title: Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity.
abstract: Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime. Adding to the sensitivity gains realized from rejecting non-modulatable background, the selective signal recovery of immobilized vs diffusing intracellular rsFastLime suggests that DM-SAFIRe can detect weak protein-protein interactions that are normally obscured by large fractions of unbound FPs.
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Yen-Cheng
authors.initials: YC
authors.lastname: Chen
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Jablonski
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Irina
authors.initials: I
authors.lastname: Issaeva
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Daisy
authors.initials: D
authors.lastname: Bourassa
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Jung-Cheng
authors.initials: JC
authors.lastname: Hsiang
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Fahrni
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Dickson
mesh_terms: D005136:Eye Proteins / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*
chemical_list: D005136:Eye Proteins; D008164:Luminescent Proteins