Stimulated Raman Spectroscopy Research Articles

Substituent Effects in the Cationic Green Fluorescent Protein Chromophore: Ultrafast Excited‐State Proton Transfer or Twisting?

Understanding the structure‐function relationships of the green fluorescent protein (GFP) chromophore is important in rationally developing new molecular tools for biological imaging and beyond. Herein, we systematically modified the GFP chromophore structure with electron‐withdrawing and ‐donating groups (EWGs and EDGs) to investigate the substituent effects on the excited‐state proton transfer (ESPT) and twisting dynamics of the cationic chromophore in solution. With key insights gained from femtosecond transient absorption and stimulated Raman spectroscopy, we reveal that the EWG substitution by –F increases photoacidity in an additive manner and leads to an ultrafast barrierless ESPT by difluorination, while the EDG substitution by –OCH3 also results in ultrafast ESPT despite the weak photoacidity as estimated by the Förster equation. We ascribe the unusually fast kinetics in methoxylated derivatives to the occurrence of a pre‐existing chromophore‐solvent complex that sets up the acceptor site for ESPT. Furthermore, the kinetic competition between ESPT and twisting pathways is crucial for the observation of ESPT in action, particularly for molecules undergoing efficient nonradiative decay in the excited state through torsional motions. Such flexible and highly engineerable molecules can enable more versatile photoswitches and sensors.

Hyper spectral resolution stimulated Raman spectroscopy with amplified fs pulse bursts

We present a novel approach for Stimulated Raman Scattering (SRS) spectroscopy in which a hyper spectral resolution and high-speed spectral acquisition are achieved by employing amplified offset-phase controlled fs-pulse bursts. We investigate the method by solving the coupled non-linear Schrödinger equations and validate it by numerically characterizing SRS in molecular nitrogen as a model compound. The spectral resolution of the method is found to be determined by the inverse product of the number of pulses in the burst and the intraburst pulse separation. The SRS spectrum is obtained through a motion-free scanning of the offset phase that results in a sweep of the Raman-shift frequency. Due to high spectral resolution and fast motion-free scanning the technique is beneficial for a number SRS-based applications such as gas sensing and chemical analysis.

Probing avoided crossings and conical intersections by two-pulse femtosecond stimulated Raman spectroscopy: Theoretical study

This study leverages two-pulse femtosecond stimulated Raman spectroscopy (2FSRS) to characterize molecular systems with avoided crossings (ACs) and conical intersections (CIs) in their low-lying excited electronic states. By simulating 2FSRS spectra of microscopically inspired ACs and CIs models, we demonstrate that 2FSRS not only delivers valuable information on the molecular parameters characterizing ACs and CIs but also helps distinguish between these two systems.

Preclinical Evaluation of NTX-301, a Novel DNA Hypomethylating Agent in Ovarian Cancer.

DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1-3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301-induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301-treated xenografts. NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

Capturing excited-state structural snapshots of evolutionary green-to-red photochromic fluorescent proteins.

Photochromic fluorescent proteins (FPs) have proved to be indispensable luminous probes for sophisticated and advanced bioimaging techniques. Among them, an interplay between photoswitching and photoconversion has only been observed in a limited subset of Kaede-like FPs that show potential for discovering the key mechanistic steps during green-to-red photoconversion. Various spectroscopic techniques including femtosecond stimulated Raman spectroscopy (FSRS), X-ray crystallography, and femtosecond transient absorption were employed on a set of five related FPs with varying photoconversion and photoswitching efficiencies. A 3-methyl-histidine chromophore derivative, incorporated through amber suppression using orthogonal aminoacyl tRNA synthetase/tRNA pairs, displays more dynamic photoswitching but greatly reduced photoconversion versus the least-evolved ancestor (LEA). Excitation-dependent measurements of the green anionic chromophore reveal that the varying photoswitching efficiencies arise from both the initial transient dynamics of the bright cis state and the final trans-like photoswitched off state, with an exocyclic bridge H-rocking motion playing an active role during the excited-state energy dissipation. This investigation establishes a close-knit feedback loop between spectroscopic characterization and protein engineering, which may be especially beneficial to develop more versatile FPs with targeted mutations and enhanced functionalities, such as photoconvertible FPs that also feature photoswitching properties.

