Fluorescence Correlation Spectroscopy Curves Research Articles

Unveiling the Influence of Brightness Heterogeneity in Fluorescence Correlation Spectroscopy of Perovskite Nanocrystals.

Nanoparticles (NPs), including perovskite nanocrystals (PNCs) with single photon purity, present challenges in fluorescence correlation spectroscopy (FCS) studies due to their distinct photoluminescence (PL) behaviors. In particular, the zero-time correlation amplitude [g2(0)] and the associated diffusion timescale (τD) of their FCS curves show substantial dependency on pump intensity (IP). Optical saturation inadequately explains the origin of this FCS phenomenon in NPs, thus setting them apart from conventional dye molecules, which do not manifest such behavior. This observation is apparently attributed to either photo-brightening or optical trapping, both lead to increased NP occupancy (N) in the excitation volume, consequently reducing the g2(0) amplitude [since g2(0) α 1/N] at high IP. However, an advanced FCS study utilizing alternating laser excitation at two different intensities dismisses such possibilities. Further investigation into single-particle blinking behaviors as a function of IP reveals that the intensity dependence of g2(0) primarily arises from the brightness heterogeneity prevalent in almost all types of NPs. This report delves into the complexities of the photophysical properties of NPs and their adverse impacts on FCS studies.

Study on Phase Separation of Fused in Sarcoma by Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation (LLPS) of fused in sarcoma (FUS) has emerged as a fundamental principle underpinning cellular function and malfunction. However, we know little about the FUS phase transition process from individual molecules to nanoscale condensates, which plays important roles in neurodegenerative diseases. Here, we propose the fluorescence correlation spectroscopy (FCS) method to quantitatively study the phase separation process of FUS protein with the fluorescent tag-enhanced green fluorescent protein (EGFP), from individual molecules to nanoscale condensates. The characteristic diffusion time (τD) of the protein condensates can be obtained from the FCS curve, which increases with the growth of the protein hydration radius. The bigger the τD value of the protein condensates, the larger the condensates formed by the phase separation of FUS. By this method, we discovered that the critical concentration for FUS to phase separation was 20 nM. We then plotted FUS phase diagrams based on τD under different concentrations of NaCl and found that both low-salt and high-salt concentrations tended to promote FUS-EGFP phase separation. Our results showed that ATP has a good inhibitory effect on FUS phase separation, and its inhibition constant IC50 was 3.2 mM. Finally, we evaluated the inhibition efficiency of single-stranded DNA sequences (ssDNA) on FUS phase separation and demonstrated that ssDNA containing three copies of TCCCCGT had relatively strong inhibition efficiency. In summary, our work provides detailed insight into the FUS phase transition process from individual molecules to nanoscale condensates at nanomolar concentrations and can be exploited for drug screening of neurodegenerative diseases.

Measuring the fluidity of the membrane of Bacillus subtilis using TIR-FCS.

Bacteria need to constantly maintain the fluidity of their membrane in response to external stimuli such as changes in temperature, growth medium, etc. So far, bacterial membrane remodeling could only be assessed indirectly via biochemical methods like fatty acid analysis or in live bacteria using environment-sensitive dyes. Fluorescence correlation spectroscopy (FCS), a gold-standard technique in membrane dynamics investigations, was however barely used to study bacterial membranes. This is likely due to an increased technical complexity, caused by the small size of bacteria and mounting difficulties, which complicates FCS measurements on confocal microscopes as they are usually performed. Here, we show that imaging FCS on a TIRF microscope (TIR-FCS) can remediate this thanks to an increased axial resolution with respect to confocal and ability to correct for sample drift using image registration. To facilitate the analysis of the large amount of data generated by TIR-FCS, we developed a simple method to automatically exclude artefactual FCS curves. As a result, we could perform high-throughput measurements of the diffusion coefficients of lipophilic dyes like Nile Red or TopFluor-sphingomyelin in the membrane of B. subtilis. Using this assay, we observed the effects of membrane remodeling in response to known stimuli like a cold shock. We also looked at the effects of the widely used membrane-perturbing drug benzyl alcohol, revealing an unexpectedly temperature-dependent fluidization effect. Finally, we used TIR-FCS to explore membrane fluidity adaptation during the sporulation process. We believe that this method has significant potential in microbiology and can easily be applied in the future to different problems, to study membrane proteins diffusion or to understand the effects of selected drugs on membrane viscosity.

