Coherent anti-Stokes Raman Scattering Signal Research Articles

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.

Imaging and quantitative analysis of atherosclerotic lesions by CARS-based multimodal nonlinear optical microscopy.

The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee. Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery. We have demonstrated the capability of CARS-based multimodal NLO microscopy to interrogate different stages of lesion development with subcellular detail to permit quantitative analysis of lipid and collagen contents.

A versatile CARS microscope for biological imaging

AbstractWe have constructed a novel, versatile Coherent anti‐Stokes Raman scattering (CARS) microscope for biological imaging. The system employs a Nd:YVO4 laser as Stokes (6 ps, 1064 nm), and a tunable optical parametric oscillators (OPOs) (5 ps, 700–1000 nm) as pump excitation, resulting in an observable range of ∼600–4000 cm−1. A second OPO can be used as a replacement pump for low wavenumber excitation (down to ∼100 cm−1). Pump and Stokes excitations are focused onto a single‐mode fibre for ease of alignment, which is connected to the input of a confocal inverted optical microscope. The epi‐detected CARS signal is filtered using a spectrometer, and the photons counted with an avalanche photodiode. This detection is preferable to using bandpass filters and photomultipliers, as it allows all wavenumbers to be imaged and ensures rejection of unwanted fluorescent and scattered light. Our microscope comprises a heater stage and perfusion cell for imaging live cells, and allows complementary biological imaging techniques (differential interference contrast (DIC), second harmonic generation, sum frequency generation, one‐ and two‐photon fluorescence, fluorescence lifetime imaging). In addition, our system features an atomic force microscope (AFM) which enables CARS imaging at 10‐nm resolution. The details and advantages of the modular imaging system are described more fully, and the latest results are presented. Copyright © 2009 John Wiley & Sons, Ltd.

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.

Tuning Gold Nanoparticle Self-Assembly for Optimum Coherent Anti-Stokes Raman Scattering and Second Harmonic Generation Response

The research and development of new substrates for use in surface-enhanced spectroscopy is primarily motivated by the ability to tune such substrates to provide maximum signal enhancement and therefore lower detection limits. We examined a series of multilayer nanoparticle (NP) arrays with between 1 and 17 NP layers using two nonlinear optical (NLO) techniques: Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG). The CARS signal of oxazine 720 was monitored at 1600 cm−1 using a 709-nm pump beam and an 800-nm stokes beam. Maximum signal was observed for 11 NP layers and is attributed to the matching of the CARS signal and the substrate surface plasmon excitation at 637 nm. The CARS signal for the 3000-cm−1 C−H stretching vibration showed maximum enhancement at 13 NP layers. The maximum SHG signal enhancement occurred at 13 NP layers, with a 50-fold overall enhancement of the SHG signal. We demonstrate that the NP-containing substrates can be tuned to provide maximum NLO respon...

Numerical study of effects of light polarization, scatterer sizes and orientations on near-field coherent anti-Stokes Raman scattering microscopy

We employ the finite-difference time-domain (FDTD) technique as a numerical approach to studying the effects of polarization, scatterers' sizes and orientations on near-field coherent anti-Stokes Raman scattering (CARS) microscopy imaging. The results show that to acquire better image contrast and larger near-field CARS signals, the scatterers with diameters of less than three-eighths of the pump field wavelength (lambda(p)) are preferable to be oriented along the polarization direction of the excitation light fields. It is also found that when the scatterers' sizes are smaller than half a wavelength of the pump field, the perpendicular polarization component of the induced near-field CARS radiations is strictly confined within the regions at the scatterer-water interface or the subsurface of scatterers, yielding a high image contrast (up to 200) with a spatial resolution of lambda(p)/16. This study indicates that perpendicular polarization components of near-field CARS microscopy could be used to uncover very fine structures of intra- and/or inter- cellular organelles in cells with nanoscale resolutions.

Single-shot interferometric approach to background free broadband coherent anti- Stokes Raman scattering spectroscopy

We introduce a single-shot interferometric approach to suppress the nonresonant background (NRB) contribution to a broadband coherent anti-Stokes Raman scattering (CARS) spectrum; this single-shot approach is conducive to rapid imaging. A pulse shaper prepares a narrowband pulse with two spectral components of differing phase. When the CARS fields generated by these two out-of-phase components are optically mixed, NRB signal is greatly reduced while a resonant CARS signal remains with minimal attenuation. We discuss and demonstrate two model schemes for the interfering pulse components: (1) two pulses with different bandwidths and the same center frequency (ps-fs scheme) and (2) two pulses with the same bandwidth and shifted center frequencies (ps-ps scheme). In both schemes, only the resonant signal from the "3-color" CARS mechanism survives. The resonant signal from "2-color" CARS mechanism vanishes along with the NRB. We discuss optimization conditions for signal intensity and shape of resonant CARS peaks. Experimental CARS spectra of c-hexane and benzonitrile demonstrate feasibility of these approaches.

