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Fluorescence imaging is currently attracting much interest as a method for intraoperative tumor detection, but most current tracers lack tumor specificity. Therefore, this technique can be further improved by tumor-specific detection. With tumor-targeted antibodies bound to a radioactive label, tumor-specific SPECT or PET is feasible in the clinical setting. The aim of the present study was to apply antibody-based tumor detection to intraoperative optical imaging, using preclinical in vivo mouse models. METHODS: Anti-vascular endothelial growth factor (VEGF) antibody bevacizumab and anti-human epidermal growth factor receptor (HER) 2 antibody trastuzumab were labeled with the near-infrared (NIR) fluorescence dye IRDye 800CW. Tumor uptake of the fluorescent tracers and their (89)Zr-labeled radioactive counterparts for PET was determined in human xenograft-bearing athymic mice during 1 wk after tracer injection, followed by ex vivo biodistribution and pathologic examination. Intraoperative imaging of fluorescent VEGF- or HER2-positive tumor lesions was performed in subcutaneous tumors and in intraperitoneal dissemination tumor models. RESULTS: Tumor-to-background ratios, with fluorescent imaging, were 1.93 ± 0.40 for bevacizumab and 2.92 ± 0.29 for trastuzumab on day 6 after tracer injection. Real-time intraoperative imaging detected tumor lesions at even the submillimeter level in intraperitoneal dissemination tumor models. These results were supported by standard histology, immunohistochemistry, and fluorescence microscopy analyses. CONCLUSION: NIR fluorescence-labeled antibodies targeting VEGF or HER2 can be used for highly specific and sensitive detection of tumor lesions in vivo. These preclinical findings encourage future clinical studies with NIR fluorescence-labeled tumor-specific antibodies for intraoperative-guided surgery in cancer patients.

Synthesis, characterization, and applications of novel thermoresponsive polymeric coatings for quantum dots (QDs) are presented. Comb-copolymers featuring hydrophobic alkyl groups, carboxylic groups and poly(N-isopropylacrylamide) (PNIPAM) side chains with molar masses ranging from 1000 g/mol to 25,400 g/mol were obtained. The amphiphilic comb-copolymers were shown to efficiently transfer the QDs to aqueous media. The PNIPAM-coated QD materials display a lower critical solution temperature (LCST). The absorbance, luminescence emission, size of the assemblies, and electrophoretic mobility were followed as a function of temperature and the reversibility of the temperature induced changes is demonstrated by cyclic heating and cooling.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy is an important technique for spectroscopy and chemically selective microscopy, but wider implementation requires dedicated versatile tunable sources. We describe an optical parametric oscillator (OPO) based on a magnesium oxide-doped periodically poled lithium niobate crystal, with a novel variable output coupler, used as a tunable coherent light source. The OPO's signal wavelength ranges from 880 to 1040 nm and its idler wavelength from 1090 to 1350 nm. We use this OPO to demonstrate high-resolution narrowband CARS spectroscopy on bulk polystyrene from 900 to 3600 cm-1, covering a large part of the molecular fingerprint region. Recording vibrational spectra using narrowband CARS spectroscopy has several advantages over spontaneous Raman spectroscopy, which we discuss. We isolate the resonant part of the CARS spectrum and compare it to the spontaneous Raman spectrum of polystyrene using the maximum entropy method of phase retrieval; we find them to be in extremely good agreement.

Controlled ensemble formation of protein-surfactant systems provides a fundamental concept for the realization of nanoscale devices with self-organizing capability. In this context, spectroscopic monitoring of pigment-containing proteins yields detailed structural information. Here we have studied the association behavior of the bacterial light-harvesting protein LH2 from Rhodobacter spheroides in an n,n-dimethyldodecylamine-n-oxide/water environment. Time-resolved studies of the excitation annihilation yielded information about aggregate sizes and packing of the protein complexes therein. The results are compared to transmission electron microscopy images of instantaneously frozen samples. Our data indicate the manifestation of different phases, which are discussed with respect to the thermodynamic equilibrium in ternary protein-surfactant-water systems. Accordingly, by varying the concentration the formation of different types of aggregates can be controlled. Conditions for the appearance of isolated LH2 complexes are defined.

