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

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 fluo-

rescent 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-dimen-

sional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5

fluorophore.