Fig. 4: Single-molecule FRET and dynamic smFLIM.
From: Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera
a Sketch of the DNA origami design used in this experiment. b Sketch of the emission process of a donor-acceptor pair in the FRET range (5.7 nm distance) where the donor has a lifetime τDA (left) and donor alone with lifetime τD (right). c Top: representative intensity traces with single-step photobleaching events. Bottom: corresponding lifetime traces sampled at 5 Hz show a short donor lifetime corresponding to the FRET case, and a long donor lifetime in the absence of FRET. d Representative intensity trace with successive acceptor and donor photobleaching, which enables a readout of the FRET and non-FRET donor lifetimes for the same molecule. Light traces in c, d sampled at 25 Hz are too noisy for a clear readout but 5 Hz sampling enables to reduce shot noise fluctuations. e Lifetime distributions obtained from all events corresponding to c (top, n = 7029) and to d (bottom, n = 413). The top inset shows experimental F-value in the range 2–6. f Single-molecule FRET histograms obtained through lifetime (horizontal) and intensity (vertical) show good agreement
