title: Single-molecule imaging at high fluorophore concentrations by local activation of dye.
abstract: Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.
authors.affiliation: Zernike Institute for Advanced Materials, Groningen, The Netherlands.
authors.forename: Hylkje J
authors.initials: HJ
authors.lastname: Geertsema
authors.affiliation: Zernike Institute for Advanced Materials, Groningen, The Netherlands.
authors.forename: Aartje C
authors.initials: AC
authors.lastname: Schulte
authors.affiliation: Zernike Institute for Advanced Materials, Groningen, The Netherlands.
authors.forename: Lisanne M
authors.initials: LM
authors.lastname: Spenkelink
authors.affiliation: Brookhaven National Laboratory, Upton, New York.
authors.forename: William J
authors.initials: WJ
authors.lastname: McGrath
authors.affiliation: Johns Hopkins School of Medicine, Baltimore, Maryland.
authors.forename: Seamus R
authors.initials: SR
authors.lastname: Morrone
authors.affiliation: Johns Hopkins School of Medicine, Baltimore, Maryland.
authors.forename: Jungsan
authors.initials: J
authors.lastname: Sohn
authors.affiliation: Brookhaven National Laboratory, Upton, New York.
authors.forename: Walter F
authors.initials: WF
authors.lastname: Mangel
authors.affiliation: Zernike Institute for Advanced Materials, Groningen, The Netherlands.
authors.forename: Andrew
authors.initials: A
authors.lastname: Robinson
authors.affiliation: Zernike Institute for Advanced Materials, Groningen, The Netherlands. Electronic address: a.m.van.oijen@rug.nl.
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D002232:Carbocyanines / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D010750:Phosphoproteins; C085321:cyanine dye 5; C076700:IFI16 protein, human; D004247:DNA