Coherent anti-Stokes Raman Scattering Signal Research Articles

Pupil-engineered coherent anti-Stokes Raman scattering spectroscopy

Coherent anti-Stokes Raman scattering (CARS) spectroscopy suffers from non-resonant background (NB) that is several orders of magnitude stronger than CARS signals. NB occupies the camera sensor dynamic range and hampers quantitative detection of weak Raman signals at low molecular concentrations. Here, we propose a novel technique to reduce NB for heterodyne CARS spectroscopy, by independently controlling the parities of the amplitude point spread functions of CARS and NB. We realize the parity control by applying the pupil engineering of the excitation and detection optical systems. A proof-of-concept experiment shows that the signal-to-background ratio (SBR) of the CARS signal to the NB can be improved as designed.

Nitrogen thermometry in an inductively coupled plasma torch using broadband nanosecond coherent anti-Stokes Raman scattering.

The development of atmospheric hypersonic flight and re-entry capabilities requires the characterization of the thermo-chemical state of representative test environments. This study demonstrates the usage of multiplex nanosecond N 2 coherent anti-Stokes Raman scattering (CARS) to measure temperatures in an atmospheric, high-temperature (>6000K), air plasma plume, generated by an inductively coupled plasma torch. These are some of the highest temperatures ever accessed via gas-phase CARS, to our knowledge. Temperatures of N 2 in the equilibrium plasma plume are determined via theoretical fits to measured CARS spectra. We discuss the practical implementation of CARS at very high temperatures, including the scaling of the N 2 CARS signal strength from 300 to 6700K, where the expected peak signal from the high-temperature plasma torch gases is two orders of magnitude less than commonly encountered in combustion environments. An intensified CCD camera enables single-laser-shot detection at temperatures as high as 6200K, by increasing sensitivity and providing a time gate against intense background luminosity. We also discuss the impacts of unwanted two-beam CARS contributions from outside the nominal three-beam measurement volume. We present mean axial and radial temperature profiles, as well as time-series data derived from both single-laser-shot and accumulated CARS spectra. The single-laser-shot precision is 1.7%-2.6% at temperatures of 3500 to 6200K. The presented results pave the way for the use of CARS at very high temperatures and the measurement of spatially resolved interface processes in high-enthalpy flows.

Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses.

Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and tissues. In narrowband CARS, two spatiotemporally superimposed picosecond pulses, pump and Stokes, illuminate the sample to interrogate a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite recent technological advancements, BCARS microscopes still struggle to image biological samples over the entire Raman-active region (400-3100 cm-1). Here, we demonstrate a robust BCARS platform that answers this need. Our system is based on a femtosecond ytterbium laser at a 1035 nm wavelength and a 2 MHz repetition rate, which delivers high-energy pulses used to produce broadband Stokes pulses by white-light continuum generation in a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20 fs duration, with narrowband pump pulses, we generate a CARS signal with a high (<9 cm-1) spectral resolution in the whole Raman-active window, exploiting both the two-color and three-color excitation mechanisms. Aided by an innovative post-processing pipeline, our microscope allows us to perform high-speed (≈1 ms pixel dwell time) imaging over a large field of view, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, paving the way for applications in histopathological settings.

Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.

Temperature measurement utilizing the vibrational CARS signal from the ground level X2Σg+ of N2+ in high-temperature flames

We analyze the generation of the vibrational coherent anti-Stokes Raman scattering (CARS) signal from the ground level X2Σg+ of N2+ and demonstrate the feasibility for gas-phase thermometry utilizing the vibrational CARS signals of N2+ in high-temperature flames. The vibrational CARS signal of N2+ is obtained using the filamentation-based and two-beam hybrid femtosecond/picosecond CARS system and is employed to extract the fitting temperatures in high-temperature flames. The accuracy and precision of the fitting temperatures from the 1000 consecutive single-shot vibrational CARS spectra of N2+ at a target temperature of 1706 K are 1.23% and 5.76%, respectively, which are similar to the fitting results of N2 in previous filamentation-based CARS thermometry and could also demonstrate that the measured temperature in the filamentation is indeed not affected by the ionization. We further introduce the CARS signal of N2+ in multiple species measurement to obtain more details about the CARS process during filamentation. This work would provide an optional target molecule for CARS thermometry and help researchers further understand the molecular dynamics of N2+ ions during the filamentation.

