Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution.

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

Fluorescence cumulants analysis with non-ideal observation profiles.

One of the challenges of fluorescence fluctuation fpectroscopy (FFS) is an adequate approximation of a brightness profile. The key feature of fluorescence intensity distribution analysis (FIDA) is a polynomial approximation of a brightness profile. A broad range of brightness profile shapes can be well described by this approximation. A different approach consisting of the introduction of additional fitting parameters, defined as a relative difference between integrals of the actual brightness profile and its Gaussian approximation, is used in photon counting histogram (PCH) analysis. It is sufficient to introduce only one additional fitting parameter (first-order correction) to get an adequate fit to the experimental data in many practical applications. In the current study, we apply these approaches to the theory of time integrated fluorescence cumulants analysis. We demonstrate that developed corrections improve results of FFS analysis applied to simulated and experimental data. The use of different brightness profile approximations and normalizations in PCH and FIDA leads to different estimates of brightness and the number of molecules, even though they represent the same physical quantities. Based on the developed theory, we derive equations that relate brightness and the number of molecules in PCH and FIDA.

Modelling of self-diffusion and relaxation time NMR in multicompartment systems with cylindrical geometry.

Multicompartment characteristics of relaxation and diffusion in a model for (plant) cells and tissues have been simulated as a means to test separating the signal into a set of these compartments. A numerical model of restricted diffusion and magnetization relaxation behavior in PFG-CPMG NMR experiments, based on Fick's second law of diffusion, has been extended for two-dimensional diffusion in systems with concentric cylindrical compartments separated by permeable walls. This model is applicable to a wide range of (cellular) systems and allows the exploration of temporal and spatial behavior of the magnetization with and without the influence of gradient pulses. Numerical simulations have been performed to show the correspondence between the obtained results and previously reported studies and to investigate the behavior of the apparent diffusion coefficients for the multicompartment systems with planar and cylindrical geometry. The results clearly demonstrate the importance of modelling two-dimensional diffusion in relation to the effect of restrictions, permeability of the membranes, and the bulk relaxation within the compartments. In addition, the consequences of analysis by multiexponential curve fitting are investigated.

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.

Initial guesses generation for Fluorescence Intensity Distribution Analysis.

The growing number of applications of Fluorescence Intensity Distribution Analysis (FIDA) demands for new approaches in data processing, aiming at increased speed and robustness. Iterative algorithms of parameter estimation, although proven to be universal and accurate, require some initial guesses (IG) of the unknown parameters. An essential component of any data processing technology, IG become especially important in case of FIDA, since even with apparently reasonable, and physically admissible but randomly chosen IG, the iterative procedure may converge to situations where the FIDA model cannot be evaluated correctly. In the present work we introduce an approach for IG generation in FIDA experiments based on the method of moments. IG are generated for the sample parameters: brightness, concentration, and for the parameters related to experimental set-up: background, observation volume profile. A number of analytical simplifications were introduced in order to increase the accuracy and robustness of the numerical algorithms. The performance of the developed method has been tested on number of simulations and experimental data. Iterative fitting with generated IG proved to be more robust and at least five times faster than with an arbitrarily chosen IG. Applicability of the proposed method for quick estimation of brightness and concentrations is discussed.

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.