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Building on our expertise in live-cell super-resolution STED microscopy and advanced opto-physiological approaches, we propose to investigate how neuron–astrocyte interactions shape synaptic signaling at the level of individual tripartite synapses. We will analyze the nanoscale organization and dynamics of peri-synaptic astrocytic processes and the surrounding extracellular space in living brain tissue, and determine how this microenvironment regulates the availability and spread of neurotransmitters and ions. By combining super-resolution imaging with biophysical measurements of molecular diffusion and synaptic activity, we aim to establish mechanistic links between the nano-anatomy of the tripartite synapse, extracellular space topology, and neuronal communication. These experiments will reveal how astrocytes and the extracellular milieu dynamically modulate synaptic function under physiological conditions.