Abstract Progress in microscopy technology has a long history of triggering major advances in neuroscience. Super-resolution microscopy (SRM), famous for shattering the diffraction barrier of light microscopy, is no exception. SRM gives access to anatomical designs and dynamics of nanostructures, which are impossible to resolve using conventional light microscopy, from the elaborate anatomy of neurons and glial cells, to the organelles and molecules inside of them. In this review, we will mainly focus on a particular SRM technique (STED microscopy), and explain a series of technical developments we have made over the years to make it practical and viable in the field of neuroscience. We will also highlight several neurobiological findings on the dynamic structure-function relationship of neurons and glia cells, which illustrate the value of live-cell STED microscopy, especially when combined with other modern approaches to investigate the nanoscale behavior of brain cells.