In situ/In vivo Optical Microspectroscopy to Probe the Emergence of Morphology.

Morphology governs function. Yet, understanding and controlling the emergence of morphology at the molecular level remains challenging. The difficulty in studying the early stage of morphology formation is due to its stochastic nature both spatially and temporally occurring at the nanoscale. This nature has been particularly detrimental for the application of optical spectroscopy. To overcome this problem, we have been developing new in situ/in vivo optical spectroscopy tools, which are label-free and non-invasive. This account highlights several examples of how optical spectroscopy can become an important tool in studying the birth of morphology.

Toward time resolved dynamic light scattering microscopy: Retrieving particle size distributions at high temporal resolutions.

Dynamic light scattering (DLS) is a widely applied technique in multiple scientific and industrial fields for the size characterization of nanoscale objects in solution. While DLS is typically applied to characterize systems under static conditions, the emerging interest in using DLS on temporally evolving systems stimulates the latent need to improve the time resolution of measurements. Herein, we present a DLS microscopy setup (micro-DLS) that can accurately characterize the size of particles from autocorrelation functions built from sub-100 ms time windows, several orders of magnitude faster than previously reported. The system first registers the arrival time of the scattered photons using a time-correlated single photon counting module, which allows the construction of the autocorrelation function for size characterization based on a time window of freely chosen position and width. The setup could characterize both monomodal (60 or 220 nm polystyrene particles; PS) and multimodal size distributions (e.g., mixture of 20 nm LUDOX and 80 nm PS) with high accuracy in a sub-100 ms time window. Notably, the width of the size distribution became narrower as a shorter time window was used. This was attributed to the ability of the system to resolve the sub-ensemble of the broad size distribution, as the broad distribution could be reconstructed by accumulating the distribution obtained by consecutive 80 ms time windows. A DLS system with high temporal resolution will accelerate the expansion of its application toward systems that evolve as a function of time beyond its conventional use on static systems.

Unveiling the Configurational Landscape of Carbamate: Paving the Way for Designing Functional Sequence-Defined Polymers.

Carbamate is an emerging class of a polymer backbone for constructing sequence-defined, abiotic polymers. It is expected that new functional materials can be de novo designed by controlling the primary polycarbamate sequence. While amino acids have been actively studied as building blocks for protein folding and peptide self-assembly, carbamates have not been widely investigated from this perspective. Here, we combined infrared (IR), vibrational circular dichroism (VCD), and nuclear magnetic resonance (NMR) spectroscopy with density functional theory (DFT) calculations to understand the conformation of carbamate monomer units in a nonpolar, aprotic environment (chloroform). Compared with amino acid building blocks, carbamates are more rigid, presumably due to the extended delocalization of π-electrons on the backbones. Cis configurations of the amide bond can be energetically stable in carbamates, whereas peptides often assume trans configurations at low energies. This study lays an essential foundation for future developments of carbamate-based sequence-defined polymer material design.

Hazardous chemical elements in cleaning cloths, a potential source of microfibres.

Although potentially hazardous chemical elements (e.g., Cu, Cr, Pb, Sb, Ti, Zn) have been studied in clothing textiles, their presence in cleaning textiles is unknown. In this study, 48 cleaning cloth products (consisting of 81 individual samples) purchased in Europe, and consisting of synthetic (petroleum-based), semi-synthetic or natural fibres or combinations of these different types, have been analysed for 16 chemical elements by X-ray fluorescence (XRF) spectrometry. Titanium was detected in most cases (median and maximum concentrations ~3700 and 12,400 mg kg-1, respectively) and Raman microspectroscopy revealed that TiO2 was present as anatase. Barium, Br, Cr, Cu, Fe and Zn were frequently detected over a range of concentrations, reflecting the presence of various additives, and Sb was present at concentrations up to about 200 mg kg-1 in samples containing polyester as catalytic residue from the polymerisation process. Lead was detected as a contaminant in four samples and at concentrations below 10 mg kg-1. Overall, the range of the chemical element profiles and concentrations was similar to those for clothing materials published in the literature, suggesting that broadly the same additives, materials and processes are employed to manufacture cloths and clothing textiles. The mechanisms by which potentially hazardous chemical elements are released into the environment with microfibres or mobilised into soluble or nano-particulate forms remain to be explored.

