The hydrogen-bonding dynamics of water to a nitrile-functionalized electrode is modulated by voltage according to ultrafast 2D IR spectroscopy.

We report the hydrogen-bonding dynamics of water to a nitrile-functionalized and plasmonic electrode surface as a function of applied voltage. The surface-enhanced two-dimensional infrared spectra exhibit hydrogen-bonded and non-hydrogen-bonded nitrile features in similar proportions, plus cross peaks between the two. Isotopic dilution experiments show that the cross peaks arise predominantly from chemical exchange between hydrogen-bonded and non-hydrogen-bonded nitriles. The chemical exchange rate depends upon voltage, with the hydrogen bond of the water to the nitriles breaking 2 to 3 times slower (>63 vs. 25 ps) under a positive as compared to a negative potential. Spectral diffusion created by hydrogen-bond fluctuations occurs on a ~1 ps timescale and is moderately potential-dependent. Timescales from molecular dynamics simulations agree qualitatively with the experiment and show that a negative voltage causes a small net displacement of water away from the surface. These results show that the voltage applied to an electrode can alter the timescales of solvent motion at its interface, which has implications for electrochemically driven reactions.

Label-Free Interference Imaging of Intracellular Trafficking.

Intracellular cargo trafficking is a highly regulated process responsible for transporting vital cellular components to their designated destinations. This intricate journey has been a central focus of cellular biology for many years. Early investigations leaned heavily on biochemical and genetic approaches, offering valuable insight into molecular mechanisms of cellular trafficking. However, while informative, these methods lack the capacity to capture the dynamic nature of intracellular trafficking. The advent of fluorescent protein tagging techniques transformed our ability to monitor the complete lifecycle of intracellular cargos, advancing our understanding. Yet, a central question remains: How do these cargos manage to navigate through traffic challenges, such as congestion, within the crowded cellular environment? Fluorescence-based imaging, though valuable, has inherent limitations when it comes to addressing the aforementioned question. It is prone to photobleaching, making long-term live-cell imaging challenging. Furthermore, they render unlabeled cellular constituents invisible, thereby missing critical environmental information. Notably, the unlabeled majority likely exerts a significant influence on the observed behavior of labeled molecules. These considerations underscore the necessity of developing complementary label-free imaging methods to overcome the limitations of fluorescence imaging or to integrate them synergistically.In this Account, we outline how label-free interference-based imaging has the potential to revolutionize the study of intracellular traffic by offering unprecedented levels of detail. We begin with a brief introduction to our previous findings in live-cell research enabled by interferometric scattering (iSCAT) microscopy, showcasing its aptitude and adeptness in elucidating intricate nanoscale intracellular structures. As we delved deeper into our exploration, we succeeded in the label-free visualization of the entire lifespan of nanoscale protein complexes known as nascent adhesions (NAs) and the dynamic events associated with adhesions within living cells. Our continuous efforts have led to the development of Dynamic Scattering-particle Localization Interference Microscopy (DySLIM), a generalized concept of cargo-localization iSCAT (CL-iSCAT). This label-free, high-speed imaging method, armed with iSCAT detection sensitivity, empowers us to capture quantitative and biophysical insights into cargo transport, providing a realistic view of the intricate nanoscale logistics occurring within living cells. Our in vivo studies demonstrate that intracellular cargos regularly contend with substantial traffic within the crowded cellular environment. Simultaneously, they employ inherent strategies for efficient cargo transport, such as collective migration and hitchhiking, to enhance overall transport rates─intriguingly paralleling the principle and practice of urban traffic management. We also highlight the synergistic benefits of combining DySLIM with chemical-selective fluorescent methods. This Account concludes with a "Conclusions and Outlook" section, outlining promising directions for future research and developments, with a particular emphasis on the functional application of iSCAT live-cell imaging. We aim to inspire further investigation into the efficient transport strategies employed by cells to surmount transportation challenges, shedding light on their significance in cellular phenomena.

Dual phase-detected infrared photothermal microscopy.

