Broadband CARS Research Articles

Non-resonant background removal in broadband CARS microscopy using deep-learning algorithms

Broadband Coherent anti-Stokes Raman (BCARS) microscopy is an imaging technique that can acquire full Raman spectra (400–3200 cm−1) of biological samples within a few milliseconds. However, the CARS signal suffers from an undesired non-resonant background (NRB), deriving from four-wave-mixing processes, which distorts the peak line shapes and reduces the chemical contrast. Traditionally, the NRB is removed using numerical algorithms that require expert users and knowledge of the NRB spectral profile. Recently, deep-learning models proved to be powerful tools for unsupervised automation and acceleration of NRB removal. Here, we thoroughly review the existing NRB removal deep-learning models (SpecNet, VECTOR, LSTM, Bi-LSTM) and present two novel architectures. The first one combines convolutional layers with Gated Recurrent Units (CNN + GRU); the second one is a Generative Adversarial Network (GAN) that trains an encoder-decoder network and an adversarial convolutional neural network. We also introduce an improved training dataset, generalized on different BCARS experimental configurations. We compare the performances of all these networks on test and experimental data, using them in the pipeline for spectral unmixing of BCARS images. Our analyses show that CNN + GRU and VECTOR are the networks giving the highest accuracy, GAN is the one that predicts the highest number of true positive peaks in experimental data, whereas GAN and VECTOR are the most suitable ones for real-time processing of BCARS images.

Tracing the Background‐Suppressed Vibrational Decay Time of a Molecular Monolayer with Time‐Resolved Surface‐Enhanced Broadband Coherent Anti‐Stokes Raman Scattering

AbstractThe sensitivity of molecular spectroscopy based on surface‐enhanced coherent anti‐Stokes Raman scattering (SECARS) is limited by the spectrally overlapping background from nonresonant four‐wave mixing (FWM). While the SECARS signal is mediated by the molecular vibrational eigenstates and exhibits long lifetime (a few picoseconds), the FWM background stems from the instantaneous electronic polarization and decays rapidly with the vanishment of excitation. Therefore, CARS and FWM can be separated by time‐resolved CARS (trCARS) using ultrashort pulsed lasers. The broad spectral bandwidth also enables broadband CARS (BCARS) for simultaneous identification of multiple vibrational signatures. This work combines trCARS and BCARS with an optimized plasmonic system to demonstrate time‐resolved surface‐enhanced BCARS) and show its capability in obtaining background‐suppressed broadband vibrational spectra from a monolayer of 4‐Aminothiophenol (4‐ATP) molecules self‐assembled on a gold grating. The vibrational dephasing time of the ring‐breathing mode for the 4‐ATP monolayer on the gold grating (≈1.00 ± 0.17 ps) is significantly shortened compared to that for 4‐ATP powder on a glass substrate (2.10 ± 0.05 ps). This work presents an effective method to suppress FWM background and investigate the vibrational dynamics of molecules in the vicinity of plasmonic nanostructures.

Toward coherent anti‐Stokes Raman scattering‐Raman optical activity for reaction monitoring in organocatalysis: Chiral vibrational signatures of the two atropisomers (R)‐ and (S)‐MOM‐BINOL in solution

AbstractCoherent anti‐Stokes Raman scattering‐Raman optical activity (CARS‐ROA) is a coherent version of spontaneous ROA spectroscopy, the latter being a well‐established chirally sensitive vibrational spectroscopic technique. A central major advantage of heterodyne‐detected CARS‐ROA over ROA is the drastic reduction of acquisition times from several hours to approximately 1 min due to the high ratio of the chiral Raman signal relative to the achiral CARS background. Here, we present the first demonstration of heterodyne‐detected CARS‐ROA of chiral molecules dissolved in an achiral solvent by using an experimental setup built from a conventional broadband CARS spectrometer. Specifically, the two atropisomers (R/S) of 2,2′‐Bis(methoxymethoxy)‐1,1′‐binaphthyl, a methoxymethyl (MOM) bis‐protected derivative of 1,1′‐Bi‐2‐naphthol (BINOL), dissolved in the achiral organic solvent dichloromethane (DCM) were characterized by employing ~1 ps, 1 mW of pump pulses and ~60 fs, 10 mW of Stokes pulses within approximately 4‐min acquisition time. MOM‐BINOL is an important precursor for the synthesis of a great variety of many chiral auxiliaries including binaphthyl phosphoric acids, which are an important class of chiral organocatalysts used in asymmetric reactions. Overall, this work is a first step toward kinetic reaction monitoring in organocatalysis by coherent ROA providing the necessary short acquisition times.