Investigation of ultrafast photoisomerization dynamics of azobenzene derivative (E)-1-phenyl-2-((triisopropylsilyl)ethynyl)diazene

When exposed to light at a specific wavelength, azobenzene and its derivatives experience a transformation from trans form to cis form through isomerization. Due to its ability to change color upon illumination, azobenzene finds extensive use in various molecular devices and functional materials. However, despite significant researches focused on practical applications, there are still ongoing investigations into the underlying mechanisms governing azobenzene’s photochemical reactions and isomerization. In this study, we employ femtosecond stimulated Raman spectroscopy (FSRS), and transient absorption spectroscopy, in conjunction with quantum chemical calculations, to elucidate the ultrafast isomerization dynamics of an azobenzene derivative known as trans-AZOTIPS ((E)-1-phenyl-2-((triisopropylsilyl)ethynyl)diazene). The results demonstrate that upon photoexcitation, rapid isomerization occurs along the C−N=N bonds via the singlet excited state S1 to hot ground state (S0*) state transition. Additionally, we explore the impact of solvent viscosity on the isomerization process and find that the duration of isomerization remains unaffected by variations in solvent viscosity. These results suggest that the isomerization pathway involves a volume-conserving motion known as “hula twist”. After that, the vibrational cooling process is obtained in S0 state.

PATH-62. RAMAN-BASED AI PLATFORM TO ACCELERATE THE DIAGNOSIS AND MANAGEMENT OF GLIOMAS VIA RAPID METHYLATION PROFILING

Abstract BACKGROUND DNA methylation profiling and subtyping of gliomas has become an integral part of the diagnosis and treatment of glioma patients but remains a challenge due to the sample preparation and time requirements of the existing methylation profiling technology. We hypothesized that machine learning and Raman spectroscopy, which creates fast molecular fingerprints of samples in situ based on chemical signatures rather than the use of exogenous dyes, can provide a fast alternative to the existing approaches for methylation profiling and classification of gliomas. METHODS Raman spectroscopy at the spatial resolution of less than 300nm was used for molecular fingerprinting of the regions of interest using 1mm2 Formalin-Fixed Paraffin-Embedded (FFPE) tissue spots from 45 patient samples with known LGm1 to LGm6 methylation subtypes. Spectral information (over 1738 frequencies) was used to construct tumor/non-tumor, IDH1WT/IDH1mut, and methylation-subtype classifiers. Oversampling was used to obtain subtype-balanced data distributions. Supervised wrapper methods and random forests were used to identify the top 20 most discriminatory Raman frequencies. Stimulated Raman spectroscopy was used to validate the findings. RESULTS We developed APOLLO - a novel platform based on spontaneous Raman spectroscopy and machine learning -- for predicting the DNA methylation subtypes of FFPE glioma tissue specimens. APOLLO discriminates tumors from non-tumor areas with 98% accuracy and discriminates IDH1mut versus IDH1WT tumors with 82% accuracy. APOLLO also achieved high discriminatory power between G-CIMP-high and G-CIMP-low molecular phenotypes (ROC of 0.75), subtypes of lower-grade IDH1mut gliomas with significantly different clinical outcomes. We determined that the Raman shifts important for discriminating IDH1mut versus IDH1wt tumors are associated with novel lipid-metabolism signatures of IDH1mut glioma. CONCLUSIONS The development of APOLLO allows fast, reliable, and accurate prediction of methylation subtypes of glioma which can speed diagnosis and, once validated on fresh tissue, can be implemented in the operating room.

