Peet Kask

Fluorescence Intensity Multiple Distributions Analysis: Concurrent Determination of Diffusion Times and Molecular Brightness

Fluorescence correlation spectroscopy (FCS) has proven to be a powerful technique with single-molecule sensitivity. Recently, it has found a complement in the form of fluorescence intensity distribution analysis (FIDA). Here we introduce a fluorescence fluctuation method that combines the features of both techniques. It is based on the global analysis of a set of photon count number histograms, recorded with multiple widths of counting time intervals simultaneously. This fluorescence intensity multiple distributions analysis (FIMDA) distinguishes fluorescent species on the basis of both the specific molecular brightness and the translational diffusion time. The combined 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 dependence of photon count number distributions on diffusion coefficients. The theory is applied to a series of photon count number histograms corresponding to different widths of counting time intervals. Although the ability of the method to determine specific brightness values, diffusion times, and concentrations from mixtures is demonstrated on simulated data, its experimental utilization is shown by the determination of the binding constant of a protein-ligand interaction exemplifying its broad applicability in the life sciences.

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.

Introduction to the Theory of Fluorescence Intensity Distribution Analysis

The primary data of a fluorescence correlation experiment is a sequence of photon counts detected from a microscopic sample volume. An essential component of the fluorescence correlation analysis is the calculation of the second order autocorrelation function of detected photons. In this way a stochastic function of photon counts is transformed into a statistical function having an expected shape from which properties of the sample can be estimated. The calculation of the autocorrelation function, however, is not the only way to extract information about the sample from the sequence of photon counts. Another approach, based on collecting the distribution of the number of photon counts for a given time interval, was introduced into fluorescence fluctuation spectroscopy by Qian and lson in 1990 [19.1].