title: A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.
abstract: Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, USA.|Department of Pediatrics, Stanford University, Stanford, California, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Chu
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, USA.|Department of Pediatrics, Stanford University, Stanford, California, USA.|Department of Neurobiology, Stanford University, Stanford, California, USA.
authors.forename: Younghee
authors.initials: Y
authors.lastname: Oh
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Sens
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Niloufar
authors.initials: N
authors.lastname: Ataie
authors.affiliation: Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
authors.forename: Hod
authors.initials: H
authors.lastname: Dana
authors.affiliation: Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Macklin
authors.affiliation: Max Planck Florida Institute, Jupiter, Florida, USA.
authors.forename: Tal
authors.initials: T
authors.lastname: Laviv
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Erik S
authors.initials: ES
authors.lastname: Welf
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Dean
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, California, USA.|Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Feijie
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, USA.
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Kim
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Clement Tran
authors.initials: CT
authors.lastname: Tang
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Hu
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.|National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Baird
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.|National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, California, USA.|Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Reto
authors.initials: R
authors.lastname: Fiolka
authors.affiliation: Max Planck Florida Institute, Jupiter, Florida, USA.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Yasuda
authors.affiliation: Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.|University of Hawaii Cancer Center, Honolulu, Hawaii, USA.
authors.forename: Ho-Leung
authors.identifier: 0000-0002-6415-1938
authors.initials: HL
authors.lastname: Ng
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, USA.|Department of Pediatrics, Stanford University, Stanford, California, USA.|Department of Neurobiology, Stanford University, Stanford, California, USA.
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Lin
mesh_terms: D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000493:pharmacokinetics; D008029:Lighting / Q000379:methods; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins