Broadband Coherent anti-Stokes Raman Scattering Research Articles

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.

Hyperspectral imaging and characterization of live cells by broadband coherent anti-Stokes Raman scattering (CARS) microscopy with singular value decomposition (SVD) analysis.

Coherent anti-Stokes Raman scattering (CARS) microscopy can be used as a powerful imaging technique to identify chemical compositions of complex samples in biology, biophysics, medicine, and materials science. In this work we developed a CARS microscopic system capable of hyperspectral imaging. By employing an ultrafast laser source, a photonic crystal fiber, and a scanning laser microscope together with spectral detection by a highly sensitive back-illuminated cooled charge-coupled device (CCD) camera, we were able to rapidly acquire and process hyperspectral images of live cells with chemical selectivity. We discuss various aspects of hyperspectral CARS image analysis and demonstrate the use of singular value decomposition methods to characterize the cellular lipid content.

High-Speed Coherent Raman Fingerprint Imaging of Biological Tissues.

An imaging platform based on broadband coherent anti-Stokes Raman scattering (BCARS) has been developed which provides an advantageous combination of speed, sensitivity and spectral breadth. The system utilizes a configuration of laser sources that probes the entire biologically-relevant Raman window (500 cm−1 to 3500 cm−1) with high resolution (< 10 cm−1). It strongly and efficiently stimulates Raman transitions within the typically weak “fingerprint” region using intrapulse 3-colour excitation, and utilizes the nonresonant background (NRB) to heterodyne amplify weak Raman signals. We demonstrate high-speed chemical imaging in two- and three-dimensional views of healthy murine liver and pancreas tissues and interfaces between xenograft brain tumours and the surrounding healthy brain matter.

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.

Quantitative, Label-Free Characterization of Stem Cell Differentiation at the Single-Cell Level by Broadband Coherent Anti-Stokes Raman Scattering Microscopy

We use broadband coherent anti-Stokes Raman scattering (BCARS) microscopy to characterize lineage commitment of individual human mesenchymal stem cells cultured in adipogenic, osteogenic, and basal culture media. We treat hyperspectral images obtained by BCARS in two independent ways, obtaining robust metrics for differentiation. In one approach, pixel counts corresponding to functional markers, lipids, and minerals, are used to classify individual cells as belonging to one of the three lineage groups: adipocytes, osteoblasts, and undifferentiated stem cells. In the second approach, we use multivariate analysis of Raman spectra averaged exclusively over cytosol regions of individual cells to classify the cells into the same three groups, with consistent results. The exceptionally high speed of spectral imaging with BCARS allows us to chemically map a large number of cells with high spatial resolution, revealing not only the phenotype of individual cells, but also population heterogeneity in the degree of phenotype commitment.

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.

Tip‐enhanced broadband CARS spectroscopy and imaging using a photonic crystal fiber based broadband light source

We report tip‐enhanced broadband coherent anti‐Stokes Raman scattering (CARS) spectroscopy and imaging based on a broadband light source using a photonic crystal fiber. Multiplexing from 900 cm–1 to 2200 cm–1 is achieved by optimizing the supercontinuum generation process at a high numerical aperture focus. By combining this system with a shear‐force microscope, semiconducting carbon nanotubes are imaged with a spatial resolution of 60 nm. Our results show that tip‐enhanced broadband CARS image exhibits a positive contrast even though the enhancement effect has not been fully optimized. Analyses of delay‐dependent tip‐enhanced broadband CARS spectra suggest the possibilities of multiple carrier interactions that uniquely occur at high intensities within the nanotubes. Copyright © 2012 John Wiley &amp; Sons, Ltd.

High-Speed Imaging of Broadband Multiplex Coherent Anti-Stokes Raman Scattering Microscopy Using a Supercontinuum Source

We combined broadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy with CARS microscopy using a femtosecond laser and a photonic crystal fiber. Broadband multiplex CARS microspectroscopy, which simultaneously measures broadband range (including significant Raman vibrational modes) from 500 to 3500 cm-1 at a high spectral resolution was subsequently performed. Also, via multiplex CARS microscopy using a pair of galvanometer mirrors we successfully demonstrated the high-speed imaging of polymer beads and a lily pollen grain for the study of cell biology dynamics. The imaging speed was 37 s per image, which is 30–40 times faster than that previously reported in broadband CARS microscopy.

