Application Of Fluorescence Correlation Spectroscopy Research Articles

Fluorescence correlation spectroscopy in living cells

Fluorescence correlation spectroscopy (FCS) is an ideal analytical tool for studying concentrations, propagation, interactions and internal dynamics of molecules at nanomolar concentrations in living cells. FCS analyzes minute fluorescence-intensity fluctuations about the equilibrium of a small ensemble (<10(3)) of molecules. These fluctuations act like a 'fingerprint' of a molecular species detected when entering and leaving a femtoliter-sized optically defined observation volume created by a focused laser beam. In FCS the fluorescence fluctuations are recorded as a function of time and then statistically analyzed by autocorrelation analysis. The resulting autocorrelation curve yields a measure of self-similarity of the system after a certain time delay, and its amplitude describes the normalized variance of the fluorescence fluctuations. By fitting the curves to an appropriate physical model, this method provides precise information about a multitude of measurement parameters, including diffusion coefficients, local concentration, states of aggregation and molecular interactions. FCS operates in real time with diffraction-limited spatial and sub-microsecond temporal resolution. Assessing diverse molecular dynamics within the living cell is a challenge well met by FCS because of its single-molecule sensitivity and high dynamic resolution. For these same reasons, however, intracellular FCS measurements also harbor the large risk of collecting artifacts and thus producing erroneous data. Here we provide a step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell. A discussion of advantages and disadvantages of one-photon versus two-photon excitation for FCS is available in Supplementary Methods online.

Review fluorescence correlation spectroscopy for probing the kinetics and mechanisms of DNA hairpin formation

This article reviews the application of fluorescence correlation spectroscopy (FCS) and related techniques to the study of nucleic acid hairpin conformational fluctuations in free aqueous solutions. Complimentary results obtained using laser-induced temperature jump spectroscopy, single-molecule fluorescence spectroscopy, optical trapping, and biophysical theory are also discussed. The studies cited reveal that DNA and RNA hairpin folding occurs by way of a complicated reaction mechanism involving long- and short-lived reaction intermediates. Reactions occurring on the subnanoseconds to seconds time scale have been observed, pointing out the need for experimental techniques capable of probing a broad range of reaction times in the study of such complex, multistate reactions.

Fluorescence based studies of endophilin B proteins and their interactions with lipid membranes

Endophilin B proteins belong to a subfamily of endophilins, cytoplasmic proteins, and are involved in membrane dynamics. During apoptosis endophilin B1 interacts with the proapoptotic protein Bax and targets mitochondria. Recently, a novel protein endophilin B2 was found that associates with B1 in yeast two-hybrid screening. Here we used confocal fluorescence microscopy to study the direct effect of endophilin B1 and B2 on lipid membrane shape deformation of giant unilamellar vesicles (GUVs) as a model for their possible effects on the mitochondrial outer membrane. We found that B2 induces massive shape transition of GUVs, manifested by generation of small vesicles, tubules, buds, and liposome fission and fusion. Under the same experimental conditions B1 does not exert a similar effect. However, after pre-incubation with Bax, B1 causes extensive tubulation of GUVs. We applied fluorescence correlation spectroscopy to determine the behavior and interactions of fluorescently-labeled B1 and B2 with Bax. We found that Bax induces aggregation/oligomerization of B1 whereas there were no measurable effects with B2. Furthermore, B2 also promotes oligomerization of B1. These results demonstrate differences between the two endophilins B in their interactions with lipid membranes and provide an assay that might be used to investigate how the interaction between endophilin B1, B2, and Bax alters membrane properties.

Internal Structure and Dynamics of Isolated Escherichia coli Nucleoids Assessed by Fluorescence Correlation Spectroscopy

The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from ∼50 kbp to ∼3 kbp).

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.

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

Fluorescence correlation studies of lipid domains in model membranes (Review)

Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.

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

AbstractIn 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.

