Fig. 3: Fluorescence trajectories and histograms of fraction of acceptor photon counts.
From: Fast three-color single-molecule FRET using statistical inference
The bin time of 1 ms is used for the trajectories and histograms. Green, orange, and red arrows indicate photobleaching of D, A1, and A2, respectively. a–c Fluorescence trajectories and acceptor photon fraction histograms of α3D at different GdmCl concentrations. 107 (2 M), 195 (2.25 M), and 215 (2.5 M) three-color DA1A2 trajectories (i.e., molecules), 40 (2 M), 190 (2.25 M), and 212 (2.5 M) two-color DA1 trajectories, and 84 (2 M), 110 (2.25 M), and 188 (2.5 M) two-color DA2 trajectories were analyzed. a Two α3D trajectories of the donor (green), acceptor 1 (orange) and acceptor 2 (red) fluorescence at 2.25 M GdmCl. In the upper trajectory, A2 fluorescence after A2 photobleaching (red arrow) that is maintained before A1 photobleaching (orange arrow) results from the leak of A1 photons into A2 channel. States assigned by the Viterbi algorithm are shown in the color bar above the upper trajectory. b Histograms of the uncorrected fractions of A1 (ε1) and A2 (ε2) from three-color segments (DA1A2). c Histograms of the uncorrected fractions of acceptor photons (ε) from two-color segments (DA1 and DA2). d–f Fluorescence trajectories and acceptor photon fraction histograms of TAD-NCBD binding at different NaCl concentrations. 140, 140, and 186 molecules were analyzed at 0, 10, and 30 mM NaCl, respectively. d Donor (green), acceptor 1 (orange) and acceptor 2 (red) fluorescence trajectories at 0 mM (upper) and 30 mM NaCl (lower). Apparent states are assigned by visual inspection as indicated in the color bar above the trajectory at 0 mM. States assigned by the Viterbi algorithm for a trajectory at 30 mM NaCl are shown in Supplementary Fig. 5. e Histograms of the uncorrected fractions of A1 (ε1) and A2 (ε2) from three-color (DA1A2)/DA1 segments. Enlarged histograms of the bound state (0.4 < ε2 < 1) are shown in the insets. f Histograms of the uncorrected fractions of acceptor photons from DA2 segments. Enlarged histograms of the bound state (0.4 < ε < 1) are shown in the insets.
