Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments

In fluorescence fluctuation polarization sensitive experiments, the limitations associated with detecting the rotational timescale are usually eliminated by applying fluorescence correlation spectroscopy analysis. In this paper, the variance of the time-averaged fluorescence intensity extracted from the second moment of the measured fluorescence intensity is analyzed in the short time limit, before fluctuations resulting from rotational diffusion average out. Since rotational correlation times of fluorescence molecules are typically much lower than the temporal resolution of the system, independently of the time bins used, averaging over an ensemble of time-averaged trajectories was performed in order to construct the time-averaged intensity distribution, thus improving the signal-to-noise ratio. Rotational correlation times of fluorescein molecules in different viscosities of the medium within the range of the anti-bunching time (1-10 ns) were then extracted using this method.