Global analysis of fluorescence fluctuation data

Over the last decade the number of applications of fluorescence correlation spectroscopy (FCS) has grown rapidly. Here we describe the development and application of a software package, FCS Data Processor, to analyse the acquired correlation curves. The algorithms combine strong analytical power with flexibility in use. It is possible to generate initial guesses, link and constrain fit parameters to improve the accuracy and speed of analysis. A global analysis approach, which is most effective in analysing autocorrelation curves determined from fluorescence fluctuations of complex biophysical systems, can also be implemented. The software contains a library of frequently used models that can be easily extended to include user-defined models. The use of the software is illustrated by analysis of different experimental fluorescence fluctuation data sets obtained with Rhodamine Green in aqueous solution and enhanced green fluorescent protein in vitro and in vivo.

Characterization of Interaction between Cationic Lipid-Oligonucleotide Complexes and Cellular Membrane Lipids Using Confocal Imaging and Fluorescence Correlation Spectroscopy

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D ODN = 1.3 ± 0.1 × 10 −6 cm 2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D CL-ODN = ∼1.5 × 10 −8 cm 2/s. Released ODN has the mean diffusion constant D = 1.1 ± 0.2 × 10 −6 cm 2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.

Application of Fluorescence Correlation Spectroscopy for Velocity Imaging in Microfluidic Devices

In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micrometer resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy (FCS). Flow velocities ranging from approximately 50 microm/s to approximately 10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion tracers. Velocity profiles and images of the flow in poly(dimethylsiloxane)-glass microchannels are presented and analyzed. Using the method, velocity images along the horizontal (top view) and vertical planes within a microdevice can be obtained. This is, to our knowledge, the first report of FCS for producing velocity maps. The high-resolution velocity maps can be used to characterize and optimize microdevice performance and to validate simulation efforts.

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 ∼200-fold smaller than that in solution. Scanning FCS provided a simple, quantitative, yet highly sensitive method to study protein-membrane interactions.

Detection of protein–DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a methodology to examine directly the translational diffusion of individual fluorescence-labeled molecules in solutions. Recent studies using FCS have quantified various bimolecular reactions without any need for amplification. To evaluate further the applicability of FCS, we studied the specific binding between proteins and DNA in crude biological samples. Using an automated FCS system that was recently developed in our laboratories and is capable of distinguishing two or more molecular species in a multicomponent analysis, we detected the binding of two representative transcription factors, activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB), in nuclear extracts of HeLa cells quantitatively with each sequence-specific DNA. The binding rates of these specific interactions were markedly augmented when cells were treated with tumor necrosis factor α which is known to activate both AP-1 and NF-κB. We also observed the pyrrolidine-dithiocarbamate-induced reciprocal regulation of these transcription factors. These results indicated that FCS is a useful tool for the analysis of complex interactions of transcription factors with DNA even in crude cellular extracts, suggesting that it is a powerful methodology for the study of a wide variety of molecular events under various experimental conditions.

Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery.

Fluorescence methods are commonly used in pharmaceutical drug discovery to assay the binding of drug-like compounds to signaling proteins and other bio-particles. For binding studies of non-fluorescent compounds, a competitive format may be used in which the binding of the compound results in displacement of another fluorescently labeled ligand. Highly-sensitive measurements within nano-liter sized open probe volumes can be accomplished using a confocal epi-illumination geometry and thus key tools for such drug-binding studies include fluorescence correlation spectroscopy (FCS) and its related techniques. This paper reviews the general protocol for application of FCS to biomolecular compound-binding assays and it focuses on methods for the reduction of experimental photon count data to obtain the normalized autocorrelation function (ACF), on theoretical models of the ACF, and on statistical and systematic errors in the experimental ACF. Results from a detailed Monte Carlo simulation of FCS, which are useful for testing theoretical models and validating short-duration assay capabilities, are discussed. An illustrative example is presented on the use of FCS to assay binding of Alexa-488-labeled Bak peptide with Bcl-x(L), which is an intracellular protein that acts to protect against programmed cell death.

Probing the Diffusion of a Dilute Dye Solution in Mesoporous Glass with Fluorescence Correlation Spectroscopy

The translational motion of rhodamine 6g dye molecules near the surface of a mesoporous glass with a disordered pore structure and a nominal pore diameter of 130 A is measured by the application of fluorescence correlation spectroscopy (FCS) to fluorescence burst data as dye molecules diffuse through the laser excitation volume. FCS analysis indicates that the motion of the rhodamine 6g molecules in the mesoporous glass deviates from simple Brownian motion with a single diffusion constant. Two models, including two-component diffusion and diffusion plus adsorption, are utilized to fit the correlation data. The multicomponent diffusion model comprised of free diffusion (with a diffusion coefficient of 4.89 × 10-7 cm2/s) and transient adsorption/desorption (desorption time of 67 ms) provides the best fit and the most plausible explanation of the correlation data. Possible explanations for the shift to multicomponent diffusion, such as restriction of the dye molecules within the pores of the glass, as well a...

