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

Abstract

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.

Authors:
 [1];  [2];  [1];  [1];  [2];  [3];  [3];  [1];  [1]
+ Show Author Affiliations
  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:
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)
OSTI Identifier:
1184512
Report Number(s):
BNL-107889-2015-JA
Journal ID: ISSN 0006-3495; 400412000
Grant/Contract Number:  
SC00112704
Resource Type:
Accepted Manuscript
Journal Name:
Biophysical Journal
Additional Journal Information:
Journal Volume: 108; Journal Issue: 4; Journal ID: ISSN 0006-3495
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; single-molecule; fluorescence microscopy; fluorescence imaging

Citation Formats

Geertsema, Hylkje J., Mangel, Walter F., Schulte, Aartje C., Spenkelink, Lisanne M., McGrath, William J., Morrone, Seamus R., Sohn, Jungsan, Robinson, Andrew, and van Oijen, Antoine M. Single-molecule imaging at high fluorophore concentrations by local activation of dye. United States: N. p., 2015. Web. doi:10.1016/j.bpj.2014.12.019.
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Geertsema, Hylkje J., Mangel, Walter F., Schulte, Aartje C., Spenkelink, Lisanne M., McGrath, William J., Morrone, Seamus R., Sohn, Jungsan, Robinson, Andrew, & van Oijen, Antoine M. Single-molecule imaging at high fluorophore concentrations by local activation of dye. United States. https://doi.org/10.1016/j.bpj.2014.12.019
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Geertsema, Hylkje J., Mangel, Walter F., Schulte, Aartje C., Spenkelink, Lisanne M., McGrath, William J., Morrone, Seamus R., Sohn, Jungsan, Robinson, Andrew, and van Oijen, Antoine M. Tue . "Single-molecule imaging at high fluorophore concentrations by local activation of dye". United States. https://doi.org/10.1016/j.bpj.2014.12.019. https://www.osti.gov/servlets/purl/1184512.
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@article{osti_1184512,
title = {Single-molecule imaging at high fluorophore concentrations by local activation of dye},
author = {Geertsema, Hylkje J. and Mangel, Walter F. and Schulte, Aartje C. and Spenkelink, Lisanne M. and McGrath, William J. and Morrone, Seamus R. and Sohn, Jungsan and Robinson, Andrew and van Oijen, Antoine M.},
abstractNote = {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.},
doi = {10.1016/j.bpj.2014.12.019},
journal = {Biophysical Journal},
number = 4,
volume = 108,
place = {United States},
year = {Tue Feb 17 00:00:00 EST 2015},
month = {Tue Feb 17 00:00:00 EST 2015}
}
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https://doi.org/10.1016/j.bpj.2014.12.019
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Carbocyanine Dyes as Efficient Reversible Single-Molecule Optical Switch
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  • Uphoff, Stephan; Holden, Seamus J.; Le Reste, Ludovic
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    Dissection of Protein Kinase Pathways in Live Cells Using Photoluminescent Probes: Surveillance or Interrogation?
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