Infrared photothermal microscopy (IPM) has recently gained considerable attention as a versatile analytical platform capable of providing spatially resolved molecular insights across diverse research fields. This technique has led to numerous breakthroughs in the study of compositional variations in functional materials and cellular dynamics in living cells. However, its application to investigate multiple components of temporally dynamic systems, such as living cells and operational devices, has been hampered by the limited information content of the IP signal, which only covers a narrow spectral window (< 1 cm-1). Here, we present a straightforward approach for measuring two distinct IPM images utilizing the orthogonality between the in-phase and quadrature outputs of a lock-in amplifier, called dual-phase IR photothermal (DP-IP) detection. We demonstrate the feasibility of DP-IP detection for IPM in distinguishing two different micro-sized polymer beads.

Ultrafast measurement of photoacoustic parameters with mid-infrared frequency comb transients.

The photoacoustic (PA) effect has been widely used in various applications, including highly sensitive spectroscopy and label-free, non-invasive imaging. In this work, we demonstrate a fast and precise measurement of PA parameters for light-absorbing liquids using mid-infrared asynchronous sampling pump-probe measurements. To simulate the observed PA oscillation signals and extract various PA parameters as a function of pump power, we derived analytical solutions of the PA wave equation driven by a train of ultrashort Gaussian pump pulses. By fitting the analytical solution to the measured PA signals using a nonlinear curve fitting method, we could measure the PA parameters, including damping rate, viscosity, and natural frequency. Furthermore, the dynamic response of thermophysical properties of the chloroform solution is also investigated by measuring the variation of the Grüneisen parameter with pump power. We anticipate that this work will open new possibilities for precise in situ measurements of the thermal properties of light-absorbing liquids.

Molecular dynamics simulation study of water structure and dynamics on the gold electrode surface with adsorbed 4-mercaptobenzonitrile.

Understanding water dynamics at charged interfaces is of great importance in various fields, such as catalysis, biomedical processes, and solar cell materials. In this study, we implemented molecular dynamics simulations of a system of pure water interfaced with Au electrodes, on one side of which 4-mercaptobenzonitrile (4-MBN) molecules are adsorbed. We calculated time correlation functions of various dynamic quantities, such as the hydrogen bond status of the N atom of the adsorbed 4-MBN molecules, the rotational motion of the water OH bond, hydrogen bonds between 4-MBN and water, and hydrogen bonds between water molecules in the interface region. Using the Luzar-Chandler model, we analyzed the hydrogen bond dynamics between a 4-MBN and a water molecule. The dynamic quantities we calculated can be divided into two categories: those related to the collective behavior of interfacial water molecules and the H-bond interaction between a water molecule and the CN group of 4-MBN. We found that these two categories of dynamic quantities exhibit opposite trends in response to applied potentials on the Au electrode. We anticipate that the present work will help improve our understanding of the interfacial dynamics of water in various electrolyte systems.

Axial profiling of interferometric scattering enables an accurate determination of nanoparticle size.

Interferometric scattering (iSCAT) microscopy has undergone significant development in recent years. It is a promising technique for imaging and tracking nanoscopic label-free objects with nanometer localization precision. The current iSCAT-based photometry technique allows quantitative estimation for the size of a nanoparticle by measuring iSCAT contrast and has been successfully applied to nano-objects smaller than the Rayleigh scattering limit. Here we provide an alternative method that overcomes such size limitations. We take into account the axial variation of iSCAT contrast and utilize a vectorial point spread function model to uncover the position of a scattering dipole and, consequently, the size of the scatterer, which is not limited to the Rayleigh limit. We found that our technique accurately measures the size of spherical dielectric nanoparticles in a purely optical and non-contact way. We also tested fluorescent nanodiamonds (fND) and obtained a reasonable estimate for the size of fND particles. Together with fluorescence measurement from fND, we observed a correlation between the fluorescent signal and the size of fND. Our results showed that the axial pattern of iSCAT contrast provides sufficient information for the size of spherical particles. Our method enables us to measure the size of nanoparticles from tens of nanometers and beyond the Rayleigh limit with nanometer precision, making a versatile all-optical nanometric technique.

