Fluorescence correlation spectroscopy in colloid and interface science

In the last two decades fluorescence correlation spectroscopy (FCS) has been increasingly applied to analyze systems and processes relevant to colloid and interface science. The method has become a routine tool to measure the hydrodynamic radii of small fluorescent molecules, macromolecules and nanoparticles, characterize their interactions and follow a possible aggregation. It was also used to study the diffusion of such species in inhomogeneous media like polymer melts, solutions, gels or porous structures. The formation kinetics and size of micelles of surfactants or block copolymers has been quantified. FCS has also been applied to characterize diffusion of tracers at fluid–liquid and solid–liquid interfaces and study the hydrodynamic boundary condition. The review is intended to summarize these applications and highlight perspectives but also limits of FCS in colloid and interface science.     

Fluorescence correlation spectroscopy evidence for structural heterogeneity in ionic liquids

In this work, we provide new experimental evidence for chain length-dependent self-aggregation in room temperature ionic liquids (RTILs) using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [C(n)MPy][Tf(2)N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decreased with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chain [C(n)MPy][Tf(2)N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [C(n)MPy](+).     

Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities

Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.     

Fluorescence correlation spectroscopy analysis of segmental dynamics in actin filaments

We adapt fluorescence correlation spectroscopy (FCS) formalism to the studies of the dynamics of semiflexible polymers and derive expressions relating FCS correlation function to the longitudinal and transverse mean-square displacements of polymer segments. The obtained relations do not depend on any specific model of polymer dynamics. We use the derived expressions to measure the dynamics of actin filaments in two experimental situations: filaments labeled at distinct positions and homogeneously labeled filaments. Both approaches give consistent results and allow to measure the temporal dependence of the segmental mean-square displacement over almost five decades in time, from approximately 40 micros to approximately 2 s. These noninvasive measurements allow for a detailed quantitative comparison of the experimental data to the current theories of semiflexible polymer dynamics. Good quantitative agreement is found between the experimental results and theories explicitly accounting for the hydrodynamic interactions between polymer segments.     

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.     

Fluorescence correlation spectroscopy of gold nanoparticles,and its application to an aptamer-based homogeneous thrombin assay

We have studied the fluorescence properties and diffusion behaviors of gold nanoparticles (GNPs) in solution by using fluorescence correlation spectroscopy (FCS) at single molecule level. The GNPs display a high photo-saturation feature. Under illumination with strong laser light, they display higher brightness per particle (BPP) despite their low quantum yields. Based on the unique fluorescence properties and diffusion behaviors of GNPs, we have developed a sensitive and homogenous thrombin assay. It is based on a sandwich strategy and is making use of GNPs to which two different aptamers are conjugated. When the differently aptamer-labeled GNPs are mixed with solutions containing thrombin, the affinity reaction causes the GNPs to form dimers or oligomers. This leads to an increase in the diffusion time of the GNPs in the detection volume that is seen in FCS. The FCS method enables sensitive detection of the change in the characteristic diffusion time of the GNPs before and after the affinity reaction. Quantitative analysis of thrombin is based on the measurement of the change in the diffusion time. Under optimal conditions, the calibration plot is linear in the 0.5 nM to 110 nM thrombin concentration range, and the detection limit is 0.5 nM. The method was successfully applied to the direct determination of thrombin in human plasma.

Analytical applications of photon correlation spectroscopy for size distribution measurements of natural colloidal suspensions: capabilities and limitations

Particles are ubiquitous in all natural systems and play an important role in the control and fate of nutrients and pollutants. Currently, only limited information is available concerning particle number and size distributions, owing to the problems involved in their experimental determination. In the present paper, limitations and optimal conditions for particle size determinations of environmental samples using photon correlation spectroscopy are studied. The detection limit, the effects of polydispersity of the sample and the refractive index value are discussed based on results obtained with synthetic colloids. The photon correlation spectroscopic determination of particle size distributions in real aquatic systems is also presented in the second part of the paper.     

