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.     

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.     

New concepts for fluorescence correlation spectroscopy on membranes

Fluorescence correlation spectroscopy (FCS) is a powerful tool to measure useful physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in model and cell membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we assess the potential and limitations of standard confocal FCS on lipid membranes and present recent developments which facilitate accurate and quantitative measurements on such systems. In particular, we discuss calibration-free diffusion and concentration measurements using z-scan FCS and two focus FCS and present several approaches using scanning FCS to accurately measure slow dynamics. We also show how surface confined FCS enables the study of membrane dynamics even in presence of a strong cytosolic background and how FCS with a variable detection area can reveal submicroscopic heterogeneities in cell membranes.     

Tackling Sample‐Related Artifacts in Membrane FCS Using Parallel SAF and UAF Detection

The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non‐membrane‐bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non‐ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample‐related artifacts in the diffusion measurement.     

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

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

Two-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.     

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.     

Photobleaching in Two‐Photon Scanning Fluorescence Correlation Spectroscopy

Two‐photon excitation in fluorescence correlation spectroscopy (FCS) is often preferred to one‐photon excitation because of reduced bulk photobleaching and photodamage, and deeper penetration into scattering media, such as thick biological specimens. Two‐photon FCS, however, suffers from lower signal‐to‐noise ratios which are directly related to the lower molecular brightness achieved. We compare standard FCS with a fixed measurement volume with scanning FCS, where the measurement volume is scanned along a circular path. The experimental results show that photobleaching is the dominant cause of the effects observed at the high excitation powers necessary for good signal‐to‐noise ratios. Theoretical calculations assuming a nonuniform excitation intensity profile, and using the concept of generalized volume contrast, provide an explanation for the photobleaching effects commonly observed in two‐photon FCS at high excitation intensities, without having to assume optical saturation. Scanning alleviates these effects by spreading the photobleaching dose over a larger area, thereby reducing the depletion of fluorescent molecules in the measurement volume. These results, which facilitate understanding of the photobleaching in FCS and of the positive effects of scanning, are particularly important in studies involving the autocorrelation amplitude g(0), such as concentration measurements or binding studies using fluorescence cross‐correlation between two labeled species.     

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).     

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.     

Formation of arenicin-1 microdomains in bilayers and their specific lipid interaction revealed by Z-scan FCS

Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.     

Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation.

A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements or diffusion coefficients with fluorescence correlation spectroscopy rather difficult. A strong difference of this behavior for continuous-wave and pulsed excitation is found.     

Size dependence of protein diffusion very close to membrane surfaces: measurement by total internal reflection with fluorescence correlation spectroscopy

The diffusion coefficients of nine fluorescently labeled antibodies, antibody fragments, and antibody complexes have been measured in solution very close to supported planar membranes by using total internal reflection with fluorescence correlation spectroscopy (TIR-FCS). The hydrodynamic radii (3-24 nm) of the nine antibody types were determined by comparing literature values with bulk diffusion coefficients measured by spot FCS. The diffusion coefficients very near membranes decreased significantly with molecular size, and the size dependence was greater than that predicted to occur in bulk solution. The observation that membrane surfaces slow the local diffusion coefficient of proteins in a size-dependent manner suggests that the primary effect is hydrodynamic as predicted for simple spheres diffusing close to planar walls. The TIR-FCS data are consistent with predictions derived from hydrodynamic theory. This work illustrates one factor that could contribute to previously observed nonideal ligand-receptor kinetics at model and natural cell membranes.     

Monitoring and Quantifying the Passive Transport of Molecules Through Patch–Clamp Suspended Real and Model Cell Membranes

Transport of active molecules across biological membranes is a central issue for the success of many pharmaceutical strategies. Herein, we combine the patch–clamp principle with amperometric detection for monitoring fluxes of redox‐tagged molecular species across a suspended membrane patched from a macrophage. Solvent‐ and protein‐free lipid bilayers (DPhPC, DOPC, DOPG) patched from single‐wall GUV have been thoroughly investigated and the corresponding fluxes measurements quantified. The quality of the patches and their proper sealing were successfully characterized by electrochemical impedance spectroscopy. This procedure appears versatile and perfectly adequate to allow the investigation of transport and quantification of the transport properties through direct measurement of the coefficients of partition and diffusion of the compound in the membrane, thus offering insight on such important biological and pharmacological issues.     

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate-dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom-like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.     

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

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

Fluorescence correlation spectroscopic studies of diffusion within the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate

Fluorescence correlation spectroscopy (FCS) measurements have been performed in order to determine the translational diffusion coefficients of three differently charged fluorescent probes (cationic: rhodamine 6G, neutral: 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4-H-pyran, DCM, and anionic: fluorescein) dissolved within the common room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF6]. These experiments demonstrate that FCS is a rapid, sensitive, precise (typical RSD from 4-8%), and low-consumption screening tool for the determination of analyte mobility within microliter ionic liquid samples.     

Fluorescence correlation spectroscopy for flow rate imaging and monitoring--optimization, limitations and artifacts

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.     

Application of fluorescence correlation spectroscopy to the measurement of local temperature in solutions under optical trapping condition

Fluorescence correlation spectroscopy (FCS) was applied to the quantitative evaluation of the local heating in small domains <1 microm in solutions under the laser trapping condition in the presence of a near-infrared (NIR) laser beam at 1064 nm. On the basis of the translational diffusion coefficient of fluorescent molecules obtained by FCS, the relationship between temperature rise and the incident NIR laser power, DeltaT/DeltaP, were determined to be 62 +/- 6, 49 +/- 7, and 23 +/- 1 deg K/W in ethylene glycol, ethanol, and water, respectively, while no remarkable temperature increase was observed for deuterated water. The value of DeltaT/DeltaP linearly increased as a function of alpha/lambda (alpha is the extinction coefficient of solvent at the wavelength and lambda is the thermal conductivity of the medium). The validity and the applicability of the present method for the measurement of the local temperature increase were discussed by comparing the present results with previous ones by other various methods.