Pump-deplete-probe and transient absorption spectroscopy are applied to carotenoids with N = 11 conjugated double bonds in solution to study the origin of recently observed transient features that have been previously assigned to new electronic states. The depletion pulse pumps the transient near-IR band, whose lifetime coincides with the fluorescence lifetime, and is hence attributed to the S 2 state. The subsequent signal of any lower-lying dark excited-state populated by internal conversion from S 2 should be affected by the depletion pulse. Correspondingly, the signal in the S 1 deactivation channel is diminished by the depleted excited population. In contrast, the S s o l * signal, purportedly reflecting an intermediate state on a competing deactivation pathway, is not affected by the depletion pulse. When comparing our results with literature data for other carotenoids, we find that the S s o l * lifetime is constant at 6.2 ′ 0.4 ps for any N ≥ 11 carotenoid; for shorter chain lengths, it is equal to the S 1 lifetime. To explain this puzzle, S s o l * is identified as a vibrationally excited ground state (S s o l * = hot S 0 ), populated by a combination of impulsive Raman scattering of the pump pulse and internal conversion (S 1 -S 0 ), and decaying by vibrational relaxation. The S s o l * state is not identical to the S T * state, which appears in the same spectral region when the carotenoid is embedded in light-harvesting complexes.

The ultrafast carotenoid band shift upon excitation of nearby bacteriochlorophyll molecules was studied in three different light harvesting complexes from purple bacteria. The results were analyzed in terms of changes in local electric field of the carotenoids. Time dependent density functional theory calculations based on known and model structures led to good agreement with experimental results, strongly suggesting that the mutual orientation of the pigment molecules rather than the type of the carotenoid molecules determines the extent of the ultrafast band shift. We further estimate that the protein induced local field nearby carotenoid molecule is about 4 or 6 MV/cm, depending on the orientation of the change of the electrical dipole in the carotenoid upon optical transition.

Using near-infrared femtosecond absorption spectroscopy, we have determined the S1 energies of the carotenoids spheroidene and rhodopin glucoside in LH2 complexes of purple bacteria. The S1 energies in the LH2 complexes yield values of 13400 ± 100 cm-1 for spheroidene and 12550 ± 150 cm-1 for rhodopin glucoside, which are very close to the S1 energies obtained for both carotenoids in solution. The 850 cm-1 difference between the S1 energies of these two carotenoids significantly affects the energy transfer pathways within the LH2 complexes. The S1 energy of spheroidene in the LH2 complex of Rhodobacter (Rb.) sphaeroides is high enough to allow efficient energy transfer from the S1 state to bacteriochlorophylls, resulting in a substantial shortening of the spheroidene S1 lifetime in the LH2 complex (1.7 ps) compared with the lifetime in solution (8.5 ps). Rhodopin glucoside, which occurs in Rhodopseudomonas (Rps.) acidophila, has an S1 energy in the LH2 complex too low for efficient S1-mediated energy tran...

LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with 11 saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S1 level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S1 energy of lycopene in n-hexane was determined to be approximately 12 500 +/- 150 cm(-1), by direct measurement of the S1-S2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S1 energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischianum, in which lycopene is the native carotenoid. The results show that the overall efficiency for Crt --> B850 energy transfer is approximately 80% in lyco-LH2 and approximately 95% in WT-LH2 of Rb. sphaeroides. The difference in overall Crt --> BChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S(1) --> BChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt S1 energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Q(y) states, energy transfer via the Crt S1 state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B800 Q(y) state, leaving only the Crt S1 --> B850 channel, strongly suggesting that Crt S1 --> BChl energy transfer is controlled by the relative Crt S1 and BChl Q(y) energies.

The multiexciton density matrix theory is utilized to achieve a microscopic description of exciton−exciton annihilation (EEA). We apply the theory to the 18 bacteriochlorophyll (BChl) molecules of the B850 ring of the light-harvesting complex LH2 of Rhodobacter sphaeroides. The simulation of the EEA process reproduces the intensity-dependent transient absorption kinetic experiments very well, and insight is obtained on microscopic parameters such as the internal conversion rate of BChl in LH2. The exciton dynamics and the different relaxation processes are visualized by constructing a multiexciton spectrogram.

Using a novel experimental approach based on near-infrared femtosecond absorption spectroscopy, we have determined the energy of the S-1 state of the carotenoid spheroidene. The energy of this state is 13 400 +/- 90 cm(-1) at both 293 and 186 K, showing that there is no temperature-induced shift of the S-1 level. A discrepancy of about 800 cm(-1) between the S-1 energy determined here and that obtained from previous fluorescence and resonance Raman measurements is explained in terms of the different conformational species coexisting in the S-1 excited state. Measurements of kinetics in the near-infrared region revealed that at least three relaxation processes take place after excitation of spheroidene into its S-2 state. Ultrafast S-2 --> S-1 internal conversion occurs within the first 300 fs, followed by vibrational cooling in the SI state, which occurs on a time scale of similar to 700 fs. The S-1 lifetime is 8 ps at 293 K, in good agreement with previous measurements of the S-1 --> S-N transition. A somewhat longer S-1 lifetime of 9.5 ps is observed at 186 K.