Application of fluorescence correlation spectroscopy to the measurement of agonist binding to a G-protein coupled receptor at the single cell level.

The A1-adenosine receptor (A1-AR) is a member of the G-protein coupled receptor superfamily, which has significant pathophysiological importance in disorders such as heart arrhythmias, asthma and stroke. Here, we have used fluorescence correlation spectroscopy (FCS) to facilitate the study of A1-AR pharmacology at the subcellular level. To this end, we have successfully designed and synthesised a fluorescently labelled A1-AR agonist, ABA-BY630. ABA-BY630 is an N6- derivative of adenosine conjugated to the red-excited fluorophore, BODIPY" 630/650. In CHO cells expressing the human A1-AR, ABA-BY630 shows strong and potent agonist activity at this receptor. Specific binding of ABA-BY630 to the A1-AR in cell membranes of living CHO cells can also be visualised using confocal microscopy. Moreover, using FCS, we can detect and quantify the binding of ABA-BY630 to the A1-AR in a small area (0.2 microm2) of the upper cell membrane. FCS measurements indicate the presence of at least two populations of receptor-ABA-BY630 complexes with diffusion times of 8 and 233 ms. The quantity of both of these complexes was significantly reduced by pre-incubation with the A1-AR antagonist DPCPX. Application of FCS in conjunction with ABA-BY630 will allow the comparison of A1-AR pharmacology in single cells from healthy and diseased tissue.

A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is becoming increasingly popular as a technique that aims at complementing live cell images with biophysical information. This article provides both a short overview over recent intracellular FCS applications and a practical guide for investigators, who are seeking to integrate FCS into live cell imaging to obtain information on particle mobility, local concentrations, and molecular interactions. A brief introduction to the principles of FCS is provided, particularly emphasizing practical aspects such as the choice of appropriate dyes and positioning of the measurement volume in the sample. Possibilities and limitations in extracting parameters from autocorrelation curves are discussed, and attention is drawn to potential artifacts, such as photobleaching and probe aggregation. The principle of dual-color cross-correlation is reviewed along with considerations for proper setup and adjustment. Practical implications of nonideal conditions including incomplete focus overlap and spectral cross-talk are considered. Recent examples of both auto- and cross-correlation applications demonstrate the potential of FCS for cell biology.

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.

Application of Fluorescence Correlation Spectroscopy: A Study of Flocculation of Rigid Rod-like Biopolymer (Schizophyllan) and Colloidal Particles

The flocculation between the rod-like biopolymer Schizophyllan and two types of colloidal particles (latex with diameter 40 nm and alumina with diameter 60 nm) has been investigated by means of fluorescence correlation spectroscopy (FCS). The concentration ratio of Schizophyllan/particle q was varied in the range 0.1∼20. Under conditions of pH about 5.7, 1 mmol·L−1 NaCl, and room temperature (22±0.5°C), the particles are strongly charged (alumina particles positively charged, latex negatively), while Schizophyllan is neutral. We observed that Schizophyllan chains flocculate with both types of particles, which suggests that the charge neutralization does not play a decisive role in these interactions. The ratio of fluorescence intensity of one floc over that of one particle, Qf/Qp, and the corresponding hydrodynamic radius (rh) of the flocs have been measured. For a Schizophyllan–latex system, Qf/Qp reached a maximum value of 5 for q=3 indicating that the flocs contained five particles on average. The corresponding value of rh was rh=455 nm. The flocculation kinetic of latex particles with Schizophyllan was too fast to be measurable by FCS. For the Schizophyllan–alumina system, Qf/Qp was stable at about 1 in the whole studied range of q but rh increased with q suggesting that many Schizophyllan chains are adsorbed on individual particles. The flocculation kinetic of this system was studied by FCS and the obtained results were compatible with those of photon correlation spectroscopy.

Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy.

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.