Fluorescence correlation spectroscopy considering the effect of photobleaching

In fluorescence correlation spectroscopy (FCS), the fluorescence intensity decay curve is used to calculate the correlation time, which characterizes the diffusion feature of the molecules. However, photobleaching, an unavoidable phenomenon that happened during fluorescence excitation, would result in fluorescence decay, and thus cause error in the calculation of the correlation time and the diffusion coefficient. To compensate this effect, a probability factor is introduced. This factor describes the probability at which a molecule is photobleached by the excitation laser during fluorescence excitation. By selecting the right value of the photobleaching factor, the effect of photobleaching on the FCS measurement can be removed.