In situ optical spectroscopy of crystallization: One crystal nucleation at a time.

While crystallization is a ubiquitous and an important process, the microscopic picture of crystal nucleation is yet to be established. Recent studies suggest that the nucleation process can be more complex than the view offered by the classical nucleation theory. Here, we implement single crystal nucleation spectroscopy (SCNS) by combining Raman microspectroscopy and optical trapping induced crystallization to spectroscopically investigate one crystal nucleation at a time. Raman spectral evolution during a single glycine crystal nucleation from water, measured by SCNS and analyzed by a nonsupervised spectral decomposition technique, uncovered the Raman spectrum of prenucleation aggregates and their critical role as an intermediate species in the dynamics. The agreement between the spectral feature of prenucleation aggregates and our simulation suggests that their structural order emerges through the dynamic formation of linear hydrogen-bonded networks. The present work provides a strong impetus for accelerating the investigation of crystal nucleation by optical spectroscopy.

Control of Intermolecular Photoinduced Electron Transfer in Deoxyadenosine-Based Fluorescent Probes.

In this work, we report on the Photoinduced Electron Transfer (PET) reaction between a donor (adenine analogue) and an acceptor (3-methoxychromone dye, 3MC) in the context of designing efficient fluorescent probes as DNA sensors. Firstly, Gibbs energy was investigated in disconnected donor-acceptor systems by Rehm-Weller equation. The oxidation potential of the adenine derivative was responsible for exergonicity of the PET reaction in separated combinations. Then, the PET reaction in donor-π-acceptor conjugates was investigated using steady-state fluorescence spectroscopy, acid-mediated PET inhibition and transient absorption techniques. In conjugated systems, PET is a favorable pathway of fluorescent quenching when an electron-rich adenine analogue (d7A) was connected to the fluorophore (3MC). We found that formation of ground-state complexes even at nm concentration range dominated the dye photophysics and generated poorly emissive species likely through intermolecular PET from d7A to 3MC. On the other hand, solution acidification disrupts complexation and turns on the dye emission. Bridging an electron-poor adenine analogue with high oxidation potential (8 d7A) to 3MC presenting low reduction potential is another alternative to prevent complex formation and produce highly emissive monomer conjugates.

Excited-State Proton Transfer in Oxyluciferin and Its Analogues.

One of the most characterized bioluminescent reactions involves the firefly luciferase that catalyzes the oxidation of the luciferin producing oxyluciferin in its first excited state. While relaxing to the ground state, oxyluciferin emits visible light with an emission maximum that can vary from green to red. Oxyluciferin exists under six different chemical forms resulting from a keto/enol tautomerization and the deprotonation of the phenol or enol moieties. The optical properties of each chemical form have been recently characterized by the investigations of a variety of oxyluciferin derivatives, indicating unresolved excited-state proton transfer (ESPT) reactions. In this work, femtosecond pump-probe spectroscopy and time-resolved fluorescence spectroscopy are used to investigate the picosecond kinetics of the ESPT reactions and demonstrate the excited state keto to enol conversion of oxyluciferin and its derivatives in aqueous buffer as a function of pH. A comprehensive photophysical scheme is provided describing the complex luminescence pathways of oxyluciferin in protic solution.

E to Z Photoisomerization of Phytochrome Cph1Δ Exceeds the Born-Oppenheimer Adiabatic Limit.

The Born-Oppenheimer adiabatic limit applies broadly in chemistry because most reactions occur on the ground electronic state. Photochemical reactions involve two or more electronic states and need not be subject to this adiabatic limit. The spectroscopic signatures of nonadiabatic processes are subtle, and therefore, experimental investigations have been limited to the few systems dominated by single photochemical outcomes. Systems with branched excited-state pathways have been neglected, despite their potential to reveal insights into photochemical reactivity. Here we present experimental evidence from coherent three-dimensional electronic spectroscopy that the E to Z photoisomerization of phytochrome Cph1 is strongly nonadiabatic, and the simulations reproduce the measured features only when the photoisomerization proceeds nonadiabatically near, but not through, a conical intersection. The results broaden the general understanding of photoisomerization mechanisms and motivate future studies of nonadiabatic processes with multiple outcomes arising from branching on excited-state potential energy surfaces.

