Molecular-level elucidation of hydration at biological membrane interfaces is of great importance for understanding biological processes. We studied ultrafast hydrogen-bond dynamics at a zwitterionic phosphatidylcholine/water interface by two-dimensional heterodyne-detected vibrational sum frequency generation (2D HD-VSFG) spectroscopy. The obtained 2D spectra confirm that the anionic phosphate and cationic choline sites are individually hydrated at the interface. Furthermore, the data show that the dynamics of water at the zwitterionic lipid interface is not a simple sum of the dynamics of the water species that hydrate to the separate phosphate and choline. The center line slope (CLS) analysis of the 2D spectra reveals that ultrafast hydrogen-bond fluctuation is not significantly suppressed around the phosphate at the zwitterionic lipid interface, which makes the hydrogen-bond dynamics look similar to that of the bulk water. The present study indicates that the hydrogen-bond dynamics at membrane interfaces is not determined only by the hydrogen bond to a specific site of the interface but is largely dependent on the water dynamics in the vicinity and other nearby moieties, through the hydrogen-bond network.

We present an overview of studies on the ultrafast dynamics of water at aqueous interfaces carried out by time-resolved vibrational sum frequency generation (VSFG) spectroscopies. This research field has been growing rapidly, stimulated by technical developments achieved recently. In this review, first, the principles and instrumentations are described for conventional VSFG, heterodyne-detected VSFG, and various IR-pump/VSFG-probe techniques, namely, time-resolved conventional VSFG, time-resolved heterodyne-detected VSFG, and their extension to two-dimensional spectroscopy. Second, the applications of these time-resolved VSFG techniques to the study of the femtosecond vibrational dynamics of water at various interfaces are discussed, in the order of silica/water, charged monolayer/water, and the air/water interfaces. These studies demonstrate that there exists water dynamics specific to the interfaces and that time-resolved VSFG spectroscopies can unambiguously detect such unique dynamics in an interface-selective manner. In particular, the most recent time-resolved heterodyne-detected VSFG and two-dimensional heterodyne-detected VSFG unveil the inhomogeneity of the hydrogen bond and relevant vibrational dynamics of interfacial water through unambiguous observation of hole-burning in the OH stretch band, as well as the subsequent spectral diffusion in the femtosecond time region. These time-resolved VSFG studies have also left several issues for discussion. We describe not only the obtained conclusive physical insights into interfacial water dynamics but also the points left unclear or controversial. A new type of experiment that utilizes UV excitation is also described briefly. Lastly, the summary and some future perspectives of time-resolved VSFG spectroscopies are given.

•Broadband stimulated Raman measurements in the deep UV region are realized.•Measurements for tryptophan, tyrosine, and glucose oxidase are demonstrated.•A way toward ultrafast structural study of amino acids inside proteins is paved.We report broadband stimulated Raman measurements in the deep ultraviolet (DUV) region, which enables selective probing of the aromatic amino acid residues inside proteins through the resonance enhancement. We combine the narrowband DUV Raman pump pulse (<10 cm−1) at wavelengths as short as 240 nm and the broadband DUV probe pulse (>1000 cm−1) to realize stimulated Raman measurements covering a >1500 cm−1 spectral window. The stimulated Raman measurements for neat solvents, tryptophan, tyrosine, and glucose oxidase are performed using 240- and 290-nm Raman pump, highlighting the high potential of the DUV stimulated Raman probe for femtosecond time-resolved study of proteins.Download high-res image (137KB)Download full-size image

The local environment within a recently developed anthracene-shelled micelle (ASM), which is a micelle-like nanocapsule composed of anthracene-embedded amphiphiles, was investigated by steady-state and time-resolved spectroscopy of an encapsulated solvatochromic fluorescent probe molecule, coumarin 153 (C153). The absorption maximum of encapsulated C153 (452 nm) is more red-shifted than that of free C153 in water, indicating a highly polar environment inside the micelle. Despite this, the fluorescence Stokes shift of encapsulated C153 (∼3700 cm−1) is substantially smaller than that of free C153 in water. Femtosecond time-resolved broadband fluorescence measurements further showed that the dynamic Stokes shift is completed within 1 ps, revealing that the reorganization of the micelle interior following photoexcitation of the C153 probe is characterized by a sub-picosecond, limited-amplitude response. The femtosecond fluorescence anisotropy data showed that the orientational diffusion of the host–guest complex is slower (860 ps) than that of the empty micelle (510 ps), suggesting that the micelle structure is flexible enough to expand when the guest molecule is accommodated and that the micelle rotates with the encapsulated guest molecule. This softness of the micelles further allows some of them to simultaneously encapsulate two C153 molecules, as evidenced by the appearance of blue-shifted, H-dimer-like absorption and fluorescence bands. Based on these steady-state and femtosecond time-resolved spectroscopic data, we discuss the electronic state of C153 and micelle structure as well as the host–guest interaction in this novel flexible synthetic nanocapsule.

Elucidation of the molecular mechanisms of protein adsorption is of essential importance for further development of biotechnology. Here, we use interface-selective nonlinear vibrational spectroscopy to investigate protein charge at the air/water interface by probing the orientation of interfacial water molecules. We measured the Im χ(2) spectra of hemoglobin, myoglobin, serum albumin and lysozyme at the air/water interface in the CH and OH stretching regions using heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy, and we deduced the isoelectric point of the protein by monitoring the orientational flip-flop of water molecules at the interface. Strikingly, our measurements indicate that the isoelectric point of hemoglobin is significantly lowered (by about one pH unit) at the air/water interface compared to that in the bulk. This can be predominantly attributed to the modifications of the protein structure at the air/water interface. Our results also suggest that a similar mechanism accounts for the modification of myoglobin charge at the air/water interface. This effect has not been reported for other model proteins at interfaces probed by conventional VSFG techniques, and it emphasizes the importance of the structural modifications of proteins at the interface, which can drastically affect their charge profiles in a protein-specific manner. The direct experimental approach using HD-VSFG can unveil the changes of the isoelectric point of adsorbed proteins at various interfaces, which is of major relevance to many biological applications and sheds new light on the effect of interfaces on protein charge.

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, an essential bioimaging tool, luminesces via excited-state proton transfer (ESPT) in which the phenolic proton of the p-hydroxybenzylideneimidazolinone chromophore is transferred to Glu222 through a hydrogen-bond network. In this process, the ESPT mediated by the low-frequency motion of the chromophore has been proposed. We address this issue using femtosecond time-resolved impulsive stimulated Raman spectroscopy. After coherently exciting low-frequency modes (<300 cm–1) in the excited state of GFP, we examined the excited-state structural evolution and the ESPT dynamics within the dephasing time of the low-frequency vibration. A clear anharmonic vibrational coupling is found between one high-frequency mode of the chromophore (phenolic CH bend) and a low-frequency mode at ∼104 cm–1. However, the data show that this low-frequency motion does not substantially affect the ESPT dynamics.

