High frequency vibrational modulations in two-dimensional electronic spectra and their resemblance to electronic coherence signatures.

In this work we analyze how nuclear coherences modulate diagonal and off-diagonal peaks in two-dimensional electronic spectroscopy. 2D electronic spectra of pinacyanol chloride are measured with 8 fs pulses, which allows coherent excitation of the 1300 cm(-1) vibrational mode. The 2D spectrum reveals both diagonal and off-diagonal peaks related to the vibrational mode. On early time scales, up to 30 fs, coherent dynamics give rise to oscillations in the amplitudes, positions, and shapes of the peaks in the 2D spectrum. We find an anticorrelation between the amplitude and the diagonal width of the two diagonal peaks. The measured data are reproduced with a model incorporating a high frequency mode coupled to an electronic two-level-system. Our results show that these anticorrelated oscillations occur for vibrational wavepackets and not exclusively for electronic coherences as has been assumed previously.

Excitonic couplings and interband energy transfer in a double-wall molecular aggregate imaged by coherent two-dimensional electronic spectroscopy.

The early stage of molecular excitonics and its quantum-kinetic dynamics in the multiband, bitubular cyanine dye aggregate C(8)O(3) at room temperature are revealed by employing two-dimensional (2D) coherent electronic spectroscopy in the visible spectral region. The sub-20 fs measurements provide a direct look into the details of elementary electronic couplings by spreading spectroscopic transitions into two frequency axes. Correlation spectra of rephasing (k(I) = -k(1) + k(2) + k(3)) and nonrephasing (k(II) = +k(1) - k(2) + k(3)) data in emission (omega(3))-absorption (omega(1)) 2D-frequency space image interband excitons into cross-peak signals and unveil the quantum-dissipative regime of exciton relaxation. Spectral streaking of cross peaks directly reveals interband dephasing and exciton population relaxation on the road to tube-to-tube energy transfer without making recourse to an a priori model. Theory and simulations, based on an effective multilevel scheme and a quantum-dissipative model with experimental pulse envelopes, explain the origin of the cross peaks, reveal the underlying sequences of electronic transitions, recover the streaking patterns of relaxing cross peaks along omega(1), and reconstruct the space-energy pathways of electronic excitation flow.

Two-dimensional electronic spectra of symmetric dimers: Intermolecular coupling and conformational states.

We study the information content of two-dimensional (2D) electronic photon-echo (PE) spectra, with special emphasis on their potential to distinguish, for waiting times T=0, between different conformations of electronically coupled symmetric dimers. The analysis is performed on the basis of an analytical formula for the frequency-domain 2D PE signal. The symmetric dimers are modeled in terms of two identical, energy-degenerate, excitonically coupled pairs of electronic states in the site representation. The spectra of conformationally weighted ensembles, composed of either two or four dimers, are compared with their one-dimensional linear absorption counterparts. In order to provide a realistic coupling pattern for the ensemble consisting of four dimers, excitonic couplings are estimated on the basis of optimized geometries and site-transition dipole moments, calculated by standard semiempirical methods for the bridged bithiophene structure 1,2-bithiophene-2-yl-ethane-1,2-dion (T2[CO]2). In the framework of our model, the highly readable 2D PE spectra can unambiguously identify spectral doublets, by relating peak heights and positions with mutual orientations of site-localized transition dipoles.

Correlation of femtosecond wave packets and fluorescence interference in a conjugated polymer: Towards the measurement of site homogeneous dephasing.

Probing electronic femtosecond (fs) coherence among segmental sites that are congested by static and dynamic site disorder and subject to structural relaxation is a big, experimental challenge in the study of photophysics of poly(p-phenylenevinylene). In this work, fs-wave-packet fluorescence interferometry experiments are presented that measure macroscopic coherent kernels and their phase-relaxation in the low-temperature, bottom-state regime of the density-of-states below the migrational threshold energy where downhill site-to-site transfer is marginal. By using freely propagating and tunable 70 fs excitation/probing pulses and employing narrow-band spectral filtering of wave packets, fluorescence interferograms with strongly damped beatings can be observed. The coherences formally follow the in-phase superpositions of two site-optical free-induction-decays and originate from distinct pairs of coherent doorway-states, different in energy and space, each of them being targeted, by two discrete quantum-arrival-states 1(alpha) and 1(beta), via independent, isoenergetic 0-->1 fluorescence transitions. The coherent transients are explained as site-to-site polarization beatings, caused by the interference of two fluorescence correlation signals. The numerical analysis of the damping regime, based upon second-order perturbational solutions, reveals the lower limit value of homogeneous dephasing in the range from T(2) approximately 100 fs to T(2) approximately 200 fs depending on the site-excitation energy of the bottom-states. The experiments enable to look into the formation of the relaxed state as a special molecular process of electron-phonon coupling and hence open-up a quite new perspective in the puzzle of multichromophore optical dynamics and structural relaxation in conjugated polymers.

Intensity-independent fluorometric detection of cellular nitric oxide release.

A new fluorescence method is introduced in which nitric oxide (NO)-derived higher-order oxygen complexes (NO(x)) are quantified at physiological pH. Detecting the fluorescence lifetime shift between 2,3-diaminonaphthalene and the NO(x)-derived protonated 2,3-naphthotriazole allows an intensity independent determination of the NO(x) concentration. The NO release from LPS and IFNgamma-stimulated murine macrophages and iNOS transfected hamster cells was quantified. The lower detection limit for NO2- was found to be 800 pmol/ml. Since the influence of static fluorescence quenching due to cellular components can be neglected, the method is applicable for clear cellular supernatants as well as turbid cellular suspensions.

