Fluorescence Correlation Spectroscopy Autocorrelation Research Articles

Cell-surface Initial Aggregation Process of Amyloid-β Peptides Studied by Fluorescence Correlation Spectroscopy

The formation of neurotoxic aggregates by amyloid-β peptide (Aβ) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of Aβ. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Fluorescence correlation spectroscopy (FCS) is a sensitive method to monitor molecular aggregation processes by measuring diffusion of fluorophore-labeled molecules. Here, we monitored the initial aggregation process of Aβ on cell membranes of SH-SY5Y neuroblastoma cells. For example, aggregation of Aβ-(1-42) was evaluated by using Aβ-(1-42) (5 μM) doped with fluorophore-Aβ-(1-42) (10 nM). A membrane-bound Aβ component was detected in FCS autocorrelation curve after 1-h incubation of the Aβs with the cells. Following incubation for ～10 h, Aβ-(1-42) formed oligomers composed of ～10 Aβ molecules. These Aβ oligomers formed on membranes did not induce activation of caspase-3, an effector caspase for apoptosis, therefore were not neurotoxic, in contrast to reported Aβ oligomers prepared in aqueous phase. Formation of larger Aβ fibrils on membranes was found to be critical for Aβ neurotoxicity. We also report that trace amounts of pyroglutaminated Aβ-(3-42), a minor species of Aβ, can enhance initial aggregation process of Aβ-(1-42) on the cell membranes. These results indicate the usefulness of FCS detection of small Aβ oligomers formed on cell surface, which can act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity.

Robustness of FCS (Fluorescence Correlation Spectroscopy) with Quenchers Present.

Inspired by recent publications doubtful of the FCS technique, we scrutinize how irreversible ("static") and reversible ("dynamic") quenching can influence the interpretation of such data. Textbook presentations often emphasize only how to analyze data in extremes, the absence of quenching or the presence of substantial quenching. Here, we consider intermediate cases where the assessment of photophysics (static quenching, blinking-like triplet-state relaxation) influence on autocorrelation curves can be delicate if dye-labeled objects diffuse on comparably rapid time scales. We used the amino acid, tryptophan, as the quencher. As our example of small-molecule dye that diffuses rapidly, we mix the quencher with the fluorescence dye, Alexa 488. The translational diffusion coefficient, inferred from fit to the standard one-component Fickian diffusion model, speeds up without the loss of quality of fit, but quenching is reflected in the fact that the data become exceptionally noisy. This reflects the bidisperse population of quenched and unquenched dyes when the time scales overlap between the processes of translational diffusion, quenching, and blinking. As our example of the large-molecule dye-labeled object that diffuses relatively slowly, we mixed the quencher with dye-labeled BSA, bovine serum albumin. Diffusion, static quenching, and blinking time scales are now separated. In spite of quenching contribution to the autocorrelation function when the delay time is relatively short, the inferred translational diffusion coefficient now depends weakly on the presence of a quencher. We conclude that when the diffusing molecule is substantially slower to diffuse than the time scale of photophysical processes of the fluorescent dye to which it is attached, the influence of quenching is self-evident and the FCS autocorrelation curves give an appropriate diffusion coefficient if correct fitting functions are chosen in the analysis.

Fluorescence Correlation Spectroscopic Studies of Particle Properties for Colloidal Ceria Abrasives Used in Chemical-Mechanical Planarization

The application of fluorescence correlation spectroscopy (FCS) in the characterization of ceria abrasives used in chemical-mechanical planarization (CMP) slurries is described. A fluorescent dye, Rhodamine 110 (R110), is used for this purpose. R110 adsorption on ceria particles is found to be pH-dependent and colloidal ceria dispersions differing in mean hydrodynamic diameter are analyzed. A Langmuir isotherm is generated for R110 adsorption on ceria, and the magnitude of the Langmuir constant is found to be four-times smaller than the corresponding constant for R110 adsorption on colloidal silica CMP abrasives. R110-labeled ceria particles are generated to quantitatively determine the particle size distribution (PSD) of abrasive ceria dispersions. The Maximum Entropy Method is used reduce the FCS autocorrelation function of R110-labeled ceria particle dispersions thereby producing a PSD. These findings indicate that ceria size distributions containing particles as small as 5 to 10 nm in diameter can be readily characterized using FCS.