Photophysical Rate Constants and Oxygen Dependence for Si and Ge Rhodamine Zwitterions.

The family of group XIV rhodamine zwitterions are fluorescence probes with carbon, silicon, germanium, or tin substituted in the 10-position of the xanthene ring. Because of their inherent near-infrared fluorescence, photostability and high quantum yields in aqueous solutions, the Si and Ge containing fluorophores in this class have become increasingly important for fluorescent labeling of proteins and biological molecules. This study fully characterizes photophysical rates derived from a model consisting of a singlet ground state, the lowest singlet excited state, and the lowest triplet excited state for two exemplar group XIV rhodamine zwitterions, one containing Si and the other Ge. Within a simple Jablonski diagram, all radiative and non-radiative rates, including intersystem crossing and triplet depopulation rates, were measured as a function of oxygen concentration. It was shown that the triplet depopulation rates are intrinsically fast in comparison with traditional xanthene containing fluorophores, probably due to the increased spin-obit coupling from the Si and Ge substitution in the xanthene ring. Dissolved oxygen increases both the intersystem crossing and triplet depopulation rates. Stern-Volmer analysis was conducted to estimate rates of quenching by oxygen. The experimental data was used to estimate the initial rates for reactive oxygen production by Si and Ge containing fluorophores in aqueous solutions containing different concentrations of dissolved O2. These estimates showed a significantly slower initial rate of reactive oxygen production in comparison with rhodamine 6G. This goes a long way to explaining their inherent photostability. Spectroscopic experiments were also conducted in 77 K viscous aqueous glasses where it was observed that the fluorescence spectra remained unchanged, and the quantum yields increased from 0.53 to 0.84 and from 0.52 to 0.89 for the Si and Ge containing fluorophores respectively; no phosphorescence was observed. All intersystem crossing and triplet depopulation rates were measured using fluorescence correlation spectroscopy (FCS) and analyzed using a new method that extrapolated the power dependence of the FCS curves to optical saturation. This method was verified using published data.

Measuring Photophysical Transition Rates with Fluorescence Correlation Spectroscopy and Antibunching.

We present a new method that combines fluorescence correlation spectroscopy (FCS) on the microsecond time scale with fluorescence antibunching measurements on the nanosecond time scale for measuring photophysical rate constants of fluorescent molecules. The antibunching measurements allow us to quantify the average excitation rate of fluorescent molecules within the confocal detection volume of the FCS measurement setup. Knowledge of this value allows us then to quantify, in an absolute manner, the intersystem crossing rate and triplet state lifetime from the microsecond temporal decay of the FCS curves. We present a theoretical analysis of the method and estimate the maximum bias caused by the averaging of all quantities (excitation rate and photophysical rates) over the confocal detection volume, and we show that this bias is smaller than 5% in most cases. We apply the method for measuring the photophysical rate constants of the widely used dyes Rhodamine 110 and ATTO 655.

Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment

Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment.

Determination of Equilibrium Constant and Relative Brightness in Fluorescence Correlation Spectroscopy by Considering Third-Order Correlations.

Fluorescence correlation spectroscopy (FCS) is a powerful tool to investigate molecular diffusion and relaxations, which may be utilized to study many problems such as molecular size and aggregation, chemical reaction, molecular transportation and motion, and various kinds of physical and chemical relaxations. This article focuses on a problem related to using the relaxation term to study a reaction. If two species with different fluorescence photon emission efficiencies are connected by a reaction, the kinetic and equilibrium properties will be manifested in the relaxation term of the FCS curve. However, the conventional FCS alone cannot simultaneously determine the equilibrium constant (K) and the relative fluorescence brightness (Q), both of which are indispensable in the extraction of thermodynamic and kinetic information from the experimental data. To circumvent the problem, an assumption of Q = 0 is often made for the weak fluorescent species, which may lead to numerous errors when the actual situation is not the case. We propose to combine the third-order FCS with the conventional second-order FCS to determine K and Q without invoking other resources. The strategy and formalism are verified by computer simulations and demonstrated in a classical example of the hairpin DNA-folding process.

What information is contained in the fluorescence correlation spectroscopy curves, and where.

