Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pK _a 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with stick boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.     

Near-field time-resolved fluorescence studies of poly(phenylmethyl silane) with subwavelength resolution

We present an example of the first time-correlated single-photon counting (TCSPC) near-field optical measurement. The aperture size of our prepared aluminun-coated fiber-optic probe was approximately 50 nm, which represents a spatial resolution of _ex/7 for our UV measurements. Near-field fluorescence decays of poly(phenylmethyl silane) in solid thin films excited in the range 325–360 nm were obtained and the steady-state excitation spectra compared with the excitation spectral information obtained in the far field. Fluorescence decays showed single exponential lifetimes ranging from 45 to 277 ps, which was dependent on the excitation wavelength and the selected near-field tip. The proximity of the metal-coated tip to the sample may be the reason for the modulation in fluorescence lifetime.     

Fluorescence Studies of Melanin by Stepwise Two-Photon Femtosecond Laser Excitation

Fluorescence of synthetic melanin in the solvents H₂O, KOH, ethylene glycol monomethyl ether, and dimethyl sulfoxide has been excited by two-photon absorption at 800 nm, using 120-fs pulses with photon flux densities of 10²⁷ cm^–2.S^–1. Compared to the one-photon (400-nm)-induced fluorescence of melanin, the overall spectral shape is red-shifted and shows a strong environment sensitivity. The decay of the two-photon-induced fluorescence (TPF) of melanin is three-exponential, with a shortest main component of about 200 ps. The results of the TPF studies in line with the unique light absorption property of melanin of a monotonously decreasing absorption spectrum between the near UV-region and the near infrared region indicate that the TPF is realized via stepwise absorption of two 800-nm photons. In comparison to the simultaneous absorption of two photons, the stepwise process needs lower photon flux densities to get a sufficient population of the fluorescent level. This stepwise process offers new possibilities of selective excitation of melanin in skin tissue in a spectral region where there is no overlap with any absorption of another fluorescent tissue component. The first results with different samples of excised human skin tissue (healthy, nevus cell nevi, malignant melanoma) suggest that fluorescence excited in this way yields information on malignant transformation.     

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Asymmetrical Distribution of Intrinsic Fluorescence Lifetimes in Proteins

Dynamic fluorescence measurements of proteins in solution are often interpreted in terms of continuous distributions of lifetimes, which reflect the intrinsic structural heterogeneity of these systems. In several cases a single Gaussian or Lorentzian symmetric distribution has been used to fit the data. In this paper we describe a new nonsymmetric Lorentzian function which contains three free parameters (the center, the left, and the right widths) like the double-discrete exponential model (the two lifetimes and one preexponential factor). Simulated data in the frequency domain have been used to compare the fits obtained with these different approaches, introducing a new parameter, , which quantitatively measures the asymmetry of the distribution or the ratio of the two preexponential factors, in the continuous and discrete models, respectively. Real measurements of a mixture of independent fluorophores, as well as of protein fluorescence decays, have also been performed and analyzed in terms of the new asymmetric function. The data have also been fitted with traditional discrete methods (such as the two- and the three-exponential decay) and with another asymmetric function, namely, the skewed Gaussian distribution.     

Rotational Diffusion of Coumarins in Alcohols: A Dielectric Friction Study

The rotational dynamics of three structurally similar polar molecules viz., coumarin 440, coumarin 151 and coumarin 450 has been studied in alcohols at room temperature using steady-state fluorescence depolarization method and time correlated single photon counting technique. The observed reorientation times of all the three probes are found to be longer than those predicted by Stokes–Einstein–Debye (SED) hydrodynamic theory with stick boundary condition and a deviation towards super-stick behavior is noted. Dielectric friction theories of Nee–Zwanzig and van der Zwan–Hynes, which treat the solute as a point dipole, overestimate the dielectric friction contribution exhibited by all the three coumarins in alcohols. Results are discussed in the light of theoretical models and the possibility of hydrogen bonding between the amino group of the probe molecules and the hydroxyl group of the alcohols.     

