Synapses are small, highly specialized structures of intercellular contact that play a crucial role in neuronal information processing and memory. While traditional fluorescence microscopy is an extremely powerful tool to study the dynamics of biological processes in vivo with molecular specificity, it has insufficient resolving power to dissect details of synaptic structure and functional organization or the structure of small subcellular components. In contrast, far-field optical super-resolution techniques provide spatial resolution at the nanoscale beyond the limit imposed by the diffraction of light. However, current super-resolution techniques are limited to thin brain preparations or to the surface of thick samples. The central aim of this proposal is to establish optical super-resolution methods for imaging chemical synapses in all layers of the cerebral cortex and in deep lying structures of the brain and to apply these techniques to timely questions in neurobiology. We will develop intravital super-resolution microendoscopy based on the stimulated emission depletion (STED) technique. This will enable intravital microscopy of arbitrary brain regions with diffraction-unlimited resolving power. In addition, we will miniaturize the setup, opening up the investigation of nanoscale structures in the brain of awake, freely moving and behaving animals. With this, we will be able to correlate for the first time synaptic structural or organizational plasticity at the nanoscale with behavioural stimuli in a living animal, including for instance in the hippocampus of an animal exposed to a learning situation. Furthermore, these methods will advance the understanding of the interplay between neurons and glia, in particular at synapses, and will help to shed light on brain structure and function in physiological states as well as in disease.