Effects of tunable excitation in carotenoids explained by the vibrational energy relaxation approach.

Carotenoids are fundamental building blocks of natural light harvesters with convoluted and ultrafast energy deactivation networks. In order to disentangle such complex relaxation dynamics, several studies focused on transient absorption measurements and their dependence on the pump wavelength. However, such findings are inconclusive and sometimes contradictory. In this study, we compare internal conversion dynamics in [Formula: see text]-carotene, pumped at the first, second, and third vibronic progression peak. Instead of employing data fitting algorithms based on global analysis of the transient absorption spectra, we apply a fully quantum mechanical model to treat the high-frequency symmetric carbon-carbon (C=C and C-C) stretching modes explicitly. This model successfully describes observed population dynamics as well as spectral line shapes in their time-dependence and allows us to reach two conclusions: Firstly, the broadening of the induced absorption upon excess excitation is an effect of vibrational cooling in the first excited state ([Formula: see text]). Secondly, the internal conversion rate between the second excited state ([Formula: see text]) and [Formula: see text] crucially depends on the relative curve displacement. The latter point serves as a new perspective on solvent- and excitation wavelength-dependent experiments and lifts contradictions between several studies found in literature.

Carotenoid-to-bacteriochlorophyll energy transfer through vibronic coupling in LH2 from Phaeosprillum molischianum.

The peripheral light-harvesting antenna complex (LH2) of purple photosynthetic bacteria is an ideal testing ground for models of structure-function relationships due to its well-determined molecular structure and ultrafast energy deactivation. It has been the target for numerous studies in both theory and ultrafast spectroscopy; nevertheless, certain aspects of the convoluted relaxation network of LH2 lack a satisfactory explanation by conventional theories. For example, the initial carotenoid-to-bacteriochlorophyll energy transfer step necessary on visible light excitation was long considered to follow the Förster mechanism, even though transfer times as short as 40 femtoseconds (fs) have been observed. Such transfer times are hard to accommodate by Förster theory, as the moderate coupling strengths found in LH2 suggest much slower transfer within this framework. In this study, we investigate LH2 from Phaeospirillum (Ph.) molischianum in two types of transient absorption experiments-with narrowband pump and white-light probe resulting in 100 fs time resolution, and with degenerate broadband 10 fs pump and probe pulses. With regard to the split Qx band in this system, we show that vibronically mediated transfer explains both the ultrafast carotenoid-to-B850 transfer, and the almost complete lack of transfer to B800. These results are beyond Förster theory, which predicts an almost equal partition between the two channels.

Anharmonic vibrational effects in linear and two-dimensional electronic spectra.

For the description of vibrational effects in electronic spectra, harmonic vibrations are a convenient and widespread model. However, spectra of larger organic molecules in solution usually exhibit signs of vibrational anharmonicity, as revealed by deviation from the mirror image symmetry between linear absorption and emission spectra of the harmonic case. For perylene and terylene, two molecules with rigid Pi-electron systems and strong vibrational-electronic coupling, we employ a simple but effective theoretical model, which introduces cubic anharmonicity in the potentials of electronic surfaces. Vibrational anharmonicity is then readily quantified based on the experimentally measured peak ratio of the first vibronic progression peaks in linear absorption and emission. This method is straightforward but not applicable if emission from the initially excited state is short lived. For such a case, we employ two-dimensional electronic spectroscopy in the visible as a comprehensive time-resolved technique for the experimental determination of the vibrational anharmonicity of pinacyanol iodide, a solvated dye molecule exhibiting ultrafast excited state isomerization. We show that the ratio between certain cross peak amplitudes in two-dimensional electronic spectra is a direct measure of vibrational anharmonicity.

Center Line Slope Analysis in Two-Dimensional Electronic Spectroscopy.

Center line slope (CLS) analysis in 2D infrared spectroscopy has been extensively used to extract frequency-frequency correlation functions of vibrational transitions. We apply this concept to 2D electronic spectroscopy, where CLS is a measure of electronic gap fluctuations. The two domains, infrared and electronic, possess differences: In the infrared, the frequency fluctuations are classical, often slow and Gaussian. In contrast, electronic spectra are subject to fast spectral diffusion and affected by underdamped vibrational wavepackets in addition to Stokes shift. All these effects result in non-Gaussian peak profiles. Here, we extend CLS-analysis beyond Gaussian line shapes and test the developed methodology on a solvated molecule, zinc phthalocyanine. We find that CLS facilitates the interpretation of 2D electronic spectra by reducing their complexity to one dimension. In this way, CLS provides a highly sensitive measure of model parameters describing electronic-vibrational and electronic-solvent interaction.

Vibronic energy relaxation approach highlighting deactivation pathways in carotenoids.