Femtosecond transient absorption measurements were performed on native and a series of reconstituted LH2 complexes from Rhodopseudomonas acidophila 10050 at room temperature. The reconstituted complexes contain chemically modified tetrapyrrole pigments in place of the native bacteriochlorophyll a -B800 molecules. The spectral characteristics of the modified pigments vary significantly, such that within the B800 binding sites the B800 Q y absorption maximum can be shifted incrementally from 800 to 670nm. As the spectral overlap between the B800 and B850 Q y bands decreases, the rate of energy transfer (as determined by the time-dependent bleaching of the B850 absorption band) also decreases; the measured time constants range from 0.9ps (bacteriochlorophyll a in the B800 sites, Q y absorption maximum at 800nm) to 8.3ps (chlorophyll a in the B800 sites, Q y absorption maximum at 670nm). This correlation between energy transfer rate and spectral blue-shift of the B800 absorption band is in qualitative agreement with the trend predicted from Forster spectral overlap calculations, although the experimentally determined rates are ∼5 times faster than those predicted by simulations. This discrepancy is attributed to an underestimation of the electronic coupling between the B800 and B850 molecules.

Using two-color pump−probe femtosecond spectroscopy, the temperature dependence of the energy transfer rate within the peripheral light-harvesting antenna (LH2) of the photosynthetic bacterium Rhodobacter sphaeroides has been measured. The energy transfer time from B800 to B850 is determined to be 0.7, 1.2, and 1.5 ps at 300, 77, and 4.2 K, respectively. These data, combined with earlier results, have been analyzed with regard to the crystal structure and spectroscopic properties of the purple bacterial LH2 complex. We conclude that the transfer within B800 occurs mainly via the incoherent Forster hopping mechanism. For B800 to B850 transfer, estimates based on the Forster formula yield considerably slower transfer times than experimentally observed, suggesting that an additional mechanism may be involved in enhancing the transfer rate. We suggest two possibilities: transfer via the upper excitonic component of B850 band and/or transfer mediated by a carotenoid molecule.

Direct studies of diradicals, the molecular species hypothesized to be archetypal of chemical bond transformations in many classes of reactions, have been made using femtosecond laser techniques with mass spectrometry in a molecular beam. These studies are aimed at "freezing" the diradicals in time and in the course of the reaction. The passage of these species through the transition-state region was observed and the effect of total energy and alkyl substitution on the rates of bond closure and cleavage was examined. The results establish the nature of these intermediates and define their existence during reactions.

We present a simple example of temporal control of the evolving dynamics in a reactive system — a unimolecular reaction with two channels. Using three femtosecond pulses, a wave packet is prepared, probed, and intercepted in the transition-state region. The femtosecond control pulse intercepts the predissociative system at a fixed time and internuclear separation, both determined by the ‘window’ of the laser pulse. Experiments are performed on the NaI system which is a prototype for studies of elementary nuclear motion and reaction dynamics along two potentials, the covalent (Na + I) and the ionic (Na^+ + I^−) channels.

Femtosecond reaction dynamics of OClO in a supersonic molecular beam are reported. The system is excited to the A 2A2 state with a femtosecond pulse, covering a range of excitation in the symmetric stretch between v1=17 to v1=11 (308–352 nm). A time-delayed femtosecond probe pulse ionizes the OClO, and OClO + is detected. This ion has not been observed in previous experiments because of its ultrafast fragmentation. Transients are reported for the mass of the parent OClO as well as the mass of the ClO. Apparent biexponential decays are observed and related to the fragmentation dynamics: OClO+hnu-->(OClO)[double-dagger]*-->ClO+O -->Cl+O2 . Clusters of OClO with water (OClO)n (H2O)m with n from 1 to 3 and m from 0 to 3 are also observed. The dynamics of the fragmentation reveal the nuclear motions and the electronic coupling between surfaces. The time scale for bond breakage is in the range of 300–500 fs, depending on v1; surface crossing to form new intermediates is a pathway for the two channels of fragmentation: ClO+O (primary) and Cl+O2 (minor). Comparisons with results of ab initio calculations are made.

