Simultaneous analysis of single-photon timing data for the one-step determination of activation energies, frequency factors and quenching rate constants. Application to tryptophan photophysics.

A general global analysis of single-photon timing data is presented in which each fluorescence decay curve can be described by a different decay law. The model parameters can be held in common within one curve and/or between related curves. Any or all parameters can be kept fixed, or they may be variable to seek optimum values. This general analysis allows the determination of activation energies, frequency factors and quenching rate constants in one step. The construction of the global mapping table which relates parameters in one experiment to those in another is explained in detail. The use and performance of this general simultaneous analysis are examined using tryptophan fluorescence decays at pH 6.0 obtained at various emission wavelengths as a function of temperature and added solute quencher. The results show that tryptophan at pH 6.0 decays as a biexponential with decay times which are independent of the analysis wavelength. The decay component with the short lifetime has a deactivation rate constant of 1.4 x 10(9) s-1 independent of temperature. The decay component with the long lifetime has an activation energy of 28 kJ/mol and a frequency factor of 3 x 10(13) s-1; its temperature-independent decay rate constant equals 1 x 10(8) s-1. Recursion formulas for a computer program to estimate activation energies, frequency factors, and decay rate constants are provided.

Fluorescence decay of 1-methylpyrene in small unilamellar l-alpha-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.

Global analysis of the time-resolved fluorescence of alpha- chymotrypsinogen A and alpha-chymotrypsin powders as a function of hydration.

The time-resolved tryptophyl fluorescence of alpha-chymotrypsin A and alpha-chymotrypsin in the crystalline state and in buffer solution at room temperature was analyzed globally. Triple-exponential decay functions are necessary to adequately describe the tryptophyl fluorescence decay surfaces of the protein powders as a function of hydration and in solution. The fluorescence lifetimes of alpha-chymotrypsinogen A (tau 1 = 0.32, tau 2 = 1.30 ns, tau 3 = 3.98 ns) and alpha-chymotrypsin(tau 1 = 0.66 n s, tau 2 = 2.26 ns, tau 3 = 5.40 ns) are constant over the entire hydration range. The spectral positions of the decay-associated spectra of the hydrated powders do not shift as a function of hydration. This indicates that the structures of the zymogen and the active enzyme are unaffected by hydration. The lifetimes of alpha-chymotrypsinogen A in phosphate buffer pH 7.4 are tau 1 = 0.37 ns, tau 2 = 1.17 ns and tau 3 = 3.44 ns while the respective values of alpha-chymotrypsin are tau 1 = 0.47 ns, tau 2 = 1.40 and tau 1 = 3.89 ns.

Photophysical properties of 2-nitro-5,10,15,20-tetra-p-tolylporphyrins.

Tetraarylporphyrins substituted with nitro groups at beta-pyrrolic positions are potential candidates for electron-accepting pigments in model systems for photosynthesis. The photophysics of 2-nitro-5,10,15,20-tetra-p-tolylporphyrin and its zinc analog have been studied in order to evaluate this potential. The ground state absorption spectrum, the triplet-triplet absorption spectrum, the fluorescence emission spectrum, and associated photophysical parameters have been determined. The molecules have short singlet lifetimes and anomalous temperature- and solvent-dependent emission spectra which are consistent with the formation of an intramolecular charge transfer state of the type P+.-NO2-. in which the nitro group is twisted about its bond to the porphyrin, relative to the ground state conformation.

A new analysis method of single molecule fluorescence using series of photon arrival times: theory and experiment.

