Dual-focus Fluorescence Correlation Spectroscopy Research Articles

Binding Constant Determined from the Angstrom-Scale Change in Hydrodynamic Radius of Transferrin upon Binding with Europium Using Dual-Focus Fluorescence Correlation Spectroscopy.

Monitoring the binding of a large fluorescently tagged molecule to a small solute by fluorescence correlation spectroscopy (FCS) is rather uncommon because the binding-related change in diffusion coefficient is very small. Here, we use a high-precision variant of FCS, namely, dual-focus FCS (2fFCS), for measuring the angstrom-scale change of the hydrodynamic radius of the bilobal metal transport protein transferrin (Tf) upon binding europium ions. Applying a sequential 1:2 complexation model, we use these measurements for determining the binding constants (K). Our results show a 0.7 Å change of the protein's hydrodynamic radius upon 1:1 Tf-Eu complex formation and a second change of 1.8 Å upon subsequent binding of a second europium ion. More than one unit variation in logK indicates an intrinsic dissimilarity in metal affinity of the C- and N-lobes of Tf, which agrees well with earlier reported ensemble spectroscopy results.

Loop formation and translational diffusion of intrinsically disordered proteins.

The conformational flexibility and dynamics of unfolded peptide chains is of major interest in the context of protein folding and protein functioning. The rate with which amino acids at different positions along the peptide chain meet sets an upper speed limit for protein folding. By using single-molecule photo-induced energy transfer spectroscopy, we have systematically measured end-to-end and end-to-internal site contact formation rates for several intrinsically disordered protein fragments (IDPs) (11 to 41 amino acids) and have also determined their hydrodynamic radius using dual-focus fluorescence correlation spectroscopy. For interpreting the measured values, we have developed a Brownian dynamics model (based on bead-rod chain dynamics in a thermal bath including hydrodynamic interactions) which quantitatively reproduces all measured data surprisingly well while requiring only two fit parameters. The model provides a complete picture of the peptides' dynamics and allows us to translate the experimental rates and radii into molecular properties of the peptides: We find a persistence length of l_{P}=5.2±1.9Å, a hydrodynamic radius of a=3.5±0.7Å per amino acid, and that excluded volume effects play an important role in the dynamics of IDPs.

The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

ABSTRACTRhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro. This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

Diffusion of rigid nanoparticles in crowded polymer-network hydrogels: dominance of segmental density over crosslinking density

Swollen polymer-network gels usually exhibit notable spatial inhomogeneity of their crosslinking density. The effect of this inhomogeneity on the permeability of the gel to small particles is of major importance in many applications such as those in analytical separation technology. To systematically address this effect, we mimic inhomogeneous polymer-network gels by dense-packed pastes of sub-micrometer-sized microgel building blocks with two distinctly different crosslinking degrees. The diffusive mobility of rigid nanoparticle tracers within these inhomogeneous pastes that contain purposely imparted densely and loosely cross-linked local domains is studied by spatially resolved dual-focus fluorescence correlation spectroscopy on a sub-micrometer length scale. The outcome of this investigation is that the sub-micrometer-scale tracer diffusivity of the tracers is not affected by the gel-matrix crosslinking density, and hence, also not by its spatial inhomogeneity. Instead, the tracer diffusion is dominantly hindered by the high density of polymer segments in the deswollen gel matrixes.

Accurate Diffusion Coefficients of Organosoluble Reference Dyes in Organic Media Measured by Dual-Focus Fluorescence Correlation Spectroscopy.

Dual-focus fluorescence correlation spectroscopy (2fFCS) is a versatile method to determine accurate diffusion coefficients of fluorescent species in an absolute, reference-free manner. Whereas (either classical or dual-focus) FCS has been employed primarily in the life sciences and thus in aqueous environments, it is increasingly being used in materials chemistry, as well. These measurements are often performed in nonaqueous media such as organic solvents. However, the diffusion coefficients of reference dyes in organic solvents are not readily available. For this reason we determined the translational diffusion coefficients of several commercially available organosoluble fluorescent dyes by means of 2fFCS. The selected dyes and organic solvents span the visible spectrum and a broad range of refractive indices, respectively. The diffusion coefficients can be used as absolute reference values for the calibration of experimental FCS setups, allowing quantitative measurements to be performed. We show that reliable information about the hydrodynamic dimensions of the fluorescent species (including noncommercial compounds) within organic media can be extracted from the 2fFCS data.

