at room temperature in several steps: acetone/EPON 1:1 for 3 h, overnight incubation in 90% EPON in acetone at RT, 36 h incubation in pure EPON at RT. Finally, specimen carriers containing infiltrated samples were placed sample-side-up on Parafilm®-covered glass slides. EPON-filled gelatin capsules were inverted on the specimen carriers and polymerized for 24 h at 60°C.
2.6.2.3. Sectioning and contrasting
Ultrathin sections (60 nm) were cut with a diamond knife (Ultra 45°, Diatome) on a Leica Ultracut UCT ultramicrotome (Leica, Vienna, Austria). Sections were collected onto a Formvar-filmed, carbon-coated, copper mesh grid. Ultrathin sections were treated 30 min with 1% UA in ddH2O, washed three times by dipping the grids seven times in ddH2O, treated 2 min with 0.3% lead citrate, washed again and dried with filter paper. For electron tomography, 200 nm thick sections were collected onto Formvar-filmed, carbon-coated copper mesh grids. To introduce fiducial markers for tomographic reconstruction, a 1:3 dilution of Protein A (ProtA) conjugated to 10 nm gold particles (Utrecht, The Netherlands) in ddH20 was applied to both sides of the grid for 1 min each. The grids were washed briefly in ddH20 and dried with filter paper.
2.6.2.4. Imaging
For two-dimensional ultrastructural analyses of synaptic morphology, electron micrographs from ultrathin sections were acquired with a transmission electron microscope (Zeiss LEO 912-Omega) operating at 80 kV. Micrographs (2048 x 2048 pixels) of synaptic profiles were acquired with a sharp:eye CCD camera (Tröndle, TRS) at 12,500 fold magnification and a pixel spacing of 0.95 nm.
For the three-dimensional electron tomographic analysis of synaptic vesicle docking/priming, fiducially-coated 200 nm-thick sections were imaged in a JEM-2100 transmission electron microscope (JEOL) operating at 200 kV. The SerialEM software enabled automated tilt series acquisition by using predictions for specimen positions during the tilt series based on the positions at previous tilts (Mastronarde, 2005). Single-axis tilt series were recorded from −60 to +60° with 1° increment binned by the factor two at 30,000 fold magnifications with an Orius SC1000 camera (Gatan). The target defocus
during image acquisition was set to -0.2-0.4 µm. The individual images in the tilt series before tomogram reconstruction had 1336 x 1336 pixels with an individual pixel spacing of 0.52 nm.
2.6.2.5. IMOD Software package
All tomographic volumes were reconstructed from their respective tilt series with the IMOD package (Kremer et al., 1996; Mastronarde, 1997). Windows version required installation of a Unix-like environment called Cygwin. All IMOD programs use the MRC image file format (*.mrc) and for an in-depth introduction and a tutorial to the IMOD software, the webpage of the Boulder laboratory for 3-D electron microscopy (http://bio3d.colorado.edu/) can be consulted. Tomogram generation was performed with the graphical user interface eTomo of the IMOD software package in a multi-step process.
Afterwards, the *.mrc file extension was renamed in *.st to be recognized as single stack by eTomo. Second, in the pre-processing step, camera artefacts, like random x-rays causing extremely dark or bright pixels were removed by replacing them with neighbouring average values (program Ccderaser). In the following steps, the image stack was coarse aligned by using the programs Tiltxcorr, Xftoxg and Newstack. Then, a fiducial model was created computationally, based on the position of gold particles that were applied to both sides of the grid before image acquisition. These were then tracked through all projections by running the program Beadtrack. In the fine alignment step, the program Tiltalign was used to solve for displacements between the different tilted views to reduce the residual error mean. In a next step, three small sample tomograms (top, middle and bottom of the volume) were created to calculate the minimal volume of the final tomogram by defining the angle around the x- and y-axes and the z-position of the section after drawing boundary lines at the end of biological material in the sample tomograms. Then, in a final alignment step by running the program Tomopitch, a full-aligned stack was produced using linear interpolation. During reconstruction, the projections were binned three times, resulting in a final isotropic voxel size of 1.55 nm.