Enhanced contrast coherent anti-Stokes Raman scattering microscopy using annular phase masks

We have experimentally demonstrated enhanced image contrast of sample fine structure in a coherent anti-Stokes Raman scattering (CARS) microscope using liquid crystal spatial light modulators as programmable Toraldo-style pupil phase masks. We demonstrated through modeling, implementation, and verification of the focal intensity and phase distributions of the excitation beams the trade-offs between improved image contrast and CARS signal levels.

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.

Elliptically polarized coherent anti-Stokes Raman scattering microscopy

We report an approach to coherent anti-Stokes Raman scattering (CARS) microscopy that significantly suppresses the nonresonant background for high-contrast vibrational imaging. We employ an elliptically polarized pump field with an aspect ratio of 1:3 of the ellipse combined with a linearly polarized Stokes field for elliptically polarized CARS (EP-CARS) microscopy. The EP-CARS technique yields a 6-7-fold improvement in signal-to-background ratios compared with normal CARS, while providing an approximately 1.25-fold improvement in resonant CARS signal compared with normal polarization-sensitive CARS microscopy. We demonstrate this technique by imaging 1.5 microm polystyrene beads in water and lipid droplets in unstained fibroblast cells.

Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy was applied to image myelinated fibers in different regions of a mouse brain. The CARS signal from the CH2 symmetric stretching vibration allows label-free imaging of myelin sheath with 3D sub-micron resolution. Compared with two-photon excited fluorescence imaging with lipophilic dye labeling, CARS microscopy provides sharper contrast and avoids photobleaching. The CARS signal exhibits excitation polarization dependence which can be eliminated by reconstruction of two complementary images with perpendicular excitation polarizations. The capability of imaging myelinated fibers without exogenous labeling was used to map the whole brain white matter in brain slices and to analyze the microstructural anatomy of brain axons. Quantitative information about fiber volume%, myelin density, and fiber orientations was derived. Combining CARS with two-photon excited fluorescence allowed multimodal imaging of myelinated axons and other cells. Furthermore, in vivo CARS imaging on an upright microscope clearly identified fiber bundles in brain subcortex white matter. These advances open up new opportunities for the study of brain connectivity and neurological disorders.

Concentration dependence in coherent Raman scattering

We investigate the concentration dependence of the femtosecond coherent anti-stokes Raman scattering (CARS) signals collected from several mixtures of molecules. We demonstrate a clear quadratic dependence of the transmitted CARS signal on the number of molecules of pyridine in water by looking at the Raman signal at 1030 cm−1. Using a backscattering configuration we study the quadratic concentration dependence in solid mixtures of dipicolinic acid, calcite, and gypsum in powder form.

Phase-controlled polarization coherent anti-Stokes Raman scattering microscopy for high-sensitivity and high-contrast molecular imaging

We report on a phase-controlled polarization coherent anti-Stokes Raman scattering (CARS) microscopy for high-sensitivity and high-contrast molecule vibrational imaging. By changing the phase difference between the two CARS signals (i.e., a weak resonant CARS and a strong nonresonant CARS signal) simultaneously generated from the same focal volume of the sample, the complete constructive (in-phase) and destructive (out-of-phase) interference CARS images can be acquired for image processing. The directly digital subtraction between the constructive and destructive interference CARS images yields a fivefold improvement in signal-to-background ratios compared with conventional CARS while providing an approximately 20-fold amplification of a resonant CARS signal compared with conventional polarization CARS imaging. We demonstrate this technique by imaging 1 μm polystyrene beads and unstained human epithelial cells in aqueous environments.

