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.

Global analysis of time-resolved fluorescence data.

In this chapter, we describe the global analysis approach for processing time-resolved fluorescence spectroscopy data of molecules in the condensed phase. Combining simultaneous analysis of data measured under different experimental conditions (spatial coordinates, temperature, concentration, emission wavelength, excitation intensity, etc.) with the fitting strategy, enabling parameter linkage and thus decreasing the total amount of estimated variables, makes global analysis more robust and more consistent compared to a sequential fit of single experimental data. We consider the main stages of the global analysis approach and provide some details that are important for its practical implementation. The application of the global approach to the analysis of time-resolved fluorescence anisotropy is demonstrated on experimental data of (enhanced) green fluorescent protein in aqueous solution.

Global analysis of autocorrelation functions and photon counting distributions.

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, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH 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. The global analysis approach is described and tested with experimental fluorescence fluctuation data of enhanced green-fluorescent protein (eGFP) and dimeric eGFP (two eGFP molecules connected by a six amino acid long linker) in aqueous buffer. Brightness values and diffusion constants are recovered with good precision elucidating novel excited-state and motional properties of both proteins.

Global analysis of fluorescence fluctuation data.

Over the last decade the number of applications of fluorescence correlation spectroscopy (FCS) has grown rapidly. Here we describe the development and application of a software package, FCS Data Processor, to analyse the acquired correlation curves. The algorithms combine strong analytical power with flexibility in use. It is possible to generate initial guesses, link and constrain fit parameters to improve the accuracy and speed of analysis. A global analysis approach, which is most effective in analysing autocorrelation curves determined from fluorescence fluctuations of complex biophysical systems, can also be implemented. The software contains a library of frequently used models that can be easily extended to include user-defined models. The use of the software is illustrated by analysis of different experimental fluorescence fluctuation data sets obtained with Rhodamine Green in aqueous solution and enhanced green fluorescent protein in vitro and in vivo.