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.     

Predissociation dynamics of Ã-state ammonia probed by two-photon excitation spectroscopy

Electronically excited ND₃(A¯) molecules have been prepared by laser two-photon excitation on theA¯¹A″₂—X¯¹′₁ transition and monitored via their resulting short-lived emission. The earlier observation of Douglas that ND₃(A¯) molecules carrying one quantum of out-of-plane bending vibration ν′₂ are least susceptible to predissociation, is confirmed. ND₃(A¯) predissociation rates are found to be both vibronic and rovibronic level dependent. Both observations may be understood by considering the likely form of the potential energy surface for ND₃(A¯) molecules in the region of the D₂N—D dissociation coordinate. At short D₂N—D separations this surface exhibits a barrier. The presence of a conical intersection (involving the ND₃ ground state surface) further out along the dissociation coordinate has a crucial influence on the magnitude of this barrier. The envisaged form of theA¯-state potential energy surface also provides a qualitative rationale for all previous experimental findings concerning electronic branching ratios and energy disposal amongst the primary photofragments arising in the photodissociation ofA¯-state ammonia.     

Selective fluorescence recovery after bleaching of single E2GFP proteins induced by two-photon excitation.

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.     

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.     

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.     

Two-photon excitation fluorescence correlation spectroscopy of diffusion for Gaussian-Lorentzian volumes

Fluorescence correlation spectroscopy (FCS) is valuable in many scientific domains where diffusion plays a fundamental role. One important experimental realization is based on fluorescence induced by two-photon excitation (TPE). In comparison with one-photon excitation (OPE), TPE-FCS defines better the interrogation volume and the background noise is sensibly reduced. Within this context and for overfilled objective lenses, the three-dimensional Gaussian (3DG) approximation, according to which the spectroscopic interaction is spatially defined by Gaussian profiles only, guarantees a simple analytical data interpretation. By contrast, the volume illuminated by the laser beam focused with partially filled objective lenses follows a Gaussian-Lorentzian (GL) distribution that is taken into account by means of numerical methods only. Here we show that contrary to common belief, the assumption of a GL volume does not hamper analytical treatment of TPE-FCS. Differences and similarities in comparison with the 3DG approximation are discussed.     

The two-photon fluorescence excitation spectrum of dibenzothiophene

The two-photon fluorescence excitation spectra of dibenzothiophene crystal (≈77 K) and solution have been investigated in the spectral region 30000–38000 cm^?1 (≈3350-2600 Å). Two electronic transitions have been found. Both the oriented gas model results applied to the crystal and the circular/linear polarization ratio to the solution claim for an ¹A₁ ← ¹A₁ assignment of the transitions. Not completely resolved vibronic structures in the crystal spectrum were observed for both transitions for which a tentative assignment to A₁ total symmetry is discussed. It has been found that the intensity of the first electronic band system arises not only from a purely electronic (i.e., Franck-Condon) mechanism, but also from its interaction with a higher state through totally symmetric vibrations.     

Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation.

A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements or diffusion coefficients with fluorescence correlation spectroscopy rather difficult. A strong difference of this behavior for continuous-wave and pulsed excitation is found.     

Polarized two-photon fluorescence excitation spectra of indole and benzimidazole

Polarized two-photon fluorescence excitation spectra of indole in hexane, benzimidazole in isopropanol, and benzimidazole cation in methanol-H₂SO₄, all at 0.2 M and 25°C are reported for the excitation range 470–600 nm, the region of their L_b, and L_a bands. Relative two-photon absorptivities are deduced by correcting for different fluorescence response and are compared to toluene's L_b band. The indole integrated absorptivity is about 10 times greater than that of toluene. The L_a band of indole appears less dominant than in one-photon but still outweighs the L_b band by a factor of 4. The two-photon polarization spectrum for indole indicates that the L_a origin lies ≈500–1000 cm⁻¹ above the L_b origin in hexane. The benzimidazoles absorb only about twice as strongly as toluene and show strong vibronic peaks; the L_a, bands are only faintly seen. Two-photon properties calculated from INDO/S CI wavefunctions with doubly excited configurations are in good agreement with those of indole, but predict the benzimidazole TPA to be several times stronger than observed. For the cation, the predicted results are nearly two orders of magnitude too high.     

Multiphoton ionization and two-photon excitation spectroscopy of nitric oxide

The multiphoton ionization and two-photon excited fluorescence action spectra of nitric oxide are compared. At low fluence the two spectra differ when more than three photons are required for ionization. This may be due to the presence of several energy acquisition routes, providing new spectroscopic information on intermediate slates.     

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.     

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.     

Level-crossing experiments by two-photon excitation

Complete orientation of the T17² P _3/2 (F=2) state by two-photon excitation allows Hanle-effect measurement in direct fluorescence as well as in the optical radiation cascade. The experiment provides estimations either of both the lifetimes of the involved excited states, or, if one of the lifetimes is reliably known, two independant determinations of the other. The measurements are reported on the background of other nonlinear Hanle-effect and level-crossing experiments.     

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.     

Excited-state symmetry and reorientation dynamics of perylenes in liquid solutions: time-resolved fluorescence depolarization studies using one- and two-photon excitation

The excited-state symmetry and molecular reorientation of perylene, 1,7-diazaperylene, and 2,5,8,11-tetra- tert-butylperylene have been studied by different fluorescence depolarization experiments. The first excited electronic singlet state was reached through one-photon excitation (OPE) and two-photon excitation (TPE). A 400 and 800 nm femtosecond laser pulse was used for this purpose, and data were collected by means of the time-correlated single-photon counting technique. It is found that the rotational correlation times for each perylene derivative are very similar in the OPE and TPE depolarization experiments. For the determination of the two-photon absorption tensor, a recently described theoretical model has been applied (Ryderfors et al. J. Phys. Chem. A 2007, 111, 11531). It was found that the two-photon process can be described by a 2 x 2 absorption tensor for which the components are solvent dependent and exhibit mixed vibronic character. In the dipole approximation this is compatible with a parity-forbidden two-photon absorption into the first excited singlet state.     

Study of binding and denaturation dynamics of IgG and anti-IgG using dual color fluorescence correlation spectroscopy

In this article, we present a systematic study on IgG and Fab fragment of anti-IgG molecules using fluorescence auto- and cross-correlation spectroscopy to investigate their diffusion characteristics, binding kinetics, and the effect of small organic molecule, urea on their binding. Through our analysis, we found that the diffusion coefficient for IgG and Fab fragment of anti-IgG molecules were 37 ± 2 μm² s⁻¹ and 56 ± 2 μm² s⁻¹, respectively. From the binding kinetics study, the respective forward (k_a) and backward (k_d) reaction rates were (5.25 ± 0.25) × 10⁶ M⁻¹ s⁻¹ and 0.08 ± 0.005 s⁻¹, respectively and the corresponding dissociation binding constant (K_D) was 15 ± 2 nM. We also found that urea inhibits the binding of these molecules at 4 M concentration due to denaturation.     

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.     

A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.