We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

An Elegant Way to Quantitatively Analyze Oligomer Formation in Solution

An Elegant Way to Quantitatively Analyze Oligomer Formation in Solution

PH-Induced Intramolecular Folding Dynamics of i-Motif DNA

Using the combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) technique, we investigate the mechanism and dynamics of the pH-induced conformational change of i-motif DNA in the bulk phases and at the single-molecule level. Despite numerous studies on i-motif that is formed from cytosine (C)-rich strand at slightly acidic pH, its detailed conformational dynamics have been rarely reported. Using the FRET technique to provide valuable information on the structure of biomolecules such as a protein and DNA, we clearly show that the partially folded species as well as the single-stranded structure coexist at neutral pH, supporting that the partially folded species may exist substantially in vivo and play an important role in a process of gene expression. By measuring the FCS curves of i-motif, we observed the gradual decrease of the diffusion coefficient of i-motif with increasing pH. The quantitative analysis of FCS curves supports that the gradual decrease of diffusion coefficient (D) associated with the conformational change of i-motif is not only due to the change in the intermolecular interaction between i-motif and solvent accompanied by the increase of pH but also due to the change of the shape of DNA. Furthermore, FCS analysis showed that the intrachain contact formation and dissociation for i-motif are 5-10 times faster than that for the open form. The fast dynamics of i-motif with a compact tetraplex is due to the intrinsic conformational changes at the fluorescent site including the motion of alkyl chain connecting the dye to DNA, whereas the slow intrachain contact formation observed from the open form is due to the DNA motion corresponding to an early stage interaction in the folding process of the unstructured open form.

Time-Resolved Three Dimensional Tracking of Individual GFP Molecules in Solution at Biologically Relevant Transport Rates

Many important cellular functions such as intracellular signaling and protein trafficking involve three dimensional molecular motion. Monitoring these processes at the single molecule level requires the ability to track individual fluorescent emitters in high background environments at biologically relevant diffusion rates, and over several microns in all three dimensions. We have previously reported a custom confocal 3D tracking fluorescence microscope design capable of tracking single quantum dots (QDs) over several microns in X, Y, and Z in high background environments (Anal. Chem., 2008, 80, 9830-9834), and followed QD labeled antibodies bound to membrane receptors on live cells (Proc. of SPIE, 2009, 7185, 71850Z). While QDs are useful for obtaining long trajectories, their use as biological labels is generally limited to receptors that can be externally labeled or to permeabilized cells. GFP labeling circumvents these limitations, and as such, is the fluorescent label of choice for many intracellular fluorescence microscopy experiments. While CCD-based methods for 2D tracking of individual GFPs in live cells are well established, 3D tracking of single GFP labeled intracellular proteins in live cells will represent a tremendous technical achievement. Towards this end, here we show our 3D tracking methods can follow the Brownian diffusion of single GFPs in glycerol-water mixtures at rates comparable to intracellular protein traffic (D ∼ 1 um2/s). Trajectory durations are limited by GFP photostability, with some trajectories lasting for seconds. Unlike CCD-based tracking schemes, the arrival time of each photon is recorded with ∼400 picoseconds accuracy, enabling time-resolved spectroscopy to be performed on the molecules tracked. In particular, we demonstrate fluorescence correlation spectroscopy (FCS) of individual GFPs, with these FCS curves being obtained as we follow individual molecules moving through 3 dimensional space.

On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope

Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to μM range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.

Brownian diffusion of gold nanoparticles in an optical trap studied by fluorescence correlation spectroscopy

The effect of thermal-induced Brownian motion on gold nanoparticles (Au NPs) in optical traps is studied by fluorescence correlation spectroscopy (FCS) method. The Brownian motion and optical trapping potential are investigated by the decay time of the FCS curve and the laser power. It is shown that that the probability of finding a gold nanoparticle in the trap depends on the ratio of the optical energy of the particle to its thermal energy. A power threshold is observed by the decay time as a function of laser power. The experimental studies show that the temperature rise does not seriously affect the average number of particles in the focal spot, but the average residence time is more sensitively affected by the temperature.

ImFCS: A software for Imaging FCS data analysis and visualization

The multiplexing of fluorescence correlation spectroscopy (FCS), especially in imaging FCS using fast, sensitive array detectors, requires the handling of large amounts of data. One can easily collect in excess of 100,000 FCS curves a day, too many to be treated manually. Therefore, ImFCS, an open-source software which relies on standard image files was developed and provides a wide range of options for the calculation of spatial and temporal auto- and cross-correlations, as well as differences in Cross-Correlation Functions (ΔCCF). ImFCS permits fitting of standard models to correlation functions and provides optimized histograms of fitted parameters. Applications include the measurement of diffusion and flow with Imaging Total Internal Reflection FCS (ITIR-FCS) and Single Plane Illumination Microscopy FCS (SPIM-FCS) in biologically relevant samples. As a compromise between ITIR-FCS and SPIM-FCS, we extend the applications to Imaging Variable Angle-FCS (IVA-FCS) where sub-critical oblique illumination provides sample sectioning close to the cover slide.