Hydrated electrons are the most fundamental anion species, consisting only of electrons and surrounding water molecules. Although hydrated electrons have been extensively studied in the bulk aqueous solutions, even their existence is still controversial at the water surface. Here, we report the observation and characterization of hydrated electrons at the air/water interface using new time-resolved interface-selective nonlinear vibrational spectroscopy. With the generation of electrons at the air/water interface by ultraviolet photoirradiation, we observed the appearance of a strong transient band in the OH stretch region by heterodyne-detected vibrational sum-frequency generation. Through the comparison with the time-resolved spectra at the air/indole solution interface, the transient band was assigned to the vibration of water molecules that solvate electrons at the interface. The analysis of the frequency and decay of the observed transient band indicated that the electrons are only partially hydrated at the water surface, and that they escape into the bulk within 100 ps.

Au–Au bond strengthening in photoexcited dimers of an Au(I) complex is captured in solution as oscillations of femtosecond absorption signals. The subsequent dynamics, when compared to the trimer's data, confirm that the bent-to-linear structural change of the trimer occurs in the first few picoseconds.

Lipid/water interaction is essential for many biological processes. The water structure at the nonionic lipid interface remains little known, and there is no scope of a priori prediction of water orientation at nonionic interfaces, either. Here, we report our study combining advanced nonlinear spectroscopy and molecular dynamics simulation on the water orientation at the ceramide/water interface. We measured χ(2) spectrum in the OH stretch region of ceramide/isotopically diluted water interface using heterodyne-detected vibrational sum-frequency generation spectroscopy and found that the interfacial water prefers an overall hydrogen-up orientation. Molecular dynamics simulation indicates that this preferred hydrogen-up orientation of water is determined by a delicate balance between hydrogen-up and hydrogen-down orientation induced by lipid–water and intralipid hydrogen bonds. This mechanism also suggests that water orientation at neutral lipid interfaces depends highly on the chemical structure of the lipid headgroup, in contrast to the charged lipid interfaces where the net water orientation is determined solely by the charge of the lipid headgroup.

Complex χ(2) spectra of buried silica/isotopically diluted water (HOD-D2O) interfaces were measured using multiplex heterodyne-detected vibrational sum frequency generation spectroscopy to elucidate the hydrogen bond structure and up/down orientation of water at the silica/water interface at different pHs. The data show that vibrational coupling (inter- and/or intramolecular coupling) plays a significant role in determining the χ(2) spectral feature of silica/H2O interfaces and indicate that the doublet feature in the H2O spectra does not represent two distinct water structures (i.e., the ice- and liquid-like structures) at the silica/water interface. The observed pH dependence of the imaginary χ(2) spectra is explained by (1) H-up oriented water donating a hydrogen bond to the oxygen atom of silanolate, which is accompanied by H-up water oriented by the electric field created by the negative charge of silanolate, (2) H-up oriented water which donates a hydrogen bond to the neutral silanol oxygen, and (3) H-down oriented water which accepts hydrogen bonds from the neutral silanol and donates hydrogen bonds to bulk water molecules. The broad continuum of the OH stretch band of HOD-D2O and a long tail in the low frequency region represent a wide distribution of strong hydrogen bonds at the silica/water interface, particularly at the low pH.

Femtosecond vibrational dynamics at the air/water interface is investigated by time-resolved heterodyne-detected vibrational sum frequency generation (TR-HD-VSFG) spectroscopy and molecular dynamics (MD) simulation. The low- and high-frequency sides of the hydrogen-bonded (HB) OH stretch band at the interface are selectively excited with special attention to the bandwidth and energy of the pump pulses. Narrow bleach is observed immediately after excitation of the high-frequency side of the HB OH band at ∼3500 cm–1, compared to the broad bleach observed with excitation of the low-frequency side at ∼3300 cm–1. However, the time-resolved spectra observed with the two different excitations become very similar at 0.5 ps and almost indistinguishable by 1.0 ps. This reveals that efficient spectral diffusion occurs regardless of the difference of the pump frequency. The experimental observations are well-reproduced by complementary MD simulation. There is no experimental and theoretical evidence that supports extraordinary slow dynamics in the high-frequency side of the HB OH band, which was reported before.

Heterodyne-detected vibrational sum frequency generation spectroscopy was applied to the water surface for measuring the imaginary part of second-order nonlinear susceptibility (Im χ(2)) spectrum in the bend frequency region for the first time. The observed Im χ(2) spectrum shows an overall positive band around 1650 cm–1, contradicting former theoretical predictions. We further found that the Im χ(2) spectrum of NaI aqueous solution exhibits an even larger positive band, which is apparently contrary to the flip-flop orientation of surface water. These unexpected observations are elucidated by calculating quadrupole contributions beyond the conventional dipole approximation. It is indicated that the Im χ(2) spectrum in the bend region has a large quadrupole contribution from the bulk water.

Bis-diimine Cu(I) complexes exhibit strong absorption in the visible region owing to the metal-to-ligand charge transfer (MLCT) transitions, and the triplet MLCT (3MLCT) states have long lifetimes. Because these characteristics are highly suitable for photosensitizers and photocatalysts, bis-diimine Cu(I) complexes have been attracting much interest. An intriguing feature of the Cu(I) complexes is the photoinduced structural change called “flattening”. Bis-diimine Cu(I) complexes usually have tetrahedron-like D2d structures in the ground (S0) state, in which two ligands are perpendicularly attached to the Cu(I) ion. With MLCT excitation, the central Cu(I) ion is formally oxidized to Cu(II), which induces the structural change to the “flattened” square-planar-like structure that is seen for usual Cu(II) complexes.In this Account, we review our recent studies on ultrafast excited-state dynamics of bis-diimine Cu(I) complexes carried out using femtosecond time-resolved optical spectroscopy. Focusing on three prototypical bis-diimine Cu(I) complexes that have 1,10-phenanthroline ligands with different substituents at the 2,9-positions, i.e., [Cu(phen)2]+ (phen = 1,10-phenanthroline), [Cu(dmphen)2]+ (dmphen = 2,9-dimethyl-1,10-phenanthroline), and [Cu(dpphen)2]+ (dpphen = 2,9-diphenyl-1,10-phenanthroline), we examined their excited-state dynamics by time-resolved emission and absorption spectroscopies with 200 fs time resolution, observed the excited-state coherent nuclear motion with 30 fs time resolution and performed complementary theoretical calculations. This combined approach vividly visualizes excited-state processes in the MLCT state of bis-diimine Cu(I) complexes.It was demonstrated that flattening distortion, internal conversion, and intersystem crossing occur on the femtosecond–early picosecond time scale, and their dynamics is clearly identified separately. The flattening distortion predominantly occurs in the S1 state on the subpicosecond time-scale, and the precursor S1 state retaining the initial undistorted structure appears as a metastable state before the structural change. This observation indicates that the traditional understanding based on the Jahn–Teller effect appears irrelevant for realistically discussing the photoinduced structural change of bis-diimine Cu(I) complexes. The lifetime of the precursor S1 state significantly depends on the substituents in the three complexes, indicating that the flattering distortion requires a longer time as the substituents at 2,9-positions of the ligands become bulkier. It is suggested that the substituents are rotated to avoid steric repulsions to achieve the flattened structure at the global minimum of the S1 state, implying the necessity of discussion based on a multidimensional potential energy surface to properly consider this excited-state structural change. After the flattening distortion, the S1 states of [Cu(dmphen)2]+ and [Cu(dpphen)2]+, which have bulky substituents, relax to the T1 state by intersystem crossing on the ∼10 ps time scale, while the flattened S1 state of [Cu(phen)2]+ relaxes directly to the S0 state on the ∼2 ps time scale. This difference is rationalized in terms of the different magnitude of the flattening distortion and relevant changes in the potential energy surfaces. Clear understanding of the ultrafast excited-state process provides a solid basis for designing and using Cu(I) complexes, such as controlling the structural change to efficiently utilize the energy of the MLCT state in solar energy conversion.