Molecular dynamics simulation of the rare amino acid LL-dityrosine and a dityrosine-containing peptide: comparison with time-resolved fluorescence.

The fluorescence of the rare amino acid LL-dityrosine, which is found in insoluble biological materials with structural features, was recently shown to decay non-exponentially (Kungl et al. (1992) J. Fluorescence 2, 63-74). Here we investigated the time-resolved fluorescence of a dityrosine-containing peptide (DCP) to study the influence of side chains on the fluorescence decay of the chromophore. The fluorescence decay of DCP was best fitted by three exponential terms including a sub-nanosecond rise term, the values of which are quite similar to the parameters obtained for the decay of free dityrosine. They were found to depend on the pH of the aqueous solution but not on the temperature. Analysis by an exponential series method revealed broad fluorescence lifetime distributions for DCP. Compared to the corresponding analysis of dityrosine transients, similar lifetime centers were found whereas the widths of the distributions were found broader for DCP. Molecular dyamics (MD) simulations of dityrosine at 300 K show that chi 1 and chi 2 side chain conformers (rotamers) of both tyrosine subunits interconvert on a picosecond timescale. The rates of interconversion were shown to depend critically upon the MD technique applied: in vacuo simulations yielded lower interconversion rates compared to stochastic dynamics (SD) and full MD (water explicitly included). However, MD simulations of the dityrosine-containing peptide revealed no interconversions of the chi 1 and chi 2 side chain rotamers of both tyrosine subunits within a 400 ps trajectory. Interconversions could be induced by raising the temperature of the system (DCP plus solvent) to 340 K. Side chain rotamers of dityrosine are not stable on a fluorescence time scale but are stable when a dityrosine-containing peptide is regarded. Nevertheless both molecules yield similar fluorescence decay patterns. We therefore conclude that the rotamer model proposed for the fluorescence decay of tyrosine and tryptophan cannot be applied to the fluorescence decay of dityrosine and peptides containing this chromophore. This should be of future interest when dityrosine is used as an intrinsic sensor to study complex dityrosine-containing macromolecules by fluorescence spectroscopy.

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.

Characterization of human immunodeficiency virus-1 (HIV-1) rev by (time-resolved) fluorescence spectroscopy.

Fluorescence spectroscopy has been applied to the single tryptophan-containing regulatory protein Rev of human immunodeficiency virus (HIV-1). The fluorescence emission was found to have a maximum at 336 nm which refers to a surrounding of the chromophore of intermediate polarity. Fluorescence transients recorded at the maximum of fluorescence were found to decay nonexponentially. A bimodal lifetime distribution is obtained from exponential series analysis (ESM) with centers at 1.7 and 4.5 ns. Two microenvironments for tryptophan are suggested to be responsible for the two lifetime distributions. No innerfilter effect occurred in a Rev solution up to a concentration of 40 µM. A data quality study of ESM analysis as function of collected counts in the peak channel maximum (CIM) showed that, for reliable reconvolution, at least 15,000 CIM are necessary. The widths of the two distributions are shown to be temperature dependent. The broadening of the lifetime distributions when the temperature is raised to 50°C is interpreted as extension of the number of conformational substates which do not interconvert on the fluorescence time scale. The thermal deactivation (temperature quenching) is reflected in a constant decrease in the center of the short-lived lifetime distribution.

L-Dityrosine: A time-resolved fluorescence investigation.

We have investigated the time-resolved fluorescence properties ofLL-dityrosine in aqueous solution. Typically, three exponential components were needed to fit the fluorescence pattern adequately, with pure decay terms for the low-intensity, high-energy state (λem = nm) but with a pronounced subnanosecond rise phase for the predominant red-edge fluorescence (λem > 380). Dual fluorescence behavior is indicative of an intramolecular precursorsuccessor pair, i.e., a consecutive intramolecular excited-state reaction. We suggest that this reaction is a torsional motion of the (deprotonated) monoanion along the biphenolic bond. Analysis of the fluorescence anisotropy decay of dityrosine yielded two rotational correlation times, the longer of which is associated with a negative preexponential term. The increase with time in the horizontally polarized component of the intensity decay is interpreted as the result of an electronic rearrangement in the excited state when the successor form of dityrosine is generated. Lifetime distributions of experimental data were probed by an unbiased exponential series method which uses a Tikhonov-type regularization function. The procedure revealed three well-separated groups of lifetimes, the short-lived ensemble forming a formally negative distribution. A photophysical model is introduced which interprets the biexponential decay of dityrosine in terms of overlapping emission signals from the precursor and the successor molecule.

Antibodies against Aspergillus fumigatus II. Identification and quantification by means of crossed immunoelectrophoresis.

Individual antigenic components of Aspergillus fumigatus were identified by crossed immunoelectrophoresis (CIE) using pooled rabbit hyperimmune antiserum. Patients' sera with enhanced precipitins against A. fumigatus showed antibodies against identical antigenic components in CIE. Identification of the antigenic components by the intermediate gel technique showed high concentrations of antibodies against a small group of antigenic components. No differences were found in the individual antigenic components involved in the precipitation reactions between groups of sera with enhanced and normal IgE levels against A. fumigatus. Crossed immunoelectrofocusing was used to determine the isoelectric point of particular components that were identified by CIE.