Early detection of apoptosis in living cells by fluorescence correlation spectroscopy

Early detection of apoptotic cells via caspase activity is demonstrated with fast response time. Fluorescence correlation spectroscopy (FCS) is used to identify the presence of a cleaved fluorogenic probe based on the fluorescence of rhodamine 110 in Jurkat cells. FCS curves are shown to be markedly different for autofluorescent (non-apoptotic) cells, whereas cells with cleaved probe showed diffusion and molecular brightness characteristic of rhodamine 110. Using FCS measurements, cells were identified as apoptotic on the basis of the presence of autocorrelated fluorescence, average molecular brightness (eta), and molecular dwell time (tau (D)). Apoptotic cells identified in this manner were detected as early as 45 min after induction. Unlike other methods with similar identification times, such as western blotting and electron microscopy, cells remain viable for further analysis. This multi-parameter approach is rapid, flexible, and does not require transfection of the cells prior to analysis, enabling apoptosis to be identified early in a wide variety of cell types.

Studying Molecular Dynamics And Interactions In Living Zebrafish Embryos By Fluorescence Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique to assess molecule dynamics and interactions on a single molecule level. It has been routinely used to harvest biomedical information both in vitro and in vivo. Numerous intracellular measurements have been reported in cytoplasm, nucleoplasm and plasma membrane. However, it is now generally accepted that the Petri-Dish based cell culture system cannot represent the essential physiological environment of a living organism and 3D cultures are only a partial solution. Here, we adapted single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) to work in a living animal model. We chose zebrafish as the optical transparency of early stage zebrafish embryo makes microscopic techniques suitable and established genetic tools enable one to easily express foreign genes within the embryo. First we investigated the penetration depth in the embryo body as tissue tends to cause light scattering and decrease signal to noise ratio if working deep beneath skin. We practiced one- and two-photon excitation and obtained FCS curves up to 80 μm and 200 μm, respectively. Then we characterized the diffusion coefficients of genetically expressed EGFP in muscle fibers and motor neurons and also investigated the mobility of a membrane expressed G protein coupled receptor-CXCR4b. Finally, we measured the dissociation constant (KD) between a small Rho-GTPase and an actin-binding scaffolding protein, in living zebrafish embryos and the results were compared with data obtain from CHO-K1 cultured cells. We showed that molecular dynamics and interactions can be studied in small living animals that provide a genuine physiological environment and questions of developmental biology on a single molecule level are directly accessible by FCS.

Using Fluorescence Correlation Spectroscopy to Study Conformational Changes in Denatured Proteins

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

Fluorescence Correlation Spectroscopy Analysis of Diffusion in a Laser Gradient Field: A Numerical Approach

Fluorescence correlation spectroscopy (FCS) is widely used in biological systems. When the laser is intense enough, such as in two-photon experiments, the trapping force due to the laser gradient field can change the diffusion behavior of the fluorescent particles and induce error in the FCS measurements. Previous studies on biased FCS are qualitative. In this article, a numerical approach is proposed to treat the problem quantitatively. By assumption of a "spherical symmetry", biased FCS curves can be calculated numerically and fitted to the experimental data to retrieve the unbiased particle number, diffusion time, and polarizability of the fluorescent particles as well as the strength of the gradient field. It has been proven using simulated FCS data that the discrepancy caused by the spherical symmetry approximation is independent of the gradient field strength; therefore it can be eliminated by a calibration.

Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting

Fluorescence correlation spectroscopy (FCS) is a powerful spectroscopic technique for studying samples at dilute fluorophore concentrations down to single molecules. The standard way of data acquisition, at such low concentrations, is an asynchronous photon counting mode that generates data only when a photon is detected. A significant problem is how to efficiently convert such asynchronously recorded photon count data into a FCS curve. This problem becomes even more challenging for more complex correlation analysis such as the recently introduced combination of FCS and time-correlated single-photon counting (TCSPC). Here, we present, analyze, and apply an algorithm that is highly efficient and can easily be adapted to arbitrarily complex correlation analysis.