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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...

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.

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.

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.

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.

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.

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.

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.

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.

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.

We perform phase-sensitive near-field scanning optical microscopy on photonic-crystal waveguides. The observed intricate field patterns are analyzed by spatial Fourier transformations, revealing several guided transverse electric (TE) and transverse magnetic (TM) like modes. Using the reconstruction algorithm proposed by Ha et al. [Opt. Lett. 34, 3776 (2009)], we decompose the measured two-dimensional field pattern in a superposition of propagating Bloch modes. This opens new possibilities to study specific modes in near-field measurements. We apply the method to study the transverse behavior of a guided TE-like mode, where the mode extends deeper in the surrounding photonic crystal when the band edge is approached

Summary by J.L. Herek. Nobel prize-winner: Professor Ahmed Zewail. In the spring of 1990, I was a young liberal arts student soon to graduate from Lawrence University in Wisconsin, dreaming of a career in chemical physics. Thanks to an elective course on applications of lasers in chemistry and physics, which also required a literature study and report, I had found that a revolution in chemical physics was underway, with many groups clamouring to conquer new territory in the study of chemical reactions: the elusive and fleeting transition state. My interest in this emerging field dictated my choices of potential graduate schools, with Caltech and the group of Ahmed Zewail at the top of my list.