N. Boens

Possibilities and limitations of the time-correlated single photon counting technique: a comparative study of correction methods for the wavelength dependence of the instrument response function

Abstract One of the principal reasons for poor fits in time-correlated single photon counting fluorescence decay measurements is the wavelength dependence of the instrument response function. Over the years several correction methods have been developed to account for or correct for this effect. The most recent and widely used procedures are critically compared in this paper. A comparison of the channel shift technique, the pseudoscatterer technique, the excitation pulse-shape mimic technique and the delta function convolution method demonstrates that the latter method is the superior one. This method requires the measurement of the fluorescence decay of a reference compound with single exponential decay kinetics under identical conditions as used for the sample. A modified functional form is used to describe the sample decay law. Since no experimental data are altered in any way, a correct residual analysis is possible. Simulations show that fluorescence lifetimes can be recovered accurately, as long as the reference decay time is sufficiently different from that of the sample. Biexponential decays can be resolved successfully when the two decay times are well separated and different from that of the reference. The difference between the standard normal variates of chi-square for single and double exponential fits can be used to distinguish between mono- and bi-exponential decays. The usefulness of the delta function convolution method is demonstrated by fluorescence decay measurements of a series of different samples with very short decay times. Fluorescence lifetimes as short as 10 ps could be resolved accurately and reliably.

Synthesis, Spectroscopy, Crystal Structure, Electrochemistry, and Quantum Chemical and Molecular Dynamics Calculations of a 3-Anilino Difluoroboron Dipyrromethene Dye

An asymmetrically substituted fluorescent difluoroboron dipyrromethene (BODIPY) dye, with a phenylamino group at the 3-position of the BODIPY chromophore, has been synthesized by nucleophilic substitution of 3,5-dichloro-8-(4-tolyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. The solvent-dependent spectroscopic and photophysical properties have been investigated by means of UV-vis spectrophotometry and steady-state and time-resolved fluorometry and reflect the large effect of the anilino substituent on the fluorescence characteristics. The compound has a low fluorescence quantum yield in all but the apolar solvents cyclohexane, toluene, and chloroform. Its emission maxima in a series of solvents from cyclohexane to methanol are red-shifted by approximately 50 nm in comparison to classic BODIPY derivatives. Its oxidation potential in dichloromethane is at ca. 1.14 V versus Ag/AgCl. The absorption bandwidths and Stokes shifts are much larger than those of typical, symmetric difluoroboron dipyrromethene dyes. The values of the fluorescence rate constant are in the (1.4-1.7) x 10(8) s(-1) range and do not vary much between the solvents studied. X-ray diffraction analysis shows that the BODIPY core is planar. Molecular dynamics simulations show that there is no clear indication for aggregates in solution.

Ratiometric, Fluorescent BODIPY Dye with Aza Crown Ether Functionality: Synthesis, Solvatochromism, and Metal Ion Complex Formation

A new pH and metal ion-responsive BODIPY-based fluorescent probe with an aza crown ether subunit has been synthesized via condensation of 4-(1,4,7,10-tetraoxa-13-aza-cyclopentadec-13-yl)-benzaldehyde with the appropriate 1,3,5,7-tetramethyl substituted boron dipyrromethene moiety. Steady-state and time-resolved fluorometries have been used to study the spectroscopic and photophysical characteristics of this probe in various solvents. 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 Catalan solvent scales are found to be the most suitable for describing the solvatochromic shifts of the fluorescence emission. Fluorescence decay profiles of the dye can be described by a single-exponential fit in nonprotic solvents, whereas two decay times are found in alcohols. Protonation as well as complex formation with several metal ions are investigated in acetonitrile as solvent via fluorometric titrations. The aza crown ether dye undergoes a reversible (de)protonation reaction (pKa = 0.09) and shows a approximately 50 nm blue shift in the excitation spectra and a 10-fold fluorescence increase upon protonation. The compound also forms 1:1 complexes with several metal ions (Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Zn(2+)), producing large blue shifts in the excitation spectra and significant cation-induced fluorescence amplifications.

PHOTOPHYSICS OF THE FLUORESCENT K+ INDICATOR PBFI

The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 1.1 x 10(9) s-1, k21 = 2.7 x 10(8) M-1s-1, k02 = 1.8 x 10(9) s-1, and k12 = 1.4 x 10(9) s-1. k01 and k02 denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state. k21 represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k12 is the first-order rate constant of dissociation of the excited K(+)-PBFI complex. From the estimated values of k12 and k21, the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (-0.7) is smaller than pKd (2.2). The effect of the excited-state reaction can be neglected in the determination of Kd and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.

