FIGURE 5.

Alexa 488 was diluted in water by a factor of 2 several times and the dual-color and single-channel PCHs of each sample were fit to their respective ideal models. Shown is the molecular brightness in detector A (A) and in detector B (B) returned from the individual fit of each sample to the ideal model and the non-ideal model (dual-color PCH only). The solid lines represent the brightness in each detection channel determined by global analysis of all dual-color PCH histograms to the non-ideal model. The number of molecules was also determined by the global fit. The ideal model fails to provide a constant brightness. It yields a brightness that is too large at low concentrations due to afterpulse effects, whereas, at high concentrations, the value is too low because of deadtime effects. Since detector A has the larger afterpulse probability of the two detectors, its difference between the single- and dual-channel brightnesses is larger than that for detector B at the lower concentrations. For each dilution step, the dual-channel histogram was fit to the non-ideal model with deadtime and afterpulses. The non-ideal model returned constant ɛ-values as expected.