Tuning the Optical Properties Through Hydrogen Bond-assisted H-aggregate Formation: The ODIN Case.

The current work focuses on the investigation of two functionalized naphthyridine derivatives, namely ODIN-EtPh and ODIN-But, to gain insights into the hydrogen bond-assisted H-aggregate formation and its impact on the optical properties of ODIN molecules. By employing a combination of X-ray and electron crystallography, absorption and emission spectroscopy, time resolved fluorescence and ultrafast pump-probe spectroscopy (visible and infrared) we unravel the correlation between the structure and light-matter response, with a particular emphasis on the influence of the polarity of the surrounding environment. Our experimental results and simulations confirm that in polar and good hydrogen-bond acceptor solvents (DMSO), the formation of dimers for ODIN derivatives is strongly inhibited. The presence of a phenyl group linked to the ureidic unit favors the folding of ODIN derivatives (forming an intramolecular hydrogen bond) leading to the stabilization of a charge-transfer excited state which almost completely quenches its fluorescence emission. In solvents with a poor aptitude for forming hydrogen bonds, the formation of dimers is favored and gives rise to H aggregates, with a consequent considerable reduction in the fluorescence emission. The urea-bound phenyl group furtherly stabilizes the dimers in chloroform.

Nanothermometer Based on Polychlorinated Trityl Radicals Showing Two-Photon Excitation and Emission in the Biological Transparency Window: Temperature Monitoring of Biological Tissues.

Nanothermometers are emerging probes as biomedical diagnostic tools. Especially appealing are nanoprobes using NIR light in the range of biological transparency window (BTW) since they have the advantages of a deeper penetration into biological tissues, better contrast, reduced phototoxicity and photobleaching. This article reports the preparation and characterization of organic nanoparticles (ONPs) doped with two polychlorinated trityl radicals (TTM and PTM), as well as studies of their electronic and optical properties. Such ONPs having inside isolated radical molecules and dimeric excimers, can be two-photon excited showing optimal properties for temperature sensing. Remarkably, in TTM-based ONPs the emission intensity of the isolated radical species is unaltered increasing temperature, while the excimer emission intensity decreases strongly being thereby able to monitor temperature changes with an excellent thermal absolute sensitivity of 0.6-3.7% K-1 in the temperature range of 278-328 K. The temperature dependence of the excimeric bands of ONPs are theoretically simulated by using electronic structure calculations and a vibronic Hamiltonian model. Finally, TTM-doped ONPs as ratiometric NIR-nanothermometers are tested with two-photon excitationwith enucleated pig eye sclera, as a real tissue model, obtaining a similar temperature sensitivity as in aqueous suspensions, demonstrating their potential as NIR nanothermometers for bio applications.

Solid state solvation: a fresh view.

The design of efficient organic electronic devices, including OLEDs, OPVs, luminescent solar concentrators, etc., relies on the optimization of relevant materials, often constituted by an active (functional) dye embedded in a matrix. Understanding solid state solvation (SSS), i.e. how the properties of the active dye are affected by the matrix, is therefore an issue of fundamental and technological relevance. Here an extensive experimental and theoretical investigation is presented shedding light on this, somewhat controversial, topic. The spectral properties of the dye at equilibrium, i.e. absorption and Raman spectra, are not affected by the matrix dynamics. Reliable estimates of the matrix polarity are then obtained from an analysis of the micro-Raman spectra of polar dyes. Specifically, to establish a reliable polarity scale, the spectra of DCM or NR dispersed in amorphous matrices are compared with the spectra of the same dyes in liquid solvents with known polarity. On the other hand, steady-state emission spectra obtained in solid matrices depend in a highly non-trivial way on the matrix polarity and its dynamics. An extensive experimental and theoretical analysis of the time-resolved emission spectra of NR in a very large time window (15 fs-15 ns) allows us to validate this dye as a good probe of the dielectric dynamics of the surrounding medium. We provide a first assessment of the relaxation dynamics of two matrices (mCBPCN and DPEPO) of interest for OLED application, unambiguously demonstrating that the matrix readjusts for at least 15 ns after the dye photoexcitation.

Amyloidogenic and non-amyloidogenic molten globule conformation of ß-lactoglobulin in self-crowded regime.

