In this Primer, we focus on the most recent advancements in stimulated emission depletion (STED) microscopy, encompassing optics, computational microscopy and probes design, which enable STED imaging to open new observation windows in challenging samples such as living cells and tissues. We showcase applications in which STED data have been essential to gain new biological insights in various cell types and model systems. Finally, we discuss what standardization will be important in our view to further advance STED imaging, including open and shareable software, analysis pipelines, data repositories and sample preparation protocols. Stimulated emission depletion microscopy opens new observation windows in challenging samples such as living cells and tissues. In this Primer, Lukinavi & ccaron;ius et al. discuss 2D and 3D stimulated emission depletion setup, including adaptive optical elements and their combination with fluorescence lifetime techniques.