Resolving the Singlet Excited State Manifold of Benzophenone by First-Principles Simulations and Ultrafast Spectroscopy.

Accurate characterization of the high-lying excited state manifolds of organic molecules is of fundamental importance for the interpretation of the rich response detected in time-resolved nonlinear electronic spectroscopies. Here, we have characterized the singlet excited state manifold of benzophenone (BP), a versatile organic photoinitiator and a well-known DNA photosensitizer. Benchmarks of various multiconfigurational/multireference (RASSCF/PT2) and time-dependent density functional theory (TD-DFT) approaches allowed assignments of experimental linear absorption signals of BP in the ultraviolet (UV) region, with unprecedented characterization of ground state absorptions in the far UV. Experimental transient absorption spectra obtained by UV-vis pump-probe spectroscopy at very short time delays are shown to be directly comparable to theoretical estimates of excited state absorptions (from the low-lying nOπ* and ππ* singlet states) in the Franck-Condon region. Multireference computations provided reliable interpretation of the PP spectra, with TD-DFT results yielding a fair agreement as long as electronic transitions featuring double excitations contributions are not involved. These results lay the groundwork for further computational studies and interpretation of experimental nonlinear electronic spectra of benzophenone in more complex systems, such as BP/DNA adducts.

Electronic Couplings in (Bio-) Chemical Processes.

During the last two decades, 2D optical techniques have been extended to the visible range, targeting electronic transitions. Since the report of the very first 2D electronic measurement (Hybl et al. in J Chem Phys 115:6606-6622, [2001]), two-dimensional electronic spectroscopy (2DES) has allowed fundamentally new insights into the structure and dynamics of condensed-phase systems (Ginsberg et al. in Acc Chem Res 42:1352-1363, 2009; Jonas in Annu Rev Phys Chem 54:425-463, 2003), producing experiments that measure correlations among electronic states of an absorbing species within complex systems. 2DES is used to investigate photo-physical phenomena involving electronic or vibrational couplings in multi-chromophoric systems [energy transfer in photosynthesis is one great example of how 2DES can disentangle various energy transfer pathways (Brixner et al. in Nature 625-628, 2005; Engel et al. in Nature 446:782-786, 2007; Collini et al. in Nature 463:644-647, 2010)], but also ultrafast photochemical processes in which the tracked molecules change permanently or are heterogeneous (Ruetzel et al. in Proc Natl Acad Sci 111:4764-4769, 2014; Consani et al. in Science 339:1586-1589, 2013). We divide this chapter according to some of the major areas that have been established thanks to 2DES in the following fields: heterogeneity of systems, excitation energy transfer mechanisms, photo-induced coherent oscillations associated with electronic and vibrational couplings, and complex chemical reactions (Fig. 1). Fig. 1 Main fields impacted by two-dimensional electronic spectroscopy (2DES) in condensed phase. The major discoveries of each field will be described in different paragraphs.

The effect of solvent relaxation in the ultrafast time-resolved spectroscopy of solvated benzophenone.

Benzophenone (BP) despite its relatively simple molecular structure is a paradigmatic sensitizer, featuring both photocatalytic and photobiological effects due to its rather complex photophysical properties. In this contribution we report an original theoretical approach to model realistic, ultra-fast spectroscopy data, which requires describing intra- and intermolecular energy and structural relaxation. In particular we explicitly simulate time-resolved pump-probe spectra using a combination of state-of-the art hybrid quantum mechanics/molecular mechanics dynamics to treat relaxation and vibrational effects. The comparison with experimental transient absorption data demonstrates the efficiency and accuracy of our approach. Furthermore the explicit inclusion of the solvent, water for simulation and methanol for experiment, allows us, despite the inherent different behavior of the two, to underline the role played by the H-bonding relaxation in the first hundreds of femtoseconds after optical excitation. Finally we predict for the first time the two-dimensional electronic spectrum (2DES) of BP taking into account the vibrational effects and hence modelling partially symmetric and asymmetric ultrafast broadening.