Fluorescence Correlation Spectroscopy in Dilute Polymer Solutions: Effects of Molar Mass Dispersity and the Type of Fluorescent Labeling.

Fluorescence correlation spectroscopy (FCS) has become an important tool in polymer science. Among various other applications the method is often applied to measure the hydrodynamic radius and the degree of fluorescent labeling of polymers in dilute solutions. Here we show that such measurements can be strongly affected by the molar mass dispersity of the studied polymers and the way of labeling. As model systems we used polystyrene and poly(methyl methacrylate) synthesized by atom transfer radical polymerization or free-radical polymerization. Thus, the polymers were either end-labeled bearing one fluorophore per chain or side-labeled with a number of fluorophores per chain proportional to the degree of polymerization.The experimentally measured autocorrelation curves were fitted with a newly derived theoretical model that uses the Schulz-Zimm distribution function to describe the dispersity in the degree of polymerization. For end-labeled polymers having a molecular weight distribution close to Schulz-Zimm, the fits yield values of the number-average degree of polymerization and the polydispersity index similar to those obtained by reference gel permeation chromatography. However, for the side-labeled polymers such fitting becomes unstable, especially for highly polydisperse systems. Brownian dynamic simulations showed that the effect is due to a mutual dependence between the fit parameters, namely, the polydispersity index and the number-average molecular weight. As a consequence, an increase of the polydispersity index can be easily misinterpreted as an increase of the molecular weight when the FCS autocorrelation curves are fitted with a standard single component model, as commonly done in the community.

A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control

We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5′ end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1 fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value.

A universal model of restricted diffusion for fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is frequently used to study the processes of restricted diffusion. The most important quantity to determine is the size of the structures that hinder the Brownian motion of the molecules. We study three qualitatively different models of restricted diffusion, widely applied in biophysics and material science: Diffusion constrained by elastic force (i), walking confined diffusion (ii), and hop diffusion (iii). They cover the diversity of statistical behaviors, from purely Gaussian (i) to sharply non-Gaussian on intermediate time scales (ii) and, additionally, discrete (iii). We test whether one can use the Gaussian approximation of the FCS autocorrelation function to interpret the non-Gaussian data. We show that (i-iii) have approximately the same mean square displacements. Using simulations, we show that the FCS data suspected of restricted diffusion can be reliably interpreted using one archetypal model (i). Even if the underlying mechanism of the restriction is different or unknown, the accuracy of fitting the confinement size is excellent, and diffusion coefficients are also estimated with a good accuracy. This study gives a physical insight into the statistical behavior of different types of restricted diffusion and into the ability of fluorescence correlation spectroscopy to distinguish between them.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3′ or 5′) end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements.

Effects of multiple scattering on fluorescence correlation spectroscopy measurements of particles moving within optically dense media

Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media.

Probing the Interaction Between Fluorophores and DNA Nucleotides by Fluorescence Correlation Spectroscopy and Fluorescence Quenching†

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa-546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B-nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye-DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.

Evaluation of influence of anisotropic diffusion near a wall in near-field fluorescence correlation spectroscopy of nanoparticles

Fluorescence correlation spectroscopy (FCS) of colloidal nanoparticles using a near-field fiber probe was numerically simulated. The near-wall dynamics was simulated by accounting for the anisotropic mobility of nanoparticles owing to hydrodynamic interaction with a wall (Stokes viscous force). By comparing the simulation results with theoretical model calculations, we found that the influence of anisotropic diffusion is insignificant in near-field FCS autocorrelation analysis.

Characterization of the Surface Contribution to Fluorescence Correlation Spectroscopy Measurements