The Effect of the Mid‐Shell Thickness on the Charge‐Carrier Dynamics of the Green‐Light Emitting InP/ZnSe/ZnS Core‐Shell Quantum Dots

AbstractUltrafast charge carrier dynamics of green‐light emitting InP/ZnSe/ZnS core–shell quantum dots (QDs) with three different ZnSe thickness of 2.0 nm (gQD‐20), 2.4 nm (gQD‐24), or 3.3 nm (gQD‐33) are interrogated by femtosecond transient absorption (TA) and femtosecond stimulated Raman spectroscopy (FSRS). Auger‐type cooling of hot electron into the band‐edge level of InP is clearly manifested in the TA spectra, giving its time constant of ≈222, 300, or 349 fs for gQD‐20, gQD‐24, or gQD‐33, respectively, indicating that the state‐filling to the 1S(e) of InP is significantly slowed down with the increase of the mid shell thickness. The global analysis of the whole TA spectra turns out to be extremely useful to explain the dynamic behavior of the heterogeneous ensemble of single and/or bi‐ (or multi‐) excitons in the presence of the Stark‐shift. The hole‐relaxation dynamics into the band‐edge 1S (h) of the core InP is revealed in the bleaching dynamics of the FSR bands corresponding to the phonon mode of InP or ZnSe, giving the estimated lifetime of 1.0–1.4 ps for gQD‐20. The hole‐relaxation dynamics by the phonon emission seem to be little sensitive to the ZnSe shell thickness.

Diffuse Reflectance-Based Femtosecond Stimulated Raman Spectroscopy of Opaque Suspensions.

By augmentation of the collection optics utilized in transmission-based femtosecond stimulated Raman spectroscopy (FSRS), two novel diffuse reflectance-based femtosecond stimulated Raman spectroscopy (drFSRS) techniques were developed. These techniques were then used to collect the Raman spectra of opaque systems, those being cyclohexane-intercalated poly(tetrafluoroethylene) microbeads and ethanol in 1% intralipid solutions. The resulting drFSRS data from the cyclohexane:PTFE system show significant distortion of the depolarization ratio of the 803 cm-1 cyclohexane peak, indicating a loss of incident pump:probe polarization in a scattering environment. The drFSRS data from the ethanol in 1% intralipid solution demonstrate less signal strength but equal spectral resolution when compared to transmission-based FSRS of the same sample. The results presented in this Technical Note demonstrate the current capabilities of collecting stimulated Raman spectra of opaque systems using drFSRS.

Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins.

Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next-generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady-state and time-resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis-trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited-state proton transfer in various LSSRFPs showcases the resolving power of wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red-emitting species. Moreover, recent progress in noncanonical RFPs with a site-specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

The Best Models of Bodipy's Electronic Excited State: Comparing Predictions from Various DFT Functionals with Measurements from Femtosecond Stimulated Raman Spectroscopy.

Density functional theory (DFT) and time-dependent DFT (TD-DFT) are pivotal approaches for modeling electronically excited states of molecules. However, choosing a DFT exchange-correlation functional (XCF) among the myriad of alternatives is an overwhelming task that can affect the interpretation of results and lead to erroneous conclusions. The performance of these XCFs to describe the excited-state properties is often addressed by comparing them with high-level wave function methods or experimentally available vertical excitation energies; however, this is a limited analysis that relies on evaluation of a single point in the excited-state potential energy surface (PES). Different strategies have been proposed but are limited by the difficulty of experimentally accessing the electronic excited-state properties. In this work, we have tested the performance of 12 different XCFs and TD-DFT to describe the excited-state potential energy surface of Bodipy (2,6-diethyl-1,3,5,7-tetramethyl-8-phenyldipyrromethene difluoroborate). We compare those results with resonance Raman spectra collected by using femtosecond stimulated Raman spectroscopy (FSRS). By simultaneously fitting the absorption spectrum, fluorescence spectrum, and all of the resonance Raman excitation profiles within the independent mode displaced harmonic oscillator (IMDHO) formalism, we can describe the PES at the Franck-Condon (FC) region and determine the solvent and intramolecular reorganization energy after relaxation. This allows a direct comparison of the TD-DFT output with experimental observables. Our analysis reveals that using vertical absorption energies might not be a good criterion to determine the best XCF for a given molecular system and that FSRS opens up a new way to benchmark the excited-state performance of XCFs of fluorescent dyes.