Energy relaxation between two electronic states of a molecule is mediated by a set of relevant vibrational states. We describe this fundamental process in a fully quantum mechanical framework based on first principles. This approach explains population transfer rates as well as describes the entire transient absorption signal as vibronic transitions between electronic states. By applying this vibronic energy relaxation approach to carotenoids, we show that ß-carotene's transient absorption signal can be understood without invoking the intensely debated S* electronic state. For a carotenoid with longer chain length, we find that vibronic energy relaxation does not suffice to explain all features in the transient absorption spectra, which we relate to an increased ground state structural inhomogeneity. Our modeling approach is generally applicable to photophysical deactivation processes in molecules. As such, it represents a well-founded alternative to data fitting techniques such as global target analysis.

Vibronic origin of long-lived coherence in an artificial molecular light harvester.

Natural and artificial light-harvesting processes have recently gained new interest. Signatures of long-lasting coherence in spectroscopic signals of biological systems have been repeatedly observed, albeit their origin is a matter of ongoing debate, as it is unclear how the loss of coherence due to interaction with the noisy environments in such systems is averted. Here we report experimental and theoretical verification of coherent exciton-vibrational (vibronic) coupling as the origin of long-lasting coherence in an artificial light harvester, a molecular J-aggregate. In this macroscopically aligned tubular system, polarization-controlled 2D spectroscopy delivers an uncongested and specific optical response as an ideal foundation for an in-depth theoretical description. We derive analytical expressions that show under which general conditions vibronic coupling leads to prolonged excited-state coherence.

Vibronic coupling explains the ultrafast carotenoid-to-bacteriochlorophyll energy transfer in natural and artificial light harvesters.

The initial energy transfer steps in photosynthesis occur on ultrafast timescales. We analyze the carotenoid to bacteriochlorophyll energy transfer in LH2 Marichromatium purpuratum as well as in an artificial light-harvesting dyad system by using transient grating and two-dimensional electronic spectroscopy with 10 fs time resolution. We find that Förster-type models reproduce the experimentally observed 60 fs transfer times, but overestimate coupling constants, which lead to a disagreement with both linear absorption and electronic 2D-spectra. We show that a vibronic model, which treats carotenoid vibrations on both electronic ground and excited states as part of the system's Hamiltonian, reproduces all measured quantities. Importantly, the vibronic model presented here can explain the fast energy transfer rates with only moderate coupling constants, which are in agreement with structure based calculations. Counterintuitively, the vibrational levels on the carotenoid electronic ground state play the central role in the excited state population transfer to bacteriochlorophyll; resonance between the donor-acceptor energy gap and the vibrational ground state energies is the physical basis of the ultrafast energy transfer rates in these systems.

Signal to noise considerations for single crystal femtosecond time resolved crystallography of the Photoactive Yellow Protein.

Femtosecond time resolved pump-probe protein X-ray crystallography requires highly accurate measurements of the photoinduced structure factor amplitude differences. In the case of femtosecond photolysis of single P63 crystals of the Photoactive Yellow Protein, it is shown that photochemical dynamics place a considerable restraint on the achievable time resolution due to the requirement to stretch and add second order dispersion in order to generate threshold concentration levels in the interaction region. Here, we report on using a 'quasi-cw' approach to use the rotation method with monochromatic radiation and 2 eV bandwidth at 9.465 keV at the Linac Coherent Light Source operated in SASE mode. A source of significant Bragg reflection intensity noise is identified from the combination of mode structure and jitter with very small mosaic spread of the crystals and very low convergence of the XFEL source. The accuracy with which the three dimensional reflection is approximated by the 'quasi-cw' rotation method with the pulsed source is modelled from the experimentally collected X-ray pulse intensities together with the measured rocking curves. This model is extended to predict merging statistics for recently demonstrated self seeded mode generated pulse train with improved stability, in addition to extrapolating to single crystal experiments with increased mosaic spread. The results show that the noise level can be adequately modelled in this manner, indicating that the large intensity fluctuations dominate the merged signal-to-noise (I/σI) value. Furthermore, these results predict that using the self seeded mode together with more mosaic crystals, sufficient accuracy may be obtained in order to resolve typical photoinduced structure factor amplitude differences, as taken from representative synchrotron results.

Distinguishing Electronic and Vibronic Coherence in 2D Spectra by Their Temperature Dependence.

Long-lived oscillations in 2D spectra of chlorophylls are at the heart of an ongoing debate. Their physical origin is either a multipigment effect, such as excitonic coherence, or localized vibrations. We show how relative phase differences of diagonal- and cross-peak oscillations can distinguish between electronic and vibrational (vibronic) effects. While direct discrimination between the two scenarios is obscured when peaks overlap, their sensitivity to temperature provides a stronger argument. We show that vibrational (vibronic) oscillations change relative phase with temperature, while electronic oscillations are only weakly dependent. This highlights that studies of relative phase difference as a function of temperature provide a clear and easily accessible method to distinguish between vibrational and electronic coherences.

