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 diffusion laws to probe the submicron cell membrane organization

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.     

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.     

The use of fluorescence correlation spectroscopy (FCS) as an alternative biomarker detection technique: a preliminary study

Biomarkers are essential part of daily medical practice. Currently, biomarkers are being used both for diagnostic and prognostic purposes. There are many approaches e.g. ELISA by which biomarker levels are detected from patient samples. However, all these approaches are laborious, time consuming and expensive. There is therefore a general need for exploring new technique which can overcome these drawbacks. Here, we present a preliminary study for detection of serum biomarkers by fluorescence correlation spectroscopy (FCS) based diagnostic technique. FCS is a technique basically used for spatial and temporal analysis of molecular interactions of extremely low-concentration biomolecules in solution. FCS is able to measure diffusion time of the fluorescent molecules passing through the open detection volume and it can also measure the average number of fluorescent molecules passing through the detection volume. Because diffusion speed is correlated with shape and molecular mass of the fluorescent molecule, this property makes it possible to study the complex formation between a small fluorescently labelled and a large unlabelled molecule. In this preliminary study, we utilize this FCS property for detection of serum biomarker. Further studies on various pathological serum samples are warranted to explore further aspects of this technique.     

Gene expression analysis using single molecule detection

Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.     

Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells

Fluorescence correlation spectroscopy (FCS) allows the study of interactions of fluorescently labeled ligand with receptors in living cells at single-molecule detection sensitivity. From the autocorrelation functions of fluorescence intensity fluctuations, the diffusion time of molecules through the confocal volume is analyzed, and from that, the molecular weights of free and bound molecules can be calculated. We have applied FCS to study the receptor diversity for the neuropeptide galanin (GAL) in cultured cells. FCS measurement of the fluorophore rhodamine-labeled GAL (Rh-GAL) has been performed in 0.2-fL confocal volume elements of the laser beam. The analysis of autocorrelation functions of Rh-GAL in solution above cells and at cell membranes demonstrates that the diffusion time of unbound Rh-GAL is 0.16 ms, whereas diffusion times of membrane-bound Rh-GAL are 22 and 700 ms. Because both of the diffusion times (22 and 700 ms) are much longer as compared to that of unbound Rh-GAL, they correspond to slow-diffusing complexes when Rh-GAL is bound to the cell membranes. Addition of excess nonlabeled GAL is accompanied by competitive displacement. Full saturation of the GAL binding is obtained at nanomolar concentrations. Scatchard analysis of binding data reveal one binding process, assuming one binding site per Rh-GAL (n = 1). On the other hand, the appearance of two diffusion times, 22 and 700 ms, suggests the existence of two subpopulations of GAL receptor complexes or two subtypes of GAL receptor not detected before. This makes an important point that FCS permits the identification of receptors, which were not possible to detect before by conventional binding techniques. The inhibitory effect of pertussis toxin on the GAL binding considers a G-protein-involved allosteric system, important for the clarification of essential steps in the G-protein-related signal transduction. This study is of pharmaceutical significance, since it will provide insights into how FCS can be used as a rapid technique for studying ligand-receptor interactions in living cells, which is one step forward for large-scale drug screening in cell cultures.     

Fluorescence correlation spectroscopy and its potential for intracellular applications

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation. Its presently most popular instrumentation by confocal-microscope setups allows for a spatial resolution of fractions of femtoliters for the measurement volumes, containing sparse or even single molecules at any time, and encourages the adaptation of the solution-based technique for cellular applications. The scope of this review is thus, to introduce the FCS technique in particular to the reader with biological background, searching for new methods for a precise quantification of physical parameters governing cellular mechanisms and dynamics, especially if high sensitivity and fast dynamic resolution are required. After a short theoretical introduction, examples are given for the so far most important experimental applications, with respect to their implementation in cellular systems. As an interesting alternative to the confocal instrumentation, two-photon excitation will be introduced, offering a number of important advantages especially in cellular systems with high-noise and low-signal levels.     

Simultaneous membrane interaction of amphipathic peptide monomers,self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.     

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.     

Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes

Bovine serum albumin (BSA) and fetal calf serum (FCS) were evaluated as protein supplements for in vitro maturation and fertilization of oocytes from cows and hamsters. BSA and low doses of FCS (0.1 or 1.0%) did not support viability or maturation of the cumulus-oocyte complex as well as higher doses of FCS (5, 10, or 20%) for either species. BSA failed to support cumulus expansion for bovine or hamster cumulus-oocyte complexes. All doses of FCS examined supported cumulus expansion in bovine cumulus-oocyte complexes, whereas the hamster complexes required at least 1.0% FCS to induce cumulus expansion. The addition of a serum filtrate, Solcoseryl, with BSA improved viability of the cumulus in the bovine but did not support cumulus expansion or completion of Meiosis I in bovine complexes. In vitro fertilization could be accomplished in media containing FCS by increasing the heparin concentration in the bovine system or reducing FCS for the hamster system. Polyspermy was increased when FCS was the protein supplement. It is not known whether this is an interaction of FCS with the sperm or oocyte. In conclusion, FCS was found necessary for follicle-stimulating-hormone (FSH)-induced cumulus expansion. It also improved cumulus cell viability and completion of the first meiotic division in complexes of both species compared with BSA.     

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments.     

Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure chemical reaction rates and diffusion coefficients of molecules in thermal equilibrium. The capabilities of FCS can be enhanced by measuring the energy, polarization, or delay time between absorption and emission of the collected fluorescence photons in addition to their arrival times. This information can be used to change the relative intensities of multiple fluorescent species in FCS measurements and, thus, the amplitude of the intensity autocorrelation function. Here we demonstrate this strategy using lifetime gating in FCS experiments. Using pulsed laser excitation and laser-synchronized gating in the detection channel, we suppress photons emitted within a certain time interval after excitation. Three applications of the gating technique are presented: suppression of background fluorescence, simplification of FCS reaction studies, and investigation of lifetime heterogeneity of fluorescently labeled biomolecules. The usefulness of this technique for measuring forward and backward rates of protein fluctuations in equilibrium and for distinguishing between static and dynamic heterogeneity makes it a promising tool in the investigation of chemical reactions and conformational fluctuations in biomolecules.     

Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross-correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.     

Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS)

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed for a straightforward detection of spatial-temporal interactions between the protein and the membrane based on the diffusion rate of the protein. To test for protein incorporation into the bilayers of the GUVs, antibodies against one specific membrane protein (NaPi II cotransporter) were labeled with ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled antibody showed marginal fluorescence enhancement on the GUV membrane bilayers (poor image contrast and resolution). With the application of scanning FCS, the binding of the antibody to the GUVs was detected directly from the analysis of diffusion rates of the fluorescent antibody. The diffusion coefficient of the antibody bound to NaPi II in the GUVs was approximately 200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.     

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.     

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.     

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple experimental systems has led to an integrated understanding of the formation, composition, regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This review discusses interactions of signaling molecules that bind directly or indirectly to LAT and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition, it focuses on how imaging studies visualize signaling assemblies as signaling clusters and demonstrate their dynamic nature and cellular fate. Finally, this review explores the function of LAT based on the interpretation of mouse models using various LAT mutants.     

Fluorescence correlation spectroscopy: molecular recognition at the single molecule level

Fluorescence correlation spectroscopy (FCS) is a fluorescence microscopy technique that allows the study of molecular interactions in extremely low volumes, at nanomolar concentrations, even when binding is not accompanied by a fluorescence change. It can be applied directly in living cells. FCS clearly considerably extends the possibilities of the classical techniques used in molecular recognition studies and can be considered to belong to a growing group of techniques that allow detection at the single molecule level. In this review, several applications of FCS, both in vitro and in vivo, will be discussed.     

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.     

Fluorescence correlation spectroscopy for ultrasensitive DNA analysis in continuous flow capillary electrophoresis

Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy (FCS). CFCE is used to resolve multiple components in a mixture of analytes, measure electrophoretic mobility shifts due to binding interactions, and study the hydrodynamic and electrostatic properties of biological molecules in solution. Often this information is obtained with greater speed and sensitivity than conventational separations-based capillary-zone electrophoresis. This paper will focus on the application of two-beam fluorescence cross-correlation spectroscopy as a versatile detection method for CFCE and explore several applications to the study of the solution properties of single-stranded DNA.