Figure 3.

Percentile-based background estimators (experimental data). (a) Background-corrected intensity trace using a median-based background estimator. Although the molecule bleaches at frame 870, the background-corrected intensity does not decay to zero, as shown by the binned histogram. The histogram includes frames after bleaching only. (b) Background pixel distribution of a blank region (inset), showing why the background-corrected intensity in figure (a) does not decay to zero. The background pixel distribution is characterized by a positive skew resulting in a lower distribution median value (red) compared with the distribution mean (black). As a result, the median background estimator underestimates the background intensity. The histogram bins pixels from 100 frames from the background aperture. (c) Squared difference (normalized at 45%) between the background pixel distribution mean and percentile for five experimental conditions differing in laser intensity and EM gain. The optimal percentile yields the value that resembles the mean and is generally between 54% and 61% (highlighted interval). The red vertical line denotes the percentile corresponding to the median value (50%). (d) Background-corrected intensity trace of the same molecule as in (a) but with a percentile-based background estimator (X = 56%). Here, the intensity decays to zero after bleaching. To see this figure in color, go online.