Fluorescence Correlation Spectroscopy Measurements Research Articles

Binding of the Alzheimer amyloid β-peptide to neuronal cell membranes by fluorescence correlation spectroscopy

The deposition of the Alzheimer amyloid β-peptide (Aβ) fibrils in brain is a key step in Alzheimer's disease. The aggregated Aβ is found to be toxic to neurons since cells die when the aggregated Aβ is added to the cell culture medium. However, target of action of Aβ to cells is unknown. We have applied the fluorescence correlation spectroscopy (FCS) technique to study the existence of a receptor or target molecule for the Alzheimer amyloid β-peptide (Aβ) in cultured human cerebral cortical neurons. FCS measurement of the fluorophore rhodamine-labeled Aβ (Rh-Aβ) shows diffusion times: 0.1 ms, 1.1 ms and 5.9 ms. Thus, 0.1 ms corresponds to the unbound Rh-Aβ, and 1.1 ms and 5.9 ms correspond to slowly diffusing complexes of Rh-Aβ bound to a kind of receptor or target molecule for Aβ. Addition of excess non-labeled Aβ is accompanied by a competitive displacement, showing that the Aβ binding is specific. Full saturation of the Aβ binding is obtained at nanomolar concentrations, indicating that the Aβ binding is of high affinity. The notion that using FCS we have found a kind of receptor or target molecule for Aβ makes an important point that Aβ kills cells possibly by affecting cell membranes via a receptor or target molecule. This study is of highly significance since it suggests that Aβ possibly affects neuronal cell membranes of Alzheimer patients via a receptor or target molecule.

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Fluorescence correlation spectroscopy in cells: Confinement and excluded volume effects

Fluorescence correlation spectroscopy (FCS) has become an important technique in biophysical research, which is also used for in vivo studies of molecular mobilities in cells. We theoretically study how confinement or exclusion of the diffusing fluorescent molecules by a spherical region influences the measured autocorrelation function in an FCS experiment. It is shown that close to the boundary of the spherical region the diffusion time can be significantly changed due to the geometric restriction of the detection volume. This is important when quantitatively evaluating and interpreting FCS measurements in cells.

Dye-substrate interactions evidenced by fluorescence correlation spectroscopy

In this report, we discuss how the nature of the substrate-water interaction may modify the free diffusion of single molecules, then influencing fluorescence correlation spectroscopy measurements. Our results show a strong influence, up to a few micrometers from the interface, of the surface polarity. This effect is assessed through the relative weight of the two dimension diffusion process observed close to the surface.

Polymer inclusion membranes: The concept of fixed sites membrane revised

The mechanism of facilitated transport of metal ions across polymer inclusion membranes (PIMs) is revised on the basis of transport flux measurements and of new data brought by techniques sensitive to local inter-molecular interactions and molecular diffusion. Cellulose triacetate (CTA) membranes built with two types of inclusion carriers: a liquid one Aliquat 336 and a crystalline one Lasalocid A, both able to carry metal ions across PIMs and supported liquid membranes (SLMs) made of the same components, have been compared. Both PIM systems show similar effects for what concern the need of a carrier threshold concentration for the occurrence of a transport flux across PIM as revealed by flux and fluorescence correlation spectroscopy (FCS) measurements, and the dependence of the chemical nature of plasticizers on the metal ion flux. These systems also present similar Raman and far IR signatures of structural evolution of PIMs with the increase of the carrier concentration within the CTA matrix. All the presented data are interpreted as concern PIMs, according to an evolution of chemical interactions between components of the polymeric membrane able to lead to a phase transition. This phase transition type of the carrier-plasticized polymer system is induced by the increase of carrier concentration in the polymer chains. The PIM progressively organizes itself like a liquid SLM because of the enhancement of preferential solvent interactions between the carrier and the plasticizer. The main conclusion of this study is that the classically adopted “hopping” transport mechanism between fixed carrier sites in a PIM does not apply to such carrier chemically unbound to polymer membrane systems.

Spatially Resolved Fluorescence Correlation Spectroscopy Using a Spinning Disk Confocal Microscope

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to ∼10 5 independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D∼10 −7 cm 2/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.

High-Affinity and Cooperative Binding of Oxidized Calmodulin by Methionine Sulfoxide Reductase

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1–DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1–DNA complex is estimated to be 0.55 μM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.

Fluorescence correlation spectroscopy shows that monomeric polyglutamine molecules form collapsed structures in aqueous solutions.