Precise phasing of 2D-electronic spectra in a fully non-collinear phase-matching geometry.

We report an experimental design for two-dimensional electronic spectroscopy (2D-ES) that avoids the need to measure notoriously weak pump-probe spectra. Retaining a fully non-collinear folded boxcar geometry, the described layout replaces pump-probe with heterodyned transient grating (het-TG). The absorptive component of the het-TG signal is measured directly, following a straightforward optimization routine. The use of het-TG achieves an improvement in signal to noise ratio by almost two orders of magnitude. As a result, 2D-ES-signals down to 0.5% can be clearly resolved.

Photoisomerisation quantum yield and non-linear cross-sections with femtosecond excitation of the photoactive yellow protein.

The quantum yield of photoisomerisation of the photoactive yellow protein (PYP) strongly depends on peak power and wavelength with femtosecond optical excitation. Using systematic power titrations and addition of second order dispersion resulting in 140, 300 and 600 fs pulse durations, the one and multi-photon cross-sections at 400, 450 and 490 nm have been assessed from transient absorption spectroscopy and additionally the Z-scan technique. Applying a target model that incorporates photoselection theory, estimates for the cross-sections for stimulated emission and absorption of the first excited state, the amount of ultrafast internal conversion and the underlying species associated dynamics have been determined. The final quantum yields for photoisomerisation were found to be 0.06, 0.14-0.19 and 0.02 for excitation wavelengths 400, 450 and 490 nm and found to increase with increasing pulse durations. Transient absorption measurements and Z-scan measurements at 450 nm, coinciding with the maximum wavelength of the ground state absorption, indicate that the photochemical quantum yield is intrinsically limited by an ultrafast internal conversion reaction as well as by stimulated emission cross-section. With excitation at 400 nm photoisomerisation quantum yield is further significantly limited by competing multi-photon excitation into excited state absorption at 385 nm previously proposed to result in photoionisation. With excitation at 490 nm the photoisomerisation quantum yield is predominantly limited further by the significantly higher stimulated emission cross-section compared to ground state cross-section as well as multi-photon processes. In addition to photoionisation, a second product of multi-photon excitation is identified and characterised by an induced absorption at 500 nm and a time constant of 2 ps for relaxation. With power densities up to 138 GW cm(-2) the measurements have not provided indication for coherent multi-photon absorption of PYP. In the saturation regime with 450 nm excitation, the limit for the photoisomerisation quantum yield was found to be 0.14-0.19 and the excited state absorption cross-section 6.1 × 10(-17) cm(2) or 0.36 times the ground state cross-section of 1.68 × 10(-16) cm(2) per molecule. This places a fundamental restriction on the maximum populations and sample penetration that may be achieved for instance in femtosecond pump-probe experiments with molecular crystals of PYP.

Pump-dump-probe and pump-repump-probe ultrafast spectroscopy resolves cross section of an early ground state intermediate and stimulated emission in the photoreactions of the Pr ground state of the cyanobacterial phytochrome Cph1.

The primary photoreactions of the red absorbing ground state (Pr) of the cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 involve C15═C16 Z-E photoisomerization of its phycocyanobilin chromophore. The first observable product intermediate in pump-probe measurements of the photocycle, "Lumi-R", is formed with picosecond kinetics and involves excited state decay reactions that have 3 and 14 ps time constants. Here, we have studied the photochemical formation of the Lumi-R intermediate using multipulse picosecond visible spectroscopy. Pump-dump-probe (PDP) and pump-repump-probe (PRP) experiments were carried out by employing two femtosecond visible pulses with 1, 14, and 160 ps delays, together with a broadband dispersive visible probe. The time delays between the two excitation pulses have been selected to allow interaction with the dominant (3 and 14 ps) kinetic phases of Lumi-R formation. The frequency dependence of the PDP and PRP amplitudes was investigated at 620, 640, 660, and 680 nm, covering excited state absorption (λ(max) = 620 nm), ground state absorption (λ(max) = 660 nm), and stimulated emission (λ(max) = 680 nm) cross sections. Experimental double difference transient absorbance signals (ΔΔOD), from the PDP and PRP measurements, required corrections to remove contributions from ground state repumping. The sensitivity of the resulting ΔΔOD signals was systematically investigated for possible connectivity schemes and photochemical parameters. When applying a homogeneous (sequentially decaying) connectivity scheme in both the 3 and 14 ps kinetic phases, evidence for repumping of an intermediate that has an electronic ground state configuration (GSI) is taken from the dump-induced S1 formation with 620, 640, and 660 nm wavelengths and 1 and 14 ps repump delays. Evidence for repumping a GSI is also seen, for the same excitation wavelengths, when imposing a target connectivity scheme proposed in the literature for the 1 ps repump delay. In contrast, using a 680 nm dump pulse, ground state formation is observed for all models examined. The ΔΔOD signals were dominated by stimulated emission, at both 1 and 14 ps delays for the longer wavelength excitation. The GSI, which is revealed by the PRP measurements and not resolved from pump-probe measurements, is found to be directly formed from the excited state of Pr, and its formation is considered using heterogeneous, homogeneous, and target models to globally fit the data.