Spectral Unmixing for Label-Free, In-Liquid Characterization of Biomass Microstructure and Biopolymer Content by Coherent Raman Imaging.

Characterization of lignocellulosic biomass microstructure with chemical specificity and under physiological conditions could provide invaluable insights to our understanding of plant tissue development, microstructure, origins of recalcitrance, degradation, and solubilization. However, most methods currently available are either destructive, are not compatible with hosting a physiological environment, or introduces exogenous probes, complicating their use for studying changes in microstructure and mechanisms of plant development, recalcitrance, or degradation in situ. To address these challenges, we here present a multi-modal chemically specific imaging technique based on coherent anti-Stokes Raman scattering (CARS) microspectroscopy with simplex maximization and entropy-based spectral unmixing enabling label-free, chemically specific characterization of plant microstructure in liquid. We describe how spatial drift of samples suspended in liquid can introduce artifacts in spectral unmixing procedures for single-frequency CARS and propose a mitigative strategy toward these effects using simultaneously acquired forward-scattered CARS signals and epi-detected autofluorescence. We further apply the technique for chemical and microstructural characterization of untreated and liquid hot water pretreated rapeseed straw by CARS and show how the framework can be extended for 3D imaging with chemical specificity. Finally, we provide examples of the intricate chemical and microstructural details recovered by this hybrid imaging technique, including discerning between primary and secondary cell walls, localization of aqueous components to cell lumina, and the presence of funnel-type pits in samples ofBrassica napus.

Cascaded amplification via three-beam double stimulated Raman scattering in benzene

As stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool for label-free imaging of biomolecules in biological systems, SRS has also received much attention in the field of nonlinear optics. Here, we enhanced the cascade SRS of benzene through an energy competition mechanism in the three-beam double SRS processes. A three-beam (pump, Stokes, and anti-Stokes beam) SRS scheme was formulated and demonstrated to simultaneously induce the parallel and cascaded SRS processes, which were related to the Raman active modes in the same molecule. Both two SRS processes generated high-order Stokes SRS through resonance enhancement of the double sample cell configuration, which effectively suppressed the CARS (Coherent anti-Stokes Raman Scattering) signal. It was shown in the experimental results that the cascaded SRS up to six orders from 562 to 780 nm were generated, and the SRS threshold of the multi-order Stokes components was lowered. The present work can potentially contribute to the development of super-resolution SRS microscopy.

Water vapor in hydrogen flames measured by time-resolved collisional dephasing of the pure-rotational N2 CARS signal

We present a novel diagnostic technique to probe water vapor (H2O) concentration in hydrogen (H2) combustion environments via the time-resolved measurement of the collisional dephasing of the pure-rotational coherent anti-Stokes Raman scattering (CARS) signal of nitrogen (N2). The rotational Raman coherence of the N2 molecules, induced by the interaction with the pump and Stokes laser fields, dephases on a timescale of hundreds of picoseconds (ps), mostly due to inelastic collisions with other molecules in atmospheric flames. In the spatial region of H2 flames where H2O is present in appreciable amount, it introduces a faster dephasing of the N2 coherence than the other major combustion species do: we use time-resolved femtosecond/picosecond (fs/ps) CARS to deduce the H2O mole fraction from the dephasing effect of its inelastic collisions with N2. The proof-of-principle is demonstrated in a laminar H2/air diffusion flame, performing sequential measurements of the collisional dephasing of the N2 CARS signal up to 360 ps. We measure the temperature and the relative O2/N2 and H2/N2 concentrations at a short probe delay, and input the results in the time-domain model to extract the H2O mole fraction from the signal decay, thus measuring the whole scalar flow fields across the flame front. We furthermore present single-shot simultaneous thermometry and absolute concentration measurements in the turbulent TU Darmstadt/DLR Stuttgart canonical ‘H3 flame’ performed by dual-probe CARS measurements obtained with a polarization separation approach. This allows us to probe the molecular coherence simultaneously at ∼20 and ∼250 ps on the basis of a single-laser-shot, and record the resulting signals in two distinct detection channels of our unique polarization-sensitive coherent imaging spectrometer. The proposed technique allows for measuring the absolute concentrations of all the major species of H2 flames, thus providing a full characterization of the flow composition, as well as of the temperature field.

