Figure 4.

Single-molecule experiment results on immobilized DNA standards. (A and B) E^∗-S_raw histograms of DNAs NoQ (A) and Q08^QSY7 (B) from TIRF experiments. (C and D) Fluorescence time-traces ($F_{G_{exc}}^{G_{em}}$ and $F_{R_{exc}}^{R_{em}}$) of DNAs NoQ (C) and Q08^QSY7 (D). Time-traces of S_raw do not include data bins where both fluorophores are photobleached. The histogram of $F_{G_{exc}}^{G_{em}}$ and S_raw are projected on the right side ($F_{R_{exc}}^{R_{em}}$ histogram is not shown). $F_{G_{exc}}^{G_{em}}$ histograms do not include data bins after photobleaching. Bleaching of probe Q (Q_off, shaded arrow) is detected when probe G intensity switches from a low state to a high state, the mean intensity value of the low state being 75–85% lower than the high state that corresponds to E_R→QSY7. When probe Q bleaches or blinks, S_raw switches from a low state (∼0.25) to a high state (∼0.5). (E and F) Histograms and fits of the bleaching lifetime distribution of the dark quenchers for DNAs Q08^QSY7 (E) and Q08^QSY21 (F). The histograms were fitted with a single-exponential decay function F (F (t) = A exp(− t/τ_Q)) with A and τ_Q the fitted parameters. The bleaching lifetimes for QSY7 and QSY21 are τ_QSY7 = 27 s and τ_QSY21 = 144 s, respectively.