Fluorescence Correlation Spectroscopy Setup Research Articles

Wavelength-dependent fluorescence fluctuation and molecular brightness analysis of a novel mEGFP-linker-mScarlet-I crowding biosensor at the single-molecule level.

Living cells are dynamic and heterogeneous microenvironments, in which macromolecular crowding impacts transport, intermolecular interactions, and kinetics. Here, we characterize a protein biosensor that has been designed to detect changes in macromolecular crowding using Förster energy transfer (FRET). We investigate the fluorescence fluctuations, molecular brightness, and translational diffusion of the mEGFP-linker-mScarlet-I construct (GE2.3) using fluorescence correlation spectroscopy (FCS) of the donor (mEGFP) in the presence and absence of the acceptor (mScarlet-I) in response to environmental crowding. Our FCS setup was calibrated using rhodamine-110, and control experiments were also carried out on the enzymatically cleaved GE2.3 to elucidate the effects of FRET on the molecular brightness at the single molecule level. Our results indicate that the molecular brightness of intact GE2.3 (PBS buffer, pH 7.4) is smaller than that of the cleaved counterpart under 488-nm excitation of the donor, due to FRET. In contrast, the molecular brightness of both cleaved and intact GE2.3 seems to be the same under the 561-nm excitation of the acceptor due to the absence of FRET. The fluctuation analyses of intact and cleaved GE2.3 sensors were also used to investigate the crowding effects on translational diffusion within the context of Stokes-Einstein model. Our single-molecule findings for FRET analysis of GE2.3 complements our ensemble studies using time-resolved fluorescence and anisotropy (rotational depolarization dynamics) because it allows us to discern the temporal-scale dependence (rotational versus translational diffusion) of potential segmental mobility of the construct as a function of crowding. Further, these studies are helping to develop a rational design strategy for environmental sensors by examining different donor-acceptor FRET pair, while refining the use of donor molecular brightness to measure FRET at the single-molecule level using a traditional FCS setup.

Quantification of protein delivery in live cells using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a quantitative single-molecule method that measures the concentration and rate of diffusion of fluorophore-tagged molecules, both large and small, in vitro and within live cells, and even within discrete cellular compartments. FCS is exceptionally well-suited to directly quantify the efficiency of intracellular protein delivery-specifically, how well different "cell-penetrating" proteins and peptides guide proteinaceous materials into the cytosol and nuclei of live mammalian cells. This article provides an overview of the procedures necessary to execute robust FCS experiments and evaluate endosomal escape efficiencies: preparation of fluorophore-tagged proteins, incubation with mammalian cells and preparation of FCS samples, setup and execution of an FCS experiment, and a detailed discussion of and custom MATLAB® script for analyzing the resulting autocorrelation curves in the context of appropriate diffusion models.

A Novel Cuvette-FCS Setup for Single Molecule Measurements of Protein Unfolding and Early Stages of Protein Aggregation

Fluorescence correlation spectroscopy (FCS) is a single molecule technique with wide spread applications in measurements of biophysical properties, e.g., hydrodynamic size and conformational dynamics of biomolecules. However, conventional FCS setups that use high numerical aperture objectives have limitations in performing certain experiments such as those requiring agitations such as stirring or heating, and those involving measurements in solvents with mismatch of refractive indices. Recently using an extra-long working distance objective (ELWD) we have developed an FCS setup that is suitable for performing measurements inside regular cuvettes. The radial resolution and the confocal volume achieved in this setup are approximately 340 nm and 1.8 fL respectively. The signal to noise ratio in the autocorrelation data and counts per molecule obtained from an aqueous solution of rhodamine B are greater than 150 and 60 kHz respectively. We demonstrate that cuvette-FCS can be coupled to accessories such as autotitrators and magnetic stirrers for fully automated measurements of unfolding of proteins mediated by chemical denaturants such as urea. Furthermore, scanning-FCS measurements in this setup allows measurements of time evolution of the size distribution of the early stage aggregates of amyloid proteins. Hence, cuvette-FCS is a suitable technique for performing single molecule measurements in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS.

