Fluorescence Correlation Spectroscopy Method Research Articles

Single-Molecule Enzymology: Studies on Glutathione-S-Transferases

An emerging concept in enzymology is the catalytic transition state ensemble: instead of the single, well-defined transition state (TS) of classical theory, enzymatic reactions progress through multiple ‘valleys’ in the energy landscape, each with its own TS. Long-lived conformations of the enzyme-substrate (ES) complex would take different pathways through the TS ensemble, and would therefore result in functional heterogeneity (i.e., a distribution of catalytic rate constants). The functional properties of enzymes are therefore completely controlled by the nature of the energetic landscape for the ES complex and the TS ensemble. We have developed TIRFM (total internal reflection fluorescence microscopy) and FCS (fluorescence correlation spectroscopy) methods to measure turnover by single immobilized enzyme molecules. We applied these methods, complemented by molecular dynamics simulations and ab initio calculations, to two isoforms of glutathione-S-transferase (GST) to examine the validity of the TS ensemble model. GST isoform A1-1 is an abundant hepatic enzyme involved in detoxification, and is extremely promiscuous in its substrate range. GST A4-4 is the predominant isoform in the heart and brain, and is specific towards lipid peroxidation products. Contrasting the behavior of these two enzymes will address fundamental questions about the mechanisms of substrate promiscuity.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Fluorescence correlation spectroscopy near individual gold nanoparticle

Dynamic behavior of fluorescent molecules near an individual gold nanoparticle is investigated experimentally by fluorescence correlation spectroscopy method. The gold particle that acts as an optical nano-antenna presents significant near-field volume reduction. The single molecule diffusion behavior is clearly observed within a reduced near-field volume due to a highly localized field enhancement. The near-field volume and fluorescence enhancement are polarization and concentration dependent and strongly depend on the properties of the gold nanoparticle. A simple approximated model is developed to fit the FCS autocorrelation curves. In principle, the single molecule analysis within the near-field volume of nanostructures could be applied to the analysis of biological membranes and intracellular processes.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

Solvent Polarity Effect on Chain Conformation, Film Morphology, and Optical Properties of a Water-Soluble Conjugated Polymer

The solvent polarity effect on chain conformation, film morphology, and photophysical properties of a nonionic water-soluble conjugated polymer (WSCP), poly[2,5-bis(diethylaminetetraethylene glycol)phenylene vinylene] (DEATG-PPV) is investigated in detail. The combination of stationary absorption and photoluminescence (PL) spectroscopy, time-resolved PL spectroscopy, and fluorescence correlation spectroscopy methods enables us to probe the chain conformation of DEATG-PPV, down to the level of a single chain when working with extremely diluted solutions. The use of correlated atomic force microscopy and confocal fluorescence lifetime imaging microscopy measurements of drop-casted DEATG-PPV films reveals the intrinsic relationship between chain conformation, film morphology, and optical properties. Depending on solvent polarity, DEATG-PPV presents extended, coiled, and collapsed chain conformations in solutions, which lead to distinct morphology and optical properties in solid films. Our work presents a pathway to control and characterize the film morphologies of WSCPs toward the optimal performance of various optoelectronic devices.

Compartmentation of ATP in Cardiomyocytes and Mitochondria Kinetic Studies and Direct Measurments

High energy demand of heart and brain cells is met by mitochondrial ATP production and energy transfer between ATP compartments mostly by creatine kinase (CK) - phosphocreatine (PCr) system. The aim of this work is to study the diffusion restrictions of ATP at the level of mitochondrial outer membrane as a basis of its compartmentation in cardiomyocytes. In a first part channelling of adenine nucleotides in mitochondria is studied via the direct transfer of the novel synthesized ATP from the adenine nucleotide translocase (ANT) to the mitochondrial creatine kinase (MtCK) by performing the complete kinetic analysis of the MtCK. A partial reconstruction of mitochondrial cytoskeletal environment was performed by incubation of heart isolated mitochondria (apparent Km for exogenous ADP = 9 ± 1 μM) with 1 μM tubulin (apparent Km for exogenous ADP = 169 ± 52 μM) without or with 20 mM creatine (apparent Km for exogenous ADP = 23 ± 6 μM). The results showed a clear restriction in adenine nucleotide diffusion in presence of tubulin by the change of the profile of respiration kinetic linearization. This restriction was found to be overcome by the presence of creatine which can activate the MtCK reaction and increase the rate of ADP/ATP turnover due to functional coupling between MtCK and ANT. In a second part the method of fluorescence correlation spectroscopy was used to study the diffusion kinetics of ATP-Alexa 647 both in solution, isolated mitochondria and cardiomyocytes. However pronounced multicomponent diffusion kinetics was found both in isolated mitochondria and permeabilised cardiomyocytes. This diffusion time seemed to be sensitive to the ordered state of cardiomyocyte and increased when the symmetry was broken in the rounded or apoptotic cells. The fluorescent ATP was use to study ATP compartmentation.