Fluorescence correlation spectroscopy studies of diffusion of a weak polyelectrolyte in aqueous solutions

We apply fluorescent correlation spectroscopy (FCS) to investigate solution dynamics of a synthetic polyelectrolyte, i.e., a weak polycarboxylic acid in aqueous solutions. The technique brings single molecule sensitivity and molecular specificity to dynamic measurements of polyelectrolyte solutions. Translational diffusion of Alexa-labeled poly(methacrylic acid), PMAA*, chains was studied in very dilute, 10(-4) mg/ml, solutions as a function of solution pH and ionic strength. The observed changes in diffusion coefficients were consistent with about twofold expansion of PMAA* coils when pH was changed from 5 to 8, and with chain contraction for alkaline metal ion concentrations from 0.01 to 0.1 M. The dependence of the hydrodynamic size of PMAA* chains on the counterion type followed the sequence: Li(+)>Na(+) approximately equal to Cs(+)>K(+). The dependence of translational diffusion on polyacid concentration was weak at the low concentration limit, but chain motions were significantly slower at higher polymer concentrations when PMAA chains overlapped. Finally, measurements of dynamics of PMAA* chains in "salt-free" solutions showed that self-diffusion of PMAA* chains significantly slowed down when PMAA concentration was increased, probably reflecting the sensitivity of PMAA* translational motions to the onset of interchain domain formation. These results illustrate the utility of the FCS technique for studying hydrodynamic sizes of polyelectrolyte coils in response to variation in solution pH or concentration of salt and polyelectrolytes. They also suggest that FCS will be a promising technique for selective observation of the dynamics of polyelectrolyte components in complex polymer mixtures.     

Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: inclusion complexes of pyronines with cyclodextrin

The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association rate constants are very similar for both guests and among the highest reported for this type of systems, although much lower than the diffusion-controlled collision rate constant. A two-step model including the formation of an encounter complex allows one to identify the unimolecular inclusion reaction as the rate-limiting step. Simulations indicate that this step may be controlled by geometrical and orientational requirements. These depend on critical molecular dimensions which are only weakly affected by the different alkyl substituents of PY and PB. Diffusion coefficients of PY and PB, of their complexes, and of rhodamine 110 are given and compared to those of similar molecules.     

Mapping vortex-like hydrodynamic flow in microfluidic networks using fluorescence correlation spectroscopy

The ability to quickly measure flow parameters in microfluidic devices is critical for micro total analysis system (μTAS) applications. Macrofluidic methods to assess flow suffer from limitations that have made conventional methods unsuitable for the flow behavior profiling. Single molecule fluorescence correlation spectroscopy (FCS) has been employed in our study to characterize the fluidic vortex generating at a T-shape junction of microscale channels. Due to its high spatial and temporal resolution, the corresponding magnitudes relative to different flow rates in the main channel can be quantitatively differentiated using flow time (τ_F) measurements of dye molecules traversing the detection volume in buffer solution. Despite the parabolic flow in the channel upstream, a heterogeneous distribution of flow has been detected across the channel intersection. In addition, our current observations also confirmed the aspect of vortex-shaped flow in low-shear design that was developed previously for cell culture. This approach not only overcomes many technical barriers for examining hydrodynamic vortices and movements in miniature structures without physically integrating any probes, but it is also especially useful for the hydrodynamic studies in polymer-glass based micro -reactor and -mixer.     

Solids, flow systems and atomic spectroscopy

An overview of major combinations and recent applicatoons of the solids/flow systems/atomic spectroscopy trinomial is presented. Several representative examples, classified according to the role played by the solids (sample or active component of a chemical reaction or separation) are described.     

Fluorescence correlation spectroscopy analysis of diffusion in a laser gradient field: a numerical approach

Fluorescence correlation spectroscopy (FCS) is widely used in biological systems. When the laser is intense enough, such as in two-photon experiments, the trapping force due to the laser gradient field can change the diffusion behavior of the fluorescent particles and induce error in the FCS measurements. Previous studies on biased FCS are qualitative. In this article, a numerical approach is proposed to treat the problem quantitatively. By assumption of a "spherical symmetry", biased FCS curves can be calculated numerically and fitted to the experimental data to retrieve the unbiased particle number, diffusion time, and polarizability of the fluorescent particles as well as the strength of the gradient field. It has been proven using simulated FCS data that the discrepancy caused by the spherical symmetry approximation is independent of the gradient field strength; therefore it can be eliminated by a calibration.     

Two-dimensional correlation analysis useful for spectroscopy, chromatography, and other analytical measurements.

Application of generalized two-dimensional (2D) correlation in various analytical fields is explored. 2D correlation is a powerful and versatile technique applicable to spectroscopy, chromatography, and other measurements. Construction of 2D spectra is relatively straightforward, requiring only a series of systematically varying analytical signals, like spectra or chromatograms, induced by an external perturbation applied to the system of interest. Perturbation can take many different forms, like change in temperature, pressure or concentration, chemical reactions, electrical or mechanical stimuli, and so on. A set of analytical signals collected under a perturbation are then converted to 2D correlation spectra, which provide rich and useful information about the presence of coordinated or independent changes among signals, as well as relative directions and sequential order of signal intensity variations. The signal resolution is also enhanced by spreading overlapped bands along the second dimension. Illustrative examples of 2D correlation are given for spectroscopic and chromatographic applications.     