Theoretical Study on the Ultrafast Selective Excitation of Surface-Enhanced Coherent Anti-Stokes Raman Scattering Based on Fano Resonance of Disk-Ring Nanostructures by Shaped Femtosecond Laser Pulses

The enhancement and selective excitation of coherent anti-Stokes Raman scattering (CARS) and the suppression of background noise are very important problems for real-time detection at the single-molecule level. Optimizing the plasmonic substrate to ensure that all the hot spots of the pump, probe, Stokes, and anti-Stokes light are at the same position is the key to increasing the CARS signal to reach the level of single-molecule detection. The selective excitation of the target CARS peak and the suppression of the other peaks are the key to improving the signal-to-noise ratio. In this paper, we present a theoretical study to control the selective excitation and enhancement of any one of the three CARS peaks using the Fano resonance of a disk-ring structure. By optimizing the modulation of the pump, Stokes, and probe pulse, one CARS peak is maximized, while the other two are suppressed to zero. Fano resonance is applied to simultaneously enhance the four surface plasmon modes of the pump, probe, Stokes, and anti-Stokes light and to ensure that all the hot spots are located at the same position by adjusting the size of the disk-ring structure. The hot spots of the four pulses are concentrated in the disk-ring gap with a deviation distance of less than 2 nm, and the intensity of the CARS is enhanced by 1.43 × 1012 times, which is much higher than the requirement of single-molecule detection. The time, frequency, and phase distribution of the input and the response of the four pulses are studied in detail. It was found that the selective excitation and the spectra of CARS are both well preserved.

Ultra-broadband three-color coherent anti-Stokes Raman scattering spectroscopy

Three-color ultrabroadband coherent anti-Stokes Raman scattering (CARS) spectroscopy in the range of 400–3500 cm−1 based on a single fiber laser is demonstrated. A 400-fs supercontinuum (SC) with spectral bandwidth between 780 and 1240 nm was generated in a facile manner, by launching 280-fs pulses from the fiber laser into an all-normal-dispersion photonic crystal fiber (PCF) with subsequent compression using a grating pair. It implements a device that can capture the CARS signal of most of the fingerprint range in real time and has a high signal-to-noise ratio. Furthermore, thanks to the excellent temporal overlap of its broad spectrum, the SC was used as both pump and Stokes in three-color broadband CARS (BCARS) spectroscopy of benzonitrile. Nine Raman modes of benzonitrile in the 400–3500 cm−1 range were detected simultaneously.

Gas detection sensitivity of hybrid fs/ps and fs/ns CARS.

Coherent anti-Stokes Raman scattering (CARS) is commonly used for thermometry and concentration measurement of major species. The quadratic scaling of CARS signal with number density has limited the use of CARS for detection of minor species, where more sensitive approaches may be more attractive. However, significant advancements in ultrafast CARS approaches have been made over the past two decades, including the development of hybrid CARS demonstrated to yield greatly increased excitation efficiencies. Yet, detailed detection limits of hybrid CARS have not been well established. In this Letter, detection limits for N2, H2, CO, and C2H4 by point-wise hybrid femtosecond (fs)/picosecond (ps) CARS are determined to be of the order of 1015 molecules/cm3. The possible benefit of fs/nanosecond (ns) hybrid CARS is also discussed.

Ultrahigh-speed multiplex coherent anti-Stokes Raman scattering microspectroscopy using scanning elliptical focal spot.

We present a beam-scanning multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy system using parallel excitation and parallel detection schemes based on an elliptical focal spot, which enables highly efficient signal acquisition even for short exposures. The elliptical focal spot was used to simultaneously observe the CARS signals of an enlarged region and reduce the peak irradiance. The developed system realized an acquisition rate of 34139 spectra/s and enabled ultrahigh-speed acquisition of a vibrational spectroscopic image, covering the fingerprintregion of 930-1 830 cm-1 with 256(x) × 256(y) × 512(spectrum) pixels in 1.92s or with 128(x) × 128(y) × 256(spectrum) pixels in 0.54s. We demonstrated ultrahigh-speed hyperspectral imaging of a mixture of polymer beads in liquid linoleic acid and living adipocytes using the developed system. All of the present demonstrations were performed with a low-peak irradiance excitation of ∼19 GW/cm2, which has been reported in previous studies to cause less photodamage to living cells. The label-free and ultrahigh-speed identification and visualization of various molecules made possible by the present system will accelerate the development of practical live-cell investigation.

