Coherent anti-Stokes Raman Scattering Signal Research Articles

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

Background and AimsEarly detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD.MethodsWe analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals.ResultsWe found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III.ConclusionsThis study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Super-Spatial- and -Spectral-Resolution in Vibrational Imaging via Saturated Coherent Anti-Stokes Raman Scattering

Coherent anti-Stokes Raman scattering (CARS) is a vibrational spectroscopic technique offering high sensitivity, chemical selectivity, and three-dimensional visualization---but with spatial resolution limited by the wave nature of light. The authors exploit the saturation of the CARS signal to break that limit, increasing both spatial and spectral resolution and reducing signal background, and thus extending the utility of CARS microscopy in materials science, biology, and beyond.

Wide-Field Vibrational Phase Contrast Imaging Based on Coherent Anti-Stokes Raman Scattering Holography**Supported by the National Natural Science Foundation of China under Grant Nos 11174019, 61322509 and 11121091, and the National Basic Research Program of China under Grant No 2013CB921904.

We propose and implement a wide-field vibrational phase contrast detection to obtain imaging of imaginary components of third-order nonlinear susceptibility in a coherent anti-Stokes Raman scattering (CARS) microscope with full suppression of the non-resonant background. This technique is based on the unique ability of recovering the phase of the generated CARS signal based on holographic recording. By capturing the phase distributions of the generated CARS field from the sample and from the environment under resonant illumination, we demonstrate the retrieval of imaginary components in the CARS microscope and achieve background free coherent Raman imaging.

Low wavenumber efficient single-beam coherent anti-Stokes Raman scattering using a spectral hole.

We demonstrate an approach to detect low wavenumber vibrational signals based on single-beam coherent anti-Stokes Raman scattering (CARS) with a spectral hole. Using a 4f pulse shaper for both pulse shaping and signal collection, we achieve an enhanced efficiency in collecting back-reflected CARS signals.

Femtosecond Coherent Anti-Stokes Raman Spectroscopy (fs-CARS) with Nitrobenzene and Nitomethane

Time-resolved coherent anti-Stokes Raman Scattering (CARS) is a nonlinear spectroscopic tool, which has gained importance with the development of ultrafast lasers due to its ability to probe ultrafast dynamical processes in molecules. Apart from the time resolution, the superiority of CARS over spontaneous Raman scattering derives from its high signal generation ability and coherent, laser like, signal emission. In this contribution, we describe the development of an experimental facility for the time-resolved coherent anti-Stokes Raman scattering spectroscopy and its application to study the vibrational dynamics of molecules containing nitro (NO2) groups. fs-CARS experiments were carried out in neat liquids of nitrobenzene, nitromethane and their mixture. A detailed mapping of the dynamics initiated by the stimulated Raman process is achieved from the spectrum of the transient CARS signal. The CARS transient shows rich beating structure corresponding to frequencies as high as 10 THz. The Fourier transform of the transient allows accurate determination of the vibrational energy differences.This methodology is capable of providing the dephasing behaviour and spectral information of the molecular system at the same time.The results demonstrate that CARS spectroscopy with femtosecond time resolution and wavenumber-resolved detection is a powerful tool for the characterization of dynamics in the electronic ground state of molecules.

Measuring the Distribution of Taurine Molecule Inside Biological Tissue via Intrinsic Molecular Vibrations using Nonlinear Raman Spectroscopy

Distributions of small molecular weight (less than 300 Da) organic compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterize the localization of large molecules such as proteins and nucleic acids, they cannot be easily applied to the cases with small molecule compounds: Fluorescent labels are relatively large compared to the target compounds and can interfere with the chemical properties of them. Raman spectroscopy is a technique to study vibrational information intrinsic to and characteristic of the chemical species of compounds. We used coherent anti-Stokes Raman scattering (CARS) spectroscopy to detect and identify a small molecule compound, taurine, in aqueous environment without labeling. Molecular species could be uniquely identified from the spectral shape of the broadband vibrational spectra of target compound. The local distribution of the compound could be determined from the spectral intensity. We have developed a phase-sensitive CARS spectroscopy capable of measuring the broadband spectrum simultaneously without losing high frequency resolution. We also utilized a time-resolved technique to remove non-resonant noise signals over a wide spectral range produced by water molecules. We combined these techniques to selectively detect resonant vibrational CARS signals from a target compound. We measured taurine inside mouse cornea tissue soaked in solution as an initial model experiment. We detected a Raman peak of taurine near 1000 wavenumber / cm inside cornea, and successfully characterized its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualize the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.