Fluorescence Correlation Spectroscopy with Nanowire Waveguide Illumination for High Concentration Conditions

Fluorescence correlation spectroscopy (FCS), a technique based on correlating the fluorescence signal from a small sample volume, has been commonly used to study biological processes such as photophysical phenomena, binding kinetics, and intracellular dynamics. In a conventional FCS setup, the sample volume is defined by a focused laser beam for excitation and a confocal pin hole for collection resulting in a size of about one femtoliter. Evanescent waves from total internal reflection have been used to obtain a smaller sample volume, as a result of the improved confinement along the longitudinal axis for the illumination. However, applying FCS under physiological conditions where the molecule concentration is typically in the µM range requires an even smaller sample volume. Here we propose to apply FCS with nanowire waveguide illumination with nanometer-sized lateral and longitudinal confinement. Nanowire waveguides, made of a dielectric nanowire with a diameter of ∼50 nm embedded inside a metal film, allow for the efficient transmission of visible light by exploiting surface waves traveling along the dielectric-metal boundary. Such waveguides can generate a lateral illumination area of about 50 nanometers in diameter. In addition, a longitudinal confinement of around 20 nanometers is achieved with the rapidly-attenuating near fields exiting from the waveguide. The strong confinement in both lateral and longitudinal directions thus lead to a sample volume on the order of one zeptoliter, allowing for FCS measurements in the µM concentration range. By combining Brownian dynamics simulations with the illumination profile obtained from finite element method simulations of a zinc oxide nanowire waveguide in a silver metal film, we numerically calculate the correlation function to demonstrate the use of this method in the study of molecular dynamics under high concentration conditions.

Building, Characterization, and Applications of Cuvette-FCS in Denaturant-Induced Expansion of Globular and Disordered Proteins.

Fluorescence correlation spectroscopy (FCS) is a single-molecule sensitive technique with widespread applications in biophysics. However, conventional microscope-based FCS setups have limitations in performing certain experiments such as those requiring agitations such as stirring or heating, and those involving measurements in solvents with the mismatch of refractive indices. We have recently developed an FCS setup that is suitable for performing measurements inside regular cuvettes. The cuvette-FCS is suitable for performing single-molecule measurements in experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS. Here we describe building and characterization of the performance of the cuvette-FCS setup in detail. Finally, we have used a natively folded protein and an intrinsically disordered protein to demonstrate and describe how cuvette-FCS can be applied conveniently to measure urea-dependent expansion of hydrodynamic size of proteins.

Detection of contactin-2 in cerebrospinal fluid (CSF) of patients with Alzheimer's disease using Fluorescence Correlation Spectroscopy (FCS)

ObjectivesAlzheimer's disease (AD) is the most common cause of dementia in the world. As many AD biomarkers occur at rather low abundances in CSF or blood, techniques of very high sensitivity and accuracy are important as diagnostic tools in the clinic. Here, we aimed to provide proof of concept of the use of a single molecule detection technique, Fluorescence Correlation Spectroscopy (FCS) for detection of novel candidate biomarkers for AD. Design and methodsFCS detects the diffusion times of the antigen-antibody complexes in highly diluted sample solutions, thus eliminating the need of large sample volumes and allows estimating the concentration of the target antigen. We developed a FCS set-up for contactin-2, a neuronal cell adhesion molecule and a ligand of beta-secretase 1 (BACE1) and amyloid precursor protein (APP), the latter proteins being important players in AD. With this method, we investigated whether contactin-2 concentrations are changed after delayed storage and in patients with Alzheimer's disease. ResultsThe FCS set-up for measuring contactin-2 in CSF had a lower limit of quantification (LLOQ) of 0.2ng/ml and intra- and inter-assay coefficients of variation (CVs) of 12.2% and 14.6% respectively. Contactin-2 levels were stable up to one week storage of CSF (n=3) at RT and 4°C. Further, contactin-2 levels were similar in probable AD patients (n=34, p=0.27) compared to patients with subjective cognitive decline (SCD) (n=11). ConclusionsFCS is a sensitive tool, which can be used for detecting biomarkers in the clinical setting using very low sample volumes (10μl) and can measure proteins in their native conformations in the body fluid.

Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size.

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin.

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

One and Two Photon Fluorescence Correlation Spectroscopy on Proteins in Glucose Solutions

Cryopreservation is a powerful technology with many applications in biomedical fields from organ preservation to cellular research. Simple sugars such as glucose and fructose are among the most widely used cryopreserving agents, yet despite their widespread use, the mechanism through which sugars protect cells on the molecular level is poorly understood. The vitrification model posits that the role of sugars are merely to inhibit ice formation altogether. The water replacement model predicts sugars preferentially bind to the surfaces of protein forming a protective layer against the denaturing ice formation. Under this paradigm, one would expect the hydrodynamic radius of diffusing protein to increase with increasing sugar concentration. In order to test this hypothesis, we have developed both one-photon and two-photon fluorescence correlation spectroscopy (FCS) to measure the hydrodynamic radius of fluorescent particles in glucose solutions. The two-photon technique was developed to investigate tryptophan-containing proteins. However, to date, we have only succeeded in measuring the diffusion of reasonably large, Avidin-coated polystyrene spheres that possess sufficient fluorescence to be reliably detected. Work is continuing on using this technique to study the diffusion of the protein hemocyanin. Given these difficulties with the limited brightness of tryptophan, the one-photon FCS setup is being used to investigate green fluorescent protein (GFP), a much smaller, but considerably brighter, fluorophore.This work was supported through funding from National Institutes of Health (grant #1R01EB009644-01).