In the coherent anti-Stokes Raman scattering (CARS) process, the emitted signal carries both amplitude and phase information of the molecules in the focal volume. These components form a vector in the complex plane, with the magnitude given by the amplitude and the angle between the vector and the real axis determined by the phase. Most CARS experiments ignore the phase component, but its detection allows for two advantages over amplitude-only CARS. First, the pure resonant response can be determined—and the non-resonant background rejected—by extracting the imaginary component of the complex response, enhancing the sensitivity of CARS measurements[1]. Second, selectivity is increased via determination of the phase and amplitude, allowing separation of individual molecular components of a sample even when their vibrational bands overlap[2]. The vibrational responses of individual molecular species trace different trajectories through the complex plane as functions of driving frequency. Chemically selective images can be made by locating the regions of the complex plane belonging to each substance at a given driving frequency. Dissolutions and chemical reactions can be followed by tracking the complex-plane positions, relative to the initial compounds, of each location in the sample. Furthermore, quantitative concentration measurements can be accurately performed, even on homogeneous solutions containing molecules with congested spectra. Figure 1 illustrates the CARS spectra of two common plastics. For each vibrational frequency the amplitude of the CARS signal is designated as the length of a vector in complex space, with the phase defined as the angle separating the vector from the horizontal axis. The horizontal and vertical axes correspond to the real and imaginary components of the χ (3) tensor, with vibrational frequencies plotted on the third axis. The real and imaginary components can be directly extracted from this plot. Figure 2 shows the complex plane trajectories of ethanol and methanol around 3000 cm -1 , as well as concentration measurements of five volumetric mixtures at two different driving frequencies. Line (I) indicates a driving frequency where only the amplitudes of the vibrational responses differ between the two molecular species, while line (II) lies at a frequency where the amplitudes are similar but the phases are well separated. In both cases the measurements agree with predicted values of the complex location of the mixture.

We review the optical properties of the FMO complex as found by spectroscopic studies of the Qy band over the last two decades. This article emphasizes the different methods used, both experimental and theoretical, to elucidate the excitonic structure and dynamics of this pigment–protein complex. Electronic supplementary material The online version of this article (doi:10.1007/s11120-010-9540-1) contains supplementary material, which is available to authorized users.

Coherent anti-Stokes Raman scattering, better known as CARS, is a powerful and versatile spectroscopic and microscopic technique, with many advantages over traditional Raman and fluorescence methods. It is immune to autofluorescence, does not require labeling, and returns molecules to their ground states, which reduces sample heating. However, CARS suffers from two negative traits compared to Raman and fluorescence measurements. First, a non-resonant background is frequently more intense than the resonant contributions in a sample. Second, CARS signals are orders of magnitude weaker than Stokes Raman lines, rendering the detection of small concentrations of weakly-resonant molecules difficult. Our current research focuses on these two problems in particular: inherent polarization-sensitivity of CARS allows isolation and rejection of the non-resonant component of CARS, though at the cost of a significant reduction in resonant signal1; heterodyning techniques provide simultaneous enhancement of CARS signals and rejection of the non-resonant background, but require explicit knowledge of the phase relations in the CARS process2. Using a homebuilt optical parametric oscillator3 we have developed a method that exploits both heterodyning and polarization sensitivity that offers resonant-only enhancement of CARS signals without and specific knowledge of the phases. This “HIP-CARS” procedure significantly simplifies the detection elements of the system and provides for future development of fast, resonant-only CARS imaging.

The real‐time dynamics of hydrogen‐atom‐transfer processes under collisionless conditions are studied using femtosecond depletion techniques. The experiments focus on the methyl salicylate system, which exhibits ultrafast hydrogen motion between two oxygen atoms due to molecular tautomerization, loosely referred to as intramolecular ‘‘proton’’ transfer. To test for tunneling and mass effects on the excited potential surface, we also studied deuterium and methyl‐group substitutions. We observe that the motion of the hydrogen, under collisionless conditions, takes place within 60 fs. At longer times, on the picosecond time scale, the hydrogen‐transferred form decays with a threshold of 15.5 kJ/mol; this decay behavior was observed up to a total vibrational energy of ∼7200 cm−1. The observed dynamics provide the global nature of the motion, which takes into account bonding before and after the motion, and the evolution of the wave packet from the initial nonequilibrium state to the transferred form along the O–H—O reaction coordinate. The vibrational periods (2π/ω) of the relevant modes range from 13 fs (the OH stretch) to 190 fs (the low‐frequency distortion) and the motion involves (in part) these coordinates. The intramolecular vibrational‐energy redistribution dynamics at longer times are important to the hydrogen‐bond dissociation and to the nonradiative decay of the hydrogen‐transferred form.

We have achieved first steps toward coherent control of excitonic energy migration in the FMO pigment-protein complex, by combining femtosecond pulse shaping with a feedback loop using an evolutionary algorithm. The experimental conditions achieved, with a rotating sample, a cryostat, and a pulse shaper, are sufficient for closed loop optimizations.