NI-78 * LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a technology routinely used intraoperatively for inspection and by neuropathology for frozen section equivalent assessment of brain tumors.

Coherent anti-Stokes Raman scattering microscopy of single nanodiamonds.

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug delivery vehicles, and contrast agents in vivo. In the quest for superior photostability and bio-compatibility, nanodiamonds (NDs) are considered one of the best choices due to their unique structural, chemical, mechanical, and optical properties. So far, mainly fluorescent NDs have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centers with stable optical properties. Here, we show that single non-fluorescing NDs exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp3 vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and ND size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of NDs internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively.

Quantitative Spectral Analysis of Coherent Anti-Stokes Raman Scattering Signals: C-H Stretching Modes of the Methyl Group.

Coherent anti-Stokes Raman scattering (CARS) vibrational spectroscopy has been extensively developed into a powerful analytical technique to study various molecules. Quantitative interpretation of CARS spectra can help to improve CARS for chemical analysis and extend its analytical applications. In this work, we quantitatively analyzed CARS signals originating from the methyl groups in poly(dimethylsiloxane) (PDMS), with the help of the bond additivity method. Experimentally, a home-built CARS spectrometer modified from a commercial sum frequency generation spectrometer was used to collect CARS spectra from a PDMS film. Theoretically, we successfully reproduced the peak intensity ratio of C-H symmetric and asymmetric stretching modes of the PDMS methyl group in different polarization combinations based on bond additivity method and Raman depolarization ratio. This research shows that bond additivity theory can help to obtain the third-order nonlinear susceptibility tensor properties probed by different polarization combinations used in CARS spectroscopy. The method developed in this work could also be applied to CARS vibrational stretching analysis of other functional groups, providing quantitative understanding of CARS spectrum for applications in spectroscopy.

Coherent anti-Stokes Raman scattering enhancement of thymine adsorbed on graphene oxide.

Coherent anti-Stokes Raman scattering (CARS) of carbon nanostructures, namely, highly oriented pyrolytic graphite, graphene nanoplatelets, graphene oxide, and multiwall carbon nanotubes as well CARS spectra of thymine (Thy) molecules adsorbed on graphene oxide were studied. The spectra of the samples were compared with spontaneous Raman scattering (RS) spectra. The CARS spectra of Thy adsorbed on graphene oxide are characterized by shifts of the main bands in comparison with RS. The CARS spectra of the initial nanocarbons are definitely different: for all investigated materials, there is a redistribution of D- and G-mode intensities, significant shift of their frequencies (more than 20 cm-1), and appearance of new modes about 1,400 and 1,500 cm-1. The D band in CARS spectra is less changed than the G band; there is an absence of 2D-mode at 2,600 cm-1 for graphene and appearance of intensive modes of the second order between 2,400 and 3,000 cm-1. Multiphonon processes in graphene under many photon excitations seem to be responsible for the features of the CARS spectra. We found an enhancement of the CARS signal from thymine adsorbed on graphene oxide with maximum enhancement factor about 105. The probable mechanism of CARS enhancement is discussed.

Determining optimum operating conditions of the polarization-maintaining fiber with two far-lying zero dispersion wavelengths for CARS microscopy.

Single femtosecond laser-based coherent anti-Stokes Raman scattering (CARS) microscopy, using a photonic crystal fiber (PCF) pumped in the near-IR to generate a supercontinuum for the Stokes source, is rapidly being adopted as a cost-effective approach. A PCF with two closely-lying zero dispersion wavelengths is a popular choice for the Stokes source, but it is often limited to imaging lipids. A polarization-maintaining PCF with two far-lying zero dispersion wavelengths offers important advantages for polarization CARS microscopy, and for CARS imaging in the fingerprint region. This PCF fiber, though commercially available, has limited use for CARS microscopy in the C-H bond region. The main problem is that the supercontinuum from this fiber is typically noisier than that from a standard PCF with two closely-lying zero dispersion wavelengths. To overcome this, we determined the optimum operating conditions for generating a low-noise supercontinuum out of a PCF with two far-lying zero dispersion wavelengths, in terms of the input parameters of the excitation pulse. We measured the relative intensity noise (RIN) of the Stokes and the corresponding CARS signal as a function of the input laser parameters in this fiber. We showed that the results of CARS imaging using this alternate fiber are comparable to those achieved using the standard fiber, for input laser pulse conditions of low average power, narrow pulse width with slightly positive chirp, and polarization direction parallel to the slow axis of the selected fiber.