Semiclassical control theory of coherent anti-Stokes Raman scattering maximizing vibrational coherence for remote detection

A semiclassical theory that describes the generation of a coherent anti-Stokes Raman scattering (CARS) signal is presented that maximizes vibrational coherence in a mode predetermined by the pump, the Stokes, and the probe chirped pulse trains and takes into account the field propagation effects in a cloud of molecules. The buildup of the anti-Stokes signal, which may be used as a molecular signature in the backward CARS signal, is demonstrated numerically. The theory is based on the solution of the coupled Maxwell's and Liouville--von Neumann equations and focuses on the quantum effects induced in the target molecules by the control pulse trains. A deep convolutional neural network technique is implemented to evaluate time-dependent phase characteristics of the control fields. The effect of decoherence induced by spontaneous decay and collisional dephasing is examined.

Nonresonant CARS Imaging of Porous and Solid Silicon Nanoparticles in Human Cells.

Coherent anti-Stokes Raman scattering (CARS), a nonlinear optical method for rapid visualization of biological objects, represents a progressive tool in biology and medicine to explore cells and tissue structures in living systems and biopsies. In this study, we report efficient nonresonant CARS imaging of silicon nanoparticles (SiNPs) in human cells as a proof of concept. As both bulk and porous silicon exhibit a high third-order nonlinear susceptibility, χ(3), which is responsible for the CARS intensity, it is possible to visualize the SiNPs without specific labels. Porous and solid SiNPs were obtained from layers of porous and nonporous silicon nanowires and mesoporous silicon. Electron microscopy and Raman spectroscopy showed that porous SiNPs consisted of ∼3 nm silicon nanocrystals (nc-Si) and pores, whereas solid nanoparticles comprised ∼30 nm nc-Si. All types of SiNPs were nontoxic at concentrations up to 500 μg/mL after 24 h of incubation with cells. We demonstrated that although nc-Si possesses a distinguished narrow Raman band of about 520 cm-1, it is possible to detect a high CARS signal from SiNPs in the epi-direction even in a nonresonant regime. 3D CARS images showed that all types of studied SiNPs were visualized as bright spots inside the cytoplasm of cells after 3-6 h of incubation because of the contrast provided by the high third-order nonlinear susceptibility of SiNPs, which is 1 × 104 to 1 × 105 times higher than that of water and typical biological media. Overall, CARS microscopy can provide localization of SiNPs within biological structures at the cellular level and can be a powerful tool for in vitro monitoring of silicon-based drug delivery systems or use SiNPs as labels to monitor various bioprocesses inside living cells.

In situ Plasmon-Enhanced CARS and TPEF for Gram staining identification of non-fluorescent bacteria

In this work, we report in situ nonlinear microscopic images on plasmon-enhanced coherent anti-Stokes Raman scattering (CARS) and plasmon-Induced two-photon excited fluorescence （TPEF)of non-fluorescent microorganism. Our unique synthesized Au@Ag nanorods provide with two distinct surface-plasmon resonance (SPR) at 400 and 800 nm, respectively, which can efficiently induce linear fluorescence signals of E. coli but also enhance the nonlinear optical spectroscopy signals of TPEF, and coherent anti-Stokes Raman scattering (CARS) imaging of E. coli and S. aureus. Furthermore, calculations with complete active space self-consistent field (CASSCF) reveals the hot electrons of SPs can efficiently induce the biological fluorescence of non-fluorescent flavin nucleotides on the surface of E. coli. This novel mechanism is expected to guide the development and application of new microbial detection reagents. Gram-negative and Gram-positive bacteria can be well distinguished by nonlinear microscopic imaging of the CARS signal at 1589 cm−1. Benefit by the strong penetrability of non-linear optical signals, it is expected to realize in situ real-time detection and classification of pathogenic microbial infections in vivo.

Molecular Multicolor Multiphoton in Vivo Bioimaging Based on a Direct Diode Pumped Ti:sapphire Oscillator

