First, staining via a membrane-bound fluorescent protein is used: expression of YFP-Channelrhodopsin-2 allows STED imaging of dendrites, axons, and synapses in living cells [in Fig. 2(a), some boutons and spines are highlighted with arrowheads and arrows, respectively]. The fluorescent signal reveals the distribution of the light-sensitive ion channel Channelrhodopsin-2 in the dendrite and in spines. This staining strategy should then allow studying correlations among synaptic plasticity, spine morphology, and ion-channel density. An alternative membrane stain for STED imaging consists of lipophilic dyes that integrate into the cell membrane.47 In contrast to staining with fluorescent proteins, no transfection or transgenic animal is needed, but the samples can easily be stained at the time of imaging. Here we use DiI, which visualizes synapses because the lipophilic dye stains pre- and postsynapses. Thus, this membrane stain enables resolving the outline of the axon and the postsynaptic dendritic spines in living cells [Fig. 2(b)]. For visualization of synapses, the DiI label has the advantage to stain, in principle, all cells, in contrast to the transfection stain, which depends on the infection rate. Notably, we noticed that cell staining was inhomogeneous with the DiI dye. The wavelengths used for uncaging (403 nm), fluorescence excitation (532 nm), and STED beam (635 nm) are well separated.