Conformational Homogeneity in the Pr Isomer of Phytochrome Cph1.

Numerous time-resolved studies of the Pr to Pfr photoisomerization in phytochrome Cph1 have revealed multiphasic excited-state decay kinetics. It remains unclear whether these kinetics arise from multiple ground-state conformational subpopulations or from a single ground-state conformation that undergoes an excited-state photoisomerization process-either branching on the excited state or relaxing through multiple sequential intermediates. Many studies have attempted to resolve this debate by fitting the measured dynamics to proposed kinetic models, arriving at different conclusions. Here we probe spectral signatures of ground-state heterogeneity of Pr. Two-dimensional electronic spectra display negligible inhomogeneous line broadening, and vibrational coherence spectra extracted from transient absorption measurements do not contain nodes and phase shifts at the fluorescence maximum. These spectroscopic results support the homogeneous model, in which the primary photochemical transformation of Pr to Lumi-R occurs adiabatically on the excited-state potential energy surface.

High-Energy Long-Lived Mixed Frenkel-Charge-Transfer Excitons: From Double Stranded (AT)n to Natural DNA.

The electronic excited states populated upon absorption of UV photons by DNA are extensively studied in relation to the UV-induced damage to the genetic code. Here, we report a new unexpected relaxation pathway in adenine-thymine double-stranded structures (AT)n . Fluorescence measurements on (AT)n hairpins (six and ten base pairs) and duplexes (20 and 2000 base pairs) reveal the existence of an emission band peaking at approximately 320 nm and decaying on the nanosecond time scale. Time-dependent (TD)-DFT calculations, performed for two base pairs and exploring various relaxation pathways, allow the assignment of this emission band to excited states resulting from mixing between Frenkel excitons and adenine-to-thymine charge-transfer states. Emission from such high-energy long-lived mixed (HELM) states is in agreement with their fluorescence anisotropy (0.03), which is lower than that expected for π-π* states (≥0.1). An increase in the size of the system quenches π-π* fluorescence while enhancing HELM fluorescence. The latter process varies linearly with the hypochromism of the absorption spectra, both depending on the coupling between π-π* and charge-transfer states. Subsequently, we identify the common features between the HELM states of (AT)n structures with those reported previously for alternating (GC)n : high emission energy, low fluorescence anisotropy, nanosecond lifetimes, and sensitivity to conformational disorder. These features are also detected for calf thymus DNA in which HELM states could evolve toward reactive π-π* states, giving rise to delayed fluorescence.

Experimental Detection of Branching at a Conical Intersection in a Highly Fluorescent Molecule.

Conical intersections are molecular configurations at which adiabatic potential-energy surfaces touch. They are predicted to be ubiquitous, yet condensed-phase experiments have focused on the few systems with clear spectroscopic signatures of negligible fluorescence, high photoactivity, or femtosecond electronic kinetics. Although rare, these signatures have become diagnostic for conical intersections. Here we detect a coherent surface-crossing event nearly two picoseconds after optical excitation in a highly fluorescent molecule that has no photoactivity and nanosecond electronic kinetics. Time-frequency analysis of high-sensitivity measurements acquired using sub-8 fs pulses reveals phase shifts of the signal due to branching of the wavepacket through a conical intersection. The time-frequency analysis methodology demonstrated here on a model compound will enable studies of conical intersections in molecules that do not exhibit their diagnostic signatures. Improving the ability to detect conical intersections will enrich the understanding of their mechanistic role in molecular photochemistry.

Resolving molecular vibronic structure using high-sensitivity two-dimensional electronic spectroscopy.

Coherent multidimensional optical spectroscopy is an emerging technique for resolving structure and ultrafast dynamics of molecules, proteins, semiconductors, and other materials. A current challenge is the quality of kinetics that are examined as a function of waiting time. Inspired by noise-suppression methods of transient absorption, here we incorporate shot-by-shot acquisitions and balanced detection into coherent multidimensional optical spectroscopy. We demonstrate that implementing noise-suppression methods in two-dimensional electronic spectroscopy not only improves the quality of features in individual spectra but also increases the sensitivity to ultrafast time-dependent changes in the spectral features. Measurements on cresyl violet perchlorate are consistent with the vibronic pattern predicted by theoretical models of a highly displaced harmonic oscillator. The noise-suppression methods should benefit research into coherent electronic dynamics, and they can be adapted to multidimensional spectroscopies across the infrared and ultraviolet frequency ranges.