Molecular insights on the ß-lactoglobulin thermal unfolding and aggregation are derived from FTIR and UV Resonance Raman (UVRR) investigations. We propose an in situ and in real-time approach that thanks to the identification of specific spectroscopic markers can distinguish the two different unfolding pathways pursued by ß-lactoglobulin during the conformational transition from the folded to the molten globule state, as triggered by the pH conditions. For both the investigated pH values (1.4 and 7.5) the greatest conformational variation of ß-lactoglobulin occurs at 80 °C and a high degree of structural reversibility after cooling is observed. In acidic condition ß-lactoglobulin exposes to the solvent its hydrophobic moieties in a much higher extent than in neutral solution, resulting on a highly open conformation. Moving from the diluted to the self-crowded regime, the solution pH and consequently the different molten globule conformation select the amyloid or non-amyloid aggregation pathway. At acidic condition the amyloid aggregates form during the heating cycle leading to the formation of transparent hydrogel. On the contrary, in neutral condition the amyloid aggregates never form. Information on the secondary structure conformational change of ß-lactoglobulin and the formation of amyloid aggregates are obtained by FTIR spectroscopy and are related to the information of the structural changes localized around the aromatic amino acid sites by UVRR technique. Our results highlight a strong involvement of the chain portions where tryptophan is located on the formation of amyloid aggregates.

From symmetry breaking to symmetry swapping: is Kasha's rule violated in multibranched phenyleneethynylenes?

The phenomenon of excited-state symmetry breaking is often observed in multipolar molecular systems, significantly affecting their photophysical and charge separation behavior. As a result of this phenomenon, the electronic excitation is partially localized in one of the molecular branches. However, the intrinsic structural and electronic factors that regulate excited-state symmetry breaking in multibranched systems have hardly been investigated. Herein, we explore these aspects by adopting a joint experimental and theoretical investigation for a class of phenyleneethynylenes, one of the most widely used molecular building blocks for optoelectronic applications. The large Stokes shifts observed for highly symmetric phenyleneethynylenes are explained by the presence of low-lying dark states, as also established by two-photon absorption measurements and TDDFT calculations. In spite of the presence of low-lying dark states, these systems show an intense fluorescence in striking contrast to Kasha's rule. This intriguing behavior is explained in terms of a novel phenomenon, dubbed "symmetry swapping" that describes the inversion of the energy order of excited states, i.e., the swapping of excited states occurring as a consequence of symmetry breaking. Thus, symmetry swapping explains quite naturally the observation of an intense fluorescence emission in molecular systems whose lowest vertical excited state is a dark state. In short, symmetry swapping is observed in highly symmetric molecules having multiple degenerate or quasi-degenerate excited states that are prone to symmetry breaking.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150 µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3 weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3 weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Time-resolved infrared absorption spectroscopy applied to photoinduced reactions: how and why.

Time-resolved infrared (IR) spectroscopy is a widely used technique in the investigation of photoinduced reactions, given its capabilities of providing structural information about the presence of intermediates and the reaction mechanism. Despite the fact that it is used in several fields since the '80s, the communication between the different scientific communities (photochemists, photobiologists, etc.) has been to date quite limited. In some cases, this lack of communication happened-and still happens-even inside the same scientific community (for instance between specialists in ultrafast ps/fs IR and those in "fast" ns/µs/ms IR). Even more surprising is the difficulty of non-specialists to understand the potential of time-resolved IR spectroscopy, despite the fact that IR spectroscopy is normally taught to all chemistry and material science students, and to several biology and physics students. This tutorial review aims at helping to solve these issues, first by providing a comprehensive but reader-friendly overview of the different techniques, and second, by focusing on five "case studies" (from photobiology, gas-phase photocatalysis, photochemistry, semiconductors and metal-carbonyl complexes). We are confident that this approach can help the reader-whichever is its background-to understand the capabilities of time-resolved IR spectroscopy to study the mechanism of photoinduced reactions.

Pressure Effects on Water Dynamics by Time-Resolved Optical Kerr Effect.

Despite water being the most common and most widely studied substance in the world, it still presents unknown aspects. In particular, water shows several thermodynamic and dynamical anomalies in the liquid and supercooled metastable phases, and the natures of these phases are still hotly debated. Here, we report measurements of water using the optical Kerr effect as a function of pressure along two isotherms, at 273 K from 0.1 to 750 MPa and at 297 K from 0.1 to 1350 MPa, reaching the supercooled metastable phase. The structural relaxation and the low frequency vibrational dynamics of water show a peculiar pressure dependence similar to that of other dynamical properties. The data analysis suggests the presence in the water phase diagram of a crossover area that divides two regions characterized by different dynamic regimes, which appear to be related to two liquid forms, one dominated by the high density water and the other by the low density water.

