Investigation of micelle formation by fluorescence correlation spectroscopy

We show that noncovalently bound dye molecules can be used as labels in single-molecule fluorescence experiments for the determination of aggregate formation in standard surfactant systems. Aqueous solutions of sulfosuccinic acid bis(2-ethylhexyl) ester sodium salt, hexadecyltrimethylammonium chloride, and pentaethylene glycol monododecyl ether have been studied by fluorescence correlation spectroscopy using commercially available dyes. The translational diffusion coefficient and the critical micelle concentrations have been determined and compare well to values reported in the literature. The respective charges of the surfactant and of the dye molecule are crucial for the effectiveness of the presented method.     

Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS) we measure the translational diffusion coefficient of asphaltene molecules in toluene at extremely low concentrations (0.03-3.0 mg/L): where aggregation does not occur. We find that the translational diffusion coefficient of asphaltene molecules in toluene is about 0.35 x 10(-5) cm(2)/s at room temperature. This diffusion coefficient corresponds to a hydrodynamic radius of approximately 1 nm. These data confirm previously estimated size from rotational diffusion studied using fluorescence depolarization. The implication of this concurrence is that asphaltene molecular structures are monomeric, not polymeric.     

A fluorescence enhanced probe for sulfur dioxide detection and fluorescence imaging in living cellsEI北大核心CSCD

基于香豆素和苯并吡啶基团,构建了用于二氧化硫(SO_(2))高效检测的荧光探针P1,其化学结构通过核磁氢谱(^(1)H NMR)、碳谱(^(13)C NMR)和高分辨质谱(HR-MS)确证。在缓冲溶液体系中,单独的探针P1具有微弱的荧光,识别SO_(2)后荧光发射强度明显增强,能够实现对SO_(2)的专一性裸眼识别,检出限为126 nmol/L。生物应用实验结果表明,该探针具有较低的细胞毒性,可用于生物活细胞中外源性SO_(2)的荧光成像。     

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.     

Dye-exchange dynamics in micellar solutions studied by fluorescence correlation spectroscopy

We investigated the dye-exchange dynamics between rhodamine 123 (R123), a mitochondrial fluorescent dye, and micelles as membrane mimetic systems. In the presence of neutral micelles (Triton X-100 and Brij 35) R123 partitions between the aqueous solution and the micellar pseudo-phase, undergoing red shift of the absorption and the emission spectra. Fluorescence correlation spectroscopy (FCS) was used to study the dynamics of these systems over an extremely wide time range and at the single-molecule level, yielding information in one and the same experiment about the diffusional dynamics of free and bound rhodamine and about the dye-exchange dynamics as well as several photophysical properties of the rhodamine bound to the micelles. It was found that the entry rate constants are diffusion-controlled, indicating that there are no geometric or orientational requirements for the association of the dye with the micelle. With respect to the dye-exchange dynamics, micelles are found to behave as soft supramolecular cages in contrast to other rigid supramolecular cavities, such as cyclodextrins. The exit rate constants depend on the surfactant and determine the stability of the binding. Single-molecule multiparameter fluorescence detection (MFD) was used to examine the fluorescence properties of individual molecules in comparison to ensembles of molecules. The MFD histograms confirm the fast dye-exchange dynamics observed by FCS and yield mean values of fluorescence lifetimes and anisotropies in agreement with those obtained in bulk measurements.     

Chemoselective reduction-based fluorescence probe for detection of hydrogen sulfide in living cells

A selective and sensitive fluorescence probe for hydrogen sulfide (H(2)S) detection was synthesized and evaluated in PBS buffer and fetal bovine serum. The effect of pH on the probe was also studied. In addition, visualization of H(2)S in Hela cells was achieved using confocal laser scanning fluorescence microscopy.     

Polymers in focus: fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy has been increasingly used in polymer science. In the present perspective, the principles of the method are briefly reviewed, and the temporal and spatial resolutions are critically discussed. Examples of recent findings are summarized, focusing on polymer solutions, environmental parameters, combination with other techniques, near-interface measurements, simulations, and modeling. Finally, desirable new developments are discussed.     

Clean and modified substrates for direct detection of living cells by surface-enhanced Raman spectroscopy

Iodide adsorption and electrochemical negative potential desorption were proposed and compared to obtain clean SERS substrates. The two methods can effectively eliminate the interference of surface impurities in the SERS detection. SERS signals of membranes of living cells with a good reproducibility have been obtained.     

Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy

The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe-PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe-PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.     

Quantification of protein-protein interactions within membranes by fluorescence correlation spectroscopy

The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation or the binding between a growth factor and its receptor during signaling. Here, we review the last developments to quantify protein/protein interactions in lipid membranes and focus on the use of fluorescence-correlation-spectroscopy approaches for that purpose.     

Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy

The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core.     

Real-time detection of dental calculus by blue-LED-induced fluorescence spectroscopy

Successful periodontal therapy requires sensitive techniques to discriminate dental calculus from healthy teeth. The aim of the present study was to develop a fluorescence-based procedure to enable real-time detection and quantification of dental calculus. Thirty human teeth--15 teeth with sub- and supragingival calculus and 15 healthy teeth--covered with a layer of physiological saline solution or blood were illuminated by a focused blue LED light source of 405 nm. Autofluorescence spectra recorded along a randomly selected line stretching over the crown-neck-root area of each tooth were utilized to evaluate a so called calculus parameter R, which was selected to define a relationship between the integrated intensities specific for healthy teeth and for calculus in the 477-497 nm (S(A)) and 628-685 nm (S(B)) wavelength regions, respectively. Statistical analysis was performed and a cut-off threshold of R=0.2 was found to distinguish dental calculus from healthy teeth with 100% sensitivity and specificity under various experimental conditions. The results of the spectral evaluation were confirmed by clinical and histological findings. Automated real-time detection and diagnostics for clinical use were implemented by a corresponding software program written in Visual Basic language. The method enables cost-effective and reliable calculus detection, and can be further developed for imaging applications.     