Conformational Relaxation Dynamics of Poly(3-hexylthiophene) Photoexcited in Solution as Studied by Femtosecond Time-Resolved Stimulated Raman Spectroscopy in 1190-1550 nm Region.

When a conjugated polymer is photoexcited in solution, its effective conjugation length in the singlet exciton state often increases through the conformational relaxation of the polymer main chain and/or hopping of the excitation. We measured femtosecond time-resolved near-IR stimulated Raman spectra of poly(3-hexylthiophene) (P3HT) photoexcited in four organic solvents for understanding the dynamics of the exciton elongation through the conformational relaxation separately from that through the exciton hopping. In the ring CC stretch frequency region, a band appears at around 1415 cm-1 and decays, while a new band rises at around 1370 cm-1. The average time constant of the change is estimated to be 8.7-19 ps and correlated almost linearly with the viscosity of the solvents. These results suggest that the main chain of P3HT in the singlet exciton state relaxes from a twisted form to a planar form in the 0-100 ps range when it surmounts an activation barrier of 5.8-7.8 kJ mol-1, generated possibly by the steric effect of the hexyl side group. When the rise of the 1370 cm-1 band is analyzed in detail, it is reproduced with two exponential rise functions with time constants of 0-3.3 and 16-22 ps. The two rise components suggest that a portion of P3HT forms a cluster in solution, while the other portion of P3HT is isolated.

High‐resolution stimulated Raman spectroscopy of low‐frequency rotational transitions: Obtention of collisional broadening coefficients in the rotational S branch of CO perturbed by H2

AbstractWe demonstrate the application of high‐resolution quasi‐continuous (q‐cw) stimulated Raman spectroscopy (SRS) to the study of low‐frequency, pure rotational transitions of molecules in diluted samples in order to obtain collisional information. The main problems encountered in the few pioneering q‐cw SRS rotational works, namely weak signals because of the small population differences between the upper and lower states of the transitions and the presence of alternating current (AC) Stark effect, have been addressed: technical improvements in the optical elements that perform the beam mixing, separation, and filtering have allowed us to use a collinear beam geometry and a triple pass configuration to enhance the Raman signals. The distortions in the line profiles caused by AC Stark effect have been measured and included in the numerical treatment of the spectra. With these extended capabilities, we have been able to register stimulated Raman transitions with wavenumbers as low as ∼ 11.5 cm−1, corresponding to the S(0) pure rotational transition of the molecule of CO. We have applied the setup to the study of collisional broadening of CO rotational lines perturbed by H2 and have obtained broadening coefficients for lines from S(0) to S(20) at 77, 180, and 298 K. A comparison between these anisotropic coefficients and those obtained from IR rovibrational experiments shows excellent agreement. This is, to our knowledge, the first demonstration of the use of rotational SRS for the obtention of collisional information in diluted gas samples.

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.

Rapid-Scan Resonant Two-Dimensional Impulsive Stimulated Raman Spectroscopy of Excited States.

