title: Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.
abstract: The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.
authors.affiliation: 1] Max Planck Institute of Neurobiology, Martinsried, Germany. [2] Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Thestrup
authors.affiliation: Max Planck Institute of Neurobiology, Martinsried, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Litzlbauer
authors.affiliation: Max Planck Institute of Neurobiology, Martinsried, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Bartholomäus
authors.affiliation: Max Planck Institute of Neurobiology, Martinsried, Germany.
authors.forename: Marsilius
authors.initials: M
authors.lastname: Mues
authors.affiliation: Max Planck Institute for Biophysical Chemistry, NMR-based Structural Biology, Göttingen, Germany.
authors.forename: Luigi
authors.initials: L
authors.lastname: Russo
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Hod
authors.initials: H
authors.lastname: Dana
authors.affiliation: Institute of Physiology II, University of Tübingen, Tübingen, Germany.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kovalchuk
authors.affiliation: Institute of Physiology II, University of Tübingen, Tübingen, Germany.
authors.forename: Yajie
authors.identifier: 0000000247984882
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Institute of Physiology II, University of Tübingen, Tübingen, Germany.
authors.forename: Georgios
authors.initials: G
authors.lastname: Kalamakis
authors.affiliation: Max Planck Institute for Biophysical Chemistry, NMR-based Structural Biology, Göttingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Laukat
authors.affiliation: Max Planck Institute for Biophysical Chemistry, NMR-based Structural Biology, Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Gene Center, Department of Biochemistry, University of Munich, Munich, Germany.
authors.forename: Gregor
authors.identifier: 0000000303130962
authors.initials: G
authors.lastname: Witte
authors.affiliation: Max Planck Institute of Neurobiology, Martinsried, Germany.
authors.forename: Anselm
authors.initials: A
authors.lastname: Geiger
authors.affiliation: Oberlin College, Oberlin, Ohio, USA.
authors.forename: Taylor
authors.identifier: 000000033018187X
authors.initials: T
authors.lastname: Allen
authors.affiliation: 1] Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. [2] The Whitman Center, Marine Biological Lab, Woods Hole, Massachusetts, USA.
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Rome
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Tsai-Wen
authors.initials: TW
authors.lastname: Chen
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Institute of Physiology II, University of Tübingen, Tübingen, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Garaschuk
authors.affiliation: Max Planck Institute for Biophysical Chemistry, NMR-based Structural Biology, Göttingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Griesinger
authors.affiliation: 1] Max Planck Institute of Neurobiology, Martinsried, Germany. [2] Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Griesbeck
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000378:metabolism*; D008213:Lymphocyte Activation; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D019209:Troponin C / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D019209:Troponin C; D002118:Calcium