Stabilization of Mixed Frenkel-Charge Transfer Excitons Extended Across Both Strands of Guanine-Cytosine DNA Duplexes.

The photoreactive pathways that may lead to DNA damage depend crucially upon the nature of the excited electronic states. The study of alternating guanine-cytosine duplexes by fluorescence spectroscopy and quantum mechanical calculations identifies a novel type of excited states that can be populated following UVB excitation. These states, denoted High-energy Emitting Long-lived Mixed (HELM) states, extend across both strands and arise from mixing between cytosine Frenkel excitons and guanine-to-cytosine charge transfer states. They emit at energies higher than ππ* states localized on single bases, survive for several nanoseconds, are sensitive to the ionic strength of the solution, and are strongly affected by the structural transition from the B form to the Z form. Their impact on the formation of lesions of the genetic code needs to be assessed.

Accurate convergence of transient-absorption spectra using pulsed lasers.

Transient-absorption spectroscopy is a common and well-developed technique for measuring time-dependent optical phenomena. One important aspect, especially for measurements using pulsed lasers, is how to average multiple data acquisition events. Here, we use a mathematical analysis method based on covariance to evaluate various averaging schemes. The analysis reveals that the baseline and the signal converge to incorrect values without balanced detection of the probe, shot-by-shot detection, and a specific method of averaging. Experiments performed with sub-7 fs pulses confirm the analytic results and reveal insights into molecular excited-state vibrational dynamics.

Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy.

Guanine-cytosine hairpins, containing a hexaethylene glycol bridge, are studied by steady-state fluorescence spectroscopy and time-correlated single photon counting; their properties are compared to those of duplexes with the same sequence. It is shown that, both in hairpins and in duplexes, base pairing induces quenching of the ππ* fluorescence, the quantum yield decreasing by at least two orders of magnitude. When the size of the systems increases from two to ten base pairs, a fluorescent component decaying on the nanosecond time-scale appears at energy higher than that stemming from the bright states of non-interacting mono-nucleotides (ca. 330 nm). For ten base pairs, this new fluorescence forms a well-defined band peaking at 305 nm. Its intensity is about 20% higher for the hairpin compared to the duplex. Its position (red-shifted by 1600 cm(-1)) and width (broader by 1800 cm(-1) FWHM) differ from those observed for large duplexes containing 1000 base pairs, suggesting the involvement of electronic coupling. Fluorescence anisotropy reveals that the excited states responsible for high energy emission are not populated directly upon photon absorption but are reached during a relaxation process. They are assigned to charge transfer states. According to the emerging picture, the amplitude of conformational motions determines whether instantaneous deactivation to the ground state or emission from charge transfer states will take place, while ππ* fluorescence is associated to imperfect base-pairing.

Mimicking conjugated polymer thin-film photophysics with a well-defined triblock copolymer in solution.

Conjugated polymers (CPs) are promising materials for use in electronic applications, such as low-cost, easily processed organic photovoltaic (OPV) devices. Improving OPV efficiencies is hindered by a lack of a fundamental understanding of the photophysics in CP-based thin films that is complicated by their heterogeneous nanoscale morphologies. Here, we report on a poly(3-hexylthiophene)-block-poly(tert-butyl acrylate)-block-poly(3-hexylthiophene) rod-coil-rod triblock copolymer. In good solvents, this polymer resembles solutions of P3HT; however, upon the addition of a poor solvent, the two P3HT chains within the triblock copolymer collapse, affording a material with electronic spectra identical to those of a thin film of P3HT. Using this new system as a model for thin films of P3HT, we can attribute the low fluorescence quantum yield of films to the presence of a charge-transfer state, providing fundamental insights into the condensed phase photophysics that will help to guide the development of the next generation of materials for OPVs.