Modelling multi-pulse population dynamics from ultrafast spectroscopy.

Current advanced laser, optics and electronics technology allows sensitive recording of molecular dynamics, from single resonance to multi-colour and multi-pulse experiments. Extracting the occurring (bio-) physical relevant pathways via global analysis of experimental data requires a systematic investigation of connectivity schemes. Here we present a Matlab-based toolbox for this purpose. The toolbox has a graphical user interface which facilitates the application of different reaction models to the data to generate the coupled differential equations. Any time-dependent dataset can be analysed to extract time-independent correlations of the observables by using gradient or direct search methods. Specific capabilities (i.e. chirp and instrument response function) for the analysis of ultrafast pump-probe spectroscopic data are included. The inclusion of an extra pulse that interacts with a transient phase can help to disentangle complex interdependent pathways. The modelling of pathways is therefore extended by new theory (which is included in the toolbox) that describes the finite bleach (orientation) effect of single and multiple intense polarised femtosecond pulses on an ensemble of randomly oriented particles in the presence of population decay. For instance, the generally assumed flat-top multimode beam profile is adapted to a more realistic Gaussian shape, exposing the need for several corrections for accurate anisotropy measurements. In addition, the (selective) excitation (photoselection) and anisotropy of populations that interact with single or multiple intense polarised laser pulses is demonstrated as function of power density and beam profile. Using example values of real world experiments it is calculated to what extent this effectively orients the ensemble of particles. Finally, the implementation includes the interaction with multiple pulses in addition to depth averaging in optically dense samples. In summary, we show that mathematical modelling is essential to model and resolve the details of physical behaviour of populations in ultrafast spectroscopy such as pump-probe, pump-dump-probe and pump-repump-probe experiments.

A structural model for apolipoprotein C-II amyloid fibrils: experimental characterization and molecular dynamics simulations.

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-ß-structure composed of two parallel ß-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-ß-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple 'letter-G-like' ß-strand-loop-ß-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-ß-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-ß-scaffold containing relatively unstructured connecting loops.

Incidence of intravascular uptake during fluoroscopically guided lumbar disc injections: a prospective observational study.

BACKGROUND: Although there are many in vivo studies evaluating lumbar disc injections, no studies have described the occurrence of intravascular uptake of contrast on discography. This phenomenon, however, has been well documented for other fluoroscopically-guided, contrast-enhanced spinal injections. OBJECTIVES: To document the phenomenon and incidence of intravascular uptake during fluoroscopically-guided, contrast-enhanced lumbar disc injections. DESIGN: A prospective, observational, community-based study. METHODS: The incidence of fluoroscopically-confirmed intravascular uptake of contrast material was documented in 160 patients representing a subset of individuals presenting to a small private community hospital for evaluation and treatment of lower back pain. These patients underwent fluoroscopically-guided lumbar disc injections for therapeutic and/or diagnostic purposes. RESULTS: A total of 280 discs from L1-2 to L5-S1 were studied. The degree of disc degeneration of the evaluated discs was scored using the Adams' scoring criteria for intervertebral disc degeneration. The incidence of intravascular uptake as well as any correlation between stage of disc degeneration and intravascular uptake was statistically evaluated. A total of 40 out of 280 discs (14.3%) demonstrated intravascular uptake. Detection of intravascular uptake usually required real-time fluoroscopy. There was no statistical correlation between the degree of disc degeneration and the incidence of intravascular uptake. No disc infections or other adverse sequelae occurred. CONCLUSION: This is the first study, to our knowledge, that addresses the incidence of intravascular uptake during lumbar disc injections in patients with low back pain. The presence of intravascular uptake may have implications both clinically and diagnostically in regards to interpretation of lumbar discography and adverse events related to loss of injected medications into the vascular system.

Spectrally resolved femtosecond two-color three-pulse photon echoes: study of ground and excited state dynamics in molecules.

We report the use of spectrally resolved femtosecond two-color three-pulse photon echoes as a potentially powerful multidimensional technique for studying vibrational and electronic dynamics in complex molecules. The wavelengths of the pump and probe laser pulses are found to have a dramatic effect on the spectrum of the photon echo signal and can be chosen to select different sets of energy levels in the vibrational manifold, allowing a study of the dynamics and vibrational splitting in either the ground or the excited state. The technique is applied to studies of the dynamics of vibrational electronic states in the dye molecule Rhodamine 101 in methanol.