We study the effective polarity of an air/liquid-mixture interface by using interface-selective heterodyne-detected electronic sum frequency generation (HD-ESFG) and vibrational sum frequency generation (HD-VSFG) spectroscopies. With water and N,N-dimethylformamide (DMF) chosen as two components of the liquid mixture, the bulk polarity of the mixture is controlled nearly arbitrarily by the mixing ratio. The effective polarity of the air/mixture interface is evaluated by HD-ESFG with a surface-active solvatochromic molecule used as a polarity indicator. Surprisingly, the interfacial effective polarity of the air/mixture interface increases significantly, when the bulk polarity of the mixture decreases (i.e. when the fraction of DMF increases). Judging from the hydrogen-bond structure at the air/mixture interface clarified by HD-VSFG, this anomalous change of the interfacial effective polarity is attributed to the interface-specific solvation structure around the indicator molecule at the air/mixture interface.

The substituent effect on the excited-state dynamics of bis-diimine Cu(I) complexes was investigated by femtosecond time-resolved absorption spectroscopy with the S1 ← S0 metal-to-ligand charge transfer (MLCT) photoexcitation. The time-resolved absorption of [Cu(phen)2]+ (phen = 1,10-phenanthroline) showed a slight intensity increase of the S1 absorption with a time-constant of 0.1–0.2 ps, reflecting the flattening distortion occurring in the S1 state. The transient absorption of the ‘flattened’ S1 state was clearly observed, although its fluorescence was not observed in the previous fluorescence up-conversion study in the visible region. The flattened S1 state decayed with a time constant of ∼2 ps, and the S0 bleaching recovered accordingly. This clarifies that the S1 state of [Cu(phen)2]+ is predominantly relaxed to the S0 state by internal conversion. The time-resolved absorption of [Cu(dpphen)2]+ (dpphen = 2,9-diphenyl-1,10-phenanthroline) showed a 0.9 ps intensity increase of the S1 absorption due to the flattening distortion, and then exhibited a 11 ps spectral change due to the intersystem crossing. This excited-state dynamics of [Cu(dpphen)2]+ is very similar to that of [Cu(dmphen)2]+ (dmphen = 2,9-dimethyl-1,10-phenanthroline). In the ultrafast pump–probe measurements with 35 fs time resolution, [Cu(phen)2]+ and [Cu(dpphen)2]+ exhibited oscillation due to the nuclear wavepacket motions of the initial S1 state, and the oscillation was damped as the structural change took place. This indicates that the initial S1 states have well-defined vibrational structures and that the vibrational coherence is retained in their short lifetimes. The present time-resolved absorption study, together with the previous time-resolved fluorescence study, provides a unified view for the ultrafast dynamics of the MLCT excited state of the Cu(I) complexes.

We report the first femtosecond time-resolved absorption study on ultrafast photoreaction dynamics of a recently discovered retinal protein, KR2, which functions as a light-driven sodium-ion pump. The obtained data show that the excited-state absorption around 460 nm and the stimulated emission around 720 nm decay concomitantly with a time constant of 180 fs. This demonstrates that the deactivation of the S1 state of KR2, which involves isomerization of the retinal chromophore, takes place three times faster than that of bacteriorhodopsin. In accordance with this rapid electronic relaxation, the photoproduct band assignable to the J intermediate grows up at ∼620 nm, indicating that the J intermediate is directly formed with the S1 → S0 internal conversion. The photoproduct band subsequently exhibits a ∼30 nm blue shift with a 500 fs time constant, corresponding to the conversion to the K intermediate. On the basis of the femtosecond absorption data obtained, we discuss the mechanism for the rapid photoreaction of KR2 and its relevance to the unique function of the sodium-ion pump.

This feature article describes our recent work on the development of interface-selective second-order nonlinear electronic spectroscopies and their application to molecular structure and dynamics at liquid interfaces. We describe three methods that measure electronically resonant second-order nonlinear optical susceptibility (χ(2)) of interfacial molecules without contribution from the bulk: electronic sum-frequency generation (ESFG) provides high-quality interfacial electronic spectra by employing the combination of narrow-band and broad-band visible/near-infrared femtosecond pulses. Time-resolved ESFG (TR-ESFG) is the extension of ESFG to time-resolved measurements and provides femtosecond time-resolved electronic spectra at interfaces. Heterodyne-detected ESFG (HD-ESFG) is the ultimate second-order nonlinear electronic spectroscopy that directly provides the real and imaginary parts of χ(2). In particular, the imaginary χ(2) spectra obtained with HD-ESFG can be directly compared to the absorption spectra of bulk solution, and their signs contain information about the absolute orientation of the interfacial molecules. These interface-selective electronic spectroscopies enable rigorous comparison between the electronic spectra of liquid interfaces and those of bulk liquids, and the application to the following topics is discussed in this article: effective polarity at the air/water interface, interfacial ultrafast dynamics, absolute alignment of interfacial molecules, pH of the air/water interface, and the origin of nonresonant background. These studies reveal unique properties of molecules at air/liquid interfaces, which is markedly different from that in the bulk from a microscopic viewpoint.

Specific counterion effects represented by Hofmeister series are important for a variety of phenomena such as protein precipitations, surface tensions of electrolytes solutions, phase transitions of surfactants, etc. We applied heterodyne-detected vibrational sum-frequency generation spectroscopy to study the counterion effect on the interfacial water at charged interfaces and discussed the observed effect with relevance to the Hofmeister series. Experiments were carried out for model systems of positively charged cetyltrimethylammonium monolayer/electrolyte solution interface and negatively charged dodecylsulfate monolayer/electrolyte interface. At the positively charged interface, the intensity of the OH band of the interfacial water decreases in the order of the Hofmeister series, suggesting that the adsorbability of halide anions onto the interface determines the Hofmeister order as previously proposed by Zhang and Cremer (Curr. Opin. Chem. Biol. 2006, 10, 658–663). At the negatively charged interfaces, on the other hand, the OH band intensity does not depend significantly on the countercation, whereas variation in the hydrogen-bond strength of the interfacial water is well correlated with the Hofmeister order of the cation effect. These results provide new insights into the molecular level mechanisms of anionic and cationic Hofmeister effects.