Label-Free Cellular Imaging by Broadband Coherent Anti-Stokes Raman Scattering Microscopy

Raman microspectroscopy can provide the chemical contrast needed to characterize the complex intracellular environment and macromolecular organization in cells without exogenous labels. It has shown a remarkable ability to detect chemical changes underlying cell differentiation and pathology-related chemical changes in tissues but has not been widely adopted for imaging, largely due to low signal levels. Broadband coherent anti-Stokes Raman scattering (B-CARS) offers the same inherent chemical contrast as spontaneous Raman but with increased acquisition rates. To date, however, only spectrally resolved signals from the strong CH-related vibrations have been used for CARS imaging. Here, we obtain Raman spectral images of single cells with a spectral range of 600–3200 cm −1, including signatures from weakly scattering modes as well as CH vibrations. We also show that B-CARS imaging can be used to measure spectral signatures of individual cells at least fivefold faster than spontaneous Raman microspectroscopy and can be used to generate maps of biochemical species in cells. This improved spectral range and signal intensity opens the door for more widespread use of vibrational spectroscopic imaging in biology and clinical diagnostics.

In situ and real time monitoring of two-photon polymerization using broadband coherent anti-Stokes Raman scattering microscopy

We demonstrate in situ and real time characterization of two-photon polymerization (TPP) by means of broadband coherent anti-Stokes Raman scattering (CARS) microscopy. The same experimental setup based on one femtosecond oscillator is used to perform both TPP and broadband CARS microscopy. We performed in situ imaging with chemical specificity of three-dimensional microstructures fabricated by TPP, and successfully followed the writing process in real time. Broadband CARS microscopy allowed discerning between polymerized and unpolymerized material. Imaging with good vibrational contrast is achieved without causing any damage to the microstructures or undesired polymerization within the sample.

CARS temperature measurement in a LOX/CH4 spray flame

Coherent anti-Stokes Raman scattering (CARS) spectroscopy has been used to investigate cryogenic liquid oxygen/gaseous methane (LOX/CH4) flames on a medium-size test facility at a pressure of 0.24 MPa and mass flow of 0.025 kg/s. Single-shot, broadband CARS spectra with simultaneous detection of the Q-branches of hydrogen and water molecules were recorded with good signal-to-noise ratio. Temperature was deduced from the H2 and H2O CARS profiles. The spatial temperature distribution in a comparatively harsh environment has been measured successfully. The measurements took place in the windowed combustion chamber of the DLR M3 test facility, aiming to provide data for validation of rocket combustor modeling. Copyright © 2010 John Wiley & Sons, Ltd.

CARS investigation of collisional shift of the hydrogen Q -branch transitions by water at high temperatures

A high-resolution (∼0.1 cm−1) spectroscopic method based on the application of a Fabry–Pérot interferometer to the spectral analysis of the coherent anti-Stokes Raman scattering (CARS) signal from an individual Raman transition was used to obtain single-shot spectra of hydrogen Q-branch transitions directly in the flame of a pulsed, high-pressure H2/O2 combustion chamber. Simultaneously with the Fabry–Pérot pattern, a broadband CARS spectrum of the complete H2Q -branch structure was recorded in order to measure the temperature of the probe volume. During every cycle of the combustion chamber, a pressure pulse together with single-shot CARS spectra, providing information on individual line shapes and medium temperature, was recorded. On the basis of the experimental data, the temperature dependences of lineshift coefficients for several Q-branch lines of hydrogen molecules under collisions with water molecules were determined in the temperature range 2100 < T < 3500 K, and an empirical ‘fitting law’ for H2H2O lineshift coefficients is proposed. Copyright © 2010 John Wiley & Sons, Ltd.

Three-color multiplex CARS for fast imaging and microspectroscopy in the entire CHn stretching vibrational region

We present a three-color multiplex coherent anti-Stokes Raman scattering (CARS) setup that facilitates a prompt recording of broadband CARS spectra along with a fast CARS imaging. With separate narrowband Stokes and probe beams being introduced in the near IR, we are able to incorporate a stable, wideband Ti:sapphire femtosecond laser as a pump beam that covers the full range of Raman shift for CHn stretching vibrational modes. Experimentally, high-resolution multiplex CARS signals are allowed to investigate molecular vibrations over the range of 2650 cm-1 - 3050 cm-1, which are spectrally integrated to construct lipid-sensitive images. It is demonstrated that the proposed implementation promises a particular benefit on CARS imaging of lipid-rich tissue structures by providing detailed information on CHn Raman-active vibrations at points of interest on the CARS images that can be obtained at high frame rates.

Broadband coherent anti-Stokes Raman scattering spectroscopy using soliton pulse trains from a photonic crystal fiber

Pulse trains of fundamental soliton pulses with different center wavelengths and delay times from a photonic crystal fiber were generated and used as Stokes optical pulses in coherent anti-Stokes Raman scattering (CARS) spectroscopy. The pulse trains were created by shaping optical pulses with a pulse shaper and their waveforms were measured by a cross-correlation frequency-resolved optical gating method. By the use of pulse trains, the time required for obtaining broadband CARS signals was reduced to be about one third compared with our previous study without using pulse trains. With this setup, broadband CARS signals between 500 and 3100 cm −1 of a single polystyrene bead sample have been measured and the most of the Raman peaks in this frequency range of samples have been observed clearly.

