Two-dimensional fluorescence correlation spectroscopy II: spectral analysis of derivatives of anthracene and pyrene in micellar solutions

Generalized two-dimensional (2D) correlation spectroscopy has been applied to the analysis of fluorescence spectra in two micellar systems: (1) a mixture of pyrene and 1,3,6,8-pyrenetetrasulfonic acid in the cationic micellar solutions of cetyltrimethylammonium chloride (CTAC) and (2) a mixture of pyrene and 9-anthracencepropionic acid in anionic micellar solutions of sodium dodecyl sulfate (SDS). Fluorescence quenching is employed as a perturbation mode for causing intensity changes in fluorescence bands (quenching perturbation). Iodide ion (I-) is used as a quencher in the former system, and cetyl pridinium chloride (CPC) is used in the latter. Vibronic bands in the complicated fluorescence spectra of the mixture of the analytes were successfully resolved. It is shown that asynchronous maps are especially useful for spectral resolution enhancement when the quenching perturbation is employed in 2D fluorescence correlation spectroscopy. Furthermore, the information about the order of response of the bands to quenching is obtained by comparing the signs of synchronous and asynchronous cross-peaks.     

Polymers in focus: fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy has been increasingly used in polymer science. In the present perspective, the principles of the method are briefly reviewed, and the temporal and spatial resolutions are critically discussed. Examples of recent findings are summarized, focusing on polymer solutions, environmental parameters, combination with other techniques, near-interface measurements, simulations, and modeling. Finally, desirable new developments are discussed.     

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.     

Resolved fluorescence of the two tryptophan residues in horse apomyoglobin

The composite fluorescence emission from the two tryptophans (W7 and W14) of horse heart apomyoglobin was explored by fluorescence quenching experiments. The fluorescence of the W7 residue is the only one involved in the quenching by iodide or trichloroethanol (TCE) titration. The fluorescence contribution of W7 is 49% of the total apomyoglobin emission, and its spectrum is red-shifted compared to the W14 emission. The fluorescence decay of Trp residues gives an average fluorescence lifetime of 2.06 ns for W14 and 2.84 ns for W7. The static fluorescence quenching by TCE was used to monitor the individual motions of the two tryptophans in apomyoglobin. The short correlation time of W7 (rho = 3 ns) explains why this residue can experience various environments without having to assume the existence of several protein conformations occurring during its lifetime emission.     

Two-dimensional correlation spectroscopy and multivariate curve resolution for the study of lipid oxidation in edible oils monitored by FTIR and FT-Raman spectroscopy

Accelerate oxidative degradation of six vegetable oils was monitored using FTIR and FT-Raman spectroscopy. Two-dimensional correlation spectroscopy and multivariate curve resolution alternating least squares (MCR-ALS) were applied to the analysis of the data. The use of hetero-spectral two-dimensional correlation of FTIR and FT-Raman data allowed the use of well established band assignments to interpret less clearly assigned spectral features. With a moving window approach it was possible to obtain simplified two-dimensional correlation maps and to detect compounds evolving with different kinetic. Simultaneous analysis of the oxidation experiments of the six different oils monitored by both spectroscopic techniques was performed using MCR-ALS. Although a complete resolution of the data was not possible, the spectral changes occurring during the oxidative degradation of the oils were described with a five-component model. The two fundamentally different chemometric approaches lead to coincident results.     

Protein matrix elasticity determined by fluorescence anisotropy of its tryptophan residues

Rotational motions of Trp residues embedded within human hemoglobin matrix have been measured by using their steady-state fluorescence anisotropy. The mean square angular displacement theta2 of Trp residues, depending on the temperature, can be expressed by W = 1/2Ctheta2 where W is the thermal energy acting on the Trp residues and C the resilient torque constant of the protein matrix. To study the external medium influencing the protein dynamics, comparative experiments were made with protein in aqueous buffer and in the presence of 32% glycerol. The data show that between 5 degrees C and 25 degrees C, external medium acts on the protein matrix elasticity.     

Two-dimensional near-ultraviolet spectroscopy of aromatic residues in amyloid fibrils: a first principles study

We report a first principles study of two dimensional electronic spectroscopy of aromatic side chain transitions in the 32-residue β-amyloid (Aβ(9-40)) fibrils in the near ultraviolet (250-300 nm). An efficient exciton Hamiltonian with electrostatic fluctuations (EHEF) algorithm is used to compute the electronic excitations in the presence of environmental fluctuations. The through-space inter- and intra-molecular interactions are calculated with high level quantum mechanics (QM) approaches, and interfaced with molecular mechanics (MM) simulations. Distinct two dimensional near ultraviolet (2DNUV) spectroscopic signatures are identified for different aromatic transitions, and the couplings between them. 2DNUV signals associated with the transition couplings are shown to be very sensitive to the change of residue-residue interactions induced by residue mutations. Our simulations suggest that 2DNUV spectra could provide a useful local probe for the structure and kinetics of fibrils.     

