Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements.

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).     

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.     

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

In this study, we demonstrate how the diffusion of probe particles in aqueous poly(vinyl alcohol) (PVA) solutions and gels is affected by: (i) the presence of cross-links, (ii) the cross-link density, (iii) the polymer concentration. We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion time of a rhodamine-based fluorescent particle (TAMRA) and TAMRA-labeled dextran in PVA solutions and gels prepared at various polymer concentrations (1% to 8.6% w/v) and cross-link densities (1/400 to 1/50 cross-link monomers per PVA monomers). The measurements indicate that the probe particles are slowed down with increasing polymer concentration and with increasing cross-link density. Also, FCS can detect differences in the diffusion times measured in “fresh” and “aged” PVA solutions. We find that FCS provides a quantitative measure of network inhomogeneities.     

Studies on bioconjugation of quantum dots using capillary electrophoresis and fluorescence correlation spectroscopy

In this paper, we systematically investigated the conjugation of quantum dots (QDs) with certain biomolecules using capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) methods. Commercial QDs and aqueous-synthesized QDs in our lab were used as labeling probes, certain bio-macromolecules, such as proteins, antibodies, and enzymes, were used as mode samples, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfo-succinimide (Sulfo-NHS) were used as linking reagents. We studied the effects of certain factors such as the isoelectric points (pIs) of bio-macromolecules and buffer pH on the bioconjugation of QDs, and found that the pIs of bio-macromolecules played an important role in the conjugation reaction. By the optimization of the buffer pH some proteins with different pIs were efficiently conjugated with QDs using EDC and Sulfo-NHS as linking agents. Furthermore, we on-line investigated the kinetic process of QDs-bioconjugation by FCS and found that the conjugation reaction of QDs with protein was rapid and the reaction process almost completed within 10 min. We also observed that QDs conjugated with proteins were stable for at least 5 days in phosphate buffer. Our work described here will be very helpful for the improvement of the QDs conjugation efficiency in bioapplications.     

Emission wavelength prediction of a full-color-tunable fluorescent core skeleton, 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one

In this paper we report on a novel fluorescent core skeleton, 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one, which we named Seoul-Fluor, having tunable and predictable photophysical properties. Using a concise and practical one-pot synthetic procedure, a 68-member library of new fluorescent compounds was synthesized with diverse substituents. In Seoul-Fluor, the electronic characteristics of the substituents, as well as their positional changes, have a close correlation with their photophysical properties. The systematic perturbation of electronic densities on the specific positions of Seoul-Fluor, guided with the Hammett constant, allows emission wavelength tunability covering the full color range. On the basis of these observations and a computational analysis, we extracted a simple first-order correlation of photophysical properties with the theoretical calculation and accurately predicted the emission wavelength of Seoul-Fluors through the rational design. In this study, we clearly demonstrate that Seoul-Fluor can provide a powerful gateway for the generation of desired fluorescent probes without the need for a tiresome synthesis and trial-and-error process.     

New concepts for fluorescence correlation spectroscopy on membranes

Fluorescence correlation spectroscopy (FCS) is a powerful tool to measure useful physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in model and cell membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we assess the potential and limitations of standard confocal FCS on lipid membranes and present recent developments which facilitate accurate and quantitative measurements on such systems. In particular, we discuss calibration-free diffusion and concentration measurements using z-scan FCS and two focus FCS and present several approaches using scanning FCS to accurately measure slow dynamics. We also show how surface confined FCS enables the study of membrane dynamics even in presence of a strong cytosolic background and how FCS with a variable detection area can reveal submicroscopic heterogeneities in cell membranes.     

