Fig. 2.

Improved photophysics of FRET-enhanced mEos3.2. a A representative trace of mEos3.2–JF₆₄₆ is shown as a montage of single frames over time. Photons detected were converted to photons emitted per on frame and summed to determine the total photon emission. The on-state time was calculated per switching event and summed to generate the total on-state time. The number of switching events was also calculated. b Histograms of total emitted photons, total on-state time, on-state time (i.e., individual track length), and total number of switching events per molecule, when performing single-molecule TIRF imaging using either mEos3.2 (filled bars) or the mEos3.2–JF₆₄₆ FRET pair (open bars) under identical imaging conditions. c The increase in total on-state time that occurred upon addition of the JF₆₄₆ dye is shown with a vertical line to illustrate that more than 20% of the mEos3.2–JF₆₄₆ molecules are still on when 99% of the mEos3.2 molecules are off. d At a single-molecule level, molecules with the longest total on-state times also had the lowest average intensity (i.e., highest FRET efficiency) measured as emitted photons/frame