A simple method for correcting baseline artifacts in nuclear magnetic resonance spectroscopy

A simple baseline correction program is described. It depends on an adjustable threshold for initiation of a data-sorting process between the signals and baseline of a computer-stored n.m.r. spectrum. An application is discussed.     

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research

A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.     

Fluorescence correlation spectroscopy of water-in-oil microemulsions: an application in specific characterisation of droplets containing biomolecules

Fluorescence correlation spectroscopy (FCS) has been successfully used to characterise water-in-oil (w/o) microemulsions. The investigated systems were stabilised by sodium bis-2-ethylhexyl sulphosuccinate (AOT) and the measured diffusion times have been related to the radii of the aggregated species, which for some systems, were separately determined by small-angle neutron scattering (SANS). We demonstrate that FCS is capable of measuring hydrodynamic radii of microemulsions rapidly and at surfactant concentrations lower than previously reported for other techniques. FCS was also used to specifically interrogate microemulsion droplets containing a fluorescently-labelled biomolecule, specifically phalloidin, a peptide fungal toxin from Amanita phalloides, and the enzyme -chymotrypsin (-CT). The microemulsion droplets are only marginally increased in size if a small peptide (phalloidin) is included in the water phase, whereas the droplet size is significantly increased when a larger protein (-CT) is included.     

Fluorescence spectroscopy in biological and medical research

Fluorescence spectroscopy is one of the most sensitive and versatile tools in medical, biological and biochemical research. Here we discuss the use of (1) polarized fluorescence spectroscopy to study the conformational dynamics of proteins, (2) fluorescence recovery after photobleaching to study lateral mobility of proteins and lipids in biological cell membranes and (3) excitation energy transfer to measure distances between interesting sites in macromolecules and biological membranes.     

Fluorescence correlation spectroscopy simulations of photophysical phenomena and molecular interactions: a molecular dynamics/monte carlo approach

Fluorescence correlation spectroscopy (FCS) is being applied increasingly to study diffusion and interactions of fluorescently labeled macromolecules in complex biological systems. Fluctuations in detected fluorescence, deltaF(t), are expressed as time-correlation functions, G(tau), and photon-count histograms, P(k;DeltaT). Here, we developed a generalized simulation approach to compute G(tau) and P(k;DeltaT) for complex systems with arbitrary geometry, photophysics, diffusion, and macromolecular interactions. G(tau) and P(k;DeltaT) were computed from deltaF(t) generated by a Brownian dynamics simulation of single-molecule trajectories followed by a Monte Carlo simulation of fluorophore excitation and detection statistics. Simulations were validated by comparing analytical and simulated G(tau) and P(k;DeltaT) for diffusion of noninteracting fluorophores in a three-dimensional Gaussian excitation and detection volume. Inclusion of photobleaching and triplet-state relaxation produced significant changes in G(tau) and P(k;DeltaT). Simulations of macromolecular interactions and complex diffusion were done, including transient fluorophore binding to an immobile matrix, cross-correlation analysis of interacting fluorophores, and anomalous sub- and superdiffusion. The computational method developed here is generally applicable for simulating FCS measurements on systems complicated by fluorophore interactions or molecular crowding, and experimental protocols for which G(tau) and P(k;DeltaT) cannot be computed analytically.     

Determination of relative rate of spectral events by novel modification of two-dimensional correlation spectroscopy

Sign of two-dimensional (2D) correlation peaks provides information on sequence of spectral events. This information is related to molecular mechanism of changes in a given system. Recently, few papers addressing the problems with interpretation of the sign of 2D correlation peaks have been published. To overcome these problems, a modification of the generalized 2D correlation method has been proposed. This method compares variations in the dynamic spectrum with a linear change at a reference point. The rates of spectral responses at individual wavenumbers are proportional to magnitudes of the peaks in the slice of asynchronous spectrum at the reference point. This way, analysis of complex 2D contour plots is replaced by a simple examination of one-dimensional (1D) slice spectrum. In spite of reduced ability of the resolution enhancement, in special cases the proposed method provides information not accessible from the classical 2D correlation analysis. At first, the principles of this method are shown with the synthetic data. Next, the influence of spectral separation, band width and position changes on the slice spectrum is evaluated. Finally, the proposed approach is applied to the experimental spectra of two hydrogen-bonded systems.     

Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.