The Cu(I) complexes having phenanthroline derivatives as ligands are known to exhibit photo-induced ‘flattening’ structural change in the metal-to-ligand charge transfer (MLCT) excited state. Our recent ultrafast spectroscopic studies of [Cu(dmphen)2]+ (dmphen = 2,9-dimethyl-1,10-phenanthroline) showed that the photo-induced structural change predominantly occurs in the S1 state on a subpicosecond time scale, with the appearance of the ‘perpendicular’ S1 state before the structural change. In this work, we carried out femto/picosecond time-resolved emission spectroscopy of [Cu(phen)2]+ (phen = 1,10-phenanthroline) and [Cu(dpphen)2]+ (dpphen = 2,9-diphenyl-1,10-phenanthroline) in dichloromethane with the S2 ← S0 photo-excitation to examine the substituent effect on the ultrafast structural change. The femtosecond time-resolved emission spectra of the two complexes exhibit ultrafast fluorescence changes that are attributed to the structural change in the S1 state after fast (50–100 fs) S2 → S1 internal conversion. By comparing with the dynamics of [Cu(dmphen)2]+, it was found that the time constant of the structural change increases as the substituents at 2- and 9- positions of the ligand become bulkier, i.e., [Cu(phen)2]+ (200 fs) < [Cu(dmphen)2]+ (660 fs) < [Cu(dpphen)2]+ (920 fs). This implies that the complex needs a longer time to flatten with the bulkier substituent. This demonstrates that the dynamics of the photo-induced structural change of Cu(I) complexes is substantially affected by the substituent of the ligand. The dynamics of the ultrafast structural change and the substituent effect are discussed with the multidimensional S1 potential energy surface of Cu(I) complexes.

Light absorption by the photoreceptor microbial rhodopsin triggers trans–cis isomerization of the retinal chromophore surrounded by seven transmembrane α-helices. Sensory rhodopsin I (SRI) is a dual functional photosensory rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (SrSRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the Sn←S1 absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial rhodopsins. The first dynamics (τ1 ∼ 54 fs) corresponds to the population branching process from the Franck–Condon region to the reactive (S1r) and nonreactive (S1nr) S1 states. The second dynamics (τ2 = 0.64 ps) is the isomerization process of the S1r state to generate the ground-state 13-cis form, and the third dynamics (τ3 = 1.8 ps) corresponds to the internal conversion of the S1nr state. The fourth component (τ3′ = 2.5 ps) is assignable to the J-decay (K-formation). This reaction scheme was further supported by the results of fluorescence spectroscopy. To investigate the protein response(s), the spectral changes of the tryptophan bands were monitored by ultraviolet resonance Raman spectroscopy. The intensity change following the K formation in the chromophore structure (τ ∼ 17 ps) was significantly small in SrSRI as compared with other microbial rhodopsins. We also analyzed the effect(s) of Cl– binding on the ultrafast dynamics of SrSRI. Compared with a chloride pump Halorhodopsin, Cl– binding to SrSRI was less effective for the excited-state dynamics, whereas the binding altered the structural changes of tryptophan following the K-formation, which was the characteristic feature for SrSRI. On the basis of these results, a primary photoreaction scheme of SrSRI together with the role of chloride binding is proposed.

We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium Rhodopseudomonas palustris, using femtosecond time-resolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH• (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH• mediates the signaling-state formation, showing that PapB is the second example of FADH•-mediated formation of the signaling state after Slr1694 (M. Gauden et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10895–10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH• was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH• was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH• in the photocycle of PapB.

Although the interface pH at a biological membrane is important for biological processes at the membrane, there has been no systematic study to evaluate it. We apply novel interface-selective nonlinear spectroscopy to the evaluation of the pH at model biological membranes (lipid/water interfaces). It is clearly shown that the pH at the charged lipid/water interfaces is substantially deviated from the bulk pH. The pH at the lipid/water interface is higher than that in the bulk when the head group of the lipid is positively charged, whereas the pH at the lipid/water interface is lower when the lipid has a negatively charged head group.Keywords: charged lipid; heterodyne detection; lipid/water interface; pH; sum frequency generation;

The interface selectivity of vibrational sum frequency generation (VSFG) spectroscopy is explained under the dipole approximation as resulting from the breakdown of inversion symmetry at the interface. From this viewpoint, VSFG is not expected to occur at the interface consisting of centrosymmetric molecules, because the inversion symmetry is preserved even at the interface. In reality, however, VSFG at the nonpolar benzene/air interface has been observed with traditional homodyne-detected VSFG. Here we report a heterodyne-detected VSFG study of the benzene/air interface. The result strongly indicates that VSFG at this interface cannot be explained within the framework of the dipole approximation. The selection rule and polarization dependence of the observed VSFG signal show that the quadrupole transition plays an essential role because of the field discontinuity at the interface. This finding implies the applicability of interface-selective VSFG to the nonpolar interfaces comprising centrosymmetric molecules, which opens a new possibility of VSFG spectroscopy.Keywords: air/liquid interface; dipole approximation; electric quadrupole; heterodyne detection; vibrational sum frequency generation;

In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution.

Fluorescence correlation spectroscopy (FCS) is a unique tool for investigating microsecond molecular dynamics of complex molecules in equilibrium. However, application of FCS in the study of molecular dynamics has been limited, owing to the complexity in the extraction of physically meaningful information. In this work, we develop a new method that combines FCS and time-correlated single photon counting (TCPSC) to extract unambiguous information about equilibrium dynamics of complex molecular systems. In this method, which we name two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), we analyze the correlation of the fluorescence photon pairs, referring to the fluorescence lifetime. We first obtain the correlations of the photon pairs with respect to the excitation–emission delay times in the form of a two-dimensional (2D) map. Then, the 2D map is converted to the correlations between different species that have distinct fluorescence lifetimes using inverse Laplace transformation. This 2D FLCS is capable of visualizing the equilibration dynamics of complex molecules with microsecond time resolution at the single-molecule level. We performed a kinetic Monte Carlo simulation of a TCPSC-FCS experiment as a proof-of-principle example. The result clearly shows the validity of the proposed method and its high potential in analyzing the photon data of dynamic systems.

Linear and nonlinear polarization-sensitive spectroscopic techniques are employed to determine the orientational distributions of solute molecules adsorbed at the air/water interface. Each solute molecule shows very broad orientational distribution with a standard deviation ranging from 16° to 21°. The experimentally determined orientational distributions are well reproduced by classical molecular dynamics simulations. The good agreement between experimental results and theoretical calculations enables us to elucidate how the orientational distribution at the air/water interface is controlled by the chemical structure of the solute molecules.

