Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions

Fluorescence correlation spectroscopy (FCS) is a powerful technique to study dynamic biomolecular processes. It allows the estimation of concentrations, diffusion coefficients, molecular interactions, and other processes causing fluctuations in the fluorescence intensity, thus yielding information about aggregation processes, enzymatic reactions, or partition coefficients. During the last years, FCS has been successfully applied to model and cellular membranes, proving to be a promising tool for the study of membrane dynamics and protein/lipid interactions. Here we describe the theoretical basis of FCS and some practical implications for its application in membrane studies. We discuss sources of potential artifacts, such as membrane undulations, positioning of the detection volume, and photobleaching. Special attention is paid to aspects related to instrumentation and sample preparation as well as data acquisition and analysis. Finally, we comment on some strategies recently developed for the specific improvement of FCS measurements on membranes.     

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.     

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.     

Fluorescence correlation spectroscopy: past, present, future

In recent years fluorescence correlation spectroscopy (FCS) has become a routine method for determining diffusion coefficients, chemical rate constants, molecular concentrations, fluorescence brightness, triplet state lifetimes, and other molecular parameters. FCS measures the spatial and temporal correlation of individual molecules with themselves and so provides a bridge between classical ensemble and contemporary single-molecule measurements. It also provides information on concentration and molecular number fluctuations for nonlinear reaction systems that complement single-molecule measurements. Typically implemented on a fluorescence microscope, FCS samples femtoliter volumes and so is especially useful for characterizing small dynamic systems such as biological cells. In addition to its practical utility, however, FCS provides a window on mesoscopic systems in which fluctuations from steady states not only provide the basis for the measurement but also can have important consequences for the behavior and evolution of the system. For example, a new and potentially interesting field for FCS studies could be the study of nonequilibrium steady states, especially in living cells.     

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.     

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope

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

Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy

Here we discuss the application of scanning fluorescence correlation spectroscopy (SFCS) using continuous wave excitation to analyze membrane dynamics. The high count rate per molecule enables the study of very slow diffusion in model and cell membranes, as well as the application of two-foci fluorescence cross-correlation spectroscopy for parameter-free determination of diffusion constants. The combination with dual-color fluorescence cross-correlation spectroscopy with continuous or pulsed interleaved excitation allows binding studies on membranes. Reduction of photobleaching, higher reproducibility, and stability compared to traditional FCS on membranes, and the simple implementation in a commercial microscopy setup make SFCS a valuable addition to the pool of fluorescence fluctuation techniques.     

Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis

We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.     

Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature.     

Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy

We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.     

Detecting amyloid-beta aggregation with fiber-based fluorescence correlation spectroscopy

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-beta molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.     

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.     

CorTectorTM SX100：一款桌面式荧光相关光谱仪的原理和应用

荧光相关光谱检测技术具有超灵敏(单分子)、快速(数秒至数分钟)和多功能(检测分子浓度、大小和相互作用)等技术优点,且无需反应物分离,因此有潜力成为一种新型均相、高敏荧光免疫检测技术,适用于在溶液中或单个活细胞内检测生物分子特性．本文首先介绍荧光相关光谱检测技术的原理和研究进展,然后结合项目团队自主研发的目前全球唯一一款可靠、易使用的桌面式荧光相关光谱仪,进一步探讨荧光相关光谱检测技术的具体实现和潜在应用．     

Detecting Amyloid-β Aggregation with Fiber-Based Fluorescence Correlation Spectroscopy

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-β molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.     

Single-molecule photobleaching: Instrumentation and applications

Single-molecule photobleaching (smPB) technique is a powerful tool for characterizing molecular assemblies. It can provide a direct measure of the number of monomers constituting a given oligomeric particle and generate the oligomer size distribution in a specimen. A major current application of this technique is in understanding protein aggregation, which is linked to many incurable diseases. Quantitative measurement of the size distribution of an aggregating protein in a physiological solution remains a difficult task, since techniques such as dynamic light scattering or fluorescence correlation spectroscopy (FCS) can provide an average size, but cannot accurately resolve the underlying size distribution. Here we describe the smPB method as implemented on a home-built total internal reflection fluorescence microscope (TIRF). We first describe the construction of a TIRF microscope, and then demonstrate the power of smPB by characterizing a solution of Amylin (hIAPP) oligomers, a 37-residue peptide whose aggregation is associated with Type II diabetes. We compare our results with FCS data obtained from the same specimen, and discuss the advantages and disadvantages of the two techniques.     

Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles

We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar vesicles (diameter approximately 100 nm) composed of phosphatidylcholine and phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large unilamellar vesicles are significantly larger than the peptide, the correlation times for the free and bound peptide could be distinguished using single color autocorrelation measurements. The molar partition coefficients calculated from the FCS measurements were comparable to those obtained from binding measurements of radioactively labeled MARCKS(151-175) using a centrifugation technique. Moreover, FCS can measure binding of peptides present at very low concentrations (1-10 nmolar), which is difficult or impossible with most other techniques. Our data indicate FCS can be an accurate and valuable tool for studying the interaction of peptides and proteins with lipid membranes.     

Detection of specific DNA sequences using dual-color two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a biomedical research tool. FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. They can also serve as a diagnostic, detecting particular molecular species with high sensitivity and specificity. We here demonstrate that dual-color FCS measurements can be applied to detect and quantify the concentration of specific non-fluorescent molecular species without requiring any modifications to the molecule of interest. We demonstrate this capability by applying dual-color two-photon fluorescence cross-correlation spectroscopy to detect single stranded gamma tubulin DNA in solution with high sensitivity. This quantification is independent of molecular size, and the methods introduced can be extended to measurements in complex environments such as within living cells.     

Recent applications of fluorescence correlation spectroscopy in live systems

Fluorescence correlation spectroscopy (FCS) is a widely used technique in biophysics and has helped address many questions in the life sciences. It provides important advantages compared to other fluorescence and biophysical methods. Its single molecule sensitivity allows measuring proteins within biological samples at physiological concentrations without the need of overexpression. It provides quantitative data on concentrations, diffusion coefficients, molecular transport and interactions even in live organisms. And its reliance on simple fluorescence intensity and its fluctuations makes it widely applicable. In this review we focus on applications of FCS in live samples, with an emphasis on work in the last 5 years, in the hope to provide an overview of the present capabilities of FCS to address biologically relevant questions.     

Fluorescence correlation spectroscopy and quantitative cell biology

Fluorescence correlation spectroscopy (FCS) analyzes fluctuations in fluorescence within a small observation volume. Autocorrelation analysis of FCS fluctuation data can be used to measure concentrations, diffusion properties, and kinetic constants for individual fluorescent molecules. Photon count histogram analysis of fluorescence fluctuation data can be used to study oligomerization of individual fluorescent molecules. If the FCS observation volume is positioned inside a living cell, these parameters can be measured in vivo. FCS can provide the requisite quantitative data for analysis of molecular interaction networks underlying complex cell biological processes.     

Fluorescence correlation spectroscopy for the detection and study of single molecules in biology

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution-based single molecule technique, fluorescence correlation spectroscopy (FCS). This time-averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual-color cross-correlation and two-photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research.