Photochemical reactions occur in the electronically excited state, which is effectively represented by a multidimensional potential energy surface (PES) with a vast degree of freedom of nuclear coordinates. The elucidation of the intricate shape of the PES constitutes an important topic in the field of photochemistry and has long been studied both experimentally and theoretically. Recently, fully time-domain resonant two-dimensional Raman spectroscopy has emerged as a potentially powerful tool to provide unique information about the coupling between vibrational manifolds in the excited state. However, the wide application of this technique has been significantly hampered by the technical difficulties associated with experimental implementation and remains challenging. Herein, we demonstrate time-domain resonant two-dimensional impulsive stimulated Raman spectroscopy (2D-ISRS) of excited states using sub-10 fs pulses based on the rapid scan of the time delay, which facilitates the efficient collection of time-domain vibrational signals with high sensitivity. As a proof-of-principle experiment, we performed 2D-ISRS of 6,13-bis(triisopropylsilylethynyl)pentacene (TIPS-pentacene) in solution. Through 2D Fourier transformation of the high-quality time-time oscillatory signal, we obtained a 2D frequency-frequency correlation map of excited-state TIPS-pentacene in the broad frequency window of 0-2000 cm-1. The data clearly resolve a number of cross peaks that signify the correlations among excited-state vibrational manifolds. The high capability of the rapid-scan-based 2D-ISRS spectrometer presented in this study enables the systematic investigation of various photochemical reaction systems, thereby further promoting the understanding and applications of this new multidimensional spectroscopy.

Torsional Wave-Packet Dynamics in 2-Fluorobiphenyl Investigated by State-Selective Ionization-Detected Impulsive Stimulated Raman Spectroscopy.

We report the creation and observation of vibrational wave packets pertinent to torsional motion in a biphenyl derivative in its electronic ground-state manifold. Adiabatically cooled molecular samples of 2-fluorobiphenyl were irradiated by intense nonresonant ultrashort laser pulses to drive impulsive stimulated Raman excitation of torsional motion. Spectral change due to the nonadiabatic vibrational excitation is probed in a state-selective manner using resonance-enhanced two-photon ionization through the S1 ← S0 electronic transition. The coherent nature of the excitation was exemplified by adopting irradiation with a pair of pump pulses: observed signals for excited torsional levels exhibit oscillatory variations against the mutual delay between the pump pulses due to wave-packet interference. By taking the Fourier transform of the time course of the signals, energy intervals among torsional levels with v = 0-3 were determined and utilized to calibrate a density functional theory (DFT)-calculated torsional potential-energy function. Time variation of populations in the excited torsional levels was assessed experimentally by measuring integrated intensities of the corresponding transitions while scanning the delay. Early time enhancement of the population (up to ∼2 ps) and gradual degradation of coherence (within ∼20 ps) appears. To explain the observed distinctive features, we developed a four-dimensional (4D) dynamical calculation in which one-dimensional (1D) quantum-mechanical propagation of the torsional motion was followed by solving the time-dependent Schrödinger equation, whereas three-dimensional (3D) molecular rotation was tracked by classical trajectory calculations. This hybrid approach enabled us to reproduce experimental results at a reasonable computational cost and provided a deeper insight into rotational effects on vibrational wave-packet dynamics.

Investigation of Excited-State Intramolecular Proton Transfer and Structural Dynamics in Bis-Benzimidazole Derivative (BBM).

The bis-benzimidazole derivative (BBM) molecule, consisting of two 2-(2'-hydroxyphenyl) benzimidazole (HBI) halves, has been synthesized and successfully utilized as a ratiometric fluorescence sensor for the sensitive detection of Cu2+ based on enol-keto excited-state intramolecular proton transfer (ESIPT). In this study, we strategically implement femtosecond stimulated Raman spectroscopy and several time-resolved electronic spectroscopies, aided by quantum chemical calculations to investigate the detailed primary photodynamics of the BBM molecule. The results demonstrate that the ESIPT from BBM-enol* to BBM-keto* was observed in only one of the HBI halves with a time constant of 300 fs; after that, the rotation of the dihedral angle between the two HBI halves generated a planarized BBM-keto* isomer in 3 ps, leading to a dynamic redshift of BBM-keto* emission.