Multiphoton microscopy (MPM) including two-photon excited fluorescence (TPEF), second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) is an important tool in biology and medicine to explore the dynamics of cells and to investigate tissue structure in living systems and biopsies. However, MPM still suffers from limited applicability due to the integration of highly sophisticated lasers. In this paper, we introduce our triple-modal multicolor MPM platform employing a single direct diode-pumped femtosecond Kerr-lens-mode-locked Ti:sapphire oscillator and acquire simultaneously intrinsic co-registered multicolor TPEF, TPEF, SHG and CARS signals in the backward direction. The complexity and by that the footprint as well the cost of the laser are considerably reduced by the direct diode-pumping scheme. In combination with an Yb fiber amplifier such a laser is suited for biomedical applications allowing for detailed morphologic and molecular spectroscopic in vivo investigation. The optimized emission wavelengths of our Ti:sapphire laser and Yb fiber amplifier provide simultaneous excitation at 805 nm, 1050 nm and 911 nm for multicontrast information from exogenous and endogenous fluorophores. We perform in vivo imaging of C. elegans and visualize metabolic changes in zebrafish larvae to investigate the tumor microenvironment with spectral focusing CARS and multicolor TPEF of green fluorescent protein.

Raman Excitation of Hydrogen Molecules to v = 1 State

Coherent anti-Stokes Raman scattering (CARS) can reveal a molecule’s vibrational spectrum to a great extent. Instantaneous interaction of Stokes and pump beams stemming from powerful pulsed lasers excites a molecule’s vibrational modes in CARS. In this technique, combining two visible laser beams could create spectra resonances relating to vibrational transitions. In this work, Raman excitation of Hydrogen molecules to v = 1 state is achieved by CARS spectroscopy. CARS measurements are successfully carried out for H2 S-branch and Q-branch transitions using our laser system. This measurement proves the feasibility of CARS experiment which could be employed to excite molecules to a specific rovibrational state. Moreover, experiments conducted for CARS signal with respect to various gas pressures differing between 200 and 600 torr for S-branch transition of H2 molecule.

Cascaded coherent anti‐Stokes Raman scattering for high‐sensitivity number density determination in the gas phase

AbstractCascaded coherent anti‐Stokes Raman scattering (CARS) signals can be efficiently generated from CARS signals when propagating collinearly with the pump/Stokes and probe beams. This effect can be seen as the CARS beam acting as the probe beam and being inelastically scattered a ‘second time’ from the Raman coherence induced along the focus of the pump/Stokes beam axis. Although much weaker, this additionally scattered signal co‐propagates with the CARS signal and may complicate the analysis of the CARS spectrum used for diagnostics in the gas phase. In particular, the occurrence of the cascaded CARS process needs to be taken into account analysing minor spectral signatures at relatively high number density of scattering molecules. Here we show how polarization control can be employed to generate CARS and cascaded CARS signals with orthogonal linear polarization and how, in this way, the cascaded CARS signals can be efficiently suppressed. However, instead of rejecting this signal, we collect both the generated CARS and cascaded CARS signals on the same detector frame, and we explore the use of these counterparts for absolute concentration measurements of the Raman‐active species. The cascaded CARS signal has exponential‐order higher sensitivity to the number density of the scattering molecules in the mixture. We demonstrate that the ratio of the CARS and the cascaded CARS signals is independent of the probe pulse energy in use, which can be a promising approach for wide‐range absolute concentration measurements in gas‐phase media.

Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy.

Engineered biomaterials provide unique functions to overcome the bottlenecks seen in biomedicine. Hence, a technique for rapid and routine tests of collagen is required, in which the test items commonly include molecular weight, crosslinking degree, purity, and sterilization induced structural change. Among them, the crosslinking degree mainly influences collagen properties. In this study, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy are used in combination to explore the collagen structure at molecular and macromolecular scales. These measured parameters are applied for the classification and quantification among the different collagen scaffolds, which were verified by other conventional methods. It is demonstrated that the crosslinking status can be analyzed from SHG images and presented as the coherency of collagen organization that is correlated with the mechanical properties. Also, the comparative analyses of SHG signal and relative CARS signal of amide III band at 1,240 cm−1 to δCH2 band at 1,450 cm−1 of these samples provide information regarding the variation of the molecular structure during a crosslinking process, thus serving as nonlinear optical signatures to indicate a successful crosslinking.

Visualization of water concentration distribution in human skin by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) microscopy

Ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging was used to visualize the distribution of water concentration in human skin ex vivo. The CARS signal of the OH stretching vibrational mode of water was found to coexist with the signal of intercellular lipids such as ceramides, which were visualized by a sharp vibrational band at 2882 cm−1. Depth-resolved CARS spectroscopic imaging of a skin sample revealed that ceramides were localized in the stratum corneum. These findings demonstrate the powerful potential of CARS spectroscopic imaging for probing pathological changes caused by anomalous water concentration distribution in human skin.