Thermoresponse of Odd-Even Effect in n-Alkanethiolate Self-Assembled Monolayers on Gold Substrates.

This study examines thermoresponse of odd-even effect in self-assembled monolayers (SAMs) of n-alkanethiolates (SCn , n=3-18) formed on template-stripped gold (AuTS ) using macro- and microscopic analytical techniques, contact angle goniometry (CAG) and vibrational sum frequency generation (VSFG) spectroscopy, respectively. Both CAG and VSFG analyses showed that the odd-even effect in liquid-like SAMs (n=3-9) disappeared upon heating at 50-70 °C, indicating that the heating led to increased structural disorder regardless of odd and even carbon numbers. In contrast, the opposite thermoresponse was observed for odd and even SCn molecules in wax- and solid-like SAMs (n=10-18). Namely, temperature-dependent orientational change of terminal CH3 relative to the surface normal was opposite for the odd and even molecules, thereby leading to mitigated odd-even effect. Our work offers important insights into thermoresponse of supramolecular structure in condensed organic matter.

Two-color infrared photothermal microscopy.

Infrared photothermal microscopy is an infrared (IR) imaging technique that enables non-invasive, non-destructive, and label-free investigations at the sub-micrometer scale. It has been applied in various research areas targeting pharmaceutical and photovoltaic materials as well as biomolecules in living systems. Despite its potency in observing biomolecules in living organisms, its practical application for cytological research has been restricted by the deficiency of molecular information from the IR photothermal signal, due to the narrow spectral width of a quantum cascade laser that is one of the most preferred IR excitation light sources for current IR photothermal imaging (IPI) techniques. Here, we address this issue by bringing modulation-frequency multiplexing into IR photothermal microscopy for developing a two-color IR photothermal microscopy technique. We demonstrate that the two-color IPI technique can be used to obtain the IR microscopic images of two discrete IR absorption bands and to distinguish two different chemical species in live cells with a sub-micrometer spatial resolution. We anticipate that the more general multi-color IPI technique and its use for metabolic studies of live cells could be realized by extending the present modulation-frequency multiplexing method.

Micro-Raman spectroscopic analysis of liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) plays a significant role in various biological processes, including the formation of membraneless organelles and pathological protein aggregation. Although many studies have found various factors that modulate the LLPS process or the liquid-to-solid phase transition (LSPT) using microscopy or fluorescence-based methods, the molecular mechanistic details underlying LLPS and protein aggregation within liquid droplets remain uncharacterized. Therefore, structural information on proteins inside liquid droplets is required to understand the mechanistic link to amyloid formation. In the present study, we monitored droplet formation related to protein fibrillation using micro-Raman spectroscopy in combination with differential interference contrast (DIC) microscopy to study the conformational change in proteins and the hydrogen-bonding (H-bonding) structure of water during LLPS. Interestingly, we found that the O-D stretching band for water (HOD in H2O) inside the droplets exhibited a distinct Raman spectrum from that of the bulk water, suggesting that the time-dependent change in the hydration environment in the protein droplets during the process of LLPS can be studied. These results demonstrate that the superior spatial resolution of micro-Raman spectroscopy offers significant advantages in investigating the molecular mechanisms of LLPS and following LSPT processes.

Molecular photothermal effects, diffusion, and sample flow in time-resolved spectroscopy and microscopy.

Time-resolved pump-probe and two-dimensional spectroscopy are widely used to study ultrafast chemical and biological processes in solutions. However, the corresponding signals at long times can be contaminated by molecular photothermal effects, which are caused by the nonradiative heat dissipation of photoexcited molecules to the surroundings. Additionally, molecular diffusion affects the transient spectroscopic signals because photoexcited molecules can diffuse away from the pump and probe beam focuses. Recently, a theoretical description of molecular photothermal effects on time-resolved IR spectroscopy was reported. In this work, I consider the molecular photothermal process, molecular diffusion, and sample flow to develop a generalized theoretical description of time-resolved spectroscopy. The present work can be used to interpret time-resolved spectroscopic signals of electronic or vibrational chromophores and understand the rate and mechanisms of the conversion of high-frequency molecular electronic and vibrational energy to solvent kinetic energy in condensed phases.