Finally, the actual tomogram was computed (program Tilt) using a back projection algorithm and was trimmed and adjusted to the right contrast in a post-processing procedure.
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2.6.2.6. Two-dimensional (2D) ultrastructural analysis of synaptic morphologyTwo-dimensional electron micrographs taken at 12,500 fold magnification were analyzed manually with the iTEM software version 5.1 (Olympus Soft Imaging Solutions GmbH).
The different parameters measured are depicted in Fig. 2.1 and comprised of the synaptic vesicle number, the presynaptic terminal area, the number of synaptic vesicles normalized to the terminal area (terminal density, Fig. 2.1 A), the synaptic vesicle cluster area, and the number of synaptic vesicles normalized to the cluster area (cluster density, Fig. 2.1.
B). Moreover, the length of the PSD (Fig. 2.1 C), the number of recycling endosomes per presynaptic terminal (Fig. 2.1 D, open arrows) and the number of LDCVs (Fig. 2.1 D, white arrows) were quantified. Synaptic vesicle terminal density and cluster density are specified as number of synaptic vesicles per 0.01 µm2 area. The PSD length is displayed in nanometers (nm).
Figure 2.1. Two-dimensional ultrastructural analysis of synaptic morphology
Ultrastructural analysis of presynaptic parameters in electron micrographs from ultrathin sections.
The number of synaptic vesicles (white crosses, A, B) within a presynaptic profile was normalized to the terminal area (blue, A) for the terminal density and to the cluster area (red, B) for the cluster density. The length of the postsynaptic density (white dotted line, C) was measured and the number of large dense core vesicles (LDCVs, white arrows, D) and endosomes (open arrows, D) was quantified. Scale Bar in D: 200 nm.
2.6.2.7. Three-dimensional (3D) electron tomographic (ET) analysis of synaptic vesicle docking
Synaptic vesicle docking analyses were performed on tomographic volumes reconstructed with an isotropic voxel size of 1.55 nm. Tomographic volumes were exported as TIFF image stacks (program tif2mrc) into ImageJ for quantitative analysis. The smallest vesicle distances from the outer leaflet of the synaptic vesicle membrane to the inner leaflet of the active zone membrane were measured using the straight line tool of the ImageJ software.
Only synaptic vesicles observed to be in physical contact with the presynaptic membrane at their midline were considered docked (0-2 nm distance). Synaptic vesicles that were close to the active zone membrane, but not in physical contact were categorized together with the membrane-attached synaptic vesicles in a second 0-4 nm bin. Moreover, membrane proximal synaptic vesicles (0-40 nm distance) and all vesicles within 100 nm of the active zone were quantified. The active zone area was calculated by measuring the active zone length on each consecutive slice using the freehand line tool in ImageJ. The obtained values were added and then multiplied by the factor of 1.55 nm (isotropic voxel size; z-dimension) to obtain the final active zone area per tomogram. The final results for the quantifications are displayed as the number of vesicles in the respective bin (0-2 nm;
0-4 nm; 0-40 nm and 0-100 nm) normalized to 0.01 µm2 active zone area. The mean synaptic vesicle diameter (d, nm) was calculated from the area (A) of the vesicle measured at its midline to the outer leaflet of the synaptic vesicle membrane using the elliptical selection tool in ImageJ (d=2√ (A/π)). As the sample sections were exposed to high electron doses during the image series acquisition, sample shrinkage in the z-dimension was commonly observed, resulting in deformed, compressed synaptic vesicles shapes. The mean synaptic vesicle volume (V) was therefore extrapolated from the mean area measured at the midline of the synaptic vesicle and the presented values therefore include the vesicular phospholipid bilayer (V= π d3/6).