Dissolution testing is a crucial part of pharmaceutical dosage form investigations and is generally performed by analyzing the concentration of the released drug in a defined volume of flowing dissolution medium. As solid-state properties of the components affect dissolution behavior to a large and sometimes even unpredictable extent there is a strong need for monitoring and especially visualizing solid-state properties during dissolution testing. In this study coherent anti-Stokes Raman scattering (CARS) microscopy was used to visualize the solid-state properties of lipid-based oral dosage forms containing the model drug theophylline anhydrate during dissolution in real time. The drug release from the dosage form matrix was monitored with a spatial resolution of about 1.5 microm. In addition, as theophylline anhydrate tends to form the less soluble monohydrate during dissolution, CARS microscopy allowed the solid-state transformation of the drug to be spatially visualized. The results obtained by CARS microscopy revealed that the method used to combine lipid and active ingredient into a sustained release dosage form can influence the physicochemical behavior of the drug during dissolution. In this case, formation of theophylline monohydrate on the surface was visualized during dissolution with tablets compressed from powdered mixtures but not with solid lipid extrudates.

Adaptive femtosecond pulse shaping in an evolutionary learning loop is applied to a bioinspired dyad molecule that closely mimics the early-time photophysics of the light-harvesting complex 2 (LH2) photosynthetic antenna complex. Control over the branching ratio between the two competing pathways for energy flow, internal conversion (IC) and energy transfer (ET), is realized. We show that by pulse shaping it is possible to increase independently the relative yield of both channels, ET and IC. The optimization results are analyzed by using Fourier analysis, which gives direct insight to the mechanism featuring quantum interference of a low-frequency mode. The results from the closed-loop experiments are repeatable and robust and demonstrate the power of coherent control experiments as a spectroscopic tool (i.e., quantum-control spectroscopy) capable of revealing functionally relevant molecular properties that are hidden from conventional techniques.

A caroteno-purpurin dyad molecule was studied by steady-state and pump-probe spectroscopies to resolve the excited-state deactivation dynamics of the different energy levels as well as the connecting energy flow pathways and corresponding rate constants. The data were analyzed with a two-step multi-parameter global fitting procedure that makes use of an evolutionary algorithm. We found that following ultrafast excitation of the donor (carotenoid) chromophore to its S2 state, the energy flows via two channels: energy transfer (70%) and internal conversion (30%) with time constants of 54 and 110 fs, respectively. Additionally, some of the initial excitation is found to populate the hot ground state, revealing another limitation to the functional efficiency. At later times, a back transfer occurs from the purpurin to the carotenoid triplet state in nanosecond timescales. Details of the energy flow within the dyad as well as species associated spectra are disentangled for all excited-state and ground-state species for the first time. We also observe oscillations with the most pronounced peak on the Fourier transform spectrum having a frequency of 530 cm(-1). The dyad mimics the dynamics of the natural light-harvesting complex LH2 from Rhodopseudomonas acidophila and is hence a good model system to be used in studies aimed to further explain previous work in which the branching ratio between the competing pathways of energy loss and energy transfer could be manipulated by adaptive femtosecond pulse shaping.

A promising photosensitizer, zinc phthalocyanine, is investigated by means of steady-state and time-resolved pump-probe spectroscopies. Spectrally resolved pump-probe data are recorded on time scales ranging from femtoseconds to nanoseconds. Global analysis yields the excited-state absorption spectra and lifetimes, as well as the pathways and efficiencies of the competing relaxation processes from the initially excited S1 state. In addition to the expected nanosecond-scale processes of fluorescence, internal conversion and inter-system crossing that follow the generally accepted kinetic scheme, we also resolve ultrafast dynamics. The nature of these fast processes and their implications to the functional pathway involving triplet formation are discussed.

Diarylethene derivatives are a promising group of photo- and electrochromic molecular switches for applications to a wide range of optoelectronic devices. Upon excitation with UV light, a cyclinzation reaction occurs to a thermally stable system, resulting in an extended π-conjugation that shifts the absorption toward the red. Cycloreversion is induced by exciting the molecules with visible light. The stability, spectral properties, switching rates, and quantum yields can be passively controlled by the choice of substituents. The goal is to actively control the switching dynamics using shaped femtosecond laser pulses. Optimization of the ring opening reaction and analysis of the optimal pump pulses will enable a better understanding of the switching mechanisms, providing details of the potential energy surfaces and the role of specific vibrational motions.

