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Title: Single-molecule imaging at high fluorophore concentrations by local activation of dye

Single-molecule fluorescence microscopy is a powerful approach to observe 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. Then, making use of short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively 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 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.
 [1] ;  [2] ;  [1] ;  [1] ;  [2] ;  [3] ;  [3] ;  [1] ;  [1]
  1. Zernike Inst. of Advanced Materials, Groningen (The Netherlands)
  2. Brookhaven National Lab. (BNL), Upton, NY (United States)
  3. John Hopkins School of Medicine, Baltimore, MD (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 0006-3495; 400412000
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Biophysical Journal
Additional Journal Information:
Journal Volume: 108; Journal Issue: 4; Journal ID: ISSN 0006-3495
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
Netherlands Organization for Scientific Research (NWO); European Research Council (ERC); National Institutes of Health (NIH)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; single-molecule; fluorescence microscopy; fluorescence imaging
OSTI Identifier: