The advent of super-resolution microscopy has created unprecedented opportunities to study the mammalian central nervous system, which is dominated by anatomical structures whose nanoscale dimensions critically influence their biophysical properties. I will present our recent methodological advances 1) to analyze dendritic spines in the hippocampus in vivo and 2) to visualize the extracellular space (ECS) of the brain. Using a two-photon–STED microscope equipped with a long working distance objective and ‘hippocampal window’ to reach this deeply embedded structure, we measured the density and turnover of spines on CA1 pyramidal neurons. Spine density was two times higher than reported by conventional two-photon microscopy; around 40% of all spines turned over within 4 days. A combination of 3D-STED microscopy and fluorescent labeling of the extracellular fluid allows super-resolution shadow imaging (SUSHI) of the ECS in living brain slices. SUSHI enables quantitative analyses of ECS structure and produces sharp negative images of all cellular structures, providing an unbiased view of unlabeled brain cells in live tissue.