Single-molecule monitoring in living cells by use of fluorescence microscopy

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.     

Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy

The dynamics of weakly bending polymers is analyzed on the basis of a Gaussian semiflexible chain model and the fluorescence correlation spectroscopy (FCS) correlation function is determined. Particular attention is paid to the influence of the rotational motion on the decay of the FCS correlation function. An analytical expression for the correlation function is derived, from which the averaged segmental mean square displacement can be determined independent of any specific model for the polymer dynamics. The theoretical analysis exhibits a strong dependence of the correlation function on the rotational motion for semiflexible polymers with typical lengths and persistence lengths of actin filaments or fd viruses. Hence, FCS allows for a measurement of the rotational motion of such semiflexible polymers. The theoretical results agree well with experimental measurements on actin filaments and confirm the importance of large relaxation times.     

Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy

A high-speed capillary electrophoresis mobility shift assay (CEMSA) for determining the binding ratios of DNA-protein complexes in solution is demonstrated. Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as they flowed continuously through a fused silica capillary under the influence of an applied electric field. Resolution of the bound and unbound complexes was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation. Data sufficient to perform the analysis was acquired in less than 10 s, compared to the minutes that are normally needed to carry out such measurement via CE separation. The binding ratios were determined with 5 to 10% precision and agreed with the results obtained by CE separation within experimental error. The resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, due to the higher mass sensitivity of the electrophoretic mobility compared to the translational diffusion coefficient. Fluorescently labeled 39-mer single stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system. The dissociation constant of the ssDNA-SSB complex was estimated to be approximately 2 nM based on the CEMSA-FCS analysis.     

Exonucleolytic degradation of high-density labeled DNA studied by fluorescence correlation spectroscopy

The exonucleolytic degradation of high-density labeled DNA by exonuclease III was monitored using two-color fluorescence correlation spectroscopy (FCS). One strand of the double stranded template DNA was labeled on either one or two base types and additionally at one end via a 5' Cy5 tagged primer. Exonucleolytic degradation was followed via the diffusion time, the brightness of the remaining DNA as well as the concentration of released labeled bases. We found a hydrolyzation rate of about 11 to 17 nucleotides per minute per enzyme (nt/min/enzyme) for high-density labeled DNA, which is by a factor of about 4 slower than for unlabeled DNA. The exonucleolytic degradation of a 488 base pair long double stranded DNA resulted in a short double stranded DNA segment of 112 ± 40 base pairs (bp) length with two single-stranded tails.     

Sizes of water-soluble luminescent quantum dots measured by fluorescence correlation spectroscopy

In this paper, fluorescence correlation spectroscopy (FCS) was applied to measure the size of water-soluble quantum dots (QDs). The measurements were performed on a home-built FCS system based on the Stokes-Einstein equation. The obtained results showed that for bare CdTe QDs the sizes from FCS were larger than the ones from transmission electron microscopy (TEM). The brightness of QDs was also evaluated using FCS technique. It was found that the stability of the surface chemistry of QDs would be significantly improved by capping it with hard-core shell. Our data demonstrated that FCS is a simple, fast, and effective method for characterizing the fluorescent quantum dots, and is especially suitable for determining the fluorescent nanoparticles less than 10 nm in water solution.     

Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.     

Extracting decay curves of the correlated fluorescence photons measured in fluorescence correlation spectroscopy

We have developed a new method to extract the decay curves of the correlated fluorescence photons from the data of fluorescence correlation spectroscopy using time-correlated single photon counting. In this method, a two-dimensional correlation map of photon pairs is generated at an absolute delay time with reference to the excitation-emission delay of each photon. Using a dye-labeled DNA as an example, we have demonstrated that the decay curve of the correlated fluorescence photons is separated from the uncorrelated background signals simply by subtracting a two-dimensional correlation map at sufficiently long delay time without additional prior information.     

Stochastic approach to data analysis in fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has emerged as a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In FCS, the experimental data is conventionally fit using standard local search techniques, for example, the Marquardt-Levenberg (ML) algorithm. A prerequisite for these categories of algorithms is the sound knowledge of the behavior of fit parameters and in most cases good initial guesses for accurate fitting, otherwise leading to fitting artifacts. For known fit models and with user experience about the behavior of fit parameters, these local search algorithms work extremely well. However, for heterogeneous systems or where automated data analysis is a prerequisite, there is a need to apply a procedure, which treats FCS data fitting as a black box and generates reliable fit parameters with accuracy for the chosen model in hand. We present a computational approach to analyze FCS data by means of a stochastic algorithm for global search called PGSL, an acronym for Probabilistic Global Search Lausanne. This algorithm does not require any initial guesses and does the fitting in terms of searching for solutions by global sampling. It is flexible as well as computationally faster at the same time for multiparameter evaluations. We present the performance study of PGSL for two-component with triplet fits. The statistical study and the goodness of fit criterion for PGSL are also presented. The robustness of PGSL on noisy experimental data for parameter estimation is also verified. We further extend the scope of PGSL by a hybrid analysis wherein the output of PGSL is fed as initial guesses to ML. Reliability studies show that PGSL and the hybrid combination of both perform better than ML for various thresholds of the mean-squared error (MSE).