A Fano-resonance plasmonic assembly for broadband-enhanced coherent anti-Stokes Raman scattering

Surface-enhanced coherent anti-Stokes Raman scattering (SECARS) technique has triggered huge interests due to the significant signal enhancement for high-sensitivity detection. Previous SECARS work has tended to focus only on the enhancement effect at a certain combination of frequencies, more suitable for single-frequency CARS. In this work, based on the enhancement factor for broadband SECARS excitation process, a novel Fano resonance plasmonic nanostructure for SECARS is studied. In addition to the 12 orders of magnitude enhancement effect that can be realized under single-frequency CARS, this structure also shows huge enhancement under broadband CARS in a wide wavenumber region, covering most of the fingerprint region. This geometrically-tunable Fano plasmonic nanostructure provides a way to realize broadband-enhanced CARS, with potentials in single-molecular monitoring and high-selectivity biochemical detection.

Removing non-resonant background from broadband CARS using a physics-informed neural network.

Broadband coherent anti-Stokes Raman scattering (BCARS) is capable of producing high-quality Raman spectra spanning broad bandwidths, 400-4000 cm-1, with millisecond acquisition times. Raw BCARS spectra, however, are a coherent combination of vibrationally resonant (Raman) and non-resonant (electronic) components that may challenge or degrade chemical analyses. Recently, we demonstrated a deep convolutional autoencoder network, trained on pairs of simulated BCARS-Raman datasets, which could retrieve the Raman signal with high quality under ideal conditions. In this work, we present a new computational system that incorporates experimental measurements of the laser system spectral and temporal properties, combined with simulated susceptibilities. Thus, the neural network learns the mapping between the susceptibility and the measured response for a specific BCARS system. The network is tested on simulated and measured experimental results taken with our BCARS system.

Multimodal approach to reveal the effect of light irradiation on chemical composition of lipofuscin granules of human RPE tissues

The accumulation of lipofuscin (LF) in tissues is considered as one a hallmark of the aging process. Usually formed in yellow-brown pigment aggregates or granules primarily composed of lipid residues and proteins, the exact chemical composition of LF varies among tissues. LF of the retinal pigment epithelium (RPE) is known to contain byproducts of the visual cycle, including one of the fluorophores in LF bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E). The last is characterized by high photoreactivity and implicates in age-related macular degeneration. Photoinduced production of reactive oxygen species (ROS) is shown among the main modes of A2E toxicity although particular photochemistry of A2E have yet to be completely identified. In this work we study changes in chemical composition of LF granules of human RPE under light irradiation using vibrational spectroscopy (femtosecond broadband CARS) and mass-spectrometry (TOF-SIMS) approach accompanied by measurements of fluorescence properties of the LF.

Vibrational Spectroscopic Characterization of 2-(2,4-Dinitrobenzyl)-pyridine (α-DNBP) in Solution by Polarization-Resolved Spontaneous Raman Scattering and Broadband CARS.

