Global Analysis of Autocorrelation Functions and Photon Counting Distributions in Fluorescence Fluctuation Spectroscopy

In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis, the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding molecular diffusion coefficients and triplet-state parameters such as fraction and decay time. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding the molecular brightness. Both FCS and PCH give information about the molecular concentration. Here we describe a global analysis protocol that simultaneously recovers relevant and common parameters in model functions of FCS and PCH from a single fluorescence fluctuation trace. Application of a global analysis approach allows increasing the information content available from a single measurement that results in more accurate values of molecular diffusion coefficients and triplet-state parameters and also in robust, time-independent estimates of molecular brightness and number of molecules.