Free volume and dynamics in a lipid bilayer.

The lateral diffusion of lipids and of small molecules inside a membrane is strictly related to the arrangement of acyl chains and to their mobility. In this study, we use FTIR and time resolved 2D-IR spectroscopic techniques to characterize the structure and dynamics of the hydrophobic region of palmitoyl-oleylphosphatidylcholine/cholesterol vesicles dispersed in water/dimethylsulfoxide solutions. By means of a non-polar probe, hexacarbonyl tungsten, we monitor the distribution of free volumes inside the bilayer and the conformational dynamics of hydrophobic tails in relation to the different compositions of the membrane or the different compositions of the solvent. Despite the important structural changes induced by the presence of DMSO in the solvating medium, the picosecond dynamics of the membrane is preserved under the different conditions.

Superheating and Homogeneous Melting Dynamics of Bulk Ice.

Homogeneous melting of crystals is a complex multistep process involving the formation of transient states at temperatures considerably higher than the melting point. The nature and persistence of these metastable structures are intimately connected to the melting process, and a precise definition of the temporal boundaries of these phenomena is not yet available. We set up a specifically designed experiment to probe by transient infrared absorption spectroscopy the entire dynamics, ranging from tens of picoseconds to microseconds, of superheating and melting of an ice crystal. In spite of a large excess of energy provided, only about 30% of the micrometric crystal liquefies in the first 20-25 ns because of the long persistence of the superheated metastable phase that extends for more than 100 ns. This behavior is ascribed to the population of low-energy states that trap a large amount of energy, favoring the formation of a metastable, likely plastic, ice phase.

A highly efficient heptamethine cyanine antenna for photosynthetic Reaction Center: From chemical design to ultrafast energy transfer investigation of the hybrid system.

The photosynthetic Reaction Center (RC) from the purple bacterium Rhodobacter sphaeroides has unique photoconversion capabilities, that can be exploited in assembly biohybrid devices for applications in solar energy conversion. Extending the absorption cross section of isolated RC through covalent functionalization with ad-hoc synthesized artificial antennas is a successful strategy to outperform the efficiency of the pristine photoenzyme under visible light excitation. Here we report a new heptamethine cyanine antenna that, upon covalent binding to RC, forms a biohybrid (hCyN7-RC) which, under white light excitation, has doubled photoconversion efficiency versus the bare photoenzyme. The artificial antenna hCyN7 successfully meets appropriate optical properties, i.e. peak position of absorption and emission maximum in the visible and NIR region respectively, large Stokes shift, and high fluorescence quantum yield, required for improving the efficiency of the biohybrid in the production of the charge-separated state in the RC. The kinetics of energy transfer and charge separation of hCyN7-RC studied via ultrafast visible and IR spectroscopies are here presented. The antenna transfers energy to RC chromophores within <10 ps and the rate of QA reduction is doubled compared to the native RC. These experiments further demonstrate hCyN7-RC, besides being an extremely efficient white light photoconverter, fully retains the charge separation mechanism and integrity of the native RC photoenzyme, thus allowing to envisage its suitability as biohybrid material in bioinspired systems for solar energy conversion.

Understanding the influence of disorder on the exciton dynamics and energy transfer in Zn-phthalocyanine H-aggregates.