Lipid/water interfaces and associated interfacial water are vital for various biochemical reactions, but the molecular-level understanding of their property is very limited. We investigated the water structure at a zwitterionic lipid, phosphatidylcholine, monolayer/water interface using heterodyne-detected vibrational sum frequency generation spectroscopy. Isotopically diluted water was utilized in the experiments to minimize the effect of intra/intermolecular couplings. It was found that the OH stretch band in the Imχ(2) spectrum of the phosphatidylcholine/water interface exhibits a characteristic double-peaked feature. To interpret this peculiar spectrum of the zwitterionic lipid/water interface, Imχ(2) spectra of a zwitterionic surfactant/water interface and mixed lipid/water interfaces were measured. The Imχ(2) spectrum of the zwitterionic surfactant/water interface clearly shows both positive and negative bands in the OH stretch region, revealing that multiple water structures exist at the interface. At the mixed lipid/water interfaces, while gradually varying the fraction of the anionic and cationic lipids, we observed a drastic change in the Imχ(2) spectra in which spectral features similar to those of the anionic, zwitterionic, and cationic lipid/water interfaces appeared successively. These observations demonstrate that, when the positive and negative charges coexist at the interface, the H-down-oriented water structure and H-up-oriented water structure appear in the vicinity of the respective charged sites. In addition, it was found that a positive Imχ(2) appears around 3600 cm–1 for all the monolayer interfaces examined, indicating weakly interacting water species existing in the hydrophobic region of the monolayer at the interface. On the basis of these results, we concluded that the characteristic Imχ(2) spectrum of the zwitterionic lipid/water interface arises from three different types of water existing at the interface: (1) the water associated with the negatively charged phosphate, which is strongly H-bonded and has a net H-up orientation, (2) the water around the positively charged choline, which forms weaker H-bonds and has a net H-down orientation, and (3) the water weakly interacting with the hydrophobic region of the lipid, which has a net H-up orientation.

Structural dynamics involving tight Au–Au bond formation of excited-state oligomers [Au(CN)2–]n was studied using picosecond/femtosecond time-resolved emission and absorption spectroscopy. With selective excitation of the trimer ([Au(CN)2–]3) in aqueous solutions, transient absorption due to the excited-state trimer was observed around 600 nm. This transient exhibited a significant intensity increase (τ = 2.1 ps) with a blue shift in the early picosecond time region. Density functional theory (DFT) and time-dependent DFT calculations revealed that the observed spectral changes can be ascribed to a structural change from a bent to a linear staggered structure in the triplet excited-state trimer. The transient absorption also exhibited a clear modulation of the peak position, reflecting coherent nuclear wave packet motion induced by photoexcitation. The frequencies of the coherent motions are 66 and 87 cm–1, in very good accord with the frequencies of two Au–Au stretch vibrations in the excited state of the trimer calculated by DFT. Time-resolved emission spectra in the subnanosecond time region showed that association of the excited-state trimer with the ground-state monomer proceeds with τ = 2.0 ns, yielding the excited-state tetramer.

We report a femtosecond time-resolved fluorescence study of cis-stilbene, a prototypical molecule showing ultrafast olefinic photoisomerization and photocyclization. The time-resolved fluorescence signals were measured in a nonpolar solvent over a wide ultraviolet-visible region with excitation at 270 nm. The time-resolved fluorescence traces exhibit non-single exponential decays which are well fit with bi-exponential functions with time constants of τA = 0.23 ps and τB = 1.2 ps, and they are associated with the fluorescence emitted from different regions of the S1 potential energy surface (PES) in the course of the structural change. Quantitative analysis revealed that the two fluorescent components exhibit similar intrinsic time-resolved spectra extending from 320 nm to 700 nm with the (fluorescence) oscillator strength of fA = 0.32 and fB = 0.21, respectively. It was concluded that the first component is assignable to the fluorescence from the untwisted S1 PES region where the molecule reaches immediately after the initial elongation of the central CC bond, while the second component is the fluorescence from the substantially twisted region around a shallow S1 potential minimum. The quantitative analysis of the femtosecond fluorescence data clearly showed that the whole isomerization process proceeds in the one-photon allowed S1 state, thereby resolving a recent controversy in quantum chemical calculations about the reactive S1 state. In addition, the evaluated oscillator strengths suggest that the population branching into the isomerization/cyclization pathways occurs in a very early stage when the S1 molecule still retains a planar Ph–CC–Ph skeletal structure. On the basis of the results obtained, we discuss the dynamics and mechanism of the isomerization/cyclization reactions of cis-stilbene, as well as the electronic structure of the reaction precursor.

We studied ultrafast structural dynamics of the chromophore of photoactive yellow protein, trans-p-coumaric acid (pCA), using newly developed ultraviolet resonance femtosecond stimulated Raman spectroscopy (UV-FSRS). The UV-FSRS data of the anionic form (pCA–) in a buffer solution showed clear spectral changes within 1 ps, followed by a spectrally uniform decay with a time constant of 2.4 ps. The observed spectral change indicates that the structural change occurs in excited pCA– from the Franck–Condon state to the S1 potential minimum in the femtosecond time region. The S1 Raman spectra exhibit spectral patterns that are similar to the ground-state spectrum, suggesting that pCA– yet retains a planar-trans conformation throughout the S1 lifetime. We concluded that S1 pCA– undergoes a femtosecond in-plane deformation, rather than a substantial Cet═Cet twist. With these femtosecond vibrational data, we discuss possible roles of the initial structural evolution of pCA in triggering the photoreceptive function when embedded in the protein.Keywords: femtosecond stimulated Raman; photoactive yellow protein; photoisomerization;

The energetically unfavorable termination of the hydrogen-bonded network of water molecules at the air/water interface causes molecular rearrangement to minimize the free energy. The long-standing question is how water minimizes the surface free energy. The combination of advanced, surface-specific nonlinear spectroscopy and theoretical simulation provides new insights. The complex χ(2) spectra of isotopically diluted water surfaces obtained by heterodyne-detected sum frequency generation spectroscopy and molecular dynamics simulation show excellent agreement, assuring the validity of the microscopic picture given in the simulation. The present study indicates that there is no ice-like structure at the surface—in other words, there is no increase of tetrahedrally coordinated structure compared to the bulk—but that there are water pairs interacting with a strong hydrogen bond at the outermost surface. Intuitively, this can be considered a consequence of the lack of a hydrogen bond toward the upper gas phase, enhancing the lateral interaction at the boundary. This study also confirms that the major source of the isotope effect on the water χ(2) spectra is the intramolecular anharmonic coupling, i.e., Fermi resonance.