In vitro detection of adrenocorticotropic hormone levels by fluorescence correlation spectroscopy immunoassay for mathematical modeling of glucocorticoid-mediated feedback mechanisms

Performing quantitative, highly sensitive measurements at a single molecule level is often necessary to address specific issues related to complex molecular and biochemical systems. For that purpose, we present a technique exploiting both the flexibility of immunoassays as well as the low operating costs and high throughput rates of the fluorescence correlation spectroscopy (FCS) method. That way we have established a quantitative measurement technique providing accurate and flexibly time resolved data of single molecules. Nanomolar changes in adrenocorticotropic hormone (ACTH) levels have been detected in a short time-frame that are caused by fast feedback actions in AtT-20 anterior pituitary glands in vitro. Especially with respect to clinical diagnostic or mathematical modeling this improved FCS setup may be of high relevance in order to accurately quantify the amounts of peptide hormones—such as ACTH—as well as signaling molecules, transcription factors, etc., being involved in intra- and extracellular reaction networks.

Understanding Ligand Interaction with Different Structures of G-Quadruplex DNA: Evidence of Kinetically Controlled Ligand Binding and Binding-Mode Assisted Quadruplex Structure Alteration

The study of ligand interaction with G-quadruplex DNA is an active research area, because many ligands are shown to bind G-quadruplex structures, showing anticancer effects. Here, we show, for the first time, how fluorescence correlation spectroscopy (FCS) can be used to study binding kinetics of ligands with G-quadruplex DNA at the single molecule level. As an example, we study interaction of a benzo-phenoxazine ligand (Cresyl Violet, CV) with antiparallel and (3 + 1) hybrid G-quadruplex structures formed by human telomeric sequence. By using simple modifications in FCS setup, we describe how one can extract the reaction kinetics from diffusion-coupled correlation curves. It is found that the ligand (CV) binds stronger, by an order of magnitude, to a (3 + 1) hybrid structure, compared to an antiparallel one. Ensemble-averaged time-resolved fluorescence experiments are also carried out to obtain the binding equilibrium constants (K) of ligand-quadruplex interactions in bulk solution for the first time, which are found to match very well with FCS results. Global analysis of FCS data provides association (k(+)) and dissociation (k(-)) rates of the ligand in the two structures. Results indicate that stronger ligand binding to the (3 + 1) hybrid structure is controlled by the dissociation rate, rather than the association rate of ligand in the quadruplexes. Circular dichroism (CD) and induced-CD spectra show that the ligand not only binds at different conformations in the quadruplexes, but also induces antiparallel structure to form a mixed-type hybrid structure in Na(+) solution. However, in K(+) solution, the ligand stabilizes the (3 + 1) hybrid structure. Molecular docking studies predict the possible differences in binding sites of the ligand inside two quadruplexes, which strongly support the experimental observations. Results suggest that different binding modes of the ligand to the quadruplex structures actually assist the alteration of structures differently.

형광입자들의 크기와 농도에 따른 형광 상관 분광함수 측정

The concentration and hydrodynamic radius of nano-sized fluorescence particles diffusing in solution were compared by using fluorescence correlation spectroscopy (FCS), which can measure the variation of the correlation function of a fluorescence signal by size and number of particles. The used nano-sized fluorescence particles are Alex Fluor 647, quantum dots, and fluorescence beads, and three kinds of sample solutions with different concentrations were prepared by dilution to 1/10 and 1/100 with distilled water for each kind of particles. The effective focal volumes were calculated by using the known diffusion coefficient of Alexa Fluor 647 particles, and the diffusion time, number of particles in focal volume, and variation of concentration according to the dilution could be measured by the FCS system. Through this study, we determined that the concentrations of arbitrarily diluted sample solutions can be measured by a home-built FCS setup in the range of 0.1 nM ~ 10 nM and that the diffusion coefficient of the quantum dot was .

Basic Fluorescence Correlation Spectroscopy Setup and Measurement

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive tool in biochemistry and biophysics. It directly obtains physical parameters such as the average number of fluorescent molecules and their diffusion time in a tiny detection area. It also provides other useful information such as the brightness of molecules. Ultimately, it can give precise information about molecular interactions in the aqueous condition. This protocol outlines the basic FCS setup and how measurements are made.

