Fluorescence intensity multiple distributions analysis: Concurrent determination of diffusion times and molecular brightness

Fluorescence correlation spectroscopy (FCS) has proven to be a powerful tec hnique with single-molecule sensitivity. Recently, it has found a complemen t in the form of fluorescence intensity distribution analysis (FIDA). Here we introduce a fluorescence fluctuation method that combines the features o f both techniques. It is based on the global analysis of a set of photon co unt number histograms, recorded with multiple widths of counting time inter vals simultaneously. This fluorescence intensity multiple distributions ana lysis (FIMDA) distinguishes fluorescent species on the basis of both the sp ecific molecular brightness and the translational diffusion time. The combi ned information, extracted from a single measurement, increases the readout effectively by one dimension and thus breaks the individual limits of FCS and FIDA. In this paper a theory is introduced that describes the dependenc e of photon count number distributions on diffusion coefficients. The theor y is applied to a series of photon count number histograms corresponding to different widths of counting time intervals. Although the ability of the m ethod to determine specific brightness values, diffusion times, and concent rations from mixtures is demonstrated on simulated data, its experimental u tilization is shown by the determination of the binding constant of a prote in-ligand interaction exemplifying its broad applicability in the life scie nces.