Multi-label in vivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins

• Published in: Cell reports (Cambridge) 2021-06, Vol.35 (9), p.109192-109192, Article 109192
• Main Authors: Willig, Katrin I., Wegner, Waja, Müller, Antonia, Clavet-Fournier, Valérie, Steffens, Heinz
• Format: Article
• Language: English
• Subjects: Animals; Cerebral Cortex - metabolism; fluorescence; Green Fluorescent Proteins - metabolism; imaging; intravital; Intravital Microscopy; Luminescent Proteins - metabolism; Mice; Mice, Inbred C57BL; mouse neocortex; multicolor; nanoscopy; Nanotechnology; photochromism; Staining and Labeling; super-resolution; synapse; Synapses - metabolism; Time-Lapse Imaging
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2021.109192

Despite the tremendous success of super-resolution microscopy, multi-color in vivo applications are still rare. Here we present live-cell multi-label STED microscopy in vivo and in vitro by combining spectrally separated excitation and detection with temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs). Triple-label STED microscopy resolves pre- and postsynaptic nano-organizations in vivo in mouse visual cortex employing EGFP, Citrine, and the RSFP rsEGP2. Combining the positive and negative switching RSFPs Padron and Dronpa-M159T enables dual-label STED microscopy. All labels are recorded quasi-simultaneously by parallelized on- and off-switching of the RSFPs within the fast-scanning axis. Depletion is performed by a single STED beam so that all channels automatically co-align. Such an addition of a second or third marker merely requires a switching laser, minimizing setup complexity. Our technique enhances in vivo STED microscopy, making it a powerful tool for studying multiple synaptic nano-organizations or the tripartite synapse in vivo. [Display omitted] •In vivo super-resolution STED microscopy of three labels in cortex of mice•Nanoscale imaging of pre- and postsynapse in vivo•Temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs)•Parallelized switching schema enables quasi-simultaneous imaging Synaptic proteins and receptors form nanostructures that cannot be resolved with conventional light microscopy. Willig et al. use in vivo STED microscopy to image three labels in the mouse cortex with nanoscale resolution. This technique enables, for example, quasi-simultaneous in vivo super-resolution imaging of pre- and postsynaptic nanostructures.