The photoinduced structural change of a prototype metal complex, [Cu(dmphen)2]+ (dmphen = 2,9-dimethyl-1,10-phenanthroline), was studied by ultrafast spectroscopy with time resolution as high as 30 fs. Time-resolved absorption measured with direct S1 excitation clearly showed spectral changes attributable to the D2d (perpendicular) → D2 (flattened) structural change occurring in the metal-to-ligand charge transfer singlet excited state (1MLCT) and the subsequent S1 → T1 intersystem crossing. It was confirmed that the two processes occur with time constants of ∼0.8 ps (structural change) and ∼10 ps (intersystem crossing), and their time scales are clearly well-separated. A distinct oscillation of the transient absorption signal was observed in the femtosecond region, which arises from the coherent nuclear motion of the perpendicular S1 state that was directly generated by photoexcitation. This demonstrated that the perpendicular S1 state has a well-defined vibrational structure and can vibrate within its subpicosecond lifetime. In other words, the S1 state stays undistorted in a short period, and the coherent nuclear motion is maintained in this state. Time-dependent density functional theory (TDDFT) calculations gave consistent results, indicating a very flat feature and even a local minimum at the perpendicular structure on the S1 potential energy surface. The vibrational assignments of the S1 nuclear wavepacket motion were made on the basis of the TDDFT calculation. It was concluded that photoexcitation induces a1 vibrations containing the Cu–ligand bond length change and a b1 vibration attributed to the ligand-twisting motion that has the same symmetry as the flattening distortion. Ultrafast spectroscopy and complementary quantum chemical calculation provided an overall picture and new understanding of the photoinduced structural change of the prototypical metal complex.

Understanding ultrafast reactions, which proceed on a time scale of nuclear motions, requires a quantitative characterization of the structural dynamics. To track such structural changes with time, we studied a nuclear wavepacket motion in photoisomerization of a prototype cyanine dye, 1,1′-diethyl-4,4′-cyanine, by ultrafast pump–dump–probe measurements in solution. The temporal evolution of wavepacket motion was examined by monitoring the efficiency of stimulated emission dumping, which was obtained from the recovery of a ground-state bleaching signal. The dump efficiency versus pump–dump delay exhibited a finite rise time, and it became longer (97 fs → 330 fs → 390 fs) as the dump pulse was tuned to longer wavelengths (690 nm → 950 nm → 1200 nm). This result demonstrates a continuous migration of the leading edge of the wavepacket on the excited-state potential from the Franck–Condon region toward the potential minimum. A slowly decaying feature of the dump efficiency indicated a considerable broadening of the wavepacket over a wide range of the potential, which results in the spread of a population distribution on the flat S1 potential energy surface. The rapid migration as well as broadening of the wavepacket manifests a continuous nature of the structural dynamics and provides an intuitive visualization of this ultrafast reaction. We also discussed experimental strategies to evaluate reliable dump efficiencies separately from other ultrafast processes and showed a high capability and possibility of the pump–dump–probe method for spectroscopic investigation of unexplored potential regions such as conical intersections.

Protein function is intimately related to the dynamics of the protein as well as to the dynamics of the solvent shell around the protein. Although it has been argued extensively that protein dynamics is slaved to solvent dynamics, experimental support for this hypothesis is scanty. In this study, measurements of fluorescence anisotropy decay kinetics have been used to determine the motional dynamics of the fluorophore acrylodan linked to several locations in a small protein barstar in its various structural forms, including the native and unfolded states as well as the acid and protofibril forms. Fluorescence upconversion and streak camera measurements have been used to determine the solvation dynamics around the fluorophore. Both the motional dynamics and solvent dynamics were found to be dependent upon the location of the probe as well as on the structural form of the protein. While the (internal) motional dynamics of the fluorophore occur in the 0.1−3 ns time domain, the observed mean solvent relaxation times are in the range of 20−300 ps. A strong positive correlation between these two dynamical modes was found in spite of the significant difference in their time scales. This observed correlation is a strong indicator of the coupling between solvent dynamics and the dynamics in the protein.

To evaluate the inhomogeneity of the solvation environment at the air/water interface, we quantitatively analyzed the shape of the electronic spectra at the air/water interface for the first time. We measured the interface-selective electronic χ(2) (second-order nonlinear susceptibility) spectra of solvatochromic coumarin molecules at the air/water interface by heterodyne-detected electronic sum frequency generation (HD-ESFG) spectroscopy. The observed imaginary χ(2) (Im[χ(2)]) spectra were well reproduced by the convolution of a line shape function with a Gaussian distribution of a frequency shift, which enabled us to quantitatively determine the peak position and bandwidth of the Im[χ(2)] spectra. The effective polarity of the air/water interface, which was determined by the peak position, was found to be dependent on each coumarin, which agreed with our previous homodyne-detected ESFG study. Interestingly, the spectra at the air/water interface showed substantially broader bandwidths than those in equally polar bulk solvents or even bulk water, indicating that the solvation environment at the air/water interface is more inhomogeneous than that in bulk solvents. At the air/water interface, the stabilization energy of the solvation not only changes with the change of the position and orientation of the surrounding solvents but also varies with the change of the position and orientation of the solute at the interface. We consider that this unique situation arising from the anisotropy along interface normal brings about a broader distribution of the stabilization energy of solvation at the air/water interface. The present work showed that the air/water interface provides a more inhomogeneous solvation environment than equally polar bulk solvents because of this broader distribution of the local solvation structure at the interface.

Cell membrane/water interfaces provide a unique environment for many biochemical reactions, and associated interfacial water is an integral part of such reactions. A molecular level understanding of the structure and orientation of water at lipid/water interfaces is required to realize the complex chemistry at biointerfaces. Here we report the heterodyne-detected vibrational sum frequency generation (HD-VSFG) studies of lipid monolayer/water interfaces. At charged lipid/water interfaces, the orientation of interfacial water is governed by the net charge on the lipid headgroup; at an anionic lipid/water interface, water is in the hydrogen-up orientation, and at the cationic lipid/water interface, water is in the hydrogen-down orientation. At the cationic and anionic lipid/water interfaces, interfacial water has comparable hydrogen bond strength, and it is analogous to the bulk water.

Fluorescence correlation spectroscopy (FCS) was extended by incorporating information of the fluorescence lifetime. This new experimental approach, called lifetime-weighted FCS, enables us to observe fluorescence lifetime fluctuations in the nano- to millisecond time region. The potential of this method for resolving inhomogeneity in complex systems was demonstrated. First, by measuring a mixture of two dye molecules having different fluorescence lifetimes, it was shown that the lifetime-weighted correlation deviates from the ordinary intensity correlation only when the system is inhomogeneous. This demonstrated that lifetime-weighted FCS is capable of detecting inhomogeneity in an ensemble-averaged fluorescence decay profile without any a priori knowledge about the system. Second, we applied this method to a dye-labeled polypeptide, a prototypical model of complex biopolymers. It was found that the ratio between the lifetime-weighted and ordinary intensity correlation changes with change of the environment around the polypeptide. This result was interpreted in terms of environment-dependent conformational inhomogeneity of the polypeptide. Delay time dependence of the ratio was found to be constant from ∼1 μs to several milliseconds, indicating that the observed inhomogeneity is persistent in the measured time scale. In combination with fluorescence intensity correlation, lifetime-weighted FCS allows us to examine conformational fluctuations of complex systems in the time region from nano- to milliseconds, being free from the translational diffusion signal.

Host−guest complexes involving M6L4 coordination cages can display unusual photoreactivity, and enclathration of the very large fluorophore bisanthracene resulted in an emissive, mechanically trapped intramolecular exciplex. Mechanically linked intramolecular exciplexes are important for understanding the dependence of energy transfer on donor−acceptor distance, orientation, and electronic coupling but are relatively unexplored. Steady-state and picosecond time-resolved fluorescence measurements have revealed that selective excitation of the encapsulated guest fluorophore results in efficient energy transfer from the excited guest to an emissive host−guest exciplex state.

