Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Camera‐based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR‐FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR‐FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR‐FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR‐FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross‐correlation functions (ΔCCF). With the determination of the PSF by ITIR‐FCS and the optimization of measurement conditions ITIR‐FCS becomes a calibration‐free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.     

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single‐particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 μm^?2) and very low surface concentrations (single‐molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well‐suited for the intermediate concentration range of about 0.1–100 μm^?2. However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z‐scan FCS, is calibration‐free for membrane measurements, but requires several experiments at different well‐controlled focusing positions. A recently established FCS method, electron‐multiplying charge‐coupled‐device‐based total internal reflection FCS (TIR‐FCS), referred to here as imaging TIR‐FCS (ITIR–FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR–FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.     

荧光关联光谱在高分子单链研究中的应用

荧光关联光谱(fluorescence correlation spectroscopy,FCS)是一项用于研究体系动力学性质的统计光谱技术,随着它被引入材料与化学研究领域,近年来取得了大量全新的研究成果.该技术在高分子科学研究中也逐渐发挥出越来越大的作用,特别是在聚合物结构和动力学方面,这表明它在高分子领域的巨大潜力.本文将从FCS的基本原理、实验技巧以及在一些具有挑战性体系中的应用等方面展开,着重介绍它在高分子溶液,如聚电解质溶液、高分子混致不溶现象,以及不同的表界面体系中取得的新成果,展示FCS区别于其他传统技术的特点和优势.     

Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise

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

Fluorescence Correlation Spectroscopy Study of Probe Diffusion in Poly(vinyl alcohol) Solutions and Gels

In this study, we demonstrate how the diffusion of probe particles in aqueous poly(vinyl alcohol) (PVA) solutions and gels is affected by: (i) the presence of cross-links, (ii) the cross-link density, (iii) the polymer concentration. We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion time of a rhodamine-based fluorescent particle (TAMRA) and TAMRA-labeled dextran in PVA solutions and gels prepared at various polymer concentrations (1% to 8.6% w/v) and cross-link densities (1/400 to 1/50 cross-link monomers per PVA monomers). The measurements indicate that the probe particles are slowed down with increasing polymer concentration and with increasing cross-link density. Also, FCS can detect differences in the diffusion times measured in “fresh” and “aged” PVA solutions. We find that FCS provides a quantitative measure of network inhomogeneities.     

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.     

Surface sticking and lateral diffusion of lipids in supported bilayers

The diffusion of fluorescently labeled lipids in supported bilayers is studied using two different methods: Z-scan fluorescence correlation spectroscopy (z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It is found that the data can be fitted consistently only when taking into account partial sticking of the labeled lipids to the supporting glass surface. A kinetic reaction-diffusion model is developed and applied to the data. We find a very slow sticking rate which, however, when neglected, leads to strongly varying estimates of the free diffusion coefficient. The study reveals a strong sensitivity of FCS on even slight binding/unbinding kinetics of the labeled molecules, which has significance for related diffusion measurements in cellular lipid membranes.     

Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements.

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).     

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

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

Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy

CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D(trans) and D(rot)) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D(rot) approximately L(-)(3) versus D(trans) approximately L(-)(1). By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D(rot) is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.     

Host�Cguest association studied by fluorescence correlation spectroscopy

Fluorescence Correlation Spectroscopy (FCS) is a powerful single molecule technique for the study of the stability and the association dynamics of supramolecular systems and, in particular, of host?Cguest inclusion complexes. With FCS the host?Cguest binding equilibrium constant is determined analysing the variation in the diffusion coefficient of the fluorescent guest or host with no need for a change in the photophysical properties of the fluorescent probe. FCS gives also access to the association/dissociation rate constants of the host?Cguest inclusion providing that the fluorescence intensity of host or guest changes upon complexation. These rate constants can be compared with that of a diffusion-controlled process estimated from the same FCS experiment allowing for a better understanding of the association dynamics. The results show that cyclodextrin cavities act as ??hard?? cages which put geometric and orientational restrictions on the inclusion of a hydrophobic guest, whereas micelles behave as ??soft?? cages without geometrical requirements. In our contribution to this special issue we review briefly the application of FCS to the study of host?Cguest inclusion complexes with an emphasis on practical aspects and relevant bibliographic references.     

