In the mammalian brain, a large network of excitable and modulatory cells efficiently processes, analyzes and stores vast amounts of information. The brain's anatomy influences the flow of neural information between neurons and glia, from which all thought, emotion and action arises. Consequently, one of the grand challenges in neuroscience is to uncover the finest structural details of the brain in the context of its overall architecture. Recent developments in microscopy and biosensors have enabled the investigation of brain microstructure and function with unprecedented specificity and resolution, dendritic spines being an exemplary case, which has provided deep insights into neuronal mechanisms of higher brain function, such as learning and memory. As diffraction-limited light microscopy methods cannot resolve the fine details of brain cells (the 'anatomical ground truth'), electron microscopy is used instead to contextualize functional signals. This approach can be quite unsatisfying given the fragility and dynamic nature of the structures under investigation. We have recently developed a method for combining super-resolution stimulated emission depletion microscopy with functional measurements in brain slices, offering nanoscale resolution in functioning brain structures. We describe how to concurrently perform morphological and functional imaging with a confocal STED microscope. Specifically, the procedure guides the user on how to record astrocytic Ca