Dynamic two-dimensional fluorescence correlation spectroscopy. Generalized correlation and experimental factors

Dynamic two-dimensional fluorescence correlation spectroscopy (2D FCS) is presented in the general form. Dynamic 2D FCS evaluates the time correlation function between two wavelength axes when an external perturbation is applied to the sample. It displays the vibronic features with similar time response functions in the synchronous correlation spectrum and the features with different time responses in the asynchronous correlation spectrum. The correlation analysis allows detailed assignments of the vibronic spectra of multicomponent samples. The emission-emission 2D FCS has proven to be able to resolve spectra with substantial overlaps, of species in equilibrium with each other, and of reacting species whose kinetic constants are linked and multiexponential. Similarly, the correlation analysis between excitation wavelengths allows the assignment of the excitation bands to fluorescent components. When a sinusoidal light source is used to excite the sample, the excitation-emission correlation requires the collection of only four spectra, two in-phase and two quadrature. The two-dimensional excitation-emission correlation analysis uncovers the association between the excitation and the emission vibronic features, enabling the complete assignment of the component spectra. The band associations and spectral assignments are facilitated by the two-dimensional phase map that is constructed from the synchronous and asynchronous correlation spectra. Spectral resolution can be optimized by varying the frequency of excitation and is not influenced by the detector phase angle used to collect the spectra. The resolution power of the 2D FCS is demonstrated with the retrieval of the anthracene emission spectrum from a pyrene-anthracene mixture when it contributes only 4% to the total fluorescence intensity.     

Investigation of micelle formation by fluorescence correlation spectroscopy

We show that noncovalently bound dye molecules can be used as labels in single-molecule fluorescence experiments for the determination of aggregate formation in standard surfactant systems. Aqueous solutions of sulfosuccinic acid bis(2-ethylhexyl) ester sodium salt, hexadecyltrimethylammonium chloride, and pentaethylene glycol monododecyl ether have been studied by fluorescence correlation spectroscopy using commercially available dyes. The translational diffusion coefficient and the critical micelle concentrations have been determined and compare well to values reported in the literature. The respective charges of the surfactant and of the dye molecule are crucial for the effectiveness of the presented method.     

Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS) we measure the translational diffusion coefficient of asphaltene molecules in toluene at extremely low concentrations (0.03-3.0 mg/L): where aggregation does not occur. We find that the translational diffusion coefficient of asphaltene molecules in toluene is about 0.35 x 10(-5) cm(2)/s at room temperature. This diffusion coefficient corresponds to a hydrodynamic radius of approximately 1 nm. These data confirm previously estimated size from rotational diffusion studied using fluorescence depolarization. The implication of this concurrence is that asphaltene molecular structures are monomeric, not polymeric.     

Extracting decay curves of the correlated fluorescence photons measured in fluorescence correlation spectroscopy

We have developed a new method to extract the decay curves of the correlated fluorescence photons from the data of fluorescence correlation spectroscopy using time-correlated single photon counting. In this method, a two-dimensional correlation map of photon pairs is generated at an absolute delay time with reference to the excitation-emission delay of each photon. Using a dye-labeled DNA as an example, we have demonstrated that the decay curve of the correlated fluorescence photons is separated from the uncorrelated background signals simply by subtracting a two-dimensional correlation map at sufficiently long delay time without additional prior information.     

Stochastic approach to data analysis in fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has emerged as a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In FCS, the experimental data is conventionally fit using standard local search techniques, for example, the Marquardt-Levenberg (ML) algorithm. A prerequisite for these categories of algorithms is the sound knowledge of the behavior of fit parameters and in most cases good initial guesses for accurate fitting, otherwise leading to fitting artifacts. For known fit models and with user experience about the behavior of fit parameters, these local search algorithms work extremely well. However, for heterogeneous systems or where automated data analysis is a prerequisite, there is a need to apply a procedure, which treats FCS data fitting as a black box and generates reliable fit parameters with accuracy for the chosen model in hand. We present a computational approach to analyze FCS data by means of a stochastic algorithm for global search called PGSL, an acronym for Probabilistic Global Search Lausanne. This algorithm does not require any initial guesses and does the fitting in terms of searching for solutions by global sampling. It is flexible as well as computationally faster at the same time for multiparameter evaluations. We present the performance study of PGSL for two-component with triplet fits. The statistical study and the goodness of fit criterion for PGSL are also presented. The robustness of PGSL on noisy experimental data for parameter estimation is also verified. We further extend the scope of PGSL by a hybrid analysis wherein the output of PGSL is fed as initial guesses to ML. Reliability studies show that PGSL and the hybrid combination of both perform better than ML for various thresholds of the mean-squared error (MSE).     

