FIGURE 4.

The molecular brightness as a function of photon count rate in a dilution experiment. Alexa 488 was diluted in dH₂O sequentially by factors of two from a concentration of ∼100 nM. In such an experiment, the brightness should remain constant although the number of molecules changes. When the experimental histograms are fit to an ideal PCH model the brightness decreases dramatically at high concentrations (squares). When the histograms are fit to a modified PCH model that incorporates dead-time effects, the brightness is constant over the entire concentration range (triangles). The dashed line indicates the brightness obtained in a global fit to the modified model of all of the experimental histograms simultaneously (ɛ = 4.18 ± 0.02 cpm).