title: A general approach to break the concentration barrier in single-molecule imaging.
abstract: Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Anna B
authors.initials: AB
authors.lastname: Loveland
authors.forename: Satoshi
authors.initials: S
authors.lastname: Habuchi
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Walter
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D004261:DNA Replication; D004058:Diffusion; D045585:Flap Endonucleases / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry
chemical_list: D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; C535245:mKikGR protein; D045585:Flap Endonucleases