Vibrational Spectroscopic Map, Vibrational Spectroscopy, and Intermolecular Interaction.

Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

Label-Free Live-Cell Imaging of Internalized Microplastics and Cytoplasmic Organelles with Multicolor CARS Microscopy.

As the bioaccumulation of microplastics (MPs) is considered as a potential health risk, many efforts have been made to understand the cellular dynamics and cytotoxicity of MPs. Here, we demonstrate that label-free multicolor coherent anti-Stokes Raman scattering (CARS) microscopy enables separate vibrational imaging of internalized MPs and lipid droplets (LDs) with indistinguishable shapes and sizes in live cells. By simultaneously obtaining polystyrene (PS)- and lipid-specific CARS images at two very different frequencies, 1000 and 2850 cm-1, respectively, we successfully identify the local distribution of ingested PS beads and native LDs in Caenorhabditis elegans. We further show that the movements of PS beads and LDs in live cells can be separately tracked in real time, which allows us to characterize their individual intracellular dynamics. We thus anticipate that our multicolor CARS imaging method could be of great use to investigate the cellular transport and cytotoxicity of MPs without additional efforts for pre-labeling to MPs.

Molecular photothermal effects on time-resolved IR spectroscopy.

Time-resolved IR pump-probe (IR-PP) and two-dimensional IR (2D-IR) spectroscopy are valuable techniques for studying various ultrafast chemical and biological processes in solutions. The time-dependent changes of nonlinear IR signals reflecting fast molecular processes such as vibrational energy transfer and chemical exchange provide invaluable information on the rates and mechanisms of solvation dynamics and structural transitions of multispecies vibrationally interacting molecular systems. However, due to the intrinsic difficulties in distinguishing the contributions of molecule-specific processes to the time-resolved IR signals from those resulting from local heating, it becomes challenging to interpret time-resolved IR-PP and 2D-IR spectra exhibiting transient growing-in spectral components and cross-peaks unambiguously. Here, theoretical considerations of various effects of vibrational coupling, energy transfer, chemical exchange, the generation of hot ground states, molecular photothermal process, and their combinations on the line shapes and time-dependent intensities of IR-PP spectra and 2D-IR diagonal peaks and cross-peaks are presented. We anticipate that the present work will help researchers using IR pump-probe and 2D-IR techniques to distinguish local heating-induced photothermal signals from genuine nonlinear IR signals.

Time-resolved spectroscopy of thioflavin T solutions: Asynchronous optical sampling method with two frequency-upconverted mode-locked lasers.

We carried out transient absorption spectroscopy of thioflavin T (ThT) molecules in various solvents employing an asynchronous optical sampling (ASOPS) scheme with dual synchronized and frequency up-converted mode-lock lasers in the near UV (NUV) spectral region. We developed a pair of synchronized femtosecond lasers with tunable center wavelengths ranging from 380 to 430 nm and spectral bandwidths of 30 nm. As a proof-of-principle experiment, we measured interferometrically detected time and frequency-resolved pump-probe signals of ThT in various solvents to study the twisted intramolecular charge transfer process of photo-excited ThT molecules. Both single-color NUV-NUV and two-color NUV-near IR (NIR) pump-probe measurements reveal that the vibronic coupling strengths of two vibrational modes with frequencies of 214 and 526 cm-1 in the excited state of ThT are reduced when ThT is dissolved in a chlorine-containing solvent, e.g., chloroform. We confirm theoretically that these vibrational modes have relatively high electric dipole moments in the excited state. As a result, the intramolecular charge transfer process of ThT in chloroform, which is driven by the solvation process of surrounding polar solvent molecules, could occur less efficiently, which results in an increase in the fluorescence quantum yield. Here, we demonstrate that the NUV-NUV and NUV-NIR ASOPS-transient absorption could be useful techniques for studying ultrafast photochemical reactions in condensed phases.

