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Noël Boens

Publications and source records attributed to Noël Boens.

17 recordsLinked to original sources
PubMed2006

Compartmental modeling of the fluorescence anisotropy decay of a cylindrically symmetric brownian rotor: Identifiability analysis.

We present the results of the deterministic identifiability analysis based on similarity transformation for models of one-state excited-state events of cylindrically symmetric rotors in isotropic environments undergoing rotational diffusion described by Brownian reorientation. Such an analysis on error-free time-resolved fluorescence (anisotropy) data can reveal whether the parameters of the considered model can be determined. The fluorescence delta-response functions I(parallel)(t) and I(perpendicular)(t), for fluorescence polarized respectively parallel and perpendicular to the electric vector of linearly polarized excitation, are used to construct, in convenient matrix form, expressions of the sum S(t) = I(parallel)(t) + 2I(perpendicular)(t), the difference D(t) = I(parallel)(t) - I(perpendicular)(t), and the time-resolved fluorescence anisotropy r(t) = D(t)/S(t). The identifiability analysis of r(t) demonstrates that the rotational diffusion coefficients D(parallel) and D(perpendicular) for rotation respectively about and perpendicular to the symmetry axis can be uniquely resolved. However, the polar and azimuthal angles defining the absorption and emission transition moments in the molecular reference frame are not individually identifiable. Nevertheless, the difference between the polar angles of these transition moments is uniquely determined.

Journal Article↗
PubMed2006

Photophysics of the fluorescent pH indicator BCECF.

The photophysical behavior of BCECF [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein]--currently the most widely used fluorescent pH indicator for near-neutral intracellular pH measurements--has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength as well as total buffer concentration on the absorbance and steady-state fluorescence has been investigated. The apparent acidity constant of the pH indicator determined by absorbance and fluorescence titration is dependent on the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton exchange of BCECF at physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the fluorescence decay times. Fluorescence decay traces collected as a function of total buffer concentration and pH were analyzed by global compartmental analysis yielding the following values of the rate constants describing excited-state dynamics of BCECF: k01 = 3.4 x 10(8) s(-1), k02 = 2.6 x 10(8) s(-1), k21 approximately 1 x 10(6) M(-1) s(-1), k12(B) = 1.4 x 10(8) M(-1) s(-1), and k21(B) = 4.3 x 10(7) M(-1) s(-1).

Journal Article↗
PubMed2006

Solvent and pH dependent fluorescent properties of a dimethylaminostyryl borondipyrromethene dye in solution.

Steady-state and time-resolved fluorescence techniques have been used to study the photophysical properties of the fluorescent BODIPY-derived dye 3-{2-[4-(dimethylamino)phenyl]ethenyl}-4,4-difluoro-8-(4-methoxyphenyl)-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene. This compound has been synthesized via a microwave-assisted condensation of p-N,N-dimethylaminobenzaldehyde with the appropriate 1,3,5,7-tetramethyl substituted borondipyrromethene unit. The fluorescence properties of the dye are strongly solvent dependent: increasing the solvent polarity leads to lower fluorescence quantum yields and lifetimes, and the wavelength of maximum fluorescence emission shifts to the red. The Catalán solvent scales are found to be the most suitable for describing the solvatochromic shifts of the fluorescence emission. These are dominated by polarity/polarizability effects, as confirmed by quantum-chemical calculations performed in the dielectric continuum approximation. Fluorescence decay profiles of the dye can be described by a single-exponential fit in most solvents investigated, while two decay times are found in alcohols. The dye undergoes a reversible protonation-deprotonation reaction in the acidic pH range with a pK(a) of 2.25 in acetonitrile solution. Fluorimetric titrations as a function of pH produce fluorescence emission enhancements at lower pH. The fluorescence excitation spectra show a hypsochromic shift from 600 nm for the neutral amine to 553 nm for the ammonium form, so that ratiometric measurements can be used to determine pK(a).

Journal Article↗
PubMed2005

Identifiability of models for intramolecular two-state excited-state processes with added quencher and coupled species-dependent rotational diffusion.