Single‐beam pumped coherent anti‐Stokes Raman scattering on carbon nanotubes

AbstractGiant optical fields locally generated in nanoscale metal structures through the local excitation of surface plasmons underlie a variety of nonlinear optical processes. Using silver and gold rough metallic films, reproducible enhancements of the Raman signal on various materials including carbon nanotubes have been demonstrated. In addition, an abnormal anti‐Stokes Raman emission, reminiscent of Coherent anti‐Stokes Raman Scattering (CARS), is accurately observed on nanometric thin films. We demonstrate that under a tight focusing of the excitation light, a CARS‐type emission, resulting from a wave mixing process between the incident laser light and Stokes Raman light generated by a SERS mechanism, is produced. The results which corroborate the CARS emission are the following: a square relation between the CARS signal intensity and the film thickness; a square relationship between the CARS signal intensity and the exciting laser intensity; a dependence of the CARS intensity on the numerical aperture (NA) of the microscope objective used for the detection of the anti‐Stokes signal; a polarization ratio in the anti‐Stokes side always greater in comparison with that measured for an usual Stokes Raman signal. We demonstrate also that this mechanism may contribute to a clear identification of the state of the observed nanotubes, i.e. in isolated or bundled forms, even when tubes are in powders. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Achievement of Narrow-Band CARS Signal by Manipulating Broad-band Laser Spectrum

We theoretically demonstrate the achievement of narrow-band coherent anti-Stokes Raman scattering (CARS) signal by manipulating broad-band probe spectrum. The narrowing of the CARS signal depends on the spectrum bandwidth of the probe beam, and thus high-resolution CARS signal for a complicated quantum system can be obtained by the simple spectrum manipulation. Furthermore, the energy-level diagram for the complicated quantum system can also be labelled by measuring the CARS signal at a given frequency.

Time- and frequency-resolved coherent anti-Stokes Raman scattering spectroscopy with sub-25fs laser pulses

In general, many different diagrams can contribute to the signal measured in broadband four-wave mixing experiments. Care must therefore be taken when designing an experiment to be sensitive to only the desired diagram by taking advantage of phase matching, pulse timing, sequence, and the wavelengths employed. We use sub-25 fs pulses to create and monitor vibrational wavepackets in gaseous iodine, bromine, and iodine bromide through time- and frequency-resolved femtosecond coherent anti-Stokes Raman scattering (CARS) spectroscopy. We experimentally illustrate this using iodine, where the broad bandwidths of our pulses, and Boltzmann population in the lower three vibrational levels conspire to make a single diagram dominant in one spectral region of the signal spectrum. In another spectral region, however, the signal is the sum of two almost equally contributing diagrams, making it difficult to directly extract information about the molecular dynamics. We derive simple analytical expressions for the time- and frequency-resolved CARS signal to study the interplay of different diagrams. Expressions are given for all five diagrams which can contribute to the CARS signal in our case.

Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy

Wide-field Coherent Anti-Stokes Raman Scattering (CARS) microscopy is employed to identify saturated and unsaturated fatty acids in micro-emulsions and cells, using the ratio between the strong -C-H CARS signal at 2850 cm(-1) and the weak signal of the =C-H vibration around 3015 cm(-1) for distinction. Quantitative CARS imaging at the =C-H resonance is challenging, since it yields only a low CARS signal, and small differences on the order of 5% in the concentration of polyunsaturated fatty lipids have to be detected. For this purpose we draw advantage of the high signal-to-noise ratio of wide-field CARS microscopy that is achieved by an excitation geometry involving a "sheet-of-light"-type illumination.

Simultaneous forward and epi-CARS microscopy with a single detector by timecorrelated single photon counting

We present a novel scheme to simultaneously detect coherent anti-Stokes Raman scattering (CARS) microscopy signals in the forward (F) and backward (epi - E) direction with a single avalanche photodiode (APD) detector using time-correlated single photon counting (TCSPC). By installing a mirror at a well-defined distance above the sample the forward-scattered F-CARS signal is reflected back into the microscope objective leading to spatial overlap of the F and E-CARS signals. Due to traveling an additional distance the F-CARS signal is time delayed relative to the E-CARS signal. TCSPC then allows for the two signals to be resolved in the time domain. This results in an efficient, simple, and compact method of CARS signal detection. We demonstrate this technique by analyzing forward and backward CARS signals obtained by imaging living adipocyte cells derived from human mesenchymal stem cells.

Thickness dependent CARS measurement of polymeric thin films without depth-profiling

Coherent anti-Stokes Raman scattering (CARS) microscopy is demonstrated to be a promising optical method for the characterization of polymer films with film thickness varying between 180 nm to 4300 nm. In case of PMMA films with a thickness of few hundreds of nanometers, the observed CARS signal was mainly associated with the interference effect of large nonresonant CARS field from glass substrate and the weak resonant field of PMMA. The dependence of resonant CARS intensity of PMMA film on film thickness is in good agreement with the theoretical prediction on a CARS field. The current work offers potential possibilities of noninvasive thickness measurement of polymeric thin film of thickness less than 180 nm by multiplex CARS microscopy without depth-profiling.

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.