Proteome-Wide Fluctuation Analysis Of S.cerevisiae

We measured over 40.000 single live yeast S. cerevisiae cells to determine the concentration and diffusion constants of more than 4100 proteins. These proteins account for more than 75% of the yeast proteome. We used Fluorescence Correlation Spectroscopy (FCS), Photon Counting Histograms (PCH), and Brightness & Number analysis (B&N) to analyze the intensity fluctuations of single molecules fused to GFP. The data was collected using a commercial FCS setup attached to a confocal microscope (ConfoCor3 and LSM 510 META, Carl Zeiss Jena GmbH, Germany) controlled by custom software.The cells were imaged in transmitted light. We acquired fluorescence images, using the avalanche photo diodes of the FCS setup. This allows us to determine the localization of proteins, the cell cycle as well as cell health.We developed a software package to automate the measurements and data analysis. We use the Open Microscopy Environment (OME) to organize our images, fluctuation measurements, and analysis results.We calculated the protein copy number per cell, and compare the noise in concentration levels between different proteins in respect to localization and biochemical pathway.We find that the diffusion coefficient for GFP is identical in nucleus and cytosol. But interestingly, most of the proteins localized in the nucleus diffuse slower than proteins localized in the cytoplasm.We will present our data and compare them to information gathered with different methods like flow-cytometry and mass-spectroscopy. We will discuss conclusions derived by complementing our data with information collected in public databases like Saccharomyces Geneome Database (www.yeastgenome.org), Yeast GFP Fusion Localization Database (yeastgfp.ucsf.edu), and the General Repository for Interaction Datasets (www.thebiogrid.org).

Improvement of Biomolecule Quantification Precision and Use of a Single-Element Aspheric Objective Lens in Fluorescence Correlation Spectroscopy

We found a way to increase the precision with which biomolecules present at concentrations below 10(-10) M can be quantified by fluorescence correlation spectroscopy (FCS). The effectiveness of the way was demonstrated experimentally by using a single-element aspheric objective lens, which was newly developed to reduce the cost of FCS instruments. In the first part of this paper, the relative standard deviation (RSD) of FCS-based concentration measurements is estimated theoretically by an analytical approximation assuming the detection volume profiles in FCS setups to be Gaussian and by molecular simulations in which more realistic profiles are calculated from physical parameters of the measurement setups. In a limit of infinitely bright molecules and zero background emission, the analytical approximation predicts that the RSD at a concentration is minimized when the mean number of molecules in a detection volume is approximately 0.5. A detection volume of the order of 10(-13) L thus gives smaller RSD values for concentrations from 10(-11) to 10(-10) M than does one of the order of 10(-15) L, which is widely used in FCS. This prediction is supported by the molecular simulations, taking into account the finite molecule brightness and background emission. In the second part of the paper, the RSD is evaluated experimentally with an FCS setup with a detection volume of 1.1 x 10(-13) L. The newly developed objective lens, serving as the bottom of the sample cell in this setup, has a large numerical aperture (0.9) without using immersion liquid. When a calibration line was made by 30-s FCS measurements of Cy3-labeled, 112-mer single-stranded DNA solutions, the RSD roughly agreed with the simulation result and was less than 0.1 for DNA concentrations from 2 x 10(-11) to 10(-10) M.

FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery

The intracellular degradation of single-stranded, double-labeled oligonucleotides (ONs) was studied by following the disappearance of Fluorescence Resonance Energy Transfer (FRET) between the rhodamine green and Cy5 fluorophores attached to respectively the 3′ and 5′ end of the ONs. The green and red fluorescence intensities upon rhodamine green excitation were monitored using the ultra-sensitive detectors of a dual-color Fluorescence Correlation Spectroscopy (FCS) instrument. The ratio of the red to green fluorescence (R/G ratio) as obtained from such FRET-FCS measurements showed to give accurate information on the integrity of the ONs, without the need for additional auto- or cross-correlation analysis of the registered fluorescence intensity fluctuations. Intracellular measurements revealed that most of the 40mer phosphodiester ONs were degraded before they entered the nucleus. For the 20mer phosphodiester ONs, this degradation occurred more slowly, and both intact and degraded ONs entered the nucleus. For the 20mer phosphorothioate ONs, no intracellular degradation was observed during the measured time period. The sensitive detection of the intracellular fluorescence by the FCS setup will be particularly useful in situations where the expected fluorescence is too low to be detected by FRET-imaging as may occur after intracellular delivery of ONs by cationic carriers.

Subwavelength patterns and high detection efficiency in fluorescence correlation spectroscopy using photonic structures

We present theoretical and experimental evidence of subwavelength resolution and high detection efficiency in fluorescence correlation spectroscopy (FCS) using photonic structures. We show that multilayered dielectric mirrors or metallic mirrors may be used in a conventional FCS setup to improve fluorescence detection and fluctuation analysis.