The photochromic molecule 2-(2,4-dinitro-benzyl)-pyridine (α-DNBP) is characterized in solution by a combination of density functional theory employing a polarizable continuum model and polarization-resolved spontaneous and nonlinear Raman spectroscopies. By the comparison of theoretically predicted wavenumber positions and depolarization ratios with the experimental spectra acquired under electronically nonresonant conditions, polarized and depolarized Raman bands are assigned. Specifically, the symmetric stretching vibrations of the two nitro groups in ortho and para positions to the pyridine ring can be experimentally differentiated, mainly because of their different Raman depolarization ratios, which supports our prediction from theory. Compared to the polarization-resolved spontaneous Raman experiments, the vibrational spectroscopic differentiation of the two nitro groups is more pronounced in time-delayed polarization-resolved coherent anti-Stokes Raman scattering experiments. Overall, this linear and nonlinear vibrational spectroscopic characterization of the CH form paves the way for the interpretation of future time-resolved pump/nonlinear Raman probe studies on the ultrafast photoinduced intramolecular proton transfer in α-DNBP involving a nitro group as an intramolecular proton acceptor.

High‐speed broadband Fourier‐transform coherent anti‐stokes Raman scattering spectral microscopy

AbstractWe demonstrate broadband Fourier‐transform coherent anti‐Stokes Raman scattering (FT‐CARS) spectral microscopy with a pixel dwell time of 42 μs, which is ~50 times shorter than the shortest‐to‐date pixel dwell time for CARS spectral microscopy. Our broadband FT‐CARS spectral microscope is composed of an FT‐CARS spectrometer, a rapid galvanometric scanner, and a high‐speed image acquisition circuit, enabling a frame rate of 2.4 fps with a pixel resolution of 100 × 100 pixels, a bandwidth of 600–1,200 cm−1, a spatial resolution of 0.95 μm, and a spectral resolution of 37 cm−1. As a proof‐of‐principle demonstration, we used the high‐speed FT‐CARS spectral microscope to perform CARS imaging of polymer beads and Haematococcus lacustris cells. Our high‐speed broadband CARS spectral microscope holds promise for studying rapid cellular dynamics, such as signaling, cell‐to‐cell communication, and molecular transport in a label‐free manner.

Comparison of two photonic crystal fibers for supercontinuum-Stokes spectral-focusing-CARS hyperspectroscopy

A popular and inexpensive experimental approach to broadband CARS microscopy involves the generation of a supercontinuum pulse from a microstructured fibre. Despite such fibres being used for CARS microscopy for over a decade, concerns about excessive spectral and polarization noise have prevented their wider adoption for this technique. In this work, the applicability of two commercially-available supercontinuum generating fibres, the NL-1.4-775-945 and the NL-PM-750 in packaged module form, are compared for spectral-focusing CARS (SF-CARS) imaging. The former has been a mainstay of SF-CARS implementations while the latter has largely found application in a related but distinct broadband CARS technique and has not been used for SF-CARS applications. We first characterize the intensity and polarization behaviour of the supercontinuum output of the fibres and demonstrate that while both can be used to produce high-quality CARS images and spectra, key operational differences between the fibres must be taken into consideration when optimizing their use for SF-CARS applications. In particular, we show via spectroscopy of a solvent sample and hyperspectral imaging of a pharmaceutical sample that the NL-1.4-775-945 is inferior for SF-CARS applications when used under fixed-power supercontinuum generation conditions. However, its operation can be significantly improved under power-tuned supercontinuum generation conditions known as spectral-surfing, an approach that does not confer the same advantage with the NL-PM-750. Furthermore, we identify a latent polarization-dependent spectral artifact in the NL-PM-750, which can degrade the CARS spectrum in time-gated techniques such as SF-CARS.

Rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectroscopy with heterodyne detection.

High-speed Raman spectroscopy has become increasingly important for analyzing chemical dynamics in real time. To address the need, rapid-scan Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy has been developed to realize broadband CARS measurements at a scan rate of more than 20,000scans/s. However, the detection sensitivity of FT-CARS spectroscopy is inherently low due to the limited number of photons detected during each scan. In this Letter, we show our experimental demonstration of enhanced sensitivity in rapid-scan FT-CARS spectroscopy by heterodyne detection. Specifically, we implemented heterodyne detection by superposing the CARS electric field with an external local oscillator (LO) for their interference. The CARS signal was amplified by simply increasing the power of the LO without the need for increasing the incident power onto the sample. Consequently, we achieved enhancement in signal intensity and the signal-to-noise ratio by factors of 39 and 5, respectively, compared to FT-CARS spectroscopy with homodyne detection. The sensitivity-improved rapid-scan FT-CARS spectroscopy is expected to enable the sensitive real-time observation of chemical dynamics in a broad range of settings, such as combustion engines and live biological cells.