Comprehensive portrait of cholesterol containing oxidized membrane

Biological membranes are under significant oxidative stress caused by reactive oxygen species mostly originating during cellular respiration. Double bonds of the unsaturated lipids are most prone to oxidation, which might lead to shortening of the oxidized chain and inserting of terminal either aldehyde or carboxylic group. Structural rearrangement of oxidized lipids, addressed already, is mainly associated with looping back of the hydrophilic terminal group. This contribution utilizing dual-focus fluorescence correlation spectroscopy and electron paramagnetic resonance as well as atomistic molecular dynamics simulations focuses on the overall changes of the membrane structural and dynamical properties once it becomes oxidized. Particularly, attention is paid to cholesterol rearrangement in the oxidized membrane revealing its preferable interaction with carbonyls of the oxidized chains. In this view cholesterol seems to have a tendency to repair, rather than condense, the bilayer.

The fast polarization modulation based dual-focus fluorescence correlation spectroscopy

We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes.

Quantifying the Diffusion of Membrane Proteins and Peptides in Black Lipid Membranes with 2-Focus Fluorescence Correlation Spectroscopy

Protein diffusion in lipid membranes is a key aspect of many cellular signaling processes. To quantitatively describe protein diffusion in membranes, several competing theoretical models have been proposed. Among these, the Saffman-Delbrück model is the most famous. This model predicts a logarithmic dependence of a protein’s diffusion coefficient on its inverse hydrodynamic radius (D ∝ ln 1/R) for small radius values. For large radius values, it converges toward a D ∝ 1/R scaling. Recently, however, experimental data indicate a Stokes-Einstein-like behavior (D ∝ 1/R) of membrane protein diffusion at small protein radii. In this study, we investigate protein diffusion in black lipid membranes using dual-focus fluorescence correlation spectroscopy. This technique yields highly accurate diffusion coefficients for lipid and protein diffusion in membranes. We find that despite its simplicity, the Saffman-Delbrück model is able to describe protein diffusion extremely well and a Stokes-Einstein-like behavior can be ruled out.

Fluorescence correlation spectroscopy (IUPAC Technical Report)

We present an overview on the applicability of fluorescence correlation spectroscopy (FCS) for the accurate determination of translational diffusion coefficients and thus, via the Stokes–Einstein relation, of molecular size. We consider several of the most common sources of optical aberrations and their impact on the outcome of conventional FCS measurements. We describe also a new variant of FCS, dual-focus FCS, which is robust against most of the considered aberrations, and we report reference values of diffusion coefficients for several fluorescent dyes across the visible spectrum.

Flow of a nematogen past a cylindrical micro-pillar

We study the flow of a nematic liquid crystal past a micron-sized cylindrical pillar within a microfluidic confinement of a rectangular cross-section. The liquid crystal molecules are anchored perpendicularly (homeotropic anchoring) to the surface of the pillar and the channel walls. Flow past the cylindrical obstacle generated topological defect structures whose nature, dimensions and morphology varied with the flow velocity and channel dimensions. On increasing the flow speed, we observed sequential evolution of a semi-integer loop, which transformed into an integer hedgehog defect, and finally equilibrated to an extended defect wall. On stopping the flow, the topological defect states reversed its sequence of appearance. Additionally, we introduce dual-focus fluorescence correlation spectroscopy as a general velocimetry technique for microfluidics of liquid crystal systems – with or without topological defect structures.

Lipid Diffusion within Black Lipid Membranes Measured with Dual‐Focus Fluorescence Correlation Spectroscopy

We present an overview of the application of dual-focus fluorescence correlation spectroscopy (2f-FCS) for the measurement of diffusion coefficients within free-standing lipid membranes. The first part gives a detailed theoretical analysis of the expected performance of 2f-FCS, in particular about the sensitivity of the method with regard to precise focus position and to aberrations caused by refractive index mismatch or cover slide thickness deviation. After describing the experimental details of the 2f-FCS setup and the preparation of free-standing black lipid membranes (BLMs), we apply the method to study the diffusion of lipids within BLMs as a function of lipid composition and of ion valency and ionic strength of the surrounding buffer.

