Detection of chemical interfaces in coherent anti-Stokes Raman scattering microscopy: D-CARS. II. Arbitrary interfaces

We address the general problem of detecting chemical interfaces arbitrarily oriented in space in coherent anti-Stokes Raman scattering (CARS) microscopy. Such a task is accomplished by using a beam reversal scheme, as recently demonstrated experimentally [J. Biomed. Opt. 16, 086006 (2011)]. We develop a full vectorial theoretical analysis of the situation and show that transverse chemical interfaces are readily highlighted without special care in the CARS signal detection. In addition, a finer analysis reveals that adequate angular analysis of the CARS far-field radiation pattern enables the detection of axial interfaces. Background-free CARS microscopy and spectroscopy are thus achievable through the combined application of excitation beam reversal and angular analysis of the CARS far-field radiation pattern. This differential CARS (D-CARS) technique is relevant for fast detection of interfaces between molecularly different media.     

Refractive effects in coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy with high sensitivity and high three- dimensional resolution has been developed for the vibrational imaging of chemical species. Due to the coherent nature of the CARS emission, it has been reported that the detection of epi-CARS and forward-CARS (F-CARS) signals depends on the size and shape of the sample. We investigate theoretically and experimentally the effects on the CARS signal of refractive index mismatches between the sample and its surroundings. Backward-CARS and F-CARS signals are measured for different polystyrene bead diameters embedded in different refractive index solvents. We show that index mismatches result in a backward-reflected F-CARS signal that generally dominates the experimentally backward-detected signal. Simulations based on geometrical and wave optics comparing forward- and backward-detected signals for polystyrene beads embedded in different index solvents confirm our findings. Furthermore, we demonstrate that the maxima of forward- and backward-detected signals are generated at different positions along the optical axis in the sample if refractive index mismatches are present between the sample and its surroundings.     

Three-dimensional transfer functions of coherent anti-Stokes Raman scattering microscopy

The three-dimensional coherent transfer function of confocal coherent anti-Stokes Raman scattering microscopy was derived theoretically. The three-dimensional optical transfer function was also derived under the weak-contrast assumption. The effect of a pinhole in front of the detector on the optical transfer function was estimated, and it was found that the cutoff frequency of the optical transfer function is independent of the pinhole. Micrometer-order spatial resolution along the optical axis was also experimentally demonstrated.     

Focus-engineered coherent anti-Stokes Raman scattering microscopy: a numerical investigation

The coherent anti-Stokes Raman scattering (CARS) signal is calculated as a function of focal-field distributions with engineered phase jumps. We show that the focal fields in CARS microscopy can be shaped such that the signal from the bulk is suppressed in the forward detection mode. We present the field distributions that display enhanced sensitivity to vibrationally resonant object interfaces in the lateral dimension. The use of focus-engineered CARS provides a simple means to detect chemical edges against the strong background signals from the bulk.     

Implementation of vibrational phase contrast coherent anti-Stokes Raman scattering microscopy

Detection of molecules using vibrational resonances in the fingerprint region for narrowband coherent anti-Stokes Raman scattering (CARS) is challenging. The spectrum is highly congested resulting in a large background and a reduced specificity. Recently we introduced vibrational phase contrast CARS (VPC-CARS) microscopy as a technique capable of detecting both the amplitude and phase of the CARS signal, providing background-free images and high specificity. In this paper we present a new implementation of VPC-CARS based on a third-order cascaded phase-preserving chain, where the CARS signal is generated at a single (constant) wavelength independent of the vibrational frequency that is addressed. This implementation will simplify the detection side considerably.     

Coherent transfer function of Fourier transform spectral interferometric coherent anti-Stokes Raman scattering microscopy

We analyze the optical resolution of Fourier transform spectral interferometric-coherent anti-Stokes Raman scattering microscopy, which extracts the complex amplitude of an image by using a spectral interferometric effect. Image-formation formulas are presented that describe the properties of the image observed by the apparatus. The image-formation properties represented by the coherent transfer function are different depending on the mode (transmission, reflection, etc.) of the microscopy.     