Fluorescence upconversion properties of a class of improved pyridinium dyes induced by two-photon absorption

We report the fluorescence upconversion properties of a class of improved pyridinium toluene-p-sulfonates having donor–π–acceptor (D–π–A) structure under two-photon excitation at 1064 nm. The experimental results show that both the two-photon excited (TPE) fluorescence lifetime and the two-photon pumped (TPP) energy upconversion efficiency were increased with the enhancement of electron-donating capability of the donor in the molecule. It is also indicated that an overlong alkyl group tends to result in a weakened molecular conjugation, leading to a decreased two-photon absorption (TPA) cross section. By choosing the donor, we can obtain a longest fluorescence lifetime of 837 ps, a highest energy upconversion efficiency of 6.1%, and a maximum TPA cross-section of 8.74×10⁻⁴⁸ cm⁴ s/photon in these dyes.     

Fluorescence lifetime-enhanced spectral characterization of human serum

Frequency-domain fluorescence lifetime techniques were used for the characterization of pooled human serum, including normal serum, hyperlipid serum, and sera that had been stripped of various components. Fluorescence lifetime measurements of normal human serum revealed lifetime components primarily in the regions of 10² ps, 1–2 ns, 4–7 ns, and 9–10 ns. Phase-resolved fluorescence spectroscopy (PRFS), a frequency-domain technique that combines spectral and lifetime information, in measurements of phase-resolved fluorescence intensity (PRFI), provided the basis for comparison of the various sera. Measurements of PRFI vs excitation wavelength and emission wavelength yield a phase-resolved excitation-emission matrix (PREEM) at a given modulation frequency. Multifrequency measurements yield a three-way excitation-emission-frequency array. The multifrequency PREEMs of the various sera were compared with each other and with the corresponding two-way excitation-emission matrices (EEMs) that are obtained using conventional, steady-state fluorescence spectroscopy. Application of matrix-based analysis techniques to the steady-state and PRFS data arrays allowed direct comparison between the two approaches. Results demonstrate the enhanced discrimination among samples that is achieved through the additional dimension of fluorescence lifetime in PRFS.     

Positron annihilation at defects in sintered high-T c perovskite superconductors

We report positron lifetime measurements in sintered superconducting YBa₂Cu₃O_7–x and GdBa₂Cu₃O_7–x oxides. It is shown that the thermal behaviour of the positron lifetime spectra strongly depends on the preparation of the ceramics. A lifetime of 190±3 ps is attributed to oxygen deficient regions. Two lifetimes of 251±7 ps and 225 ±5 ps are attributed to a cation vacancy presenting a temperature dependent atomic arrangement. The lifetime transition (251225ps) occurs during decreases in temperature across the resistivity superconducting transition. This lifetime change indicates that the volume of the cation vacancy decreases in the superconducting state.     

Microstructural evolution of ZnS during sintering monitored by optical and positron annihilation techniques

Positron lifetime and optical absorption techniques were employed to track the microstructural evolution of polycrystalline ZnS grown by Chemical Vapor Deposition (CVD). As grown material and material treated with Hot Isostatic Pressure (HIP) was sintered at temperatures ranging from 400 to 1000°C for 2–18 h. A 290 ps defect lifetime could be resolved in all samples, while an additional longer lifetime (=430 ps) was found only in samples annealed at low temperatures. This component gradually disappeared during annealing at 800°C. Associated with the disappearance of the long-lived component, the apparent bulk lifetime of the material changed from 235 to 215 ps. A 215±2 ps bulk parameter was also found for HIP-treated material annealed at temperatures greater than 400°C and hence is taken to represent the delocalized state of the positrons in ZnS. Optical absorption measurements showed that annealing at 800°C also caused the absorption profiles of the CVD and HIP samples to converge. The rate of the bulk lifetime transition correlates with the absorption changes. The observed sharpening of the absorption profile is attributed to a decrease in scattering from grain boundaries and voids, and a decrease in absorption from point defects. The 430 ps lifetime is believed to be due to trapping at voids and grain boundaries, while the 290 ps lifetime likely is due to a monovacancy stabilized as a small complex.Paper presented at the 132nd WE-Heraeus-Seminr on Positron Studies of Semiconductor Defects, Halle, Germany, 29 August to 2 September 1994     

Quantum Mayer Graphs for Coulomb Systems and the Analog of the Debye Potential

Within the Feynman–Kac path integral representation, the equilibrium quantities of a quantum plasma can be represented by Mayer graphs. The well known Coulomb divergencies that appear in these series are eliminated by partial resummations. In this paper, we propose a resummation scheme based on the introduction of a single effective potential that is the quantum analog of the Debye potential. A low density analysis of shows that it reduces, at short distances, to the bare Coulomb interaction between the charges (which is able to lead to bound states). At scale of the order of the Debye screening length ^–1 _D, approaches the classical Debye potential and, at large distances, it decays as a dipolar potential (this large distance behaviour is due to the quantum nature of the particles). The prototype graphs that result from the resummation obey the same diagrammatical rules as the classical graphs of the Abe–Meeron series. We give several applications that show the usefulness of to account for Coulombic effects at all distances in a coherent way.     