Theoretical description for nonlinear dynamic light scattering based on stimulated Raman effect (NLDLS‐SRS)

AbstractDynamic light scattering (DLS) is a method to analyze the diffusion constant and mesostructure of colloidal systems based on refractive index fluctuations. For example, DLS evaluates particle size distribution, particle drift velocity, and the typical length of the cooperative diffusion in gels. However, DLS inherently lacks chemical sensitivity since it detects the spatial Fourier component of the refractive index at the scattering vector (q). This work theoretically demonstrates the feasibility of nonlinear dynamic light scattering (NLDLS) of stimulated Raman effect that reflects molecular vibration. Specifically, we show that by interfering with two pump beams (in the pump‐probe method terminology), it is possible to extract the spatial Fourier component of the imaginary part of the third‐order nonlinear susceptibility. Here, the phase modulation of either of the two beams is effective for mode‐selective detection of the Fourier component. The difference in the wavevectors of these beams corresponds to q in DLS. The result is the same as DLS in mesostructural sensitivity but has a molecular vibration selectivity. The proposed assay in the present paper enables qualitative and chemical‐structure analysis of the colloidal systems without labeling and size differentiation in a mesopic regime.

Photogrammetry of Ultrafast Excited-State Intramolecular Proton Transfer Pathways in the Fungal Pigment Draconin Red.

Proton transfer processes of organic molecules are key to charge transport and photoprotection in biological systems. Among them, excited-state intramolecular proton transfer (ESIPT) reactions are characterized by quick and efficient charge transfer within a molecule, resulting in ultrafast proton motions. The ESIPT-facilitated interconversion between two tautomers (PS and PA) comprising the tree fungal pigment Draconin Red in solution was investigated using a combination of targeted femtosecond transient absorption (fs-TA) and excited-state femtosecond stimulated Raman spectroscopy (ES-FSRS) measurements. Transient intensity (population and polarizability) and frequency (structural and cooling) dynamics of -COH rocking and -C=C, -C=O stretching modes following directed stimulation of each tautomer elucidate the excitation-dependent relaxation pathways, particularly the bidirectional ESIPT progression out of the Franck-Condon region to the lower-lying excited state, of the intrinsically heterogeneous chromophore in dichloromethane solvent. A characteristic overall excited-state PS-to-PA transition on the picosecond timescale leads to a unique "W"-shaped excited-state Raman intensity pattern due to dynamic resonance enhancement with the Raman pump-probe pulse pair. The ability to utilize quantum mechanics calculations in conjunction with steady-state electronic absorption and emission spectra to induce disparate excited-state populations in an inhomogeneous mixture of similar tautomers has broad implications for the modeling of potential energy surfaces and delineation of reaction mechanisms in naturally occurring chromophores. Such fundamental insights afforded by in-depth analysis of ultrafast spectroscopic datasets are also beneficial for future development of sustainable materials and optoelectronics.

Photoexcited Plasmon-Driven Ultrafast Dynamics of the Adsorbate Probed by Femtosecond Time-Resolved Surface-Enhanced Time-Domain Raman Spectroscopy.

Metal nanoparticles have high potential in light-harvesting applications by transferring absorbed photon energy to the adsorbates. However, photoexcited plasmon-driven ultrafast dynamics of the adsorbate on metal nanoparticles have not been clearly understood. We studied ultrafast plasmon-driven processes of trans-1,2-bis(4-pyridyl)ethylene (BPE) adsorbed on gold nanoparticle assemblies (GNAs) using time-resolved surface-enhanced impulsive stimulated Raman spectroscopy (TR-SE-ISRS). After photoexciting the localized surface plasmon resonance (LSPR) band of the GNAs, we measured femtosecond time-resolved surface-enhanced Raman spectra of the adsorbate, which exhibited transient bleach in the Raman signal and following biphasic recovery that proceeds on the time scale of a few tens of picoseconds. The TR-SE-ISRS data were analyzed with singular value decomposition, and the obtained species-associated Raman spectra indicated that photoexcitation of the LSPR band alters chemical interaction between BPE and the GNAs on an ultrafast time scale; initial steady-state BPE is recovered through a precursor state that has weaker interaction with the GNAs.