title: The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators.
abstract: We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements; however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.
authors.forename: Jung-Hwa
authors.identifier: 0000-0002-9041-3081
authors.initials: JH
authors.lastname: Cho
authors.affiliation: Biophysics Program, University of Michigan , 930 N. University, Room 4028, Ann Arbor, Michigan 48109, United States.
authors.forename: Carter J
authors.identifier: 0000-0001-6697-9475
authors.initials: CJ
authors.lastname: Swanson
authors.forename: Jeannie
authors.initials: J
authors.lastname: Chen
authors.forename: Ang
authors.initials: A
authors.lastname: Li
authors.affiliation: Department of Genetics, Cell Biology & Development, University of Minnesota Twin Cities , 4-130 MCB, 420 Washington Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Lisa G
authors.initials: LG
authors.lastname: Lippert
authors.affiliation: Department of Ophthalmology, University of Florida , 2000 SW Archer Rd, Rm R3-128, Gainesville, Florida 32611, United States.
authors.forename: Shannon E
authors.initials: SE
authors.lastname: Boye
authors.forename: Kasey
authors.initials: K
authors.lastname: Rose
authors.affiliation: Department of Genetics, Cell Biology & Development, University of Minnesota Twin Cities , 4-130 MCB, 420 Washington Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Sivaraj
authors.initials: S
authors.lastname: Sivaramakrishnan
authors.forename: Cheng-Ming
authors.initials: CM
authors.lastname: Chuong
authors.affiliation: Department of Biomedical Engineering, Zilkha Neurogenetic Institute, University of Southern California , Room 323, Keck School of Medicine, Los Angeles, California 90089, United States.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Chow
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D002645:Chickens; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein