Figure 5.

Effect of background on smFRET histograms. (a) Comparison of FRET histograms of the same sample in regions of varying density, determined using five different background estimators (experimental data). The histogram of the sparse molecular regions (left) contains 229 double-stranded DNA molecules. The histogram of dense molecular regions (right) contains 65 molecules. Dense regions are defined as molecular regions in which neighbors contribute photons to the background aperture (7–8 pixels; see Fig. 2, d and f), whereas sparse regions consist of isolated molecules (see Fig. 2, b and c). The FRET histograms contain only molecules in which both the donor and acceptor bleach in one step, and only frames obtained before the first bleaching event. (b) E_mean (μ) and E_std.dev. (σ) obtained from a normal distribution fit of each of the histograms in (a). To see this figure in color, go online.