Two distinguishable fluorescent modes of 1-anilino-8-naphthalenesulfonate bound to human albumin

We study the interaction of 1-anilino-8-naphthalenesulfonate (ANS) with human (HSA) and bovine serum albumin (BSA) by phase and modulation fluorescence spectroscopy. We determined that both HSA and BSA show one or two distinguishable fluorescent sites, depending of the ANS/serum albumin ratio. At above a 11 ANS/HSA molar ratio, the steady-state emission spectra for ANS can be resolved in two components: component 1, emitting with a lifetime (₁) of 16 ns and a _1max of 478 nm, with a quantum yield (_f1) of 0.67, and component 2, with a lifetime (₂) of 2–4 ns and a _2max of 483 nm, with an average quantum yield (_f2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantK _as1=0.87×10⁶M^–1 and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aK _as2=0.079×10⁶M^–1 and a capacity of 2.34 mol of ANS/mol of HSA. Analysis of fluorescence lifetime distributions shows that the rigidity of the fluorophore environment at site 1 changes when site 2 is occupied. These findings suggest an interconnection between the two sites and that ligands can stabilize the protein's globular structure. To assess the identity of the ANS binding sites we used diazepam as a marker of the site located at the IIIA HSA subdomain and aspirin as a marker of sites located at the IIIA and IIA HSA subdomains. Both ligands displace ANS only from site 1, suggesting that it corresponds to the binding site located at the IIIA sub-domain of the protein. We determined that theK _as values for diazepam and aspirin are 0.113× 10⁶ and 0.021×10⁶ M ^–1 respectively.     

The application of a raman-shifted tunable KrF excimer laser for laser-induced fluorescence combustion diagnostics

Four wavelength extensions have been investigated by stimulated Raman scattering in hydrogen or deuterium gas of the 248 nm fundamental output of a narrow-band tunable KrF excimer laser. They have been used to acquire laser-induced fluorescence spectra of NO and OH in flames at atmospheric pressure. NO is detected in relatively high rotational states within the -band electronic system at 225 nm. OH was excited at 291.5 nm in the (1–0), at 313 nm in the (1–1) and at 268.5 nm in the predissociative (3–1) band of the ²–² electronic band system, respectively. Prospects of 2D imaging for concentration and temperature measurements in flames using these wavelength extensions are discussed.     

Two-photon predissociative fluorescence of H2O by a KrF laser for concentration and temperature measurement in flames

Two-photon laser-induced predissociative fluorescence (LIPF) of H₂O is examined as a potential measurement technique of H₂O concentration and temperature in flames. Two-photons of 248 nm light from a narrowband KrF laser excite H₂O to the highly predissociative state in a hydrogen-air flame. The subsequent bound-free emission is observed from 400–500 nm in the flame at temperatures of 1000–2000 K and is found to be free of fluorescence interference from other flame species. This LIPF signal is not affected by collisional quenching due to the short lifetime of the predissociative state (2.5 ps). Broadband laser dispersion spectra, narrowband laser dispersion spectra, laser excitation spectra and probability density functions of the H₂O fluorescence are obtained in the hydrogen flame. The H₂O LIPF signal is found to be temperature sensitive and a two-line LIPF technique is needed for concentration and temperature measurement. The accuracy of a two-line LIPF technique for H₂O concentration and temperature measurement is determined.     