Protein Adsorption onto FePt Nanoparticles Investigated by Dual-Focus Fluorescence Correlation Spectroscopy

Upon exposure to biological fluids such as blood plasma, nanoparticles (NPs) adsorb proteins and other biomolecules from the fluid. The formation of the so-called ‘protein corona’ is a dynamic process: The composition of the corona depends on the respective concentrations of the proteins in the fluid and on their affinities to the NP. Understanding the structural and dynamic properties of the corona at the molecular level is a prerequisite to the safe use of NPs in industrial or medical applications.By using dual-focus fluorescence correlation spectroscopy (2fFCS), we have studied the adsorption of human blood serum proteins onto polymer-coated, fluorescently labeled FePt NPs (∼12 nm diameter) carrying negatively charged carboxyl groups on their surfaces. For all proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers. Indeed, the absolute increase in hydrodynamic radius can be correlated with the molecular dimensions of the proteins. The apparent dissociation coefficients measuring the affinity of the proteins to the nanoparticles differed by about four orders of magnitude. These variations can be explained in terms of the electrostatic properties of the protein surfaces.

Quantifying the Diffusion of Membrane Proteins and Peptides in Lipid Bilayers

Membrane proteins play a key role in cellular processes, e.g. ion transport into and out of the cell as well as signal transduction between cells. Protein diffusion in lipid membranes is important in this regard, since the kinetics of most protein interactions inside the membrane are diffusion limited.Protein diffusion can be investigated accurately with dual-focus fluorescence correlation spectroscopy (2f-FCS). In the present study, we measured the diffusion of lipids and the SNARE protein Synaptobrevin-2 in free-standing lipid bilayers (Black Lipid Membranes, BLM) and investigated the dependence of the diffusion coefficient on mono- and divalent ions.Diffusion of proteins and peptides within lipid bilayers is described by the Saffman-Delbruck model. It predicts a logarithmic dependence of the protein's diffusion coefficient on its hydrodynamic radius. In recent publications, however, this has been both challenged and supported.To test the validity of the Saffman-Delbruck model, we reconstituted proteins of different size into the lipid bilayer, e.g. the SNARE protein Syntaxin, the potassium channel KcsA and the chloride channel EcClC. We found that the Saffman-Delbruck model is only applicable for proteins with a significantly larger hydrodynamic radius compared to the lipids. The diffusion of proteins in the size range of the lipids, however, is better described with a Stokes-Einstein-like model, where the protein's diffusion coefficient is inversely proportional to its hydrodynamic radius.

Characterization of protein adsorption onto FePt nanoparticles using dual-focus fluorescence correlation spectroscopy

Using dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter) carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces.

The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3

STAT3 is an important transcription factor involved in immunity and cancer. In response to cytokine stimulation, STAT3 becomes phosphorylated on a single tyrosine residue. Tyrosine-phosphorylated STAT3 accumulates in the nucleus, binds to specific DNA response elements and induces gene expression. Unphosphorylated, latent STAT3 shuttles constitutively between cytoplasm and nucleus. We analysed the importance of previously identified putative nuclear localization sequences (NLS) and nuclear export sequences (NES) for nucleocytoplasmic shuttling of latent STAT3 using STAT3-deficient cells reconstituted with fluorescently labelled STAT3 mutants. Mutation of a putative NLS or NES sequence did not impair nucleocytoplasmic shuttling of latent STAT3. We were also interested in the structural requirements for dimerization of unphosphorylated STAT3 and its relevance for nucleocytoplasmic shuttling. By native gel electrophoresis and dual-focus fluorescence correlation spectroscopy (2f-FCS) we identified the N-terminal domain (amino acids 1-125) to be essential for formation of unphosphorylated STAT3 dimers but not for assembly of tyrosine-phosphorylated STAT3 dimers. In resting cells, the monomeric N-terminal deletion mutant (STAT3-ΔNT) shuttles faster between the cytoplasm and nucleus than the wild-type STAT3, indicating that dimer formation is not required for nucleocytoplasmic shuttling of latent STAT3. STAT3-ΔNT becomes phosphorylated and dimerizes in response to interleukin-6 stimulation but, surprisingly, does not accumulate in the nucleus. These results highlight the importance of the N-terminal domain in the formation of unphosphorylated STAT3 dimers and nuclear accumulation of STAT3 upon phosphorylation.

A comprehensive framework for fluorescence cross-correlation spectroscopy

Dual-colour fluorescence cross-correlation spectroscopy is a powerful method of studying binding between labelled biomolecules in vitro as well as in vivo. However, numerous artefacts and experimental complexities complicate quantitative measurements. Here, we show that a combination of dual-colour fluorescence correlation spectroscopy (FCS) with dual-focus FCS avoids artefacts due to chromatic aberrations or saturation and circumvents the calibration of the detection volumes. In addition, we present a comprehensive mathematical framework that allows us to accurately analyse correlation curves even in the presence of spectral cross-talk, incomplete or stochastic labelling, multiple binding sites, a fluorescent background and depletion due to photobleaching. We demonstrate the merits of this approach using dual-colour dual-focus scanning FCS, which allows binding measurements on membranes not affected by membrane movements.

