEI, Eindhoven, The Netherlands) Tilt series of digital images within an angular range of 65° to +65° and a tilt increment of 1° were recorded with the help of the Xplore 3D software (FEI company) using a Eagles 4K-CCD camera (FEI Company; chip size: 4.096 × 4.096 pixels). In order to reconstruct the volume of the 200–300 nm thick sections into virtual slices (thickness 0.39–0.46 nm corresponding to a microscope magnification of 29.000× or 25.000× respectively), we used the IMOD software (Boulder Laboratory for 3D Electron Microscopy of Cells, University of Colorado, USA). For 3D-modeling, the structures of interest in each slice were traced with colored contours that were merged in the Z-axis with the help of the Amira 5.3 software (Mercury Computer Systems, Merignac, Cedex, France).

300 nm thick sections were transferred to copper grids, and analyzed stained with uranyl acetate and lead citrate. Colloidal gold particles (10 nm) were then deposited on both surfaces of these sections for use as fiducial markers during subsequent image alignment. Single and dual axis tomography was performed in a Tecnai 20 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) at 200 kV by acquisition of tilt series at a tilt range of ±65° with an increment of 1° using either a single high-tilt holder (FEI Company) or a high-tilt rotation holder (Gatan, Inc., Pleasanton, CA) and an EAGLE 4k CCD camera (FEI Company; chip size: 4,096 x 4,096 pixels). Tilt series data were digitally recorded automatically with the Xplore3D software (FEI Company) which allows compensating dislocations of the region of interest during tilting. The digital images were stored into stack files (*.mrc). Tilt series were aligned using 10 nm gold fiducials and reconstructed by means of the weighted back projection (WBP) algorithm using the IMOD software (Boulder Laboratory for 3D Electron Microscopy of Cells, University of Colorado, USA).