Unexpected Acid-Triggered Formation of Reversibly Photoswitchable Stenhouse Salts from Donor-Acceptor Stenhouse Adducts

Donor-acceptor Stenhouse adducts (DASAs) are reversibly photoswitchable dyes, which are able to interconvert between a red/NIR absorbing triene-like state and a colorless cyclic state. Although optically attractive for multiple applications, their low solubility and lack of photoswitching in water impede their use in aqueous environments. We developed water-soluble DASAs based on indoline as donor and methyl, or trifluoromethyl, pyrazolone-based acceptors. In acetonitrile, photophysical analysis and photochemical studies, accounted with a three-state kinetic model, confirmed the reversible photoswitching mechanism previously proposed. In water, the colorless cyclic state is a thermodynamic sink at neutral pH values. In contrast, in acidic conditions, we observed a fast scrambling of DASAs’ end-group resulting in the in situ formation of Stenhouse salts (StS), which are in turn capable of reversible photoswitching. We believe that this unexpected result is of interest not only for the future design of DASAs with improved stability, but also for further development and applications of StS as photoswitchable probes.     

Role of the tracer in characterizing the aggregation behavior of aqueous block copolymer solutions using fluorescence correlation spectroscopy

The hydrodynamic radii of micelles formed by amphiphilic poly(2-alkyl-2-oxazoline) diblock copolymers in aqueous solution determined using fluorescence correlation spectroscopy (FCS) depend on the nature of the fluorescent tracer used. We have compared the values of the hydrodynamic radii of the unimers and the micelles as well as the critical micelle concentrations (CMC), using as tracers (1) the identical diblock copolymers being fluorescence-labeled at the hydrophilic or the hydrophobic block terminus [Bonné et al. Colloid Polym Sci (2004) 282:833–843], and (2) a low molar mass fluorescence dye, rhodamine 6G. Whereas similar values for the CMC were found for both probes, the hydrodynamic radius of micelles is significantly underestimated using a free dye as a tracer in FCS, especially near the CMC. We attribute this discrepancy to the fast exchange of the dye between micelles and solution.     

Two rhodamine lactam modulated lysosome-targetable fluorescence probes for sensitively and selectively monitoring subcellular organelle pH change

Be a powerful technique for convenient detection of pH change in living cells, especially at subcellular level, fluorescent probes has attracted more and more attention. In this work, we designed and synthesized three rhodamine lactam modulated fluorescent probes RS1, RS2 and RS3, which all respond sensitively toward weak acidity (pH range 4–6) via the photophysical property in buffer solution without interference from the other metal ions, and they also show ideal pKa values and excellent reversibility. Particularly, by changing the lone pair electrons distribution of lactam-N atom with different conjugations, RS2 and RS3 exhibit high quantum yield, negligible cytotoxicity and excellent permeability. They are suitable to stain selectively lysosomes of tumor cells and monitor its pH changes sensitively via optical molecular imaging. The above findings suggest that the probes we designed could act as ideal and easy method for investigating the pivotal role of H⁺ in lysosomes and are potential pH detectors in disease diagnosis through direct intracellular imaging.     

Host�Cguest association studied by fluorescence correlation spectroscopy

Fluorescence Correlation Spectroscopy (FCS) is a powerful single molecule technique for the study of the stability and the association dynamics of supramolecular systems and, in particular, of host?Cguest inclusion complexes. With FCS the host?Cguest binding equilibrium constant is determined analysing the variation in the diffusion coefficient of the fluorescent guest or host with no need for a change in the photophysical properties of the fluorescent probe. FCS gives also access to the association/dissociation rate constants of the host?Cguest inclusion providing that the fluorescence intensity of host or guest changes upon complexation. These rate constants can be compared with that of a diffusion-controlled process estimated from the same FCS experiment allowing for a better understanding of the association dynamics. The results show that cyclodextrin cavities act as ??hard?? cages which put geometric and orientational restrictions on the inclusion of a hydrophobic guest, whereas micelles behave as ??soft?? cages without geometrical requirements. In our contribution to this special issue we review briefly the application of FCS to the study of host?Cguest inclusion complexes with an emphasis on practical aspects and relevant bibliographic references.     

Green fluorescent protein based pH indicators for in vivo use: a review

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.     

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.     

Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M⁻¹, with dissociation constants faster or equal to 10³ s⁻¹, which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 10⁴ M⁻¹. In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of ²⁴¹Am³⁺, where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper.     

Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration.

Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function.     

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.     