Application of a high power Yb fiber‐based laser compatible with commercial optical parametric oscillator for coherent anti‐stokes raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical analysis at a subcellular level, frequently used for imaging lipid dynamics in living cells. We report a high-power picosecond fiber-based laser and its application for optical parametric oscillator (OPO) pumping and CARS microscopy. This fiber-based laser has been carefully characterized. It produces 5 ps pulses with 0.8 nm spectral width at a 1,030 nm wavelength with more than 10 W of average power at 80 MHz repetition rate; these spectral and temporal properties can be slightly modified. We then study the influence of these modifications on the spectral and temporal properties of the OPO. We find that the OPO system generates a weakly spectrally chirped signal beam constituted of 3 ps pulses with 0.4 nm spectral width tunable from 790 to 930 nm optimal for CARS imaging. The frequency doubling unconverted part is composed of 7-8 ps pulses with 0.75 nm spectral width compatible with CARS imaging. We also study the influence of the fiber laser properties on the CARS signal generated by distilled water. In agreement with theory, we find that shorter temporal pulses allow higher peak powers and thus higher CARS signal, if the spectral widths are less than 10 cm(-1) . We demonstrate that this source is suitable for performing CARS imaging of living cells during several hours without photodamages. We finally demonstrate CARS imaging on more complex aquatic organisms called copepods (micro-crustaceans), on which we distinguish morphological details and lipid reserves.

Fourier‐based approach to overcome anomalies in high‐resolution spectra of vibrational CARS measurements of gases

In this work, we report on an unconventional experimental procedure useful for thermometric measurements on the basis of high‐resolution vibrational coherent anti‐Stokes Raman scattering (CARS) generated using conventional lasers. The high spectral dispersion of 0.02 cm−1/pixel is achieved by means of some specific spectral arrangements (single‐mode pump laser and diffraction grating providing spectra at the sixth order) and is further strengthened by the use of a relay lens system mounted before the charge‐coupled device camera. Surprisingly, at the high spectral dispersion of our measurements, a significant and persistent thermometric inaccuracy is observed. The effect arises from an inevitable spectral modulation of defined frequency hidden in the main CARS signal of the gas under study, and to secure a good thermometric evaluation, we describe a Fourier approach that is experimentally demonstrated for the typical nitrogen CARS spectrum measured at room temperature. Copyright © 2014 John Wiley & Sons, Ltd.

Contrast enhancement using silica microspheres in coherent anti-Stokes Raman spectroscopic imaging

Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful imaging technique that can provide chemical information of organic and nonorganic materials through vibrational spectroscopy. However, its contrast is not sufficient for monitoring thin film materials. In this study, silica microspheres were employed for enhancing the signal contrast in CARS imaging. One layer of optically transparent silica microspheres was self-assembled onto polymer grating samples to enhance the CARS signals. The highest contrast enhancement factor of 12.5 was achieved using 6.1-μm-diameter microspheres. Finite-difference time-domain method (FDTD) simulation was conducted to simulate the contrast enhancement with silica microspheres of different diameters.

Time- and frequency-dependent model of time-resolved coherent anti-Stokes Raman scattering (CARS) with a picosecond-duration probe pulse

The hybrid femtosecond∕picosecond coherent anti-Stokes Raman scattering (fs∕ps CARS) technique presents a promising alternative to either fs time-resolved or ps frequency-resolved CARS in both gas-phase thermometry and condensed-phase excited-state dynamics applications. A theoretical description of time-dependent CARS is used to examine this recently developed probe technique, and quantitative comparisons of the full time-frequency evolution show excellent accuracy in predicting the experimental vibrational CARS spectra obtained for two model systems. The interrelated time- and frequency-domain spectral signatures of gas-phase species produced by hybrid fs∕ps CARS are explored with a focus on gas-phase N2 vibrational CARS, which is commonly used as a thermometric diagnostic of combusting flows. In particular, we discuss the merits of the simple top-hat spectral filter typically used to generate the ps-duration hybrid fs∕ps CARS probe pulse, including strong discrimination against non-resonant background that often contaminates CARS signal. It is further demonstrated, via comparison with vibrational CARS results on a time-evolving solvated organic chromophore, that this top-hat probe-pulse configuration can provide improved spectral resolution, although the degree of improvement depends on the dephasing timescales of the observed molecular modes and the duration and timing of the narrowband final pulse. Additionally, we discuss the virtues of a frequency-domain Lorentzian probe-pulse lineshape and its potential for improving the hybrid fs∕ps CARS technique as a diagnostic in high-pressure gas-phase thermometry applications.