Halorhodopsin is a retinal protein that acts as a light-driven chloride pump in the Haloarchaeal cell membrane. A chloride ion is bound near the retinal chromophore, and light-induced all-trans → 13-cis isomerization triggers the unidirectional chloride ion pump. We investigated the primary ultrafast dynamics of Natronomonas pharaonis halorhodopsin that contains Cl−, Br−, or I− (pHR-Cl−, pHR-Br−, or pHR-I−) using ultrafast pump−probe spectroscopy with ∼30 fs time resolution. All of the temporal behaviors of the Sn ← S1 absorption, ground-state bleaching, K intermediate (13-cis form) absorption, and stimulated emission were observed. In agreement with previous reports, the primary process exhibited three dynamics. The first dynamics corresponds to the population branching process from the Franck−Condon (FC) region to the reactive (S1r) and nonreactive (S1nr) S1 states. With the improved time resolution, it was revealed that the time constant of this branching process (τ1) is as short as 50 fs. The second dynamics was the isomerization process of the S1r state to generate the ground-state 13-cis form, and the time constant (τ2) exhibited significant halide ion dependence (1.4, 1.6, and 2.2 ps for pHR-Cl−, pHR-Br−, and pHR-I−, respectively). The relative quantum yield of the isomerization, which was evaluated from the pump−probe signal after 20 ps, also showed halide ion dependence (1.00, 1.14, and 1.35 for pHR-Cl−, pHR-Br−, and pHR-I−, respectively). It was revealed that the halide ion that accelerates isomerization dynamics provides the lower isomerization yield. This finding suggests that there is an activation barrier along the isomerization coordinate on the S1 potential energy surface, meaning that the three-state model, which is now accepted for bacteriorhodopsin, is more relevant than the two-state model for the isomerization process of halorhodopsin. We concluded that, with the three-state model, the isomerization rate is controlled by the height of the activation barrier on the S1 potential energy surface while the overall isomerization yield is determined by the branching ratios at the FC region and the conical intersection. The third dynamics attributable to the internal conversion of the S1nr state also showed notable halide ion dependence (τ3 = 4.5, 4.6, and 6.3 ps for pHR-Cl−, pHR-Br−, and pHR-I−). This suggests that some geometrical change may be involved in the relaxation process of the S1nr state.

In situ characterization of surface denaturation of a protein was realized by newly developed interface-selective multiplex electronic sum frequency generation spectroscopy. The observed electronic spectra of cytochrome c at the air/water interface exhibited a broad feature, which demonstrated coexistence of the nativelike and denatured protein at the interface. This situation of the mixed conformation at the air/water interface did not change in the acidic condition of pH = 2 where the protein was completely denatured in the bulk water. In sharp contrast, only native spectrum was observed at the silica/water interface.

Abstract

Understanding a chemical reaction ultimately requires the knowledge of how each atom in the reactants moves during product formation. Such knowledge is seldom complete and is often limited to an oversimplified reaction coordinate that neglects global motions across the molecular framework. To overcome this limit, we recorded transient impulsive Raman spectra during ultrafast photoisomerization of cis-stilbene in solution. The results demonstrate a gradual frequency shift of a low-frequency spectator vibration, reflecting changes in the restoring force along this coordinate throughout the isomerization. A high-level quantum-chemical calculation reproduces this feature and associates it with a continuous structural change leading to the twisted configuration. This combined spectroscopic and computational approach should be amenable to detailed reaction visualization in other photoisomerizing systems as well.

The dynamics and mechanism of the double proton transfer reaction of the 7-azaindole dimer was investigated in solution by excitation wavelength dependence in steady-state and femtosecond time-resolved fluorescence spectroscopy. Femtosecond measurements in the UV region revealed that the dynamics of the dimer fluorescence exhibits remarkable change as the excitation wavelength was scanned from 280 to 313 nm. The fluorescence showed a biexponential decay (0.2 and 1.1 ps) with 280-nm excitation, whereas it exhibited a single exponential decay (1.1 ps) with 313-nm excitation (the red-edge of the dimer absorption). This observation clearly indicates that the 0.2-ps component is irrelevant to the proton transfer. In the visible region, we found that the tautomer fluorescence rises in accordance with the decay of the dimer fluorescence with a common time constant of 1.1 ps. This finding unambiguously denies the appearance of any intermediate species in between the dimer and tautomer excited states, indicating that the double proton transfer reaction is essentially a single-step process. We conclude that the double proton transfer of the 7-azaindole dimer in solution proceeds in the concerted manner from the lowest excited state with the 1.1-ps time constant. On the basis of the experimental data obtained, we discuss the long-lasting concerted versus step-wise controversy for the double proton transfer mechanism in solution.

Imaginary χ(2) spectra of HOD at air/charged surfactant/aqueous interfaces highly resemble the IR spectrum of the bulk liquid HOD, showing no indication of the “ice-like” structure. Clearly, the hydrogen bond structures at highly charged interfaces are not like ice but very similar to the structure in the bulk.

We study the effective polarity of an air/liquid-mixture interface by using interface-selective heterodyne-detected electronic sum frequency generation (HD-ESFG) and vibrational sum frequency generation (HD-VSFG) spectroscopies. With water and N,N-dimethylformamide (DMF) chosen as two components of the liquid mixture, the bulk polarity of the mixture is controlled nearly arbitrarily by the mixing ratio. The effective polarity of the air/mixture interface is evaluated by HD-ESFG with a surface-active solvatochromic molecule used as a polarity indicator. Surprisingly, the interfacial effective polarity of the air/mixture interface increases significantly, when the bulk polarity of the mixture decreases (i.e. when the fraction of DMF increases). Judging from the hydrogen-bond structure at the air/mixture interface clarified by HD-VSFG, this anomalous change of the interfacial effective polarity is attributed to the interface-specific solvation structure around the indicator molecule at the air/mixture interface.

We report a femtosecond time-resolved fluorescence study of cis-stilbene, a prototypical molecule showing ultrafast olefinic photoisomerization and photocyclization. The time-resolved fluorescence signals were measured in a nonpolar solvent over a wide ultraviolet-visible region with excitation at 270 nm. The time-resolved fluorescence traces exhibit non-single exponential decays which are well fit with bi-exponential functions with time constants of τA = 0.23 ps and τB = 1.2 ps, and they are associated with the fluorescence emitted from different regions of the S1 potential energy surface (PES) in the course of the structural change. Quantitative analysis revealed that the two fluorescent components exhibit similar intrinsic time-resolved spectra extending from 320 nm to 700 nm with the (fluorescence) oscillator strength of fA = 0.32 and fB = 0.21, respectively. It was concluded that the first component is assignable to the fluorescence from the untwisted S1 PES region where the molecule reaches immediately after the initial elongation of the central CC bond, while the second component is the fluorescence from the substantially twisted region around a shallow S1 potential minimum. The quantitative analysis of the femtosecond fluorescence data clearly showed that the whole isomerization process proceeds in the one-photon allowed S1 state, thereby resolving a recent controversy in quantum chemical calculations about the reactive S1 state. In addition, the evaluated oscillator strengths suggest that the population branching into the isomerization/cyclization pathways occurs in a very early stage when the S1 molecule still retains a planar Ph–CC–Ph skeletal structure. On the basis of the results obtained, we discuss the dynamics and mechanism of the isomerization/cyclization reactions of cis-stilbene, as well as the electronic structure of the reaction precursor.