The photophysics of 9(19),16(17),23(24)-tri-tert-butyl-2-[ethynyl-(4-carboxymethyl)phenyl]phthalocyaninatozinc(ii) and its H-aggregates is studied in different solvents by means of ultrafast non-linear optical spectroscopy and computational modeling. In non-coordinating solvents, both stationary and time-resolved spectroscopies highlight the formation of extended molecular aggregates, whose dimension and spectral properties depends on the concentration. In all the explored experimental conditions, time-resolved transient absorption experiments show multi exponential decay of the signals. Additional insights into the excited state relaxation mechanisms of the system is obtained with 2D electronic spectroscopy, which is employed to compare the deactivation channels in the absence or presence of aggregates. In ethanol and diethylether, where only monomers are present, an ultrafast relaxation process among the two non-degenerate Q-states of the molecule is evidenced by the appearance of a cross peak in the 2D-maps. In chloroform or CCl4, where disordered H-aggregates are formed, an energy transfer channel among aggregates with different composition and size is observed, leading to the non-radiative decay towards the lower energy dark state of the aggregates. Efficient coupling between less and more aggregated species is highlighted in two-dimensional electronic spectra by the appearance of a cross peak. The kinetics and intensity of the latter depend on the concentration of the solution. Finally, the linear spectroscopic properties of the aggregate are reproduced using a simplified structural model of an extended aggregate, based on Frenkel Hamiltonian Calculations and on an estimate of the electronic couplings between each dimer composing the aggregate computed at DFT level.

Solvent Effects on the Actinic Step of Donor-Acceptor Stenhouse Adduct Photoswitching.

Donor-acceptor Stenhouse adducts (DASAs) are negative photochromes that switch with visible light and are highly promising for applications ranging from smart materials to biological systems. However, the strong solvent dependence of the photoswitching kinetics limits their application. The nature of the photoswitching mechanism in different solvents is key for addressing the solvatochromism of DASAs, but as yet has remained elusive. Here, we employ spectroscopic analyses and TD-DFT calculations to reveal changing solvatochromic shifts and energies of the species involved in DASA photoswitching. Time-resolved visible pump-probe spectroscopy suggests that the primary photochemical step remains the same, irrespective of the polarity and protic nature of the solvent. Disentangling the different factors determining the solvent-dependence of DASA photoswitching, presented here, is crucial for the rational development of applications in a wide range of different media.

Tailoring Photoisomerization Pathways in Donor-Acceptor Stenhouse Adducts: The Role of the Hydroxy Group.

Donor-acceptor Stenhouse adducts (DASAs) are a rapidly emerging class of visible light-activatable negative photochromes. They are closely related to (mero)cyanine dyes with the sole difference being a hydroxy group in the polyene chain. The presence or absence of the hydroxy group has far-reaching consequences for the photochemistry of the compound: cyanine dyes are widely used as fluorescent probes, whereas DASAs hold great promise for visible light-triggered photoswitching. Here we analyze the photophysical properties of a DASA lacking the hydroxy group. Ultrafast time-resolved pump-probe spectroscopy in both the visible and IR region show the occurrence of E-Z photoisomerization on a 20 ps time scale, similar to the photochemical behavior of DASAs, but on a slower time scale. In contrast to the parent DASA compounds, where the initial photoisomerization is constrained to a single position (next to the hydroxy group), 1H NMR in situ-irradiation studies at 213 K reveal that for nonhydroxy DASAs E-Z photoisomerization can take place at two different bonds, yielding two distinct isomers. These observations are supported by TD-DFT calculations, showing that in the excited state the hydroxy group (pre)selects the neighboring C2-C3 bond for isomerization. The TD-DFT analysis also explains the larger solvatochromic shift observed for the parent DASAs as compared to the nonhydroxy analogue, in terms of the dipole moment changes evoked upon excitation. Furthermore, computations provide helpful insights into the photoswitching energetics, indicating that without the hydroxy group the 4π-electrocyclization step is energetically forbidden. Our results establish the central role of the hydroxy group for DASA photoswitching and suggest that its introduction allows for tailoring photoisomerization pathways, presumably both through (steric) fixation via a hydrogen bond with the adjacent carbonyl group of the acceptor moiety, as well as through electronic effects on the polyene backbone. These insights are essential for the rational design of novel, improved DASA photoswitches and for a better understanding of the properties of both DASAs and cyanine dyes.

Shedding Light on the Photoisomerization Pathway of Donor-Acceptor Stenhouse Adducts.

Donor-acceptor Stenhouse adducts (DASAs) are negative photochromes that hold great promise for a variety of applications. Key to optimizing their switching properties is a detailed understanding of the photoswitching mechanism, which, as yet, is absent. Here we characterize the actinic step of DASA-photoswitching and its key intermediate, which was studied using a combination of ultrafast visible and IR pump-probe spectroscopies and TD-DFT calculations. Comparison of the time-resolved IR spectra with DFT computations allowed to unambiguously identify the structure of the intermediate, confirming that light absorption induces a sequential reaction path in which a Z-E photoisomerization of C2-C3 is followed by a rotation around C3-C4 and a subsequent thermal cyclization step. First and second-generation DASAs share a common photoisomerization mechanism in chlorinated solvents with notable differences in kinetics and lifetimes of the excited states. The photogenerated intermediate of the second-generation DASA was photo-accumulated at low temperature and probed with time-resolved spectroscopy, demonstrating the photoreversibility of the isomerization process. Taken together, these results provide a detailed picture of the DASA isomerization pathway on a molecular level.

