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.

Simulation of autocorrelation function and photon counting distribution in fluorescence fluctuation spectroscopy.

In modern fluorescence fluctuation spectroscopy, the autocorrelation function and photon counting distribution are two widely used statistical characteristics of the measured fluctuating fluorescence intensity signal. Applying special analysis methods such as fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) to these properties, it is possible to recover values of different parameters of fluorescent molecules such as the concentration, diffusion coefficient, molecular brightness, and kinetic rate constants. The development of new analysis methods is senseless without testing their validity, accuracy, and robustness.

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.

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.

Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells.

Fluorescence correlation spectroscopy (FCS) and photon-counting histogram (PCH) analysis use the same experimental fluorescence intensity fluctuations, 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 analysis yielding information about the molecular brightness of fluorescent species.

A mathematical model of actin filament turnover for fitting FRAP data.

A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations.

Pore-scale dispersion in electrokinetic flow through a random sphere packing.

The three-dimensional velocity field and corresponding hydrodynamic dispersion in electrokinetic flow through a random bulk packing of impermeable, nonconducting spheres are studied by quantitative numerical analysis. First, a fixed bed with interparticle porosity of 0.38 is generated using a parallel collective-rearrangement algorithm.

FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein.

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles.

FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association.

A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state Förster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems.

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.

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.

Artificial neural network modification of simulation-based fitting: application to a protein-lipid system.

Simulation-based fitting has been applied to data analysis and parameter determination of complex experimental systems in many areas of chemistry and biophysics. However, this method is limited because of the time costs of the calculations. In this paper it is proposed to approximate and substitute a simulation model by an artificial neural network during the fitting procedure.