Stimulated emission depletion (STED) microscopy has an extremely high (nanoscale) resolution, but this technique has not been applied in vivo in rodents. Here, the authors used STED microscopy to study neuron dynamics in vivo in the somatosensory cortex of adult mice. The images showed structures as small as <70 nm. Moreover, serial imaging revealed morphological changes and movement in the head and neck regions of dendritic spines over minutes. STED and other forms of nanoscopy could be used to investigate spine dynamics in brain development and function as well as in disease processes.