We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length (N) by measuring the scaling of translational diffusion times (tau(D)) for the peptide series (Gly)-(Gln)(N)-Cys-Lys(2) in aqueous solution. We find that tau(D) scales with N as tau(o)N(nu) and therefore ln(tau(D)) = ln(tau(o)) + nuln(N). The values for nu and ln(tau(o)) are 0.32 +/- 0.02 and 3.04 +/- 0.08, respectively. Based on these observations, we conclude that water is a polymeric poor solvent for polyglutamine. Previous studies have shown that monomeric polyglutamine is intrinsically disordered. These observations combined with our fluorescence correlation spectroscopy data suggest that the ensemble for monomeric polyglutamine is made up of a heterogeneous collection of collapsed structures. This result is striking because the preference for collapsed structures arises despite the absence of residues deemed to be hydrophobic in the sequence constructs studied. Working under the assumption that the driving forces for collapse are similar to those for aggregation, we discuss the implications of our results for the thermodynamics and kinetics of polyglutamine aggregation, a process that has been implicated in the molecular mechanism of Huntington's disease.

Improvement of Biomolecule Quantification Precision and Use of a Single-Element Aspheric Objective Lens in Fluorescence Correlation Spectroscopy

We found a way to increase the precision with which biomolecules present at concentrations below 10(-10) M can be quantified by fluorescence correlation spectroscopy (FCS). The effectiveness of the way was demonstrated experimentally by using a single-element aspheric objective lens, which was newly developed to reduce the cost of FCS instruments. In the first part of this paper, the relative standard deviation (RSD) of FCS-based concentration measurements is estimated theoretically by an analytical approximation assuming the detection volume profiles in FCS setups to be Gaussian and by molecular simulations in which more realistic profiles are calculated from physical parameters of the measurement setups. In a limit of infinitely bright molecules and zero background emission, the analytical approximation predicts that the RSD at a concentration is minimized when the mean number of molecules in a detection volume is approximately 0.5. A detection volume of the order of 10(-13) L thus gives smaller RSD values for concentrations from 10(-11) to 10(-10) M than does one of the order of 10(-15) L, which is widely used in FCS. This prediction is supported by the molecular simulations, taking into account the finite molecule brightness and background emission. In the second part of the paper, the RSD is evaluated experimentally with an FCS setup with a detection volume of 1.1 x 10(-13) L. The newly developed objective lens, serving as the bottom of the sample cell in this setup, has a large numerical aperture (0.9) without using immersion liquid. When a calibration line was made by 30-s FCS measurements of Cy3-labeled, 112-mer single-stranded DNA solutions, the RSD roughly agreed with the simulation result and was less than 0.1 for DNA concentrations from 2 x 10(-11) to 10(-10) M.

Fiber-optic fluorescence correlation spectrometer

Fluorescence correlation spectroscopy (FCS) is a sensitive technique used to probe size, concentration, flow velocity, and reaction kinetics in a dilute solution. Conventional FCS spectrometers achieve this sensitivity at the cost of using bulky optics. We demonstrate a technique that utilizes a single-mode optical fiber of 3.3 microm mode field diameter to perform FCS measurements. We demonstrate that the technique has adequate sensitivity to perform FCS measurements on fluorescent beads of 13 nm radius, and that the results agree with theoretical predictions. Our method potentially allows FCS to be extended to remote and in vivo applications.

CHARACTERIZATION OF POLY(ACRYLIC ACID) DIFFUSION DYNAMICS ON THE GRAFTED SURFACE OF POLY(ETHYLENE TEREPHTHALATE) FILMS BY FLUORESCENCE CORRELATION SPECTROSCOPY

Poly(acrylic acid) (PAAc) is a commonly used polymer grafted on poly(ethylene terephthalate) films for the immobilization of bioactive molecules that directly interact with living cells or tissues for the maintenance of their viability and functionality. The diffusion property of the grafted PAAc on the surface is a critical parameter related to the density, length of polymer chains, and ionic strength of the solution. Fluorescence correlation spectroscopy (FCS) is able to measure the diffusion coefficient of fluorescent particles in solution with single molecule sensitivity and specificity. It was used as an effective tool to detect diffusion dynamics of Atto 565 molecules, a good indicator for viscosity of PAAc, in both aqueous polymer solutions and polymer grafted film surfaces immersed in solution. In this work we determine the polymer chain length under different polymerization conditions in solution and deduce the solution viscosity by FCS measurements of Atto 565 as tracer molecule. By using the same tracer on the grafted polymer films we can infer the viscosity of these grafted layers under a variety of conditions, including the PAAc chain length, the UV exposure time during polymerization, the ionic strength, and the pH value of the immersed solution.