The parameters describing the kinetics of excited-state processes can possibly be recovered by analysis of the fluorescence decay surface measured as a function of the experimental variables. The identifiability analysis of a photophysical model assuming errorless time-resolved fluorescence data can verify whether the model parameters can be determined and may lead to the minimal experimental conditions under which this is possible. In this work, we used the method of similarity transformation to investigate the identifiability of three kinetic models utilized to describe the time-resolved fluorescence of reversible intramolecular two-state excited-state processes in isotropic environments: (1) model without added quencher, (2) model with added quencher, (3) model with added quencher coupled with species-dependent rotational diffusion described by Brownian reorientation. Without a priori information, model 1 is not identifiable. For model 2, two sets of quenching rate constants and combinations of excited-state deactivation/exchange rate constants are possible, but they cannot be allocated to a specific excited-state species. For both sets, upper and lower limits on the excited-state deactivation/exchange rate constants can be obtained. For model 3, both spherically and cylindrically symmetric rotors, with no change in the principal axes of rotation in the latter, are considered. The fluorescence delta-response functions I(parallel)(t) and I(perpendicular)(t), for fluorescence polarized parallel and perpendicular, respectively, to the electric vector of linearly polarized excitation, are used to define the sum S(t) identically equal to I(parallel)(t) + 2 I(perpendicular)(t) and the difference D(t) identically equal to I(parallel)(t) - I(perpendicular)(t). The identifiability analysis is performed using the S(t) and D(t) functions. Also for model 3, two sets of kinetic parameters (i.e., quenching rate constants, combinations of deactivation/exchange rate constants, and rotational diffusion coefficients) exist, but these parameters cannot be assigned unequivocally to a specific species. For the three models, an infinite number of alternative spectroscopic parameters associated with excitation and emission are found.

Diffusion↗
PubMed2005

Identifiability of models for fluorescence quenching in aqueous micellar systems.

The first deterministic identifiability analysis is presented for four commonly used kinetic models of fluorescence quenching of an excited probe in aqueous micelles: A) model with immobile quenchers, B) model with mobile quenchers, C) an extension of model B in which exchange of quenchers both via the aqueous phase and during micelle collisions is taken into account, and D) model with probe migration. It is shown that these specific models for fluorescence decay of an excited probe solubilized in a micelle and quenched by molecules or ions that are Poisson-distributed over the micelles, resulting in the generalized four-parameter equation f(t)=A(1) exp{-A(2)t-A(3)[1-A(4)t]}, are uniquely identifiable in terms of four descriptive A parameters. Moreover, each model also can be uniquely identified in terms of the underlying rate constants and micellar concentration or mean micellar aggregation number. This means that these parameters can be extracted in a unique way from time-resolved fluorescence quenching experiments on a probe in micelles. For each model the recommended analysis approach is given.

Journal Article↗
PubMed2005

Photophysical properties of BODIPY-derived hydroxyaryl fluorescent pH probes in solution.

The photophysical properties of five fluorescent pH probes derived from 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene with phenolic or naphtholic subunits at position 8 and with substituents having different electron driving forces at positions 3 and 5 have been investigated in several organic solvents, by means of absorption, steady-state, and time-resolved fluorimetry. For each compound, the fluorescence quantum yield and lifetime are lower in solvents with higher polarity, owing to an increase in the rate of nonradiative deactivation. The rate constants for radiative deactivation, k(f), are nearly constant for all dyes in all solvents studied [k(f)=(1.7+/-0.2)x10(8) s(-1)]. In aqueous solution, these probes undergo a reversible protonation-deprotonation in the near-neutral to basic pH range, producing intensity increases with lower pH. The pK(a) values of the indicators are between 7.5 and 9.3, depending on the substitution pattern on positions 3, 5, and 8. The difference between the absorption and excitation spectra as a function of pH is indicative of the presence of two species in aqueous solution: the phenol- or naphthol-based indicator and its conjugate base.