Studies of divacancy in Si using positron lifetime measurement

The charge state dependence of positron lifetime and trapping at divacancy (V₂) in Si doped with phosphorus or boron has been studied after 15 McV electron irradiation up to a fluence of 8.0×10¹⁷ e/cm². The positron trapping cross sections for V ₂ ^2– , V ₂ ^– and V ₂ ⁰ at 300 K were about 6×10^–14, 3×10^–14 and 0.1–3×10^–14 cm², respectively. For V ₂ ⁺ , however, no positron trapping was observed. The marked difference in the cross sections comes from Coulomb interaction between the positron and the charged divacancy. The trapping rates for V ₂ ⁰ and V ₂ ^2– have been found to increase with decreasing temperature in the temperature range of 10–300 K. These results are well interpreted by a two-stage trapping model having shallow levels with energy of 9 meV (V ₂ ⁰ ) and 21 meV (V ₂ ^2– ). The appearance of a shallow level for V ₂ ⁰ can not be explained by a conventional Rydberg state model. The lifetime (290–300 ps) in V ₂ ⁰ is nearly constant in the temperature range from 10 to 300 K, while that in V ₂ ^2– increases from 260 ps at 10 K to 320 ps at 300 K. The lifetime (260 ps) in V ₂ ^2– is shorter than that in V ₂ ⁰ at low temperature, which is due to the excess electron density in V ₂ ^2– . At high temperature, however, the longer lifetime of V ₂ ^2– than that of V ₂ ⁰ is attributed to lattice relaxation around V ₂ ^2– .     

Spectral-Luminescent Properties of Deposited Rhodamine 6G Films

We have investigated the absorption and fluorescence spectra of Rhodamine 6G thin (20–100 nm) films vacuum-deposited on solid dielectric substrates: quartz and borosilicate glasses and polymer films. By the absorption spectra several molecular absorption centers — monomers, dimers, and complex associates — have been revealed. For the first time luminescence of vacuum-deposited Rhodamine 6G films has been obtained and investigated. We classified it as fluorescence of plane dimers and dimers of the house-of-cards type and also as luminescence of complex associates. The dependence of the spectral composition of the films on the excitation wavelength in the 293–353 K temperature range has been studied. It has been proposed that amorphous Rhodamine 6G films obtained by the vacuum deposition method contain crystalline zones and microclusters of adsorbed water molecules.     

Observation of new laser transitions and saturation effects in optically pumped NO

We report investigations of an NO laser employing specially profiled magnetic fields of up to 3.4T, and F₂ pump laser intensities as great as 20 MW cm^–2. We have observed laser oscillation at 226 nm on a rotational branch of the B'-X/it(3–11) band of NO for the first time, in addition to the previously reported oscillation at 218 nm on the B'-X/it(3–10) band. We have also observed visible laser emission on a rotational branch of the B ²-B ² II(3–1) band of NO. Saturation of the NO laser pulse energy with pump intensity has been observed, the total NO laser pulse energy having been increased to 490 J. The possibility of increasing the NO laser pulse energy towards 1 mJ per transition is discussed.     

New Series of Dyes for Flashlamp-Excited Lasers

A new series of laser compounds which are derivatives of 7H-benzo[4, 5]imidazo[1, 2-c]chromeno[3, 2–e]pyrimidine have been developed and their working characteristics are presented. It is shown that the new compounds fluoresce with a high quantum yield (0.83–0.97) in the green spectral region; their methanol solutions gave effective lasing upon flashlamp excitation with a pulse duration of about 2.0 sec at the level 0.5. In a nonselective cavity they emit laser radiation in the range 540–570 nm. The influence of the molecular structure of the investigated dyes on their fluorescence and laser characteristics is discussed.     

Time-Resolved Flavin Adenine Dinucleotide Fluorescence Study of the Interaction Between Immobilized Glucose Oxidase and Glucose

Time-resolved fluorescence experiments have shown that flavin adenine dinucleotide (FAD) fluorescence emission of sol–gel immobilized glucose oxidase (GOD) exhibits a three-exponential decaying behaviour characterized by long- (about 2.0–3.0 ns), intermediate- (about 300 ps) and short- (less than 10 ps) lifetime, each one being characteristic of a peculiar conformational state of the FAD structure. In the present work time-resolved fluorescence is used to monitor FAD signals in the time interval immediately following the addition of glucose at various concentrations in order to detect the conformational changes occurring during the interaction between sol–gel immobilized GOD and glucose. The analysis of time-dependent fluorescence emission signal has shown that the FAD conformational state changes during the process from a configuration with a prevalence of the state characterized by the long lifetime to a configuration with increased contribution from the process with the intermediate lifetime. The time needed to complete this configuration change decreases with the concentration of added glucose. The results here reported indicate that time-resoled fluorescence can be extremely useful for a better understanding of solid phase biocatalysis that is particularly important in light of their clinical and biotechnological applications.