title: A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.
abstract: The introduction of calcium ion (Ca(2+)) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca(2+) dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650-1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca(2+) indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca(2+) indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca(2+) and glutamate imaging in organotypic cultures.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Jiahui
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Ahmed S
authors.initials: AS
authors.lastname: Abdelfattah
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, Neuroengineering Program, McGill University, Montreal, Quebec, Canada H3A 2B4.
authors.forename: Loïs S
authors.initials: LS
authors.lastname: Miraucourt
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, Neuroengineering Program, McGill University, Montreal, Quebec, Canada H3A 2B4.
authors.forename: Elena
authors.initials: E
authors.lastname: Kutsarova
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Araya
authors.initials: A
authors.lastname: Ruangkittisakul
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Hang
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Klaus
authors.initials: K
authors.lastname: Ballanyi
authors.affiliation: Department of Physics, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Wicks
authors.affiliation: Department of Physics, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Drobizhev
authors.affiliation: 1] Department of Physics, Montana State University, Bozeman, Montana 59717, USA [2] National Institute of Chemical Physics and Biophysics, Tallinn, Estonia 12618.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Rebane
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, Neuroengineering Program, McGill University, Montreal, Quebec, Canada H3A 2B4.
authors.forename: Edward S
authors.initials: ES
authors.lastname: Ruthazer
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D015394:Molecular Structure; D061848:Optical Imaging; D015202:Protein Engineering; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011522:Protons; D000097573:Red Fluorescent Protein