Determining the localization of proteins within a cell and their possible interactions is highly relevant to understand their functionality. Nevertheless, subcellular structures of interest in neurobiology, most importantly synapses with their pre- and postsynaptic compartments, are usually smaller than the resolution limit of conventional light microscopy. Indeed, diffraction of light limits to roughly half of the wavelength of light the resolution of a conventional light microscope. In this regard, super-resolution light microscopy (SRLM) techniques have emerged, achieving even more than ten times the resolution of conventional light microscopy, thus allowing to resolve subsynaptic structures also in situ. Importantly, stimulated emission depletion (STED) microscopy has been extensively used to image in situ the nanoscale organization of presynaptic compartments, such as the area of the presynaptic plasma membrane where synaptic vesicles fuse to release neurotransmitters, the so-called active zone. In this article, we outline a method to determine the localization of active zone scaffolding key players relative to voltage-gated calcium channels within the presynaptic active zone by time-gated STED (gSTED) microscopy in situ.