Fluorescence correlation spectroscopy (FCS) is a sophisticated and an accurate analytical technique used to study the diffusion of molecules in a solution at the single-molecule level. FCS is strongly affected by many factors such as the stability of the excitation power, photochemical processes, mismatch between the refractive indices, and variations in the cover glass thickness. We have studied FCS near the surface of a cover glass by using rhodamine 123 as a fluorescent probe and have observed that the surface has a strong influence on the measurements. The temporal autocorrelation of FCS decays with two characteristic times when the confocal detection volume is positioned near the surface of the cover glass. As the position of the detection volume is moved away from the surface, the FCS autocorrelation becomes one-component decaying; the characteristic time of the decay is the same as the faster-decaying component in the FCS autocorrelation near the surface. This observation suggests that the faster component can be attributed to the free diffusion of the probe molecules in the solution, while the slow component has its origin from the interaction between the probe molecules and the surface. We have characterized the surface contribution to the FCS measurements near the surface by changing the position of the detection volume relative to the surface. The influence of the surface on the diffusion of the probe molecules was monitored by changing the chemical properties of the surface. The surface contribution to the temporal autocorrelation of the FCS strongly depends on the chemical nature of the surface. The hydrophobicity of the surface is a major factor determining the surface influence on the free diffusion of the probe molecules near the surface.

Anomalous transport resolved in space and time by fluorescence correlation spectroscopy

A ubiquitous observation in crowded cell membranes is that molecular transport does not follow Fickian diffusion but exhibits subdiffusion. The microscopic origin of such a behaviour is not understood and highly debated. Here we discuss the spatio-temporal dynamics for two models of subdiffusion: fractional Brownian motion and hindered motion due to immobile obstacles. We show that the different microscopic mechanisms can be distinguished using fluorescence correlation spectroscopy (FCS) by systematic variation of the confocal detection area. We provide a theoretical framework for space-resolved FCS by generalising FCS theory beyond the common assumption of spatially Gaussian transport. We derive a master formula for the FCS autocorrelation function, from which it is evident that the beam waist of an FCS experiment is a similarly important parameter as the wavenumber of scattering experiments. These results lead to scaling properties of the FCS correlation for both models, which are tested by in silico experiments. Further, our scaling prediction is compatible with the FCS half-value times reported by Wawrezinieck et al. [Biophys. J. 89, 4029 (2005)] for in vivo experiments on a transmembrane protein.

How does sensitivity to dynamical heterogeneity in supercooled colloidal liquids depend on tracer size?

Few experimental investigations have examined the dependence of probe length scale on the evolution of dynamical heterogeneity in supercooled liquids. In this study, we use fluorescent tracer probes, which are smaller than that of the constituent unlabeled particles in the matrix suspension, to investigate the tracer size sensitivity to the onset of dynamical heterogeneity in model hard-sphere colloidal suspensions close to the glass transition. The dynamics of poly(methyl methacrylate) (PMMA) tracer particles of radii a(S) = 137 nm embedded into constituent PMMA particles of a(L) = 212 and 637 nm with bulk volume fraction phi varied from 0.50 to 0.57 is examined using fluorescence correlation spectroscopy (FCS). Upon increasing phi for the largest tracer size ratio explored, f (= a(S)/a(L)) = 0.646, the onset of dynamic heterogeneity is observed from the broad distribution of characteristic decay times of the tracers, which is extracted from the measured FCS autocorrelation functions. The strong coupling between tracer size and its sensitivity to dynamical heterogeneity is reflected in the dynamic susceptibility, where dynamic correlation lengths are immeasurable for the smallest size ratio and show a marked increase to five tracer particle diameters for the largest size ratio.

Stick-and-Diffuse and Caged Diffusion: A Comparison of Two Models of Synaptic Vesicle Dynamics

Two models were recently proposed to enable us to understand the dynamics of synaptic vesicles in hippocampal neurons. In the caged diffusion model, the vesicles diffuse in small circular cages located randomly in the bouton, while in the stick-and-diffuse model the vesicles bind and release from a cellular cytomatrix. In this article, we obtain analytic expressions for the fluorescence correlation spectroscopy (FCS) autocorrelation function for the two models and test their predictions against our earlier FCS measurements of the vesicle dynamics. We find that the stick-and-diffuse model agrees much better with the experiment. We find also that, due to the slow dynamics of the vesicles, the finite experimental integration time has an important effect on the FCS autocorrelation function and demonstrate its effect for the different models. The two models of the dynamics are also relevant to other cellular environments where mobile species undergo slow diffusionlike motion in restricted spaces or bind and release from a stationary substrate.