Coacervation of α-elastin studied by ultrafast nonlinear infrared spectroscopy.

Elastin is the main protein to confer elasticity to biological tissues, through the formation of a hierarchical network of fibres. α-Elastin, a soluble form of the protein, is widely used in studies of the biosynthesis of human elastic tissue and exhibits coacervation in solution. This process involves the association of α-elastin molecules through a liquid-liquid phase transition, which is reversible unless the temperature is driven sufficiently high to induce the formation of insoluble aggregates. The thermodynamics of this process have attracted interest over many years and in the present work we used ultrafast nonlinear infrared spectroscopy of the amide I protein backbone vibration to resolve the secondary structural changes occurring during coacervation and probe the protein dynamics on a picosecond time scale. Four classes of carbonyl oscillators with distinct absorption peaks were revealed and, through narrowband excitation, vibrational and anisotropy decays could be distinguished. Analysis of the vibrational lifetimes and anisotropy decay times of these bands characterized the conformational changes and revealed the structural bases of the coacervation process.

Pressure Dependence of Hydrogen-Bond Dynamics in Liquid Water Probed by Ultrafast Infrared Spectroscopy.

Clarifying the structure/dynamics relation of water hydrogen-bond network has been the aim of extensive research over many decades. By joining anvil cell high-pressure technology, femtosecond 2D infrared spectroscopy, and molecular dynamics simulations, we studied, for the first time, the spectral diffusion of the stretching frequency of an HOD impurity in liquid water as a function of pressure. Our experimental and simulation results concordantly demonstrate that the rate of spectral diffusion is almost insensitive to the applied pressure. This behavior is in contrast with the previously reported pressure-induced speed up of the orientational dynamics, which can be rationalized in terms of large angular jumps involving sudden switching between two hydrogen-bonded configurations. The different trend of the spectral diffusion can be, instead, inferred considering that the first solvation shell preserves the tetrahedral structure with pressure and the OD stretching frequency is only slight perturbed.

Excitation Dynamics in Hetero-bichromophoric Calixarene Systems.

In this work, the dynamics of electronic energy transfer (EET) in bichromophoric donor-acceptor systems, obtained by functionalizing a calix[4]arene scaffold with two dyes, was experimentally and theoretically characterized. The investigated compounds are highly versatile, due to the possibility of linking the dye molecules to the cone or partial cone structure of the calix[4]arene, which directs the two active units to the same or opposite side of the scaffold, respectively. The dynamics and efficiency of the EET process between the donor and acceptor units was investigated and discussed through a combined experimental and theoretical approach, involving ultrafast pump-probe spectroscopy and density functional theory based characterization of the energetic and spectroscopic properties of the system. Our results suggest that the external medium strongly determines the particular conformation adopted by the bichromophores, with a direct effect on the extent of excitonic coupling between the dyes and hence on the dynamics of the EET process itself.

Identification of the Excited-State C═C and C═O Modes of trans-ß-Apo-8'-carotenal with Transient 2D-IR-EXSY and Femtosecond Stimulated Raman Spectroscopy.

Assigning the vibrational modes of molecules in the electronic excited state is often a difficult task. Here we show that combining two nonlinear spectroscopic techniques, transient 2D exchange infrared spectroscopy (T2D-IR-EXSY) and femtosecond stimulated Raman spectroscopy (FSRS), the contribution of the C═C and C═O modes in the excited-state vibrational spectra of trans-ß-apo-8'-carotenal can be unambiguously identified. The experimental results reported in this work confirm a previously proposed assignment based on quantum-chemical calculations and further strengthen the role of an excited state with charge-transfer character in the relaxation pathway of carbonyl carotenoids. On a more general ground, our results highlight the potentiality of nonlinear spectroscopic methods based on the combined use of visible and infrared pulses to correlate structural and electronic changes in photoexcited molecules.