Figure 1.
A custom-built positioning device to mount ribbons of serial sections onto a hydrophylized silicon wafer.
A: Frame carrying an angled micromanipulator and forceps for wafer positioning. B: Wafer positioning frame mounted onto the ultramicrotome. C: Positioning of the wafer in the diamond knife boat (blue part). Note the extended size of the knife boat. D: Close-up showing the position of the wafer close to the edge of the diamond knife. A tissue block mounted on the microtome arm is visible on the left. E: A silicon wafer strip glued to the SEM sample holder within the sample chamber of the SEM. F: Close-up showing the wafer mounted on sample holder. The wafer contains two ribbons (black stripes). A jumper link was used to establish a conductive connection between the wafer and the sample holder (arrows).
Figure 2.
High resolution images can be obtained from ribbons of serial sections via SEM.
A: Low magnification SEM image of a ribbon mounted on a silicon wafer. B: Low magnification images of sections cut in trapezoid shape to determine the orientation of the tissue. The sections are evenly space by resin throughout the ribbon. C: Intermediate magnification SEM images that allow the identification of landmarks in the tissue like blood vessels (asterisks) and axon bundles (arrows). D: Intermediate magnification overview image of a MNTB principle neuron. Boxed area is shown at high magnification in E. E, F: High magnification (10,000fold in E, 20,000fold in F) SEM images of a calyx of Held synapse segment. Boxed area in E is shown at higher magnification in F.
Figure 3.
S3EM provides TEM-like images of cellular ultrastructure.
A, B: SEM images of subcellular structures acquired at 10,000fold magnification (3.7 nm/pixel). Mitochondria (*), synaptic vesicle clusters (+), active zone (black arrow head), plasma membrane (white arrow head), postsynaptic density (thick arrow), ribosomes (thin arrow).
Figure 4.
S3EM enables the acquisition of high resolution and high quality electron micrographs from large tissue volumes.
A: Consecutive sections of a segment of the calyx of Held synapse (coH) and the corresponding MNTB principle cell (postsyn.) at 10,000fold magnification (3.7 nm/pixel). Cellular protrusions can be followed through the tissue (asterisks), and cellular compartments and fine structure can be recognized. Boxed areas are shown in detail at smaller scale in B–E. B: Detailed image of a synaptic release site, characterized by the cluster of synaptic vesicles and the electron-dense postsynaptic density. Scale as in D. C: Detailed image of a mitochondrion. The cristae are clearly visible. Scale as in D. D: Detailed image of a synaptic vesicle cluster. E: Detailed image of a part of the synaptic cleft.
Figure 5.
3D reconstruction of a calyx of Held segment based on SEM images obtained from 100 consecutive sections.
A: Full reconstruction of a 31.7 µm3 calyx segment. The innervation side is orientated to the front. Red: plasma membrane; yellow: postsynaptic densities; blue: mitochondria; green: synaptic vesicles. B: Close up showing only plasma membrane and postsynaptic densities (colours as in A). C: Close up showing plasma membrane, postsynaptic densities and synaptic vesicles (colours as in A). D: 3D reconstruction of a single release site (colours as in A). E: Histogram of the distribution of distances between the presynaptic membrane and the synaptic vesicle membranes based on the release site shown in D.