Ultrafast Continuum IR Generation and Its Application in IR Spectroscopy.

The spectral range of femtosecond time-resolved infrared spectroscopy is limited by the bandwidth of mid-IR pulses (100~400 cm-1) generated from the combination of Ti:Sapphire amplifier, Optical Parametric Amplifier (OPA), and Difference Frequency Generation (DFG). To overcome this limitation, we implement a compact continuum mid-IR source producing ultrafast pulses that span the frequency range from 1000 to 4200 cm-1 (from 10 to 2.4 µm), which utilize the mixing of fundamental, second-harmonic, and third-harmonic of 800 nm pulse in the air. After building an IR spectrometer with continuum IR and a monochromator, we found that the distortion of the measured IR spectrum originated from the contamination of higher-order diffraction. We used bandpass filters to eliminate the higher-order contributions and correct the measured IR spectrum. We further characterized the spectral properties of fundamental, second-harmonic, and third-harmonic fields after the plasmonic filamentation process, which helps to improve the efficiency of the continuum IR generation. Using the generated continuum IR pulses, we measured the IR absorption spectrum of a water-benzonitrile mixture, which was found to be consistent with the spectrum obtained with a commercial FT-IR spectrometer. The present work will be useful for the efficient generation of continuum IR pulses for IR pump-probe and two-dimensional IR spectroscopy experiments in the future.

Solvation Structure around Li+ Ions in Organic Carbonate Electrolytes: Spacer-Free Thin Cell IR Spectroscopy.

Organic carbonate electrolytes are widely used materials for lithium-ion batteries. However, detailed solvation structures and solvent coordination numbers (CNs) of lithium cations in such solutions have not been accurately described nor determined yet. Because transmission-type IR spectroscopy is not of use for measuring the carbonyl stretch modes of electrolytes due to their absorption saturation problem, we here show that simple spacer-free thin cell IR spectroscopy can provide quantitative information on the number of solvating carbonate molecules around each lithium ion. We could estimate the solvent (carbonate) CNs of lithium ions in dimethyl carbonate, ethyl methyl carbonate, diethyl carbonate, propylene carbonate, and butylene carbonate over a wide range of lithium salt concentrations accurately, and they are compared with the previous results obtained with attenuated total reflection IR spectroscopy technique. We anticipate that our spacer-free thin cell approach will potentially be used to investigate the solvation dynamics, chemical exchange process, and vibrational energy transfers between solvating carbonate molecules in lithium salt electrolytes when combined with time-resolved IR spectroscopy.

Real-Time Reaction Monitoring with In Operando Flow NMR and FTIR Spectroscopy: Reaction Mechanism of Benzoxazole Synthesis.

In operando observation of reaction intermediates is crucial for unraveling reaction mechanisms. To address the sensitivity limitations of commercial ReactIR, a flow cell was integrated with a Fourier transform infrared (FTIR) spectrometer yielding a "flow FTIR" device coupled with an NMR spectrometer for the elucidation of reaction mechanisms. The former device detects the low-intensity IR peaks of reaction intermediates by adjusting the path length of the FTIR sample cell, whereas the flow NMR allows the quantitative analysis of reaction species, thus offsetting the limitations of IR spectroscopy resulting from different absorption coefficients of the normal modes. Using the flow NMR and FTIR device, the controversial mechanism of benzoxazole synthesis was conclusively determined by spectroscopic evaluation of the reaction intermediates. This system enabled the accurate acquisition of previously elusive kinetic data, such as the reaction time and rate-determining step. The implementation of reaction flow cells into NMR and FTIR systems could be widely applied to study various reaction mechanisms, including dangerous and harsh reactions, thus avoiding contact with potentially harmful reaction intermediates.

Vibrational Lifetime of the SCN Protein Label in H2O and D2O Reports Site-Specific Solvation and Structure Changes During PYP's Photocycle.