Systematic Exploration of Furoindolizine-Based Molecular Frameworks towards a Versatile Fluorescent Platform

The photophysical behaviors of fluorescent molecules largely determine their major utility in biological studies. Despite their well-defined characteristics, classical fluorophores have often been challenged by their limited synthetic methodology and tunability in adjusting intrinsic optical properties. A novel heterocyclic core equipped with modular functional groups could offer the flexibility to control its photophysical properties with a minimum synthetic effort. By conducting a systematic analysis guided by quantum calculations, we proposed the furoindolizine-based molecular framework as a unique fluorescent platform capable of providing versatile photophysical properties with minimal structural modification. A broad tunability of furoindolizine derivatives′ photophysical properties such as emission wavelength, Stokes shift, fluorescent brightness, and charge transfer characteristics was achieved through synergistic interaction between two functional moieties. Furthermore, this modular platform led to live-cell imaging probes with two distinct optical features simply by reorganizing a pair of functional moieties.     

Dual-focus fluorescence correlation spectroscopy of colloidal solutions: influence of particle size

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring diffusion coefficients of small fluorescent molecules at pico- to nanomolar concentrations. Recently, a modified version of FCS, dual-focus FCS (2fFCS), was introduced that significantly improves the reliability and accuracy of FCS measurements and allows for obtaining absolute values of diffusion coefficients without the need of referencing again a known standard. It was shown that 2fFCS gives excellent results for measuring the diffusion of small molecules. However, when measuring colloids or macromolecules, the size of these objects can no longer be neglected with respect to the excitation laser focus. Here, we analyze how 2fFCS data evaluation has to be modified for correctly taking into a count these finite size effects. We exemplify the new method of measuring the absolute size of polymeric particles with simple and complex fluorophore distributions.     

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate-dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom-like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.     

Fluorescence correlation spectroscopy studies of diffusion of a weak polyelectrolyte in aqueous solutions

We apply fluorescent correlation spectroscopy (FCS) to investigate solution dynamics of a synthetic polyelectrolyte, i.e., a weak polycarboxylic acid in aqueous solutions. The technique brings single molecule sensitivity and molecular specificity to dynamic measurements of polyelectrolyte solutions. Translational diffusion of Alexa-labeled poly(methacrylic acid), PMAA*, chains was studied in very dilute, 10(-4) mg/ml, solutions as a function of solution pH and ionic strength. The observed changes in diffusion coefficients were consistent with about twofold expansion of PMAA* coils when pH was changed from 5 to 8, and with chain contraction for alkaline metal ion concentrations from 0.01 to 0.1 M. The dependence of the hydrodynamic size of PMAA* chains on the counterion type followed the sequence: Li(+)>Na(+) approximately equal to Cs(+)>K(+). The dependence of translational diffusion on polyacid concentration was weak at the low concentration limit, but chain motions were significantly slower at higher polymer concentrations when PMAA chains overlapped. Finally, measurements of dynamics of PMAA* chains in "salt-free" solutions showed that self-diffusion of PMAA* chains significantly slowed down when PMAA concentration was increased, probably reflecting the sensitivity of PMAA* translational motions to the onset of interchain domain formation. These results illustrate the utility of the FCS technique for studying hydrodynamic sizes of polyelectrolyte coils in response to variation in solution pH or concentration of salt and polyelectrolytes. They also suggest that FCS will be a promising technique for selective observation of the dynamics of polyelectrolyte components in complex polymer mixtures.     

Sol-gel-derived micron scale optical fibers for chemical sensing

We report for the first time on the preparation of organically-doped room temperature processed sol-gel-derived micron scale optical fibers as platforms for chemical- and bio-sensors. Micron scale optical fibers are drawn from fluorescent dye-doped tetraethoxysilane (TEOS)-derived sol-gel solution processed under ambient conditions. Such a simple methodology to entrap organic and even bioactive species within the optical fiber offers many advantages over more conventional ways of immobilizing organic probes for the development of optical sensors. Specifically, we report on the photophysical properties of fluorescein (a pH sensitive fluorescent dye) and rhodamine 6G (R6G; laser dye) entrapped within sol-gel-derived optical fibers. We present the preliminary results on the viability of such doped optical fibers for chemical sensing. Our results demonstrate that a fluorescein-doped sol-gel-derived optical fiber responds to ammonia and acid vapors with a response time of 1–2 seconds.