Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye
| Autor: | Seamus Morrone, Walter F. Mangel, Andrew Robinson, Antoine M. van Oijen, Aartje C. Schulte, Jungsan Sohn, Lisanne M. Spenkelink, William J. McGrath, Hylkje Geertsema |
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| Rok vydání: | 2015 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Fluorophore Chemistry Biophysics Nuclear Proteins DNA Fluorescence in the life sciences Carbocyanines Phosphoproteins Fluorescence Single Molecule Imaging Bimolecular fluorescence complementation chemistry.chemical_compound Biochemistry Microscopy Fluorescence Fluorescence microscope Proteins and Nucleic Acids Macromolecule Fluorescent Dyes Protein Binding |
| Zdroj: | Biophysical Journal. 108(4):949-956 |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2014.12.019 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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