Heterodyne interferometric polarization coherent anti‐Stokes Raman scattering (HIP‐CARS) spectroscopy

We demonstrate a new technique that combines polarization sensitivity of the coherent anti‐Stokes Raman scattering (CARS) response with heterodyne amplification for background‐free detection of CARS signals. In this heterodyne interferometric polarization CARS (HIP‐CARS), the major drawbacks of polarization and heterodyne CARS are rectified. Using a home‐built picosecond optical parametric oscillator, we are able to address vibrational stretches between 600 and 1650 cm⁻¹ and record continuous high‐resolution Raman equivalent HIP‐CARS spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical imaging of glucose by CARS microscopy

Glucose is one of the most fundamental molecules within life and bioengineering sciences. Present understanding of its role in cellular and bioengineering processes relies primarily on invasive, large‐scale biochemical analysis, providing no spatial information on glucose pools or fluxes. This work identifies an emerging microscopy technique based on coherent anti‐Stokes Raman scattering (CARS), which fulfills the need of quantitative imaging of glucose at the single‐cell level with submicrometer resolution. No sample preparation with reporter molecules is required, ensuring that the low‐weight metabolite is studied under natural conditions. The potential of CARS microscopy is illustrated by quantitatively mapping glucose fluxes and distributions in a microfluidic bioreactor and in lipid‐bilayer vesicles, the latter as a model for glucose transmembrane transport. Furthermore, the metabolic response to a glucose pulse was monitored in living yeast cells. This study signifies a new era within CARS microscopy for its use of monitoring carbohydrates, in particular glucose which is one of the most abundant molecules in nature. Copyright © 2010 John Wiley & Sons, Ltd.     

Herman–Wallis correction in vibrational CARS of oxygen

Light molecules are subject to vibration–rotation (VR) interaction, which implies corrections to the rigid rotor approximation and, in particular, corrections to spectral line intensities are related to the so‐called Herman–Wallis (HW) factor. This problem is outlined here for the spectral response of some medium‐weight diatomics in the gas phase and probed by means of vibrational coherent anti‐Stokes Raman scattering (CARS) used for diagnostic reasons in combustion science. However, different from other works on this subject, we specialized our analysis to oxygen and, since the peculiarity of its anti‐bonding molecular orbital, we find that the VR coupling is responsible for deviations that compete with the effect of Raman line widths typical of collisional environments of hot gases at room pressure. The HW correction is ultimately demonstrated to affect O₂ CARS thermometry in such a manner that the accuracy for measurements at high temperatures can be improved. Copyright © 2011 John Wiley & Sons, Ltd.     

CARS thermometry revisited in lightof the intramolecular perturbation

The rigid rotor approximation (RRA) is commonly assumed in the Raman cross section used in thermometric analysis based on coherent anti‐Stokes Raman scattering (CARS). In this paper, we discuss instead the role of the coupling between molecular vibrations and rotations in view of the alterations found in the amplitude of CARS signals of basic molecules and, in the end, we demonstrate that the deviation of a few percent from the RRA results in corrections to the measured temperature that are comparable to the thermometric accuracy of very well‐known Q‐branch CARS measurements on nitrogen, which is unanimously regarded as the fundamental molecule in CARS thermometry. Copyright © 2010 John Wiley & Sons, Ltd.     

Time‐resolved investigation of the ν1 ro‐vibrational Raman band of H2CO with fs‐CARS

The technique of femtosecond time‐resolved coherent anti‐Stokes scattering (fs‐CARS) is used to investigate the strongly perturbed ν₁ ro‐vibrational Raman band of formaldehyde (H₂CO). The time‐dependent signal is simulated using a ‘Watson‐’Hamiltonian in A‐type reduction and Raman theory for asymmetric rotors. The results are compared with the experimental data. The fs‐CARS method measures the evolution of the polarization in a molecular ensemble via superposition of many states and is sensitive to spectral irregularities or line shifts of the involved transitions. ‘Coriolis’ interactions play a major role in the analysis of the ν₁ band of formaldehyde. We successfully simulate the fs‐CARS transient signal from the ν₁ band of formaldehyde including a model for multiple ‘Coriolis’ interactions, without the necessity of describing the complete interaction between all the vibrational levels. ‘Coriolis’ coupling coefficients and energy shifts are derived from the experiment by a least‐square fit. The results are discussed and compared to literature values. Copyright © 2006 John Wiley & Sons, Ltd.     

