Figure 1.

Five different background estimators applied to the same molecule (experimental data). (a) Gaussian profile fitting-based background estimator. The inset shows an example of the result of a profile fit. (b–e) Aperture-based background estimators. The background aperture (highlighted ring in the upper-left inset in (b) showing donor and acceptor fluorescence spots (top and bottom images, respectively)) overlaps a neighboring molecule in the donor channel. Traces shown are the raw background-uncorrected donor intensity (green), raw acceptor intensity (red), backgrounds (black), and FRET efficiencies (blue). The FRET histograms (bottom) only include frames until bleaching of the acceptor. The donor bleaches at 142 s and the acceptor bleaches at 122 s. Aperture-based background intensities were determined using (b) the local mean background, (c) the intensity after bleaching, (d) the local median background, and (e) the LSP background. To see this figure in color, go online.