Vector analysis of the coherent anti-Stokes Raman scattering signals generated under the tightly focused condition

In a coherent anti-Stokes Raman scattering (CARS) microscope, when samples with different shapes and dimensions are excitated by collinearly introduced and tightly focused Gaussian beams, the microscopic structure will be determined by the spatial distributions of generated CARS signals. Therefore, we build a theoretical model for CARS signals from spherical sample under the tightly focused condition. The intensity and phase distributions of tightly focused linear polarization Gaussian beams are analyzed with vector wave equations. The vector wave equation of CARS signals is derived from Green's function. The far-field CARS radiation patterns of spherical scatters with different diameters are simulatively calculated. Theoretical analysis and simulative calculation results show that the intensities of forward and backward CARS signals from the small spherical sampler are similar. The images with high contrast can be obtained by backward detection method from an objective with a high numerical aperture. For big spherical samplers, intensities of CARS signals are greatly increased. The emission direction is mainly concentrated in a spatial angle. The forward CARS signals can be effectively collected by an objective with low numerical aperture.

Theoretical and experimental study on the multi-color broadband coherent anti-Stokes Raman scattering processes

In order to exactly distinguish and quantitatively analyze the different or unknown components in a mixture, the global molecular CARS spectra information should be obtained simultaneously with a broad-band coherent anti-Stokes Raman scattering (CARS) spectroscopy in supercontinuum. In a broad-band CARS spectroscopy, two-and three-color CARS processes are generated due to different functions of effective spectroscopic components in supercontinuum. Firstly, we theoretically analyzed the generation conditions of CARS signals and the relationships between their intensity and power of excitation lights in the two types of CARS process with the broad-band excitation. On this basis, the two types of CARS process are achieved with a home-built broad-band CARS spectroscopic system, respectively. Using the functional fitting analysis of the obtained CARS spectral signals of benzonitrile, the relationships between CARS signals and excitation lights are experimentally verified in two different kinds of CARS process. Further optimizations of broad-band time-resolved CARS spectroscopic and microscopic systems, for simultaneously obtaining the global CARS spectral signals of samples, can be achieved under the guidance of theoretical and experimental results.

Intracavity Phase-matched Coherent Anti-stokes Raman Spectroscopy for Trace Gas Detection

We present a novel, cavity-enhanced spectroscopic technique based on a phase-matched Raman process to detect trace quantities of gas. The essence of this technique is the careful control of cavity dispersion to satisfy the phase-matching condition of coherent anti-Stokes Raman scattering (CARS) enhanced in a high-finesse optical cavity. A 6000-fold improvement of the CARS signal is observed under optimized conditions, indicating that this is a promising tool to quantify Raman-active molecules with an extremely low detection limit.

Multimodal mapping of human skin

The combination of coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging--referred to as multimodal imaging--provides complementary contrast based on molecular vibrations, the structure of various tissue components and endogenous fluorophores, respectively. To present a comprehensive overview of the appearance of human skin in multimodal imaging. Multimodal imaging of unstained skin cross-sections of 32 individuals was performed using a laser scanning microscope and picosecond laser pulse for excitation. The epidermis, dermis and subcutis are distinguishable in all three applied modalities, but are unveiled best in multimodal images. While the subcutis is dominated by the CARS signal, predominately SHG and the secondary TPEF signal detect the dermis. In contrast, no SHG signal is detected in the epidermis, whereas CARS and TPEF show equal contributions. Additionally, the appearance of the major skin appendages is described, i.e. the hair follicle, sebaceous and sweat glands, and blood vessels belonging to the vascular system. All four investigated functional units show a characteristic morphochemistry in TPEF and CARS, allowing identification of further subunits, e.g. the major components of the hair follicle, while the SHG signal delineates the localization of the functional units. Multimodal imaging is a powerful tool to investigate human skin by providing high contrast based on the molecular constitution. It is therefore suggested that multimodal imaging has a high potential in application to dermatological research and clinical diagnostics of various skin alterations.

Lipid droplet pattern and nondroplet-like structure in twofatmutants ofCaenorhabditis elegansrevealed by coherent anti-Stokes Raman scattering microscopy

Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.