Microphotonic control of single molecule fluorescence correlation spectroscopy using planar optofluidics

We demonstrate the implementation of fluorescence correlation spectroscopy (FCS) on a chip. Full planar integration is achieved by lithographic definition of sub-picoliter excitation volumes using intersecting solid and liquid-core optical waveguides. Concentration dependent measurements on dye molecules with single molecule resolution are demonstrated. Theoretical modeling of the FCS autocorrelation function in microstructured geometries shows that the FCS behavior can be controlled over a wide range by tailoring the micro-photonic environment. The ability to perform correlation spectroscopy using silicon photonics without the need for free-space microscopy permits implementation of numerous diagnostic applications on compact planar optofluidic devices.

Combining Small Angle Neutron Scattering (SANS) and Fluorescence Correlation Spectroscopy (FCS) Measurements To Relate Diffusion in Agarose Gels to Structure

Small angle neutron scattering (SANS) and fluorescence correlation spectroscopy (FCS) measurements were carried out on agarose hydrogels to link their microscopic structure to the diffusivity of solutes at different scales. SANS allowed for the determination of the distribution of void volumes within the gels. They were shown to be compatible with a random network of cylindrical fibers as described by the Ogston model. FCS measured solute diffusivity in spaces similar in size to the void volumes, and thus, the results reflected the gel heterogeneity. Solute diffusivity was predicted by modeling the gel as microscopic geometrical cells. Variations in the diffusivity of solutes of different sizes could be predicted from the structural parameters of the gel using theory, taking into account obstruction by cylindrical cells and solute hydrodynamics. Prediction of the FCS autocorrelation functions for solutes from a cell model demonstrated a lack of sensitivity of this technique for multicomponent analysis.

Ligand binding by estrogen receptor beta attached to nanospheres measured by fluorescence correlation spectroscopy

Although many indirect methods have been chosen to study the system of estrogen receptor ligand binding, an ideal method is fluorescence correlation spectroscopy (FCS). FCS is nondestructive to the sample, uses very small sample volumes, and operates well within physiological concentration ranges. The methodology was developed to biotinylate the estrogen receptor beta-ligand binding domain (ERbeta-LBD) using biotin with a very short spacer and to then attach this protein to a 40 nm neutravidin-coated bead (nanosphere). Diffusional FCS data were obtained for a fluorescently labeled coactivator peptide, steroid receptor coactivator peptide-1 (A-SRC-1(2)), in the absence and presence of bead-bound ERbeta-LBD. Data were also acquired in the presence of one of the endogenous ligands for ERbeta, 17beta-estradiol, and with tamoxifen. The bead strategy resulted in a decreased receptor diffusion coefficient and consequent increase in the decay time of the FCS autocorrelation functions for receptor-bound, labeled SRC-1(2). Thus, free and bound coactivators were much more readily distinguished by FCS. Discrimination between the fluorescently labeled unbound and bound species could be determined in autocorrelation functions obtained in as few as 30 s. The advantage of using FCS with the ERbeta-LBD: bead methodology is the ability to obtain reliable and reproducible data in a short time frame.

Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 μs to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as ∼80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (∼4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS.

Noise on Fluorescence Correlation Spectroscopy

The time dependence of the noise and the signal-to-noise (SN) ratio of the fluorescence correlation spectroscopy (FCS) autocorrelation function is obtained from replica measurements of standard dextran solutions. The noise dependence on the delay time is fitted by a hyperbolic function with two fitting parameters. The dependence of these parameters on concentration, fluorescence intensity, and accumulation time is obtained experimentally. The behavior of SN at zero delay time agrees well with the theoretical predictions reported in the literature. The obtained data are useful for the quantitative evaluation of the FCS data fits, as well as for simulation of the FCS autocorrelation functions.

Theory of sample translation in fluorescence correlation spectroscopy

New applications of the technique of fluorescence correlation spectroscopy (FCS) require lateral translation of the sample through a focused laser beam (Peterson, N.O., D.C. Johnson, and M.J. Schlesinger, 1986, Biophys. J., 49:817-820). Here, the effect of sample translation on the shape of the FCS autocorrelation function is examined in general. It is found that if the lateral diffusion coefficients of the fluorescent species obey certain conditions, then the FCS autocorrelation function is a simple product of one function that depends only on transport coefficients and another function that depends only on the rate constants of chemical reactions that occur in the sample. This simple form should allow manageable data analyses in new FCS experiments that involve sample translation.