Broadband coherent Raman spectroscopy running at 24,000 spectra per second.

We present a Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy technique that achieves broadband CARS measurements at an ultrahigh scan rate of more than 20,000 spectra/s – more than 20 times higher than that of previous broadband coherent Raman scattering spectroscopy techniques. This is made possible by an integration of a FT-CARS system and a rapid-scanning retro-reflective optical path length scanner. To demonstrate the technique’s strength, we use it to perform broadband CARS spectroscopy of the transient mixing dynamics of toluene and benzene in the fingerprint region (200–1500 cm−1) with spectral resolution of 10 cm−1 at a record high scan rate of 24,000 spectra/s. Our rapid-scanning FT-CARS technique holds great promise for studying chemical dynamics and wide-field label-free biomedical imaging.

Determination of 3D molecular orientation by concurrent polarization analysis of multiple Raman modes in broadband CARS spectroscopy.

A theoretical description is presented about a new analysis method to determine three-dimensional (3D) molecular orientation by concurrently analyzing multiple Raman polarization profiles. Conventional approaches to polarization Raman spectroscopy are based on single peaks, and their 2D-projected polarization profiles are limited in providing 3D orientational information. Our new method analyzes multiple Raman profiles acquired by a single polarization scanning measurement of broadband coherent anti-Stokes Raman scattering (BCARS). Because the analysis uses only dimensionless quantities, such as intensity ratios and phase difference between multiple profiles, the results are not affected by sample concentration and the system response function. We describe how to determine the 3D molecular orientation with the dimensionless observables by using two simplified model cases. In addition, we discuss the effect of orientational broadening on the polarization profiles in the two model cases. We find that in the presence of broadening we can still determine the mean 3D orientation angles and, furthermore, the degree of orientational broadening.

Beam-Scanning Broadband Cars Microscopy for Rapid Tissue Imaging

A beam-scanning approach is demonstrated to improve signal-to-noise ratio and to reduce photo-induced sample damages in nonlinear hyperspectral imaging, such as broadband coherent anti-Stokes Raman scattering (BCARS) microscopy. Signals generated on a fast scanning axis are transferred to the slit of a monochromator, then onto the vertical axis of a CCD. This configuration acquires the whole BCARS spectra on the fast scanning axis at once, reducing the overall data transfer time. The fast scanning speed by a resonant beam scanner at 5 kHz greatly reduces sample damage from accumulative photo excitation, allowing for a higher excitation laser power to generate a stronger nonlinear signal. The optical characteristics related with this configuration and parameters are discussed and a few examples that demonstrate the advantage of the beam-scanning method for BCARS tissue imaging are presented.

Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy.

We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times.

Exploring, tailoring, and traversing the solution landscape of a phase-shaped CARS process

Pulse shaping techniques are used to improve the selectivity of broadband CARS experiments, and to reject the overwhelming background. Knowledge about the fitness landscape and the capability of tailoring it is crucial for both fundamental insight and performing an efficient optimization of phase shapes. We use an evolutionary algorithm to find the optimal spectral phase of the broadband pump and probe beams in a background-suppressed shaped CARS process. We then investigate the shapes, symmetries, and topologies of the landscape contour lines around the optimal solution and also around the point corresponding to zero phase. We demonstrate the significance of the employed phase bases in achieving convex contour lines, suppressed local optima, and high optimization fitness with a few (and even a single) optimization parameter.