Vicarious Nucleophilic Substitution of α-Hydrogen of BODIPY and Its Extension to Direct Ethenylation

Direct, oxidizer-free substitution of the 3-hydrogen of BODIPY derivatives has been established through a vicarious nucleophilic substitution procedure. This methodology has been combined with a reversible Michael addition on nitrostyrenes to provide a novel, highly efficient entry to the valuable 3-styrylated BODIPY dyes.

On the use and the performance of the delta function convolution method for the estimation of fluorescence decay parameters

Abstract The delta function convolution method (DFCM) is a rigorous and convenient procedure for the analysis of time-correlated single photon counting experiments. DFCM deconvolutes the sample decay by the decay of a monoexponential reference compound to circumvent the wavelength dependence of the instrument. In this report, the accuracy of the parameter recovery by DFCM is investigated in a systematic way on simulated data sets. Mono- and biexponential sample decays are considered in combination with different reference lifetimes. The effect on the parameter recovery when a multiexponentially decaying reference is erroneously taken as being monoexponential is discussed. The formulae for DFCM using a multiexponential reference are derived. Recursion formulae for a computer analysis program in the case of a (multi-)exponential sample decay and a monoexponential reference are provided.

Fluorescence decay of pyrene in small and large unilamellar L, alpha-dipalmitoylphosphatidylcholine vesicles above and below the phase transition temperature.

The fluorescence decays of pyrene in small and large unilamellar L,α-dipalmitoylphosphatidylcholine vesicles have been investigated as a function of probe concentration and temperature. When the molar ratio of pyrene to phospholipid equals 1:3000, no excimer emission is observed and the fluorescence decays are mono-exponential. When this ratio is equal to or higher than 1:120, excimer formation is observed.Above the phase transition temperature the observed fluorescence decays of monomer and excimer can be adequately described by a bi-exponential function. The monomer decays can be equally well fitted to a decay law which takes into account a time-dependence in the probe diffusion rate constant. The fluorescence decay kinetics are compatible with the excimer formation scheme which is valid in an isotropic medium. The excimer lifetime and the (apparent) rate constant of excimer formation have been determined as a function of probe concentration at different temperatures above the phase transition temperature. The activation energy of excimer formation is found to be 29.4±1.3 kJ/mol. In small unilamellar vesicles the diffusion constant associated with the pyrene excimer formation process varies from 8.0x10-7 cm2/s at 40°C to 2.2x10-6 cm2/s at 70°C.Below the phase transition temperature the monomer decays can be described by a decay law which takes into account a time dependence of the rate constant of excimer formation. The lateral diffusion coefficient of pyrene calculated from the decay fitting parameters of the monomer region varies from 4.0x10-9 cm2/s at 20°C to 7.9x10-8 cm2/s at 35°C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.

Photochemistry of bichromophoric compounds: scope and expectations

Abstract Excitation of molecules containing more than one chromophore leads to intramolecular interactions such as excimer-, exciplex- and product formation. It is shown in this paper that these interactions can occur even if the chain connecting them contains several (more than three) units. Different factors affecting these interactions and the relation between the photophysical and photochemical aspects in these systems is stressed. If an intramolecular interaction is made impossible intermolecular reaction leads to high molecular weight products. The principles and the scope of this photopolymerization process is discussed.

Fluorescence decay of 1-methylpyrene in small unilamellar l-α-dimyristoylphosphatidylcholine vesicles: A temperature and concentration dependence study

The fluorescence decay kinetics of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles above the phase transition temperature has been studied as a function of concentration and temperature. When the 1-methylpyrene/phospholipid ratio equals 1:2000 no excimer is observed and the fluorescence decay is monoexponential. When this ratio is equal to or higher than 1 200, excimer is observed and the monomer and excimer decays can be adequately described by two exponential terms. The deviation of the monomer decays from monoexponentiality cannot be described by a model where the diffusion-controlled excimer formation is time dependent. The observed decays are compatible with the excimer formation scheme which is valid in an isotropic medium. The activation energy of excimer formation is found to be 29-9 +/-1.4 kJ mol . The (apparent) excimer formation constant and the excimer lifetime at different temperatures have been determined. The diffusion coefficient associated with the excimer formation process varies between 2 x 10(-10) m(2)/s at 70 degrees C to 4 x 10(-11) m(2)/s at 25 degrees C.

Photophysics of the fluorescent Ca2+ indicator Fura-2

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.