Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy.

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences

Calculation of Photon-Count Number Distributions via Master Equations

Fitting of photon-count number histograms is a way of analysis of fluorescence intensity fluctuations, a successor to fluorescence correlation spectroscopy. First versions of the theory for calculating photon-count number distributions have assumed constant emission intensity by a molecule during a counting time interval. For a long time a question has remained unanswered: to what extent is this assumption violated in experiments? Here we present a theory of photon-count number distributions that takes account of intensity fluctuations during a counting time interval. Theoretical count-number distributions are calculated via a numerical solution of Master equations (ME), which is a set of differential equations describing diffusion, singlet-triplet transitions, and photon emission. Detector afterpulsing and dead-time corrections are also included. The ME-theory is tested by fitting a series of photon-count number histograms corresponding to different lengths of the counting time interval. Compared to the first version of fluorescence intensity multiple distribution analysis theory introduced in 2000, the fit quality is significantly improved. It is discussed how a theory of photon-count number distributions, which assumes constant emission intensity during a counting time interval, may also yield a good fit quality. We argue that the spatial brightness distribution used in calculations of the fit curve is not the true spatial brightness distribution. Instead, a number of dynamic processes, which cause fluorescence intensity fluctuations, are indirectly taken into account via the profile adjustment parameters