Observing hydrogen silsesquioxane cross‐linking with broadband CARS

AbstractBroadband coherent anti‐Stokes Raman scattering microscopy (CARS microscopy) is used for real‐time measurements of in situ cross‐linking of the photoresist material hydrogen silsesquioxane (HSQ). Using samples baked at different temperatures, and therefore cross‐linked to varying degrees, characteristic Raman bands are identified for the open ‘cage’ structure (345 cm−1, 454 cm−1 and 562 cm−1) as well as the cross‐linked ‘network’ structure (484 cm−1). Cross‐linking can also be induced by absorption of high intensity near‐infrared (IR) laser light (∼1013 W cm−2) and followed in real time by monitoring the characteristic Raman bands. Using the time resolution of CARS to follow the HSQ reaction during the laser exposure, a two‐stage kinetic process is observed and associated time constants of 20 ± 10 s and 3 ± 1 min are determined for the IR laser‐induced cross‐linking in films of ∼1 µm thickness. Copyright © 2009 John Wiley & Sons, Ltd.

CARS spectroscopy of radio-frequency discharge plasma in hydrogen

The population of the vibrational and rotational levels of hydrogen is studied by the narrow-and broadband CARS spectroscopy in capacitive and inductive-capacitive radio-frequency discharge plasmas. Computational codes are developed to analyze and process CARS spectra of hydrogen obtained under conditions of disturbance of thermodynamic equilibrium over internal degrees of freedom of molecules. To interpret the measurement results, a model is developed, which makes it possible to calculate the vibrational temperature in radio-frequency discharge plasmas. It is shown that the broadband CARS spectrometer in combination with the software package developed appreciably reduces the time necessary to determine the vibrational and rotational temperatures.

Chemical Imaging by Single Pulse Interferometric Coherent Anti-Stokes Raman Scattering Microscopy

A single pulse interferometric coherent anti-Stokes Raman (CARS) spectroscopy method is used to obtain broadband CARS spectra and microscopy images of liquid and polymer samples. The pump, Stokes, and probe pulses are all selected inside a single broadband ultrafast pulse by a phase- and polarization-controlled pulse shaping technique and used to generate two spectral interference CARS signals simultaneously. The normalized difference of these two signals provides an amplified background-free broadband resonant CARS spectrum over the 400-1500 cm(-1) range with 35 cm(-1) spectral resolution. Chemically selective microscopy images of multicomponent polymer and liquid samples are investigated with this new CARS method. Multiplex CARS spectra at 10,000 spatial points are measured within a few minutes, and used to construct chemically selective microscopy images with a spatial resolution of 400 nm. The spectral bandwidth limits, sensitivity, homodyne amplification advantages, spatial resolution, depolarization, chromatic aberration, and chemical imaging aspects of this new technique are discussed in detail.

NO Reburn and Formation Chemistry in Methane Diffusion Flames

Abstract We have applied time-resolved picosecond linear laser-induced fluorescence to obtain spatially resolved profiles of NO in laminar methane/air and methane/nitric oxide/air counterflow diffusion flames at atmospheric pressure. Temperature profiles have been measured with broadband CARS of nitrogen. Flames at strain rates from 59 s−1 to 269 s−1 were studied to characterize the strain rate dependence of the NO concentration. The work shows that NO concentrations decrease with increasing strain rate. Comparisons have been made with predicted NO levels obtained using two different chemical kinetic mechanisms (Lindstedt and co-workers and GRI-Mech. 3.0). Computed concentrations are shown to be in good agreement with experimental data. The addition of up to 600 ppm NO to the fuel did permit an assessment of differences in the reburn chemistry between the two mechanisms.

CARS study of linewidths of the Q-branch of hydrogen molecules at high temperatures in a pulsed high-pressure H2—O2 combustion chamber

The results of measurements of individual line widths of the Q-branch of a hydrogen molecule and the corresponding coefficients of broadening caused by collisions with water molecules at T = 2700 K in a repetitively pulsed high-pressure (50–200 atm) hydrogen-oxygen combustion chamber are presented. CARS spectra of individual Q1—Q7 hydrogen lines, pressure pulses, and the broadband CARS spectra of the entire Q-branch of hydrogen are recorded simultaneously during a single laser pulse. The shape of line profiles was analysed using a Fabry—Perot interferometer. The temperature in the volume being probed was determined from the 'broadband' CARS spectra. The entire body of the experimental results gives information on the spectral linewidths, temperature and pressure in the combustion chamber during CARS probing.