Absorption↗
PubMed2005

A highly potassium-selective ratiometric fluorescent indicator based on BODIPY azacrown ether excitable with visible light.

[structure: see text] A potassium-selective fluorescent BODIPY-linked azacrown ether chemosensor has been synthesized using novel substitution reactions of 3,5-dichloroBODIPY. The indicator absorbs and emits light in the visible wavelength range. The dissociation constant Kd for the complex between K+ and the probe is 0.5 mM in acetonitrile. Quantum chemical calculations show that the experimental hypsochromic shifts in absorption and fluorescence upon potassium binding are due to complexation, which induces a large conformational change of the sensor.

Boron Compounds↗
PubMed2005

Photophysical properties of borondipyrromethene analogues in solution.

The photophysical properties of seven new 8-(p-substituted)phenyl analogues of 4,4-difluoro-3,5-dimethyl-8-(aryl)-4-bora-3a,4a-diaza-s-indacene (derivatives of the well-known fluorophore BODIPY) in several solvents have been studied by means of absorption and steady-state and time-resolved fluorimetry. For each compound, the fluorescence quantum yield and lifetime are lower in solvents with higher polarity owing to an increase in the rate of nonradiative deactivation. Increasing the electron withdrawing strength of the p-substituent on the phenyl group in position 8 also leads to lower fluorescence quantum yields and lifetimes. When the p-substituent on the phenyl group in position 8 is a tertiary amine [8-(4-piperidinophenyl), 8-(4-N,N-dimethylaminophenyl), and 8-(4-morpholinophenyl)], the low quantum yields of these compounds in more polar solvents can be rationalized by the inversion of the energy levels of an apolar, highly fluorescent and a polar, nonfluorescent excited state, where charge transfer from the tertiary amine to the BODIPY unit occurs. These amine analogues can be protonated at low pH in aqueous solution. Fluorescence titrations yielded pK(a) values of their conjugate ammonium salts which are in agreement with the electron donating tendency of the amine group: piperidino (4.15) > dimethylamino (2.37) > morpholino (1.47), with the pK(a) values in parentheses. The rate constant of radiative deactivation (k(f)) is the same for all compounds in all solvents studied (k(f) = 1.4 x 10(8) s(-1)).

Journal Article↗
PubMed2005

Compartmental modeling of reversible intermolecular two-state excited-state processes coupled with rotational diffusion or with added quencher.

Analysis of related time-resolved fluorescence measurements can possibly lead to the determination of the kinetic parameters of excited-state processes. A deterministic identifiability analysis on an error-free fluorescence decay data surface has to be executed to verify whether the parameters of a particular model can be determined and may point to the minimal experimental conditions under which this will become possible. In this work, similarity transformation is chosen as an identifiability analysis approach because it also gives the explicit relationships between the true and alternative model parameters. Results are presented for two kinetic models of a reversible intermolecular two-state excited-state process in isotropic environments: (a) with coupled species-dependent rotational diffusion described by Brownian reorientation and (b) with added quencher. For model a, both spherically and cylindrically symmetric rotors, with no change in the principal axes of rotation in the latter, are considered. The fluorescence delta-response functions I(parallel)(t) and I(perpendicular)(t), for fluorescence polarized respectively parallel and perpendicular to the electric vector of linearly polarized excitation, are used to define the sum S(t) = I( parallel)(t) + 2 I( perpendicular)(t) and the difference D(t) = I(parallel)(t) - I(perpendicular)(t) function. The identifiability analysis is carried out using the S(t) and D(t) functions. The analysis involving S(t) shows that two physically acceptable possible solutions for the overall rate constants of the excited-state process exist. Inclusion of information from polarized fluorescence measurements on the rotational kinetic behavior contained in D(t) results in the unique set of rate constants and rotational diffusion coefficients when the rotational diffusion coefficients are different. For model b, it is shown that addition of quencher plays formally the same role as rotational diffusion as far as the identification is concerned. When the quenching rate constants are different, the rate constants of a reversible intermolecular two-state excited-state process with added quencher can be uniquely determined.

