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

Katrin I Willig; Waja Wegner; Antonia Müller; Valérie Calvet-Fournier; Heinz Steffens

doi:10.1016/j.celrep.2021.109192

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

Cell Rep. 2021 Jun 1;35(9):109192. doi: 10.1016/j.celrep.2021.109192.

Authors

Katrin I Willig¹, Waja Wegner², Antonia Müller³, Valérie Calvet-Fournier⁴, Heinz Steffens²

Affiliations

¹ Optical Nanoscopy in Neuroscience, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany; Max Planck Institute of Experimental Medicine, Göttingen, Germany; Cluster of Excellence "Multiscale Bioimaging: From Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Göttingen, Germany. Electronic address: kwillig@em.mpg.de.
² Optical Nanoscopy in Neuroscience, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany; Max Planck Institute of Experimental Medicine, Göttingen, Germany.
³ Optical Nanoscopy in Neuroscience, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany; Max Planck Institute of Experimental Medicine, Göttingen, Germany; Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.
⁴ Optical Nanoscopy in Neuroscience, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany; Max Planck Institute of Experimental Medicine, Göttingen, Germany; Göttingen Graduate Center for Neurosciences, Biophysics, und Molecular Biosciences (GGNB), Göttingen, Germany.

PMID: 34077731
DOI: 10.1016/j.celrep.2021.109192

Abstract

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.

Keywords: fluorescence; imaging; intravital; mouse neocortex; multicolor; nanoscopy; photochromism; super-resolution; synapse.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cerebral Cortex / metabolism
Green Fluorescent Proteins / metabolism
Intravital Microscopy*
Luminescent Proteins / metabolism*
Mice
Mice, Inbred C57BL
Nanotechnology
Staining and Labeling*
Synapses / metabolism
Time-Lapse Imaging

Substances

Luminescent Proteins
enhanced green fluorescent protein
Green Fluorescent Proteins