In situ visualization of paclitaxel distribution and release by coherent anti-Stokes Raman scattering microscopy

Visualization of three-dimensional distribution of drug molecules and subsequent changes during the release process is critical for understanding drug delivery mechanisms as well as designing tailor-made release profiles. This study utilized coherent anti-Stokes Raman scattering (CARS) imaging to examine paclitaxel distribution in various polymer films with lateral resolution of 0.3 microm and depth resolution of 0.9 microm. Raman bands in the CH stretch vibration and fingerprint regions were used to distinguish paclitaxel from the polymers. The detection sensitivity was measured to be 29 mM by imaging paclitaxel molecules dissolved in N,N-dimethylformamide solution. Release of paclitaxel from a polymer matrix was monitored at an acquisition speed of 1 frame/s. Our results show that CARS microscopy can be used effectively for in situ imaging of native drug molecules in a delivery system.     

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.     

Multiplex H2 coherent anti-Stokes Raman scattering thermometry with a modeless laser

The use of a modeless laser as the Stokes source for multiplex coherent anti-Stokes Raman scattering in molecular hydrogen is reported. The elimination of noise associated with mode competition in conventional standing wave lasers is shown to result in reliable and accurate single-shot thermometry of H(2) in a microwave-assisted diamond chemical vapor deposition plasma reactor. Single-shot temperatures are recorded with a precision of 7.3%. Possible improvements to this precision are discussed and applications of the technique for on-line process monitoring are briefly presented.     

Gas-phase thermometry using delayed-probe-pulse picosecond coherent anti-Stokes Raman scattering spectra of H2

We report the development and application of a simple theoretical model for extracting temperatures from picosecond-laser-based coherent anti-Stokes Raman scattering (CARS) spectra of H2 obtained using time-delayed probe pulses. This approach addresses the challenges associated with the effects of rotational-level-dependent decay lifetimes on time-delayed probing for CARS thermometry. A simple procedure is presented for accurate temperature determination based on a Boltzmann distribution using delayed-probe-pulse vibrational CARS spectra of H2; this procedure requires measurement at only a select handful of probe-pulse delays and requires no assumptions about sample environment.     

Immuno-surface-enhanced coherent anti-stokes Raman scattering microscopy: immunohistochemistry with target-specific metallic nanoprobes and nonlinear Raman microscopy

Immunohistochemistry (IHC) is one of the most widely used staining techniques for diagnostic purposes. The selective localization of target proteins in tissue specimens by conventional IHC is achieved with dye- or enzyme-labeled antibodies in combination with light microscopy. In this contribution, we demonstrate the proof-of-principle for IHC based on surface-enhanced coherent Raman scattering for contrast generation. Specifically, antibody-labeled metallic nanoshells in conjunction with surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy are employed for the selective, sensitive, and rapid localization of the basal cell protein p63 in normal prostate tissue. Negative control experiments were performed in order to confirm the selective binding of the target-specific metal nanoprobes and to disentangle the role of plasmonic (metal) and molecular (Raman reporter) resonances in this plasmon-assisted four-wave mixing technique.     

Dual-pump coherent anti-Stokes Raman scattering measurements of nitrogen and oxygen in a laminar jet diffusion flame

Dual-pump coherent anti-Stokes Raman scattering (CARS) has been demonstrated for the simultaneous measurement of gas-phase temperature and concentrations of molecular nitrogen and oxygen. A polarization technique was used to vary the relative intensities of the two CARS signals and expand the dynamic range of the relative concentration measurements. Detailed temperature and oxygen mole fraction measurements were performed in the stabilization region of a hydrogen-nitrogen jet diffusion flame. These results indicate that there is a region below the nozzle exit where significant amounts of oxygen are found on the fuel side of the peak flame temperature profile.     

Simultaneous coherent anti-Stokes Raman scattering and two-dimensional laser Rayleigh thermometry in a contained technical swirl combustor

The simultaneous application of vibrational coherent anti-Stokes Raman scattering (CARS) and the two-dimensional (2D) UV laser Rayleigh technique is reported for the investigation of a highly turbulent swirl frame inside a contained technical combustor. The CARS technique has been used to determine accurate temperature values at one point within the 2D Rayleigh-probed combustion field. These values were necessary to normalize the Rayleigh data to overcome influences of absorption effects along the detection path of the Rayleigh-scattered light through the exhaust gas volume and by the sealing window of the combustion chamber. At several different downstream positions, 500 simultaneous measurements with the point and with the 2D technique were performed to cover the whole combustion field. These data can be used for both the evaluation of 2D temperature structures in single frames and for the calculation of temperature probability density functions from the Rayleigh data at one single camera pixel over 500 frames. With this information, characterization of a highly turbulent flame is possible.     

