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Abstract

Release of synaptic vesicles (SV) is a process that is orchestrated by proteins present in the presynaptic terminus called the active zone (AZ). Knowledge of the placement of proteins is necessary to understand how SV release occurs. There is limited information on the location of the AZ proteins from studies of biochemical assays or immuno-electron microscope. Developments in fluorescence light microscopy are capable of reaching subnanometer resolution and therefore can be used to image multiple proteins of the AZ. For instance, a technique called direct Stochastic Optical Reconstruction Microscopy (dSTORM) can reach a resolution of 20 nm in the x-y plane, which is an order of magnitude greater than conventional light microscope. This work is devoted to developing techniques, which enables the use of dSTORM on thick brain tissue samples. In this respect, two thick tissue handling techniques have been explored, namely tomoSTORM and Tokuyasu’s ultracryotomy. Using tomoSTORM, we could construct a super-resolution 3D structure of the calyx of Held synapse. In addition, we also demonstrate multicolor capability by being able to localize the abundantly distributed mitochondria to the synaptic compartment of the calyx of Held. Due to antibody staining limitations, Tokuyasu’s ultracryotomy was explored. Using this approach we gathered dual-color super-resolution data in the calyx of Held on the distribution of Bassoon with respect to Piccolo. In agreement with the standardresolution microscopy, overview image of Bassoon and Piccolo show that both proteins exist together in the majority of the AZs. In addition we can show at the nanoscopic level in a given AZ that the two proteins not only exist as separate entities but are also found to be colocalized. We also gathered data on the distribution of Septin 5 and Piccolo and found that at P7 Septin 5 and Piccolo colocalize while at P17 they do not colocalize. This observation is consistent with the finding that Septin 5 may cluster voltage gated calcium channels at P7 at the AZ. In addition, as dSTORM is limited to photoswitching of 2 dyes, efforts were made to extend this. To this extent, we show efficient photoswitching of phalloidin conjugated to ATTO 488, TRITC and BODIPY 650. 8 In summary, this thesis is focused on adapting dSTORM to thick tissue samples and developing multicolor photoswitching probes to explore multiple protein distribution in the synaptic compartments of mammalian brain tissue.