Two-dimensional near-infrared correlation spectroscopy study the methanol in acidic pH region

The effect of pH on the structure of methanol was investigated by FT-NIR (Fourier transform near-infrared) spectroscopy and generalized two-dimensional (2D) correlation spectroscopy. pH perturbed 2D correlation spectra are calculated for the spectra in the 5,500-4,000, 7,500-5,500 cm(-1) regions at different pH values. We observed that the stretching of CH(3) was shifted because of the direct interaction of the CH(3) group of methanol with the OH group of water, the change of free OH is more sensitive to pH than the cyclic dimmer and CH.O.     

The decay of the fluorescence anisotropy of tryptophan residues in fungal lipase fromHumicola lanuginosa

Pulse nanosecond fluorescence anisotropy decay has been used to study the mobility of tryptophan residues within fungal lipase fromHumicola lanuginosa. The decay of emission anisotropy of protein in native, inhibited and mutated form has been investigated in buffered water and 50% v/v glycerol solutions. The rotational motions of the lipase were analyzed in terms of two different kinetic models. It was found that the fluorescence emission anisotropy decay can best be desribed with two rotational correlation times: 0.63 and 5.45 ns in water and 0.98 and 10.70 ns and in 50% v/v glycerol solution. Using the same experimental conditions the decay of inhibited and mutatedH. lanuginosa lipase showed a similar biexponential character. These results are interpreted in terms of local or segmental motion arising from a mass of about 1083 daltons which corresponds to the ‘lid’-helix fragment of the enzyme.     

Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.     

Dual-focus fluorescence correlation spectroscopy of colloidal solutions: influence of particle size

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring diffusion coefficients of small fluorescent molecules at pico- to nanomolar concentrations. Recently, a modified version of FCS, dual-focus FCS (2fFCS), was introduced that significantly improves the reliability and accuracy of FCS measurements and allows for obtaining absolute values of diffusion coefficients without the need of referencing again a known standard. It was shown that 2fFCS gives excellent results for measuring the diffusion of small molecules. However, when measuring colloids or macromolecules, the size of these objects can no longer be neglected with respect to the excitation laser focus. Here, we analyze how 2fFCS data evaluation has to be modified for correctly taking into a count these finite size effects. We exemplify the new method of measuring the absolute size of polymeric particles with simple and complex fluorophore distributions.     

Frequency-domain fluorescence spectroscopy, a new method for the resolution of complex fluorescence emission

The fluorescence emission from complex chemical and biological samples can be resolved by measuring the frequency-response of the emission, which is now possible from 1 to 2000 MHz. The frequency-response allows determination of the components in a mixture, construction of time-resolved emission spectra, and measurement of the dynamic and hydrodynamic properties of biological macromolecules. The instrumentation is relatively simple, and data acquisition times can be short. At present, this method may be superior to direct measurements of time-resolved fluorescence emission.     

Dye-exchange dynamics in micellar solutions studied by fluorescence correlation spectroscopy

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.     

The residence probability: single molecule fluorescence correlation spectroscopy and reversible geminate recombination

The residence probability of a freely diffusing particle within an open d-dimensional ball is calculated as a function of time, for an initial distribution that is either a spherical delta function or uniform within the sphere. The latter is equivalent to the autocorrelation function (ACF) of fluorescence correlation spectroscopy (FCS) when utilizing near-field scanning optical microscopy (NSOM) probes. Starting from the general equation for the Laplace transform of the residence probability, we solve it in Laplace space for any dimensionality, inverting it into the time domain in one- and three-dimensions. The short- and long-time asymptotic behaviors of the residence probability are derived and compared with the exact results. Approximations for the two-dimensional ACF are discussed, and a new approximation is derived for the NSOM-FCS ACF. Also of interest is an analytic expression for a three-dimensional ACF, which could be useful for two-photon FCS. Analogy with the binding probability for reversible geminate recombination suggests that more information could be extracted from the long-time tails in FCS experiments.