Application of dual-focus fluorescence correlation spectroscopy to microfluidic flow-velocity measurement

Several methods exist to measure and map fluid velocities in microfluidic devices, which are vital to understanding properties on the micro- and nano-scale. Fluorescence correlation spectroscopy (FCS) is a method traditionally exploited for its ability to measure molecular diffusion coefficients. However, several reports during the past decade have shown that FCS can also be successfully used to measure precise flow rates in microfluidics with very high spatial resolution, making it a competitive alternative to other common flow-measurement methods. In 2007 we introduced a modified version of conventional FCS that overcomes many of the artifacts troubling the standard technique. Here we show how the advantages of this method, called dual-focus FCS, extend to flow measurements. To do so, we have measured the velocity flow profile along the cross-section of a square-bore microfluidic channel and compared the result to the theoretical prediction.

Dual-Focus Fluorescence Correlation Spectroscopy: Measuring Translational and Rotational Diffusion of Biomolecules

We present numerous applications of the recently developed dual-focus fluorescence correlation spectroscopy (2fFCS) to the measurement of translational diffusion of proteins and protein complexes. The method is applied to measuring Ca2+-binding curves of wild-type and mutant of the proteins calmodulin and recoverin. 2fFCS is also capable of quantifying the conformational flexibility of macromolecular complexes, which is exemplified on the peptide binding of the major histocompatibility complex I (MHC I). Furthermore, we extended 2fFCS to measure fluorescence correlation at the nanosecond time scale, allowing also for measuring rotational diffusion. We performed a comparative study of translational and rotational diffusion (and related hydrodynamics size) of proteins, and present results for several globular proteins (BSA, human serum albumin, aldolase, ovalbumin). In all cases, measurements are performed at pico- to nanomolar sample concentrations, and with an accuracy of determining hydrodynamic size of a few percent. For performing 2fFCS measurements, a protein or protein complex needs only unspecific fluorescent labeling which is easily accomplished with commercially available dyes. Thus it is hoped that 2fFCS becomes a widely used and easy to handle technique in biophysical research.

Dual-Focus Confocal Microscopy for Flow and Brightness Measurements

Fluorescence correlation spectroscopy (FCS) is a confocal microscopy method mostly used to measure the dynamic properties of molecules in solution and in biological membranes. In 2007, Dertinger et al. introduced a new two-beam confocal method called dual-focus fluorescence correlation spectroscopy (2f-FCS). [1] This development has been extremely important to the field because it overcomes major artifacts that limit conventional FCS as a quantitative measurement technique. In this poster we present two applications of 2f-FCS. One is measuring the velocity profile in a microfluidic channel with high accuracy and sub-micrometer spatial resolution. Second, we present a scheme to measure the absolute brightness of single fluorescent molecules, and thereby directly observe the heterogeneity intrinsic to biological systems.View Large Image | View Hi-Res Image | Download PowerPoint Slide Measured velocity profile in a 300-by-300 micrometer-squared capillary.1. Dertinger, T., et al., ChemPhysChem, 2007. 8(3): p. 433-443.

Study of Layer-by-Layer Films on Thermoresponsive Nanogels Using Temperature-Controlled Dual-Focus Fluorescence Correlation Spectroscopy

While a few studies have reported on the layer-by-layer (LbL) assembly of polyelectrolytes on soft and porous templates, none have really demonstrated direct proof that the layers are actually on the template. Thermoresponsive nanogels present challenges that render a quantitative proof of successful polyelectrolyte deposition extremely difficult. Additionally, the fate of the polyelectrolyte has never been investigated during the phase transition of the coated nanogel. Here, the auto- and cross-correlation functions of a labeled polyelectrolyte assembled via the LbL technique onto soft and porous thermoresponsive labeled nanogels using dual-focus fluorescence correlation spectroscopy (2f-FCS) are presented. Performing 2f-FCS as a function of temperature, hydrodynamic radii of nanogels coated with various numbers of layers are determined, which are found to be in excellent agreement with values obtained from dynamic light scattering. This study presents irrefutable quantitative evidence of successful LbL assembly on thermoresponsive nanogels and demonstrates that the layers are not stripped off during the phase transition of the nanogels. Forster Resonance Energy Transfer (FRET) detection also supports our findings.