Experimental comparison of single-shot broadband vibrational and dual-broadband pure rotational coherent anti-Stokes Raman scattering in hot air

Broadband vibrational and dual-broadband pure rotational coherent anti-Stokes Raman scattering (CARS) have been compared in a high-temperature oven, in which the accuracy and single-shot precision of gas temperature and relative O(2)- and N(2)-concentration measurements in hot air were probed over a temperature range that is typical for many combustion processes. To ensure a realistic comparison, we used nearly the same experimental setup for both CARS techniques. Besides temperature information, dual-broadband pure rotational CARS offers the possibility of achieving simultaneous single-shot concentration measurements. The comparison shows that this technique also has significant advantages in temperature evaluation over a large temperature range in comparison with vibrational CARS.     

Broadband coherent anti-Stokes Raman spectroscopy characterization of polymer thin films

Broadband coherent anti-Stokes Raman spectroscopy (CARS) is demonstrated as an effective probe of polymer thin film materials. A simple modification to a 1 kHz broad bandwidth sum frequency generation (SFG) spectrometer permits acquisition of CARS spectra for polymer thin films less than 100 nm thick, a dimension relevant to organic electronic device applications. CARS spectra are compared to the conventional Raman spectra of polystyrene and the resonance-enhanced Raman spectra of poly(3-hexylthiophene). The CARS spectra obtained under these conditions consistently demonstrate enhanced signal-to-noise ratio compared to the spontaneous Raman scattering. The sensitivity of the CARS measurement is limited by the damage threshold of the samples. The dielectic properties of the substrate have a dramatic effect on the detected signal intensity. For ultrathin films, the strongest signals are obtained from fused silica surfaces. Similar to surface-enhanced Raman scattering (SERS), Au also gives a large signal, but contrary to SERS, no surface roughening is necessary.     

Estimation of spatial averaging of temperatures from coherent anti-Stokes Raman spectroscopy

Spatial averaging is a potential problem in the application of coherent anti-Stokes Raman spectroscopy (CARS) to combustion diagnostics when varying temperatures and composition are present at spatial scales smaller than the typical 1-2-mm spatial resolution of CARS. The observed CARS spectrum is then a mixture of the hot and cold components. We show that simulated, spatially averaged spectra, generated by the incoherent addition of intensities, can be significantly different from those obtained by the coherent addition of the electric field amplitudes of the component spectra. The analyses of these simulated, spatially averaged CARS spectra demonstrate that the use of theoretical CARS spectra, generated by the addition of intensities of the hot and cold components of a binary gas mixture, can lead to errors in the estimated flame temperatures.     

Development of multipoint vibrational coherent anti-Stokes Raman spectroscopy for flame applications

A novel technique for coherent anti-Stokes Raman spectroscopy (CARS) measurements in multiple points is presented. In a multipass cavity the pump and Stokes laser beams are multiply reflected and refocused into a measurement volume with an adjustable number of separated points along a line. This optical arrangement was used in a vibrational CARS setup with planar BOXCARS phase-matching configuration. The CARS spectra from spatially separated points were recorded at different heights on a CCD camera. Measurements of temperature profiles were carried out in the burned gas zone of a premixed one-dimensional flame to demonstrate the applicability of this method for temperature measurements in high-temperature regions. The ability to measure in flames with strong density gradients was demonstrated by simultaneous measurements of Q-branch spectra of N2 and CO in a Wolfhard-Parker burner flame. Interference phenomena found in multipoint spectra are discussed, and possible solutions are proposed. Merits and limitations of the technique are discussed.     

Pure rotational coherent anti-Stokes Raman spectroscopy in mixtures of CO and N2

We present a model for quantitative measurements in binary mixtures of nitrogen and carbon monoxide by the use of dual-broadband rotational coherent anti-Stokes Raman spectroscopy. The model has been compared with experimental rotational coherent anti-Stokes Raman scattering spectra recorded within the temperature range of 294-702 K. Temperatures and concentrations were evaluated by spectral fits using libraries of theoretically calculated spectra. The relative error of the temperature measurements was 1-2%, and the absolute error of the CO concentration measurements was <0.5% for temperatures < or =600 K. For higher temperatures, the gas composition was not chemically stable, and we observed a conversion of CO to CO2. The influence of important spectroscopic parameters such as the anisotropic polarizability and Raman line-broadening coefficients are discussed in terms of concentration measurements. In particular, it is shown that the CO concentration measurement was more accurate if N2-CO and CO-N2 line-broadening coefficients were included in the calculation. The applicability of the model for quantitative flame measurements is demonstrated by measuring CO concentrations in ethylene/air flames.