Synaptic transmission is regulated by nanoscale assembly of synaptic molecules. Stimulated emission depletion (STED) microscopy is a modality of superresolution microscopy that allows visualizing such nanoscale molecular distribution. However, photobleaching severely limits time-series imaging by STED microscopy in living neurons. In this study, we aimed to develop a fluorescence labeling method that circumvents the limitation due to photo-bleaching in STED microscopy. Our method is based on a fusion protein tag that reversibly binds a small organic dye and turns on its fluorescence emission. We reasoned that this reversibility in binding enables everlasting imaging because the bleached dye should be continuously displaced by a fresh dye. Accordingly, we prepared expression constructs of the protein tag fused with synaptic molecules, RimBP2, CAST, and Rim1a. When expressed in cultured hippocampal neurons, the synaptic localization of these molecules was visualized under a standard fluorescence microscope. We successfully performed STED imaging of RimBP2 and CAST with the spatial resolution of less than 100 nm at 0.2 Hz for 10 min. Thus, our fluorescence labeling method enables live STED microscopy in neurons which will be useful to unveil the dynamic feature of nanoscale molecular distribution underlying synaptic function.