Motivated originally by the goal of steering a photoreaction into desired product channels, the concept of coherent control is to adapt the spectral and temporal characteristics of the excitation light to the inherent molecular resonances and dynamics, such that these can be selectively addressed and manipulated. In the last decade, the ultrafast dynamics of many atomic and molecular quantum systems in the gas and condensed phase have been controlled successfully. Motivations in chemistry are now 1) to perform spectroscopy by coherent control, which requires a deeper understanding of control mechanisms, 2) to treat more complex, biological photoreactions, and 3) the pragmatic use of coherent control techniques, for example, for pulse compression or enhanced contrast in multiphoton microscopy. As examples for 1) and 2) we review here the combined effort and interplay of conventional spectroscopy and coherent control experiments, applied to the energy flow in the light-harvesting complex LH2 from bacterial photosynthesis. Closed-loop control experiments allowed the characteristic coupling frequency of internal conversion in the carotenoid in LH2 to be extracted. Open-loop three-pulse control experiments, on the other hand, could directly observe an anticipated Raman-excited carotenoid ground state. As a variant of difference spectroscopy, coherent control has thus served to gain complementary spectroscopic knowledge about the energy flow in carotenoids by comparing natural to manipulated dynamics. Finally, we propose future coherent control experiments on the electronic state structure of carotenoids and discuss prospects of coherent control for other biological chromophores.

We introduce alternative femtosecond spectroscopy techniques to study unattributed carotenoid deactivation signals in broadband transient absorption. These are shaped VIS excitation, nonlinear IR excitation, or insertion of an additional IR depletion pulse to manipulate excited state population.

The standard three-level scheme of carotenoids consists of the ground state S 0 (lAg ‑ in idealized C 2h symmetry), the absorbing bright state S 2 (1B u + ) and the intermediate dark state S 1 (2Ag - ). Internal conversion from S 2 via S 1 to the ground state has long been thought to be the dominant deactivation channel for the biologically relevant carotenoids with N=9-13 conjugated double bonds. According to this three-level model, after absorption of light in the blue-green region by the S 2 state (1Bu + ) the population decays through two channels: ultra fast (100-200 fs) internal conversion to the S 1 state and direct fluorescence to the ground state with almost negligible quantum yield. The involvement of 1Bu + and 3Ag - dark states in deactivation energy flow is yet to be found. In addition, the role of state referred to as S* giving a spectral signature on the red side of ground state absorption is still unknown. This chapter discusses the nature of the transient absorption signal attributed to S* on a series of β-carotene derivatives with conjugation lengths N = 9, 11, 13, 15.

The function of a light-harvesting antenna complex is to harvest sunlight and, through a series of energy transfer steps both within and between complexes, to make the energy available to the reaction centre, where it is used to fuel the primary dark reactions of photosynthesis. Harvested excitation energy from the absorption of blue-green light in carotenoids undergoes ultrafast partitioning between excitation energy transfer (EET) to bacteriochlorophyll (BChl) and losses by internal conversion (IC) within the carotenoid (Car). The classical scheme of the energy flow describes the carotenoid IC as a sequential process from the absorbing Car S 2 state, 1B u + in the idealised C 2h symmetry, to the lower-lying Car S 1 (2Ag - ) state and further on to the ground state Car S 0 (lAg - ). The EET channel takes roughly half of the excitation energy directly from Car S 2 to the BChls, which ultimately resides in the lowest-lying singlet state of the complex, BChl B850 Qy. This chapter presents additional data in order to extract a characteristic ‘‘control’’ frequency and establish a detailed picture of the laser-chromophore interaction and the ensuing dynamics. It demonstrates the use of coherent control as a valuable tool for the spectroscopy of complex molecules.

A new channel of excitation energy deactivation in bacterial light harvesting was recently discovered, which leads to carotenoid triplet population on an ultrafast timescale. Here we show that this mechanism is also active in LH2 of Rhodopseudomonas acidophila through analysis of transient absorption data with an evolutionary target analysis. The algorithm offers flexible testing of kinetic network models with low a priori knowledge requirements. It applies universally to the simultaneous fitting of target state spectra and rate constants to time-wavelength-resolved data. Our best-fit model reproduces correctly the well-known cooling and decay behavior in the S1 band, but necessitates an additional, clearly distinct singlet state that does not exchange with S1, promotes ultrafast triplet population and participates in photosynthetic energy transfer.

Quantum control of photochemistry exploits molecular interferences by adjusting the phases of multiple quantum pathways to different product channels via adapted excitation light. Experiments implement a learning loop in which molecular feedback iteratively refines the excitation pulse until an optimum solution directs the photoreaction towards a desired channel [1]. So far, feedback control has been absent from the world of biology. Furthermore, only few examples have proven explicitly the role of molecular coherence. Here we report feedback-optimised coherent control of biological function, namely regulation of the energy flow pathways in the 125 kDa light-harvesting antenna complex LH2 from Rhodospeudomonas acidophila, a photosynthetic purple bacterium.