Up to now, single molecule fluorescence experiments were performed by dividing the time into a set of intervals and to observe the number of fluorescence photons arriving in each interval. It is obvious that the detected photons carry less information than the arrival times of the photons themselves. From the arrival times, one can still calculate the number of photons in any user-defined interval; whereas, when only the number of photons in an interval are recorded, information about their positions in time is lost. Therefore, we present a new analysis method of single molecule fluorescence data based on the positions in time of the detected fluorescence photons. We derive mathematically different statistical characteristics describing the single molecule fluorescence experiment assuming an immobilized molecule. The theory of point processes using the generating functionals formalism is ideally suited for a consistent description, linking the statistical characteristics of the excitation and detected photons to the statistical characteristics of the single motionless molecule. We then use computer-generated data sets mimicking the single molecule fluorescence experiment to explore the parametric estimation of mono- and bi-exponential single molecule impulse response functions (SMIRFs) via the following statistical characteristics: the probability density distributions (pdd) of the single and first photocount time positions in a user-defined detection interval, the probability distribution of the number of photocounts per user-defined detection interval, the time correlation function and the pdd of the time interval between two consecutive photocounts. It is shown that all of the above characteristics ensure a satisfactory recovery of the decay time of mono-exponential SMIRFs for a broad range of excitation intensities and widths of user-defined detection intervals. For bi-exponential SMIRFs, the selection of the experimental conditions is more critical and dependent on the detection procedure. At lower excitation intensities it is advantageous to use the pdds of the single and first photocount time occurrences in the user-defined detection interval. To show the practical usefulness of the new analysis method, series of photon arrival times from immobilized single molecules of DiI and rhodamine 6G were analyzed to estimate triplet lifetimes and intersystem crossing yields.

Triplet states as non-radiative traps in multichromophoric entities: single molecule spectroscopy of an artificial and natural antenna system.

Energy transfer in antenna systems, ordered arrays of chromophores, is one of the key steps in the photosynthetic process. The photophysical processes taking place in such multichromophoric systems, even at the single molecule level, are complicated and not yet fully understood. Instead of directly studying individual antenna systems, we have chosen to focus first on systems for which the amount of chromophores and the interactions among the chromophores can be varied in a systematic way. Dendrimers with a controlled number of chromophores at the rim fulfill those requirements perfectly. A detailed photophysical study of a second-generation dendrimer, containing eight peryleneimide chromophores at the rim, was performed 'J. Am. Chem. Soc., 122 (2000) 9278'. One of the most intriguing findings was the presence of collective on/off jumps in the fluorescence intensity traces of the dendrimers. This phenomenon can be explained by assuming a simultaneous presence of both a radiative trap (energetically lowest chromophoric site) and a non-radiative trap (triplet state of one chromophore) within one individual dendrimer. It was shown that an analogue scheme could explain the collective on/off jumps in the fluorescence intensity traces of the photosynthetic pigment B-phycoerythrin (B-PE) (Porphyridium cruentum). The different values of the triplet lifetime that could be recovered for a fluorescence intensity trace of B-PE were correlated with different intensity levels in the trace, suggesting different chromophores acting as a trap as function of time.

Fluorescence lifetime fluctuations of single molecules probe the local environment of oligomers around the glass transition temperature.

Single molecule fluorescence experiments have been performed on a BODIPY-based dye embedded in oligo(styrene) matrices to probe the density fluctuations and the relaxation dynamics of chain segments surrounding the dye molecules. The time-dependent fluorescence lifetime of the BODIPY probe was recorded as an observable for the local density fluctuations. At room temperature, the mean fraction of holes surrounding the probes is shown to be unaffected by the molecular weight in the glassy state. In contrast, the free volume increases significantly in the supercooled regime. These observations are discussed in the framework of the entropic theories of the glass transition.

Energy dissipation in multichromophoric single dendrimers.

Single-molecule spectroscopy of well-chosen dendritic multichromophoric systems allows investigation of fundamental photophysical processes such as energy or electron transfer in much greater detail than the respective ensemble measurements. In dendrimers with multiple chromophores, energy hopping and transfer to the chromophore with the energetically lowest S(1) state was observed. If more than one chromophore is in an excited state in one molecule, annihilation, either singlet-triplet or singlet-singlet, can occur. In the latter case, a higher singlet state is populated opening new deactivation pathways. In the presence of an electron donor, reversible electron transfer could be observed, and the rate constants of forward and backward electron transfer were established. The value of these rate constants fluctuates time-correlated with the rotational motion of the dendrimer arms and the mobility of the embedding matrix.

Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin.

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.

Fluorescence quenching with lindane in small unilamellar L,alpha-dimyristoylphosphatidylcholine vesicles.