The application of vibrational labels such as thiocyanate  (-S-C≡N) for studying protein structure and dynamics is thriving. Absorption spectroscopy is usually employed to obtain wavenumber and line shape of the label. An observable of great significance might be the vibrational lifetime, which can be obtained by pump probe or 2D-IR spectroscopy. Due to the insulating effect of the heavy sulfur atom in the case of the SCN label, the lifetime of the C≡N oscillator is expected to be particularly sensitive to its surrounding as it is not dominated by through-bond relaxation. We therefore investigate the vibrational lifetime of the SCN label at various positions in the blue light sensor protein Photoactive Yellow Protein (PYP) in the ground state and signaling state of the photoreceptor. We find that the vibrational lifetime of the C≡N stretching mode is strongly affected both by its protein environment and by the degree of exposure to the solvent. Even for label positions where the line shape and wavenumber observed by FTIR are barely changing upon activation of the photoreceptor, we find that the lifetime can change considerably. To obtain an unambiguous measure for the solvent exposure of the labeled site, we show that it is imperative to compare the lifetimes in H2O and D2O. Importantly, the lifetimes shorten in H2O as compared to D2O for water exposed labels, while they stay largely the same for buried labels. We quantify this effect by defining a solvent exclusion coefficient (SEC). The response of the label's vibrational lifetime to its solvent exposure renders it a suitable universal probe for protein investigations. This applies even to systems that are otherwise hard to address, such as transient or short-lived states, which could be created during a protein's working cycle (as here in PYP) or during protein folding. It is also applicable to flexible systems (intrinsically disordered proteins), protein-protein and protein-membrane interactions.

Three-dimensional interferometric scattering microscopy via remote focusing technique.

Interferometric scattering (iSCAT) microscopy enables us to track nm-sized objects with high spatial and temporal resolutions and permits label-free imaging of biomolecules. Its superb sensitivity, however, comes at a cost by several downsides, such as slow three-dimensional imaging and limited vertical tracking. Here, we propose a new method, Remote Focusing-iSCAT (RF-iSCAT) microscopy, to visualize a volume specimen by imaging sections at different depths without translation of either the objective lens or sample stage. We demonstrate the principle of RF-iSCAT by determining the z-position of submicrometer beads by translating the reference mirror instead. RF-iSCAT features an unprecedentedly long range of vertical tracking and permits fast but vibration-free vertical scanning. We anticipate that RF-iSCAT would enhance the utility of iSCAT for dynamics study.

Two-dimensional IR spectroscopy reveals a hidden Fermi resonance band in the azido stretch spectrum of ß-azidoalanine.

Azido stretch modes in a variety of azido-derivatized nonnatural amino acids and nucleotides have been used as a site-specific infrared (IR) probe for monitoring changes in their conformations and local electrostatic environments. The vibrational bands of azide probes are often accompanied by complex line shapes with shoulder peaks, which may arise either from incomplete background subtraction, Fermi resonance, or multiple conformers. The isotope substitution in the infrared probe has thus been introduced to remove Fermi resonances without causing a significant perturbation to the structure. Here, we synthesized and labeled the mid-N atoms of aliphatic azide derivatives with 15N to study the effects of isotope labelling on their vibrational properties. The FT-IR spectra of the aliphatic azide with asymmetric lineshape became a single symmetric band upon isotope substitution, which might be an indication of the removal of the hidden Fermi resonance from the system. We also noticed that the 2D-IR spectrum of unlabeled aliphatic azide has cross-peaks, even though it is not apparently identifiable. The 1D slice spectra obtained from the 2D-IR spectra reveal the existence of a hidden Fermi resonance peak. Furthermore, we show that this weak Fermi resonance does not produce discernible oscillatory beating patterns in the IR pump-probe spectrum, which has been used as evidence of the Fermi resonance. Therefore, we confirm that isotope labelling combined with 2D-IR spectroscopy is the most efficient and incisive way to identify the origin of small shoulder peaks in the linear and nonlinear vibrational spectra of various IR probe molecules.