Coherent light sources have been widely used in control schemes that exploit quantum interference effects to direct the outcome of photochemical processes. The adaptive shaping of laser pulses is a particularly powerful tool in this context: experimental output as feedback in an iterative learning loop refines the applied laser field to render it best suited to constraints set by the experimenter. This approach has been experimentally implemented to control a variety of processes, but the extent to which coherent excitation can also be used to direct the dynamics of complex molecular systems in a condensed-phase environment remains unclear. Here we report feedback-optimized coherent control over the energy-flow pathways in the light-harvesting antenna complex LH2 from Rhodopseudomonas acidophila, a photosynthetic purple bacterium. We show that phases imprinted by the light field mediate the branching ratio of energy transfer between intra- and intermolecular channels in the complex's donor acceptor system. This result illustrates that molecular complexity need not prevent coherent control, which can thus be extended to probe and affect biological functions.

In this work, we have determined the energy of the S, state of the carotenoid spheroidene. The energy of this state is 13,400 ± 90 cm-1 at both 293 K and 186 K, showing that there is no temperature-induced shift of the SI level. A discrepancy of about 800 cm-1 between the S1 energy determined here and that obtained from previous fluorescence and resonance Raman measurements is explained in terms of the different conformational species co-existing in the S1 excited state.

The critical stage in a chemical reaction — the progression through the transition state from reagents to products — occurs in less than a picosecond (10^(−12)s). Using laser pulses of femtosecond (10^(−15)s) duration it is possible to probe the nuclear motions throughout formation and break-up of the transition state. The coherence and very short duration of these femtosecond pulses provides a means to influence the course of the reaction during this stage if the time resolution is made sufficiently short. Here we describe a demonstration of such control of a chemical reaction on the femtosecond timescale. Using two sequential coherent laser pulses, we can control the reaction of iodine molecules with xenon atoms to form the product XeI by exciting the reactants through the transition state, in a two-step process. The yield of product XeI is modulated as the delay between the pulses is varied, reflecting its dependence on the nuclear motions of the reactants.

Singlet-singlet annihilation is used to study exciton delocalization in the light harvesting antenna complex LH2 (B800-B850) from the photosynthetic purple bacterium Rhodobacter sphaeroides. The characteristic femtosecond decay constants of the high intensity isotropic and the low intensity anisotropy kinetics of the B850 ring are related to the hopping time tau(h) and the coherence length N(coh) of the exciton. Our analysis yields N(coh) = 2.8+/-0.4 and tau(h) = 0.27+/-0.05 ps. This approach can be seen as an extension to the concept of the spectroscopic ruler.

Carrier (exciton) multiplication in colloidal InAs/CdSe/ZnSe core−shell quantum dots (QDs) is investigated using terahertz time-domain spectroscopy, time-resolved transient absorption, and quasi-continuous wave excitation spectroscopy. For excitation by high-energy photons (∼2.7 times the band gap energy), highly efficient carrier multiplication (CM) results in the appearance of multi-excitons, amounting to ∼1.6 excitons per absorbed photon. Multi-exciton recombination occurs within tens of picoseconds via Auger-type processes. Photodoping (i.e., photoinjection of an exciton) of the QDs prior to excitation results in a reduction of the CM efficiency to ∼1.3. This exciton-induced reduction of CM efficiency can be explained by the twofold degeneracy of the lowest conduction band energy level. We discuss the implications of our findings for the potential application of InAs QDs as light absorbers in solar cells.

IN chemical reactions, the dynamics of the transition from reagents to products can be described by the trajectories of particles (or rigorously, of quantum mechanical wave packets) moving on a potential-energy surface. Here we use femtosecond pulsed laser techniques to follow directly the evolution in space and time of such trajectories during the breakage of a chemical bond in the dissociation of sodium iodide. The bond breakage can be described in terms of the time evolution of a single reaction coordinate, the internuclear separation. As the velocities of the separating fragments are typically of the order of a kilometre per second, a time resolution of a few tens of femtoseconds is required to view the motions on a molecular distance scale of less than an angstrom. The resolution obtained here permits the direct visualization of the wave packet's motion and provides snapshots of the trajectories along the reaction coordinate.