Quantitative,comparable coherent anti‐Stokes Raman scattering (CARS) spectroscopy: correcting errors in phase retrieval

Coherent anti‐Stokes Raman scattering (CARS) microspectroscopy has demonstrated significant potential for biological and materials imaging. To date, however, the primary mechanism of disseminating CARS spectroscopic information is through pseudocolor imagery, which explicitly neglects a vast majority of the hyperspectral data. Furthermore, current paradigms in CARS spectral processing do not lend themselves to quantitative sample‐to‐sample comparability. The primary limitation stems from the need to accurately measure the so‐called nonresonant background (NRB) that is used to extract the chemically sensitive Raman information from the raw spectra. Measurement of the NRB on a pixel‐by‐pixel basis is a nontrivial task; thus, surrogate NRB from glass or water is typically utilized, resulting in error between the actual and estimated amplitude and phase. In this paper, we present a new methodology for extracting the Raman spectral features that significantly suppresses these errors through phase detrending and scaling. Classic methods of error correction, such as baseline detrending, are demonstrated to be inaccurate and to simply mask the underlying errors. The theoretical justification is presented by re‐developing the theory of phase retrieval via the Kramers–Kronig relation, and we demonstrate that these results are also applicable to maximum entropy method‐based phase retrieval. This new error‐correction approach is experimentally applied to glycerol spectra and tissue images, demonstrating marked consistency between spectra obtained using different NRB estimates and between spectra obtained on different instruments. Additionally, in order to facilitate implementation of these approaches, we have made many of the tools described herein available free for download. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.     

A quantitative approach to evaluate the problem of coherence of spectral components of the third‐order susceptibility generating coherent anti‐Stokes Raman signals

Precise interpretation of spectral measurements is central to the development of the full extent of the applicative potential of coherent anti‐Stokes Raman spectroscopy (CARS). One recognized problem that jeopardizes the achievement of high precision is the determination of the best spectral convolution over the relevant bandwidths when degeneracy of laser frequencies is involved. Although the analytical solutions of CARS signals generated by pump and Stokes lasers with standard (i.e. Gaussian or Lorentzian) lineshapes are well known, research in this field has overlooked the criterion on how to discern coherence between spectral components of the third‐order nonlinear susceptibility. Understandably, the ordinary approach is based on an intuitive comparison between the spectral width σ₁ of the pump laser with respect to the width Γ of the relevant Raman transitions. More precisely, if σ₁ ≪ Γ, then the spectral synthesis can be obtained in the limit of narrowband pump; otherwise spectral coherence has to be included in the calculation leading to problematic spectral analysis. In an attempt to clarify this qualitative criterion better, the present work demonstrates that the limit between the two opposite regimes can have a clearer and neater definition than that accepted so far. In this case, this paper shows that for nonoverlapping Raman transitions determined by a Lorentzian susceptibility, the issue is governed by the analytic function erfc(Γ/σ₁)/σ₁, which depends uniquely on the ratio Γ/σ₁. The unitary limit of this function for σ₁ ≪ Γ justifies the incoherent or the narrowband‐pump approach. Copyright © 2006 John Wiley & Sons, Ltd.     

Measurement of the third‐order nonlinear optical susceptibility χ(3) for the 1002‐cm–1 mode of benzenethiol using coherent anti‐Stokes Raman scattering with continuous‐wave diode lasers

The components of the third‐order nonlinear optical susceptibility χ⁽³⁾ for the 1002‐cm^–1 mode of neat benzenethiol have been measured using coherent anti‐Stokes Raman scattering with continuous‐wave diode pump and Stokes lasers at 785.0 and 852.0 nm, respectively. Values of 2.8 ± 0.3 × 10^–12, 2.0 ± 0.2 × 10^–12, and 0.8 ± 0.1 × 10^–12 cm·g^–1·s² were measured for the xxxx, xxyy, and xyyx components of |3χ⁽³⁾|, respectively. We have calculated these quantities using a microscopic model, reproducing the same qualitative trend. The Raman cross‐section σ_RS for the 1002‐cm^–1 mode of neat benzenethiol has been determined to be 3.1 ± 0.6 × 10^–29 cm² per molecule. The polarization of the anti‐Stokes Raman scattering was found to be parallel to that of the pump laser, which implies negligible depolarization. The Raman linewidth (full‐width at half‐maximum) Γ was determined to be 2.4 ± 0.3 cm^–1 using normal Stokes Raman scattering. The measured values of σ_RS and Γ yield a value of 2.1 ± 0.4 × 10^–12 cm·g^–1·s² for the resonant component of 3χ⁽³⁾. A value of 1.9 ± 0.9 × 10^–12 cm·g^–1·s² has been deduced for the nonresonant component of 3χ⁽³⁾. Copyright © 2011 John Wiley & Sons, Ltd.     

Coherent molecular vibrational dynamics of CH2 stretching modes in polyethylene studied by femtosecond‐CARS

We have studied the coherent molecular vibrational dynamics of CH₂ stretching modes in polyethylene by time‐resolved femtosecond coherent anti‐Stokes Raman spectroscopy. We observed that the coherent vibrational relaxation of symmetric CH₂ stretching modes in polyethylene at room temperature is much faster than that previously measured in polyvinyl alcohol. In addition, it was detected that, at low temperature, the coherent vibrational relaxation of the symmetric stