Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.     

Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes

In this paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined.     

An array of planar apertures for near-field fluorescence correlation spectroscopy

We have developed a method of performing near-field fluorescence correlation spectroscopy via an array of planarized circular apertures of 50 nm diameter. This technique provides 1 μs and 60 nm resolution on proximal samples, including live cells, without incorporating a scanning probe or pulsed lasers or requiring penetration of the sample into the aperture. Millions of apertures are created in an array within a thin film of aluminum on a coverslip and planarized to achieve no height distinction between the apertures and the surrounding metal. Supported lipid bilayers and plasma membranes from live cells adhere to the top of this substrate. We performed fluorescence correlation spectroscopy to demonstrate the sub-diffraction-limited illumination with these devices.     

Measuring diffusion in cell membranes by fluorescence correlation spectroscopy

Fluorescence Correlation Spectroscopy (FCS) can measure diffusion on the cell surface with unparalleled sensitivity. In appropriate situations, this can be the most sensitive and accurate method for measuring receptor interaction and oligomerization. Here we attempt to describe FCS in sufficient detail so that the reader is able to judge when there is a compelling reason to choose this technique, understand the basic theory behind it, construct a FCS spectrometer in the laboratory, and analyze the data to obtain a meaningful estimate of the physical parameters.     

Incorporation of integrins into artificial planar lipid membranes: characterization by plasmon-enhanced fluorescence spectroscopy

An optimized peptide-tethered artificial lipid membrane system has been developed. Integrins (cell adhesion receptors) were functionally incorporated into this membrane model and integrin-ligand interactions were analyzed by surface plasmon-enhanced fluorescence spectroscopy (SPFS). The transmembrane receptors alpha(v)beta(3) and alpha(1)beta(1) of the integrin superfamily were incorporated into a lipid-functionalized peptide layer by vesicle spreading. Consecutive layer formations were monitored by surface plasmon spectroscopy (SPS). Orientation and accessibility of the membrane receptor alpha(v)beta(3) was reliably assessed by specific and reproducible binding of selective antibodies. Moreover, full retention of the functional properties of this receptor was verified by specific and reversible binding of natural ligands. Functional integrity of incorporated integrins was maintained over a time period of 72 h. The integrin/extracellular matrix ligand complexes, whose formations are known to depend on the presence of divalent cations, were lost upon addition of ethylenediaminetetraacetate. Therefore, regeneration of the surface for further binding experiments with minimized unspecific ligand association was possible. These results demonstrate that integrins can be functionally incorporated into peptide-tethered artificial membranes. In combination with the SPS/SPFS method, this artificial membrane system provides a reliable experimental platform for investigation of isolated membrane proteins under experimental conditions resembling those of their native environment.     

Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.     

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.     

Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.     

Rebinding of IgE Fabs at haptenated planar membranes: measurement by total internal reflection with fluorescence photobleaching recovery

In previous work, a general analytical theory for ligand rebinding at cell surfaces was developed for a reversible bimolecular reaction between ligands in solution and receptors on a membrane surface [Lagerholm, B. C., and Thompson, N. L. (1998) Biophys. J. 74, 1215-1228]. This theory can be used to predict theoretical forms for data obtained by using total internal reflection with fluorescence photobleaching recovery (TIR-FPR) [Thompson, N. L., Burghardt, T. P., and Axelrod, D. (1981) Biophys. J. 33, 435-454]. Thus, one method by which the rebinding theory can be tested is to use TIR-FPR. In the work described herein, the reversible kinetics of mouse monoclonal anti-dinitrophenyl (DNP) IgE Fabs at substrate-supported planar membranes composed of 25 mol % DNP-conjugated phosphatidylethanolamine and 75 mol % dipalmitoylphosphatidylcholine have been examined by using TIR-FPR. Data were obtained as a function of the Fab solution concentration. Higher Fab concentrations reduce rebinding (and increase the fluorescence recovery rate) because different Fab molecules compete for the same surface-binding sites. Data were also obtained for solutions containing different volume fractions of glycerol. In these measurements, higher glycerol concentrations increase rebinding (and decrease the fluorescence recovery rate) because the solution viscosity is increased and the Fab diffusion coefficient in solution is decreased. The TIR-FPR data were quantitatively compared with theoretical predictions which follow from the general theory for rebinding at the membrane surface. The data were consistent with the theoretical predictions and, therefore, provide experimental verification of the previously developed theory.     

High-pressure fluorescence correlation spectroscopy

We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence of these beam distortions by FCS and photon-counting histogram (PCH) analysis and identify the optimal position for fluorescence fluctuation experiments in the capillary. At this position within the capillary, FCS and photon-counting histogram experiments are described by the same equations as used in standard FCS experiments. We report the first experimental realization of fluorescence fluctuation spectroscopy under high pressure. A fluorescent dye was used as a model system for evaluating the properties of the capillary under pressure. The autocorrelation function and the photon count distribution were measured in the pressure range from 0 to 300 MPa. The fluctuation amplitude and the diffusion coefficient show a small pressure dependence. The changes of these parameters, which are on the order of 10%, are due to the pressure changes of the viscosity and the density of the aqueous medium.     

Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy.

The hybridization of fluorescently tagged 18mer deoxyribonucleotides with complementary DNA templates was analysed by fluorescence correlation spectroscopy (FCS) in a droplet under an epi-illuminated fluorescence microscope at the level of single molecules. The interaction can be monitored by the change in the translational diffusion time of the smaller (18mer) primer when binding to the bigger (7.5 kb) DNA containing the complementary sequence. The hybridization process in the presence of template M13mp18 ssDNA was monitored in a small volume (2 x 10(-16)I) at various temperatures. The Arrhenius plot of the association rate constant shows that the activation energy was 38.8 kcal/mol, but the hybridization process may involve several components. The titration experiment suggested that approximately 2 primers can be associated with one template DNA at 40 degrees C. Results of a simple homology search for the sequences complementary to the primer indicate the existence of additional sites of lower specificity.     

Supercritical angle fluorescence correlation spectroscopy

We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.     

Position-sensitive scanning fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.     

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.     

Theory of sample translation in fluorescence correlation spectroscopy.

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

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.