Chemically selective imaging by spectral phase shaping for broadband CARS around 3000 cm^−1

Chemically selective microscopy based on broadband coherent anti-Stokes Raman scattering (CARS) is demonstrated on a mixed sample of 4 μm diameter polystyrene (PS) and poly (methyl methacrylate) (PMMA) beads. The CARS signal from the PS or the PMMA beads is enhanced or suppressed, depending on the phase profile applied to the broadband spectrum. Using a combination of negative and positive (sloped) π-phase steps the purely nonresonant background signal is removed.

Broadband Coherent Anti-Stokes Raman Scattering Spectroscopy Using Pulse-Shaper-Controlled Variable-Wavelength Soliton Pulses from a Photonic Crystal Fiber

Fundamental soliton pulses from a photonic crystal fiber (PCF) were used as variable-wavelength optical pulses in coherent anti-Stokes Raman scattering (CARS) spectroscopy using a single un-amplified laser oscillator in a microscopy setup. Both the center wavelengths and the delay times of soliton pulses were controlled simultaneously by shaping the input pulse of a PCF by the use of a pulse shaper and these parameters were switched automatically in measurement. Broadband CARS signals between 500 and 3100 cm-1 of polymer samples have been measured and the most of the Raman peaks in this frequency range of samples were observed clearly by this method.

Characterization of three-color CARS in a two-pulse broadband CARS spectrum

We demonstrate that a broadband coherent anti-Stokes Raman scattering (CARS) spectrum generated with a typical two-pulse scheme contains two distinct, significant signals: '2-color' CARS, where the pump and probe are provided by a narrowband pulse and the continuum pulse constitutes the Stokes light, and '3-color' CARS, where the pump and Stokes are provided by two different frequency components in the continuum pulse and the narrowband pulse serves as the probe. The CARS spectra from the two different mechanisms show distinct characteristics in Raman shift range, laser power dependence, and chirping dependence. We discuss the potential for a 3-color CARS signal to cover the fingerprint region with reduced photodamage of live cells. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States.

Improving Signal-To-Interference Ratio in Rich Hydrocarbon—Air Flames Using Picosecond Coherent Anti-Stokes Raman Scattering

There is growing interest in the use of short-pulse lasers for coherent anti-Stokes Raman scattering (CARS) to minimize non-resonant background (NRB) contributions in a variety of applications. Using time-coincident picosecond (ps) pump and Stokes beams and a time-delayed ps probe beam, we show that a three orders of magnitude reduction in NRB interference can be achieved in rich hydrocarbon-air flames while preserving 60% to 80% of the CARS signal. This represents a significant improvement in signal-to-interference ratio compared with previous measurements in room temperature air and is attributable to reduced rates of collisional dephasing and relaxation at flame temperatures. Measurements within the flame zone of a laminar flat-flame burner are used to investigate the characteristics of time-coincident and probe-delayed broadband ps N(2)-CARS spectra for C(2)H(4)-air equivalence ratios of 0.5 to 1.2. Up to three ro-vibrational bands of N(2) are excited with each laser shot using 135 ps pump and 106 ps Stokes beams, and the CARS signal is generated using a 135 ps probe beam delayed by 165 ps. The enhanced signal-to-interference ratio achieved in the current work is one to two orders of magnitude higher than that previously achieved using polarization-selection techniques without sensitivity to the effects of birefringence caused by density gradients or test cell windows. Moreover, the use of a 135 ps laser source in this study enables frequency domain "broadband" CARS with sufficient resolution to extract ro-vibrational spectral features under various flame conditions. The effect of probe delay and NRB suppression on characteristics of these broadband CARS spectra are investigated, and evidence of preferential collisional dephasing and relaxation of different ro-vibrational transitions is not detected. This is a promising but preliminary result to be investigated further in future work.

Broadband coherent anti-Stokes Raman scattering spectroscopy of nitrogen using a picosecond modeless dye laser

Broadband picosecond coherent anti-Stokes Raman scattering (CARS) spectroscopy of nitrogen is demonstrated using 145-ps pump and probe beams and a 115-ps Stokes beam with a spectral bandwidth of 5 nm. This is, to our knowledge, the first demonstration of broadband CARS using subnanosecond lasers. The short temporal envelope of the laser pulses and the broadband spectral nature of the Stokes beam will enable nonresonant-background-free, single-shot, or time-dependent spectroscopy in high-pressure or hydrocarbon-rich environments. Successful correlation of room-temperature broadband picosecond N2 CARS with a theoretical spectrum is presented.