Two-colors Photo-Switching of E222Q-GFPMut2 Mutant by Fluorescence Correlation Spectroscopy

GFP mutants display complex photodynamics whose properties can be tuned even by single mutations of the chromophore or the protein backbone. Some GFP mutants can be photo-activated (paGFP) or photo-switched (E222Q mutants). Although photo-activable mutants are valuable tools in nanoscopy studies and have been already applied in this field, the photo-switching behavior of some GFP mutants has not been yet exploited in biological imaging.We report here the characterization of the two-color enhancement of the E222Q mutant of the GFPMut2 protein aimed to its application in cellular imaging. The anionic fluorescence output is enhanced when the GFP is irradiated simultaneously at 390-440 nm. By fine tuning the 488 nm direct anionic excitation and the UV-blue irradiation, the GFP mutant emission can be enhanced up to 2.5 times. The maximum switching efficiency occurs at 420 nm and display a marked pH dependence.Moreover, we have characterized the activation time of this process by modulating the irradiation or excitation beams. By means of Fluorescence Correlation Spectroscopy methods under modulated irradiation in the UV-blue range of the spectrum, we are able to measure the activation times of the switching process that lie in the 10-100 ms range. We present a simple two states model and analyze it by Laplace Transform methods to obtain a validation of the proposed model and a direct estimate of the activation times.Finally we discuss possible applications of this behavior in fluorescence imaging and direct studies of intracellular dynamic processes.

A comparative study on ganglioside micelles using electronic energy transfer, fluorescence correlation spectroscopy and light scattering techniques

Ganglioside (G(M1)) micelles have been studied by means of three different techniques: fluorescence correlation spectroscopy (FCS), electronic energy transfer, as monitored by time-resolved fluorescence spectroscopy, as well as static and dynamic light scattering. The aggregation numbers obtained, 168 +/- 4, remain constant over a wide range of G(M1) concentrations (0.764-156 muM), are very consistent when using different donor-acceptor energy transfer pairs and have served as reference values in tests of the FCS method. It is recommended to calibrate the focal volume by using known dye concentrations. For this the rhodamine dye, 5-TAMRA, turns out to be most suitable. It is also shown that FCS provides correct values of the aggregation numbers, provided that the focal volume is calibrated by using updated values of the diffusion constant of Rhodamine 6G. These results also support recent methodological advances in FCS.

Oversulfated chondroitin sulfate‐E binds to BMP‐4 and enhances osteoblast differentiation

Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization.

Minimally invasive determination of mRNA concentration in single living bacteria

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to ∼40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 microm(-2)) and very low surface concentrations (single-molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concentration range of about 0.1-100 microm(-2). However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several experiments at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.

Fluctuation correlation spectroscopy and photon histogram analysis of light scattered bygold nanospheres

Fluorescence correlation spectroscopy (FCS) is a valuable tool in biological research. In recentyears there has been growing interest in using light scattered from metallic colloids in placeof organic fluorophores. Metallic colloids display optical cross sections for scattering thatare orders of magnitude brighter than fluorophores. We used the FCS method to study thescattering properties of varying sizes of gold colloids 38–100 nm in diameter. The opticalcross sections of the gold colloids increase rapidly with size, as can be seen by both theG(0) value of the autocorrelation function and the scattering intensity distributions. In mixturesof different size gold colloids the autocorrelation function is dominated by thelarger (brighter) colloids, even when present at a small fractional population.We show that it is possible to detect one 100 nm gold colloid in the presence of103–104 smaller 39 nm diameter colloids. Because the scattering cross sections of colloids willincrease with aggregation, we believe that FCS can be used to detect a small number ofassociated bio-labeled colloids in the presence of a much larger population ofnon-associated colloids.

Palmitoylation is required for efficient Fas cell death signaling

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death.

New fluorescence correlation spectroscopy (FCS) suitable for the observation of anomalous diffusion in polymer solution: Time and space dependences of diffusion coefficients

We propose a new method of fluorescence correlation spectroscopy (FCS) enabling direct detection of anomalous diffusion as the distance dependence of diffusion coefficients (DDDC), namely, sampling-volume-controlled (SVC)-FCS. In discussing the results of SVC-FCS measurement of molecular diffusion in aqueous hyaluronan (HA) solutions in this paper, we suggest the use of “local anomalous diffusion” based on the differential of the mean-square displacement (MSD) which is convenient for understanding the total lineshapes of the observed diffusion coefficient as a function of the diffusion distance or the diffusion time.