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.     

Dynamics of Biological Polyelectrolytes

Summary: Biological polymers and structures, including proteins and DNAs, can be made in essentially monodisperse form. Proteins usually have well-defined shapes. Duplex oligonucleotides are rigid and rodlike, and longer DNAs are semiflexible coils. The DNAs also constitute a homologous series. The dynamics of both proteins and DNAs can be studied by readily available techniques such as dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). These systems can thus be used as model systems to elucidate elusive charge effects on the dynamics of macromolecules in solution (polyelectrolyte effects) for both rigid and semiflexible polymers. We present here as examples the results of measurements of mutual and self-diffusion coefficients dynamics of a rodlike oligonucleotide as functions of polymer concentration and the concentration of added salt (which screens the charges).     

Noncovalent cell surface engineering: incorporation of bioactive synthetic glycopolymers into cellular membranes

The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. Here we demonstrate that cell surfaces can be engineered to display synthetic bioactive polymers at defined densities by exogenous membrane insertion. The polymers were designed to mimic native cell-surface mucin glycoproteins, which are defined by their dense glycosylation patterns and rod-like structures. End-functionalization with a hydrophobic anchor permitted incorporation into the membranes of live cultured cells. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Their diffusion properties mirrored those of many natural membrane-associated biomolecules. Furthermore, the membrane-bound glycopolymers were internalized into early endosomes similarly to endogenous membrane components and were capable of specific interactions with protein receptors. This system provides a platform to study cell-surface phenomena with a degree of chemical control that cannot be achieved using conventional biological tools.     

Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).     

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.     

Vibrational spectroscopy of water in hydrated lipid multi-bilayers. II. Two-dimensional infrared and peak shift observables within different theoretical approximations

In a previous report, we calculated the infrared absorption spectrum and both the isotropic and anisotropic pump-probe signals for the OD stretch of isotopically dilute water in dilauroylphosphatidylcholine (DLPC) multi-bilayers as a function of the lipid hydration level. These results were then compared to recent experimental measurements and are in generally good agreement. In this paper, we will further investigate the structure and dynamics of hydration water using molecular dynamics simulations and calculations of the two-dimensional infrared and vibrational echo peak shift observables for hydration water in DLPC membranes. These observables have not yet been measured experimentally, but future comparisons may provide insight into spectral diffusion processes and hydration water heterogeneity. We find that at low hydration levels the motion of water molecules inside the lipid membrane is significantly arrested, resulting in very slow spectral diffusion. At higher hydration levels, spectral diffusion is more rapid, but still slower than in bulk water. We also investigate the effects of several common approximations on the calculation of spectroscopic observables by computing these observables within multiple levels of theory. The impact of these approximations on the resulting spectra affects our interpretation of these measurements and reveals that, for example, the cumulant approximation, which may be valid for certain systems, is not a good approximation for a highly heterogeneous environment such as hydration water in lipid multi-bilayers.     

Combined atomic force microscopy and fluorescence correlation spectroscopy measurements to study the dynamical structure of interfacial fluids

We have studied the dynamic structure of thin (approximately a few nanometers) liquid films of a nearly spherical, nonpolar molecule tetrakis(2-ethylhexoxy)silane (TEHOS) by using a combination of atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS). Ultra-sensitive interferometer-based AFM was used to determine the stiffness (force gradient) and the damping coefficient of the liquid film. The experiments show oscillations in the damping coefficient with a period of approximately 1 nm, which is consistent with the molecular dimension of TEHOS as well as previous X-ray reflectivity measurements. Additionally, we performed FCS experiments for direct determination of the molecular dynamics within the liquid film. From the fluctuation autocorrelation curve, we measured the translational diffusion of the probe molecule embedded within the fluid film formed on a solid substrate. The autocorrelation function was best fitted with two components, which indicate that the dynamics are heterogeneous in nature. However, the heterogeneity is not as pronounced as had been previously observed for molecularly thin liquid films sandwiched between two solid substrates.     

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.