Diffusion↗
PubMed2005

Synthesis and spectroscopic characterisation of BODIPY based fluorescent off-on indicators with low affinity for calcium.

Two fluorescent off-on Ca2+ indicators based on APTRA (o-aminophenol-N,N,O-triacetic acid) as low-affinity ligand for Ca2+ and BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) as a fluorophore were synthesized. The new BODIPY-APTRA compounds absorb in the visible spectrum, with absorption maxima from 505 nm to 570 nm, and have fluorescence spectra that span the visible spectrum, with emission maxima ranging from 525 nm to 625 nm dependent on the substituents at the alpha-positions to the nitrogen atoms. The indicators show a large increase of the fluorescence quantum yield upon increasing Ca2+ concentration. The ground-state dissociation constants Kd estimated at 20 degrees C in 100 mM KCl aqueous buffered solution, pH 7.20, for the two complexes with Ca2+ were found to be around 100 microM.

Journal Article↗
PubMed2005

Experimental design for the estimation of photophysical parameters of the two-state excited-state proton-exchange reaction in the presence of pH buffer.

The excited-state proton-exchange reaction of commonly used fluorescent pH probes at physiological pH becomes reversible upon addition of pH buffer. Using computer-generated fluorescence decay surfaces, we investigated under which experimental conditions (pH, buffer concentration, and excitation and emission wavelengths) the rate constants describing the excited-state processes (k(ij)) and the spectral parameters related to excitation ((~)b(1)) and emission ((~)c(1)) can be accurately and precisely estimated by global compartmental curve fitting. It was found that a minimum of three fluorescence decay traces should be collected for the pH probe in the presence of buffer. These three decays should be characterized by at least two different pH values and at least two different buffer concentrations. In addition to these three traces, a minimum of one trace corresponding to the pH probe without buffer has to be recorded. Furthermore, for the accurate estimation of k(ij), (~)b(1), and (~)c(1), at least two of these traces should be collected at the same pH and excitation and emission wavelengths. The experimental conditions should be chosen in such a way that decays with unambiguous biexponential character are obtained. For fluorescent pH probes with pK(a) approximately equal to 7 that are responsive in the near-neutral pH range, it is advisable to use buffers with pK(B)(a) values comparable to or higher than the pK(a) of the probe. Because the changes in the decay times are already apparent with a small quantity of buffer, there is no need to use excessively high buffer concentrations. From a practical point of view, the best experimental design is attained when one combines in a single fluorescence decay surface traces originating from samples characterized by different pH values at the same buffer concentration with traces characterized by different buffer concentrations at the same pH and decays of samples without buffer measured at several pH values.

Buffers↗
PubMed2005

BODIPY-based hydroxyaryl derivatives as fluorescent pH probes.

[structures: see text] Seven new 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes with phenolic or naphtholic subunits on position 8 and with substituents having different electron driving forces on positions 3 and 5 were synthesized. Their absorption and steady-state fluorescence properties were investigated as a function of solvent. The novel compounds, with the exception of 4,4-difluoro-8-(4-hydroxyphenyl)-3,5-bis-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene, are characterized by absorption maxima in the range 493-515 nm and small (400-600 cm(-1)) Stokes shifts. The exceptional dye has absorption maxima in the 570-580 nm region and fluorescence emission maxima around 610-620 nm, depending on the solvent. In aqueous solution, the dyes show a large fluorescent enhancement upon increasing the acidity of the solution. They can be used in aqueous solution as fluorescent pH probes excitable with visible light, with pKa values ranging from 7.5 to 9.3, depending on the substitution pattern on positions 3, 5, and 8.

Boron Compounds↗
PubMed2005

Determination of photophysical parameters from photon arrival time trajectories in single molecule fluorescence spectroscopy.