The description of chemical reaction dynamics often assumes that vibrational modes are well coupled (ergodic) and redistribute energy rapidly with respect to the course of the reaction. To experimentally probe nonergodic, nonstatistical behavior, studies of a series of reactions induced by femtosecond activation for molecules of varying size but having the same reaction coordinates [CH 2 − (CH 2 ) n − 2 − C = O † → products, with n = 4, 5, 6, and 10] were performed. Comparison of the experimental results with theoretical electronic structure and rate calculations showed a two to four orders of magnitude difference, indicating that the basic assumption of statistical energy redistribution is invalid. These results suggest that chemical selectivity can be achieved with femtosecond activation even at very high energies.

The organization of bacteriochlorphyll a (BChl) and carotenoid pigments within the peripheral light harvesting complex of purple bacteria, LH2, has been partially generalized using the 2.5 A resolution crystal structure obtained for Rps. acidophila (1) and is illustrated in Figure 1. Two types of BChl molecules, distinguished by their binding sites and separated by ~20 A, are labeled according to their peak absorption wavelengths as B850 and B800. Carotenoid pigments are observed to “snake” between the BChl molecules. The first, fully resolved in the crystallographic structure, spans the membrane on the inside of the ring with the B800 BChl located at approximately the center of the conjugated region. The head of this carotenoid lies near the N terminus of the protein, where several polar residues can be found. The second carotenoid has been only partially resolved, with the head located on the opposite end of the membrane below the B850 BChls.

The primary events in bacterial photosynthesis involve the capture of light energy by light harvesting (LH) antenna complexes, followed by rapid and efficient energy transfer to a reaction centre (RC), where the absorbed energy is “trapped”. In most species of purple bacteria, there are two types of light harvesting complex. The first type, LH1, is intimately associated with the RC to form a “core” complex. Arranged more peripherally to this and present in variable amounts is the second type of complex called LH2. Both types of LH complex are built on a similar modular principle, in which the light-absorbing pigments bacteriochlorophyll a (Bchla) and carotenoid molecules are non-covalently bound to two low Mr hydrophobic apoproteins α and β. The native complexes are oligomers of these monomeric units. The crystal structure of the LH2 complex from Rps. acidophila 10050 has recently been determined to a resolution of 2.5 A° by McDermott et al1. The complex was observed to be an α9β9 oligomer, with each monomeric unit containing an α and β apoprotein, 3 Bchla molecules and 1 (or 2?) carotenoid (rhodopin-glucoside) molecules. The 9 α apoproteins form a hollow cylinder with the 9 β apoproteins arranged radially outside them. Eighteen Bchla molecules are sandwiched between the α and β apoprotein helices and form a continous overlapping ring. These molecules absorb at approximately 850 nm (B850) and have their bacteriochlorin rings perpendicular to the membrane surface. A further nine Bchla molecules are positioned more peripherally in the complex, situated between the β apoproteins.

At low temperature a new dynamic feature has been observed in LH2 at longer delays by Chachisvilis et al (1, 2). The stimulated emission/bleaching band broadens and splits into two bands in about 3 ps. The new band is located at about 870 nm and it continues to move further to the red and broadens on the tens of picoseconds time scale. Early 4.2 K steady state fluorescence measurements of a LH1-less mutant of Rb. sphaeroides showed an unusually large Stokes’ shift, which was explained by an emission from a minor long-wavelength component of B850 (3). On the other hand recent time resolved data were interpreted as a stimulated emission from the lowest exciton component of a disordered ring of B850 (4, 5), whereas the slower phases of the dynamics were assigned to the transfer among inhomogeneously distributed rings (5). The zero phonon hole action spectra have also been interpreted as to position the lowest exciton component of B850 at 870 nm (6). The effect is much less pronounced in LH1 suggesting that it may be related to the tendency of LH2 to form very large aggregates (7, 8) or to some LH2-specific impurity. In this work we try to identify the origin of this emission.

Transient absorption spectra and kinetics are used for detailed studies of energy transfer dynamics within the B850 ring of the LH2 antenna of the purple bacterium Rb. sphaeroides. The initial exciton relaxation was found to occur within the first 100 fs and no temperature dependence of this process was observed. Transient absorption spectra of the LH2 antenna complex at 6 K revealed a new spectral band around 880 nm as a result of energy distribution of the lowest exciton states of B850 rings at cryogenic temperatures.

In this communication, we present our first femtosecond (fs), real-time studies of the retro-Diels-Alder reactions of norbornene (NB) and norbornadiene (NBD), products of the cycloaddition of cyclopentadiene with ethylene and acetylene, respectively. The reaction path, together with transition state (TS) structures, is displayed in Figure 1 (bottom). The experiments were designed with the following considerations in mind. First, the reactions were studied in a molecular beam in order to isolate the elementary processes, free of solvent perturbations. Second, we examined the dynamics of the reaction from a well-defined initial geometry, a precursor of Diels-Alder products.