Fig. 1.
Two-dimensional frequency histogram of single-molecule data of a mixture of 3 distinct samples with interfluorophore distances of 5, 11, and 19 bp, where frequency increases in gray scale from white to black. The ratio of donor and acceptor fluorescence (FD/FA) and the donor anisotropy (rD) are both plotted against τD(A). FD and FA are determined from green and red signals by correcting for background counts, BG and BR, detection efficiencies, gG and gR, and spectral cross-talk α according to equation 3 in ref. 5. (Typical values are BG = 2.9 kHz, BR = 0.9 kHz, gG = 0.36 and gR = 0.53 and α = 0.019). The direct acceptor excitation is negligible. (A) The red curve shows the FRET relationship FD/FA = τD(A)φFD/((τD(0) − τD(A))·φFA) with τD(0) = 4.1 and the fluorescence quantum yields of the donor and acceptor ΦFD = 0.8 and ΦFA = 0.43, respectively. (B) The red curve shows the Perrin equation r = r0/(1 + τ/ρ) with a fundamental anisotropy r0 = 0.375 and a mean rotational correlation time ρ = 0.63 ns.