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H 2O 2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded β-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR–FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.

Scanning Fluorescence Correlation Spectroscopy: A Tool for Probing Microsecond Dynamics of Surface-Bound Fluorescent Species

In this report, a combined imaging and fluorescence correlation spectroscopy (FCS) method is described and its ability to characterize microsecond fluctuations in the fluorescence emission of a sample is demonstrated. A sample scanning laser confocal microscope is operated in the customary way while recording the time that each photon is detected with a time resolution of 50 ns using a low-cost counting board. The serial data stream of photon detection times allows access to fluorescence signal fluctuations that can be used to characterize dynamics using correlation methods. The same data stream is used to generate images of the sample. Using the technique, we demonstrate that it is possible to characterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC16 and ATTO 520. Results are similar to, but deviate slightly from, a model that has been frequently used for extracting singlet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration, or in our case the areal density of coverage, of fluorescent species can also be obtained. Many single-molecule fluorescence experiments aim to extract kinetics from intensity trajectories; this method may be used as a rapid and convenient technique for characterization of surface-linked or thin-film samples prior to performing the more time and effort intensive single-molecule measurements. Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also be applied for measuring conformational changes or interaction kinetics for species immobilized on a surface. One possible scenario is measurements of the frequency and duration of association of ligand-receptor pairs where a fluorescently labeled component is freely diffusing and the other is surface immobilized. Given that microarrays of custom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of the surface-linked species with a freely diffusing species could be a useful extension to what has already become an extremely important tool for characterizing the interactions of biomolecules.

Fluorescence correlation spectroscopy: a new tool for quantification of molecular interactions.

Fluorescence correlation spectroscopy (FCS) provides a powerful method to measure molecular dynamics and interactions in a wide variety of experimental systems and environments. In this article we focus on the use of FCS methods to quantify molecular interactions, including the use of diffusion analysis and molecular counting. Both autocorrelation and cross-correlation FCS measurements are discussed.

Measurement of diffusion of fluorescent molecules in living cells

The possibilities of the method of fluorescence correlation spectroscopy for studying the molecular dynamics in living cells are demonstrated. The method provides point measurements of extremely low concentrations of fluorescent molecules and their diffusion coefficients with a high time resolution in a microscopic volume, which is especially important in pharmacological investigations. A biological model of the interaction of liposomes with a cellular membrane is considered. The diffusion coefficients of fluorescent molecules are measured directly in the living cell cytoplasm.

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.

Two-Photon Fluorescence Polarization Anisotropy Decay on Highly Diluted Solutions by Phase Fluorometry

We report here the phase-sensitive measurements of two-photon fluorescence polarization anisotropy (FPA) on highly diluted solutions. We first describe the characterization of the response of the two-photon microscope to the light polarization and its test by means of measurements of the FPA on rhodamine 6G as a function of the viscosity. Further, we report the study of the FPA at high dilutions on a globular protein, the beta-lactoglobulin B (BLG) labeled with Alexa 532. The number of molecules (from 0.4 to 17 molecules per excitation volume) is measured by means of fluorescence correlation spectroscopy (FCS). The average rotational and translational diffusion coefficients measured with the FPA and FCS methods are in good agreement with the protein size and do not show a substantial dependence on the protein concentration, despite the very low signal (≌3 times the background) observed for highly diluted solutions.

Applications of fluorescence correlation spectroscopy: measurement of size-mass relationship of native and denatured schizophyllan.

Diffusion dynamics of a polysaccharide, schizophyllan has been studied by fluorescence correlation spectroscopy (FCS). Several different sizes of nondenatured and denatured schizophyllan have been labeled with rhodamine 6G in borate buffer. The length of the nondenatured schizophyllan was calculated from FCS data by using the Broersma's relationship for rod-like macromolecules. The obtained length was close to that obtained by atomic force microscopy (AFM) measurements. Denatured schizophyllan possesses a random coil conformation. Its hydrodynamic radius R(h) was measured by FCS. The relationship between R(h) and the molecular mass M has been studied and the scaling relationship R(h)--M(0.59) has been obtained, which is in agreement with the random coil model with excluded volume effect. The persistence length q(denat) of the denatured schizophyllan was determined by Hearst's relationship, to be equal to 5.16 +/- 0.75 (nm). The work demonstrates the utility of FCS method for dynamics investigations of biopolymers especially in diluted regime (concentration lower than 10(-8)M could be measured) where other techniques could not be used.