Gery Barmettler

We collected Tokuyasu ultrathin sections on silicon wafers. After washing in PBS and fixation with glutaraldehyde (0.1% in PBS) we applied methylcellulose to the wafers, centrifuged them, dried at 40 °C for 10 minutes and brought directly to the scanning electron microscope. Low voltage (1.5 keV) imaging of the samples enabled identification of synaptic vesicles, synaptic clefts, mitochondria, presynaptic and postsynaptic membranes (Fig. 1).

This method combines super-resolved protein localization with context information to determine the precise position of proteins in an organelle. We demonstrate here the completion of the experiment to visualize the expression of Tom20 in the outer membrane of mitochondria, and its relation to other organelles like nuclei or outer segments of the photoreceptor in the larval zebrafish retina. Tokuyasu cryo-sectioning requires some training to acquire well-preserved sections.