The Cu(I) complexes having phenanthroline derivatives as ligands are known to exhibit photo-induced ‘flattening’ structural change in the metal-to-ligand charge transfer (MLCT) excited state. Our recent ultrafast spectroscopic studies of [Cu(dmphen)2]+ (dmphen = 2,9-dimethyl-1,10-phenanthroline) showed that the photo-induced structural change predominantly occurs in the S1 state on a subpicosecond time scale, with the appearance of the ‘perpendicular’ S1 state before the structural change. In this work, we carried out femto/picosecond time-resolved emission spectroscopy of [Cu(phen)2]+ (phen = 1,10-phenanthroline) and [Cu(dpphen)2]+ (dpphen = 2,9-diphenyl-1,10-phenanthroline) in dichloromethane with the S2 ← S0 photo-excitation to examine the substituent effect on the ultrafast structural change. The femtosecond time-resolved emission spectra of the two complexes exhibit ultrafast fluorescence changes that are attributed to the structural change in the S1 state after fast (50–100 fs) S2 → S1 internal conversion. By comparing with the dynamics of [Cu(dmphen)2]+, it was found that the time constant of the structural change increases as the substituents at 2- and 9- positions of the ligand become bulkier, i.e., [Cu(phen)2]+ (200 fs) < [Cu(dmphen)2]+ (660 fs) < [Cu(dpphen)2]+ (920 fs). This implies that the complex needs a longer time to flatten with the bulkier substituent. This demonstrates that the dynamics of the photo-induced structural change of Cu(I) complexes is substantially affected by the substituent of the ligand. The dynamics of the ultrafast structural change and the substituent effect are discussed with the multidimensional S1 potential energy surface of Cu(I) complexes.

The substituent effect on the excited-state dynamics of bis-diimine Cu(I) complexes was investigated by femtosecond time-resolved absorption spectroscopy with the S1 ← S0 metal-to-ligand charge transfer (MLCT) photoexcitation. The time-resolved absorption of [Cu(phen)2]+ (phen = 1,10-phenanthroline) showed a slight intensity increase of the S1 absorption with a time-constant of 0.1–0.2 ps, reflecting the flattening distortion occurring in the S1 state. The transient absorption of the ‘flattened’ S1 state was clearly observed, although its fluorescence was not observed in the previous fluorescence up-conversion study in the visible region. The flattened S1 state decayed with a time constant of ∼2 ps, and the S0 bleaching recovered accordingly. This clarifies that the S1 state of [Cu(phen)2]+ is predominantly relaxed to the S0 state by internal conversion. The time-resolved absorption of [Cu(dpphen)2]+ (dpphen = 2,9-diphenyl-1,10-phenanthroline) showed a 0.9 ps intensity increase of the S1 absorption due to the flattening distortion, and then exhibited a 11 ps spectral change due to the intersystem crossing. This excited-state dynamics of [Cu(dpphen)2]+ is very similar to that of [Cu(dmphen)2]+ (dmphen = 2,9-dimethyl-1,10-phenanthroline). In the ultrafast pump–probe measurements with 35 fs time resolution, [Cu(phen)2]+ and [Cu(dpphen)2]+ exhibited oscillation due to the nuclear wavepacket motions of the initial S1 state, and the oscillation was damped as the structural change took place. This indicates that the initial S1 states have well-defined vibrational structures and that the vibrational coherence is retained in their short lifetimes. The present time-resolved absorption study, together with the previous time-resolved fluorescence study, provides a unified view for the ultrafast dynamics of the MLCT excited state of the Cu(I) complexes.

Elucidation of the molecular mechanisms of protein adsorption is of essential importance for further development of biotechnology. Here, we use interface-selective nonlinear vibrational spectroscopy to investigate protein charge at the air/water interface by probing the orientation of interfacial water molecules. We measured the Imχ(2) spectra of hemoglobin, myoglobin, serum albumin and lysozyme at the air/water interface in the CH and OH stretching regions using heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy, and we deduced the isoelectric point of the protein by monitoring the orientational flip-flop of water molecules at the interface. Strikingly, our measurements indicate that the isoelectric point of hemoglobin is significantly lowered (by about one pH unit) at the air/water interface compared to that in the bulk. This can be predominantly attributed to the modifications of the protein structure at the air/water interface. Our results also suggest that a similar mechanism accounts for the modification of myoglobin charge at the air/water interface. This effect has not been reported for other model proteins at interfaces probed by conventional VSFG techniques, and it emphasizes the importance of the structural modifications of proteins at the interface, which can drastically affect their charge profiles in a protein-specific manner. The direct experimental approach using HD-VSFG can unveil the changes of the isoelectric point of adsorbed proteins at various interfaces, which is of major relevance to many biological applications and sheds new light on the effect of interfaces on protein charge.

The local environment within a recently developed anthracene-shelled micelle (ASM), which is a micelle-like nanocapsule composed of anthracene-embedded amphiphiles, was investigated by steady-state and time-resolved spectroscopy of an encapsulated solvatochromic fluorescent probe molecule, coumarin 153 (C153). The absorption maximum of encapsulated C153 (452 nm) is more red-shifted than that of free C153 in water, indicating a highly polar environment inside the micelle. Despite this, the fluorescence Stokes shift of encapsulated C153 (∼3700 cm−1) is substantially smaller than that of free C153 in water. Femtosecond time-resolved broadband fluorescence measurements further showed that the dynamic Stokes shift is completed within 1 ps, revealing that the reorganization of the micelle interior following photoexcitation of the C153 probe is characterized by a sub-picosecond, limited-amplitude response. The femtosecond fluorescence anisotropy data showed that the orientational diffusion of the host–guest complex is slower (860 ps) than that of the empty micelle (510 ps), suggesting that the micelle structure is flexible enough to expand when the guest molecule is accommodated and that the micelle rotates with the encapsulated guest molecule. This softness of the micelles further allows some of them to simultaneously encapsulate two C153 molecules, as evidenced by the appearance of blue-shifted, H-dimer-like absorption and fluorescence bands. Based on these steady-state and femtosecond time-resolved spectroscopic data, we discuss the electronic state of C153 and micelle structure as well as the host–guest interaction in this novel flexible synthetic nanocapsule.

Au–Au bond strengthening in photoexcited dimers of an Au(I) complex is captured in solution as oscillations of femtosecond absorption signals. The subsequent dynamics, when compared to the trimer's data, confirm that the bent-to-linear structural change of the trimer occurs in the first few picoseconds.