Triplet blinking is a phenomenon observed commonly in single molecule fluorescence spectroscopy. At high to moderate excitation intensities one can distinguish bright (on) and dark (off) periods in the fluorescence intensity trajectory caused by sojourns into the nonemissive triplet state. In this work, we focus on triplet blinking of an immobilized molecule in the low excitation regime, where a threshold between on and off intensity levels cannot be set, and, therefore, a standard on/off analysis of fluorescence intensity trajectories is not possible. In the low excitation regime triplet blinking parameters can be recovered from photon arrival time trajectories, i.e., records of individual photon arrival time. We use computer-generated data to compare the recovery of the triplet blinking parameters from the intensity correlation function (ICF) and the histogram of interarrival time. We have found that the ICF offers a better statistics for the recovery of the triplet blinking parameters.

Journal Article↗
PubMed2005

Absorption and emission study of 2',7'-difluorofluorescein and its excited-state buffer-mediated proton exchange reactions.

2',7'-Difluorofluorescein (Oregon Green 488) is a new fluorescein-based dye, which has found many applications, above all in biochemistry and neurosciences, and its use has become very popular in the last years. In recent years, we have been investigating the excited-state proton exchange reactions of fluorescein and the effect of suitable proton acceptors and donors which promote these reactions. The excited-state proton transfer reactions may appreciably influence the fluorescence results when using these dyes. We present steady-state emission evidence that acetate buffer species promote an excited-state proton transfer between neutral, monoanionic, and dianionic forms of 2',7'-difluorofluorescein. The time course of the excited species in this reaction was characterized through time-resolved fluorescence measurements, and the kinetics of the reaction was solved by using the global compartmental analysis. A previous identifiability study on the compartmental system set the conditions to design the fluorescence decay surface. This is the first experimental system, studied within this kinetic model, solved under identifiability conditions through global compartmental analysis. The recovered rate constant values for deactivation were 2.94 x 10(8) s(-1) for the monoanion and 2.47 x 10(8) s(-1) for the dianion, whereas the rate constant values of the buffer-mediated excited-state reaction were 9.70 x 10(8) and 1.79 x 10(8) M(-1) s(-1) for the deprotonation and protonation, respectively. With these values, a pK(a) = 4.02 was obtained. In this work, we additionally provide an absorption study, including acid-base equilibria, determination of ground-state pK(a) values (1.02, 3.61, and 4.69), and recovery of molar absorption coefficients of every prototropic species, including absorption and NMR evidence for the existence of three tautomers in neutral species. Steady-state emission spectra of 2',7'-difluorofluorescein in aqueous solution are also described, where the strong photoacid behavior of the cation is noteworthy.

Absorption↗
PubMed2005

Pitfalls and their remedies in time-resolved fluorescence spectroscopy and microscopy.

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.

Journal Article↗
PubMed2004

Identifiability analysis of models for reversible intermolecular two-state excited-state processes coupled with species-dependent rotational diffusion monitored by time-resolved fluorescence depolarization.

A deterministic identifiability analysis of the kinetic model for a reversible intermolecular two-state excited-state process with species-dependent rotational diffusion described by Brownian reorientation is presented. The cases of both spherically and cylindrically symmetric rotors, with no change in the principal axes of rotation on interconversion in the latter case, are specifically considered. The identifiability analysis is carried out in terms of compartmental modeling based on the S(t) identical with I( parallel)(t)+2I( perpendicular)(t) and D(t) identical with I( parallel)(t)-I( perpendicular)(t) functions, where I( parallel)(t) and I( perpendicular)(t) are the delta-response functions for fluorescence, polarized, respectively, parallel and perpendicular to the electric vector of linearly polarized excitation. It is shown that, from polarized time-resolved fluorescence data collected at two concentrations of coreactant and three appropriately chosen emission wavelengths, (a) a unique set of rate constants for the overall excited-state process is always obtained by making use of polarized measurements and (b) the rotational diffusion constants and geometrical factors associated with the different anisotropy decay components can be uniquely determined and assigned to each species. The geometrical factors are determined by the absorption and emission transitions in the two rotating species. For spherical rotors, these factors depend directly on the relative orientations of the transition moments, while for cylindrically symmetric rotors they depend on the orientations with respect to each other and to the symmetry axis.

Algorithms↗