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.

Wormlike lipid/DNA micelles in a non-polar solvent

The phase behavior of DOPE/DOTAP-DNA complexes in phase-separated oil(dodecane)/water mixtures was explored using Small Angle X-Ray Scattering (SAXS) and Fluorescence Correlation Spectroscopy (FCS). Inverse micelles of DNA with cationic-lipid coating were found in the oil phase. Varying the ratio between cationic and neutral lipids a transition from wormlike to spherical structures is observed for both long ( approximately 75000bp) and short (30-1246bp) DNA. In contrast to lipid/DNA complexes in the water phase, there is no indication of condensed liquid-crystalline structures in the non-polar phase. In fact, FCS measurements on short DNA oligomers complexed with cationic lipid in alkane give clear evidence for monomeric inverse micelles of DNA. Dilution series revealed a critical lower concentration of lipids and DNA for observing lipid/DNA micelles.

Accounting for Triplet and Saturation Effects in FCS Measurements

Fluorescence correlation spectroscopy (FCS) is an increasingly important tool for determining low concentrations and dynamics of molecules in solution. Oftentimes triplet transitions give rise to fast blinking effects, which are accounted for by including an exponential term in the fitting of the autocorrelation function (ACF). In such cases, concomitant saturation effects also modify the amplitude and shape of the remaining parts of the ACF. We review studies of triplet and saturation effects in FCS and present a simple procedure to obtain more accurate results of particle concentrations and diffusional dynamics in experiments where triplet kinetics are evident, or where moderate laser powers approaching saturation levels are used, for example, to acquire sufficient photon numbers when observation times are limited. The procedure involves use of a modified function for curve-fitting the ACF, but there are no additional fitting parameters. Instead, a simple calibration of the total fluorescence count rate as a function of relative laser power is fit to a polynomial, and the non-linear components of this fit, together with the relative laser power used for the FCS measurement, are used to specify the magnitude of additional terms in the fitting function. Monte Carlo simulations and experiments using Alexa dyes and quantum dots, with continuous and pulsed laser excitation, demonstrate the application of the modified fitting procedure with first order correction terms, in the regime where distortions in the ACF due to photobleaching and detector dead time are small compared to those of fluorescence saturation and triplet photophysics.

Coil–globule transition in the denatured state of a small protein

Upon transfer from strongly denaturing to native conditions, proteins undergo a collapse that either precedes folding or occurs simultaneously with it. This collapse is similar to the well known coil-globule transition of polymers. Here we employ single-molecule fluorescence methods to fully characterize the equilibrium coil-globule transition in the denatured state of the IgG-binding domain of protein L. By using FRET measurements on freely diffusing individual molecules, we determine the radius of gyration of the protein, which shows a gradual expansion as the concentration of the denaturant, guanidinium hydrochloride, is increased all the way up to 7 M. This expansion is observed also in fluorescence correlation spectroscopy measurements of the hydrodynamic radius of the protein. We analyze the radius of gyration measurements using the theory of the coil-globule transition of Sanchez [Sanchez, I. C. (1979) Macromolecules 12, 980-988], which balances the excluded volume entropy of the chain with the average interresidue interaction energy. In particular, we calculate the solvation energy of the denatured protein, a property that is not readily accessible in other experiments. The dependence of this energy on denaturant concentration is nonlinear, contrasting with the common linear extrapolation method used to describe denaturation energy. Interestingly, a fit to the binding model of chemical denaturation suggests a single denaturant binding site per protein residue. The size of the denatured protein under native conditions can be extrapolated from the data as well, showing that the fully collapsed state of protein is only approximately 10% larger than the folded state.

Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Delta, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Delta apoprotein (apoCph1Delta) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Delta altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Delta and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø <120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

Membrane-Bound Basic Peptides Sequester Multivalent (PIP 2), but Not Monovalent (PS), Acidic Lipids

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.

Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N4,N9-dioleoylspermine and N1-cholesteryl spermine carbamate. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. N4,N9-dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 x 10(-12) m2/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio = 1.0-1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N1-cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5-2.5) with D = 1.3 x 10(-12) m2/s and PN value of 5.2. N4,N9-dioleoylspermine is a more efficient DNA-condensing agent than N1-cholesteryl spermine carbamate. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.