The lateral mobility and lipid-water partition of the pesticide lindane was studied by fluorescence quenching of N-isopropylcarbazole (NIPC) and L,alpha-palmitoyl-beta-(N-carbazolyl) undecanoylphosphatidylcholine (PCUPC) in liposomes of dimyristoylphosphatidylcholine at 50 degrees C. In isotropic solvents the quenching reaction was highly inefficient. A scheme for dynamic quenching, in which the monomolecular quenching rate constant is small, was valid. In lipid bilayers the same scheme was applied to describe the quenching results but the rate constant of the back-reaction of the excited complex to quencher and excited probe was of comparable magnitude to the monomolecular quenching rate constant. This phenomenon results in biexponential decays of the fluorescent probe in the presence of quencher. All the rate constants of the scheme could be determined. Stern-Volmer plots at different membrane concentrations were obtained from fluorescence intensity and decay time measurements. From these plots the true bimolecular quenching rate constant, Kq, and the rate constant for lateral diffusion, kd, were determined: kq[NIPC] = 3.2 +/- 0.5 x 10(8) M-1 s-1, kq[PCUPC] = 1.9 +/- 0.4 x 10(8) M-1 s-1, kd[NIPC] = 6.6 +/- 0.8 x 10(8) M-1 s-1. The smaller value of kq compared to kd for the quenching reaction of NIPC with lindane indicates that this quenching reaction is not diffusion controlled. The lateral diffusion coefficient D of lindane was found to be 1.7 +/- 0.2 x 10(-6) cm2/s in dimyristoylphosphatidylcholine vesicles at 50 degrees C. The partition coefficient of lindane in these lipid bilayers is very high (greater than 2000).

A new calibration equation for ratiometric fluorescent ion indicators: Application to fura-2.

The absolute values of intracellular ion concentrations as monitored by specific fluorescent indicators are determined by using calibration curves obtained underin vitro andin vivo conditions. In the derivation of the calibration curve by Grynkiewicz et al [(1985)J. Biol. Chem 260, 3440] it is implicitly assumed that the observed fluorescence signal is directly related to the concentrations of the free dye and the dye-ion complex in the ground state. We modified the calibration equation so that ion binding and dissociation in the excited state are taken into account. The extended calibration equation assumes the knowledge of the rate constants in the excited state. Expressions for the calibration curve assuming the absence or presence of an excited-state reaction are compared for the Ca(2+) indicator Fura-2. The excited-state rate constants are determined by global compartmental analysis of time-resolved fluorescence decays of Fura-2 collected at various excitation and emission wavelengths using different Ca(2+) concentrations. It is found that for Fura-2 there is negligible interference of the excited-state reaction so that the original calibration can used.

Fluorescence study of a field-induced director reorientation in a liquid crystalline polyacrylate.

The electric field-induced director reorientation is investigated by fluorescence spectroscopy and turbidimetry. The dynamics of this reorientation are studied as a function of temperature, applied voltage, and frequency.

Fluorescence study of field-induced director reorientations in low mass liquid crystalline compounds.

The Frederickz transition and the dynamic scattering deformation in 4-alkyloxy-4'-cyanobiphenyl compounds are investigated by fluorescence spectroscopy and turbidimetry. The main characteristics of the Frederickz deformation, reversibility and threshold voltage, in the nematic and the smectic A phase are investigated by means of fluorescence spectroscopy. Furthermore, the dynamics of the Frederickz transition are studied as a function of temperature and applied voltage. The results obtained with both techniques are compared and correlate well.

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.

Inhibition or initiation of a radical polymerization reaction by an ultraviolet-induced enzymatic process.

alpha-Chymotrypsin was modified to a light-controllable enzyme derivative by acylating active serine 195 residue with a cinnamoyl group or analogue. Upon UV irradiation the acylgroup could be isomerized, leading to release of the inhibiting group. Enzymatic activity could thus be regulated by means of UV light. A full 100% inhibition of the enzymatic activity could not be reached by the cinnamoyl derivative. Only posttreatment with diisopropylfluorophosphate yields a fully inactive enzyme derivative. The shelf-life of the inhibited enzyme was rather poor. Only freeze-dried samples could be used for several months without significant recovery of activity. Adapting the sensitivity of the system to visible light seems limited to the size of an enzyme's active site. Combination of the enzymatic system producing an inhibitor or an initiator with a polymerization reaction can result in a photographic process with a higher amplification factor.

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, alpha-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 8.0 X 10(-7) cm2/s at 40 degrees C to 2.2 X 10(-6) cm2/s at 70 degrees 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 4.0 X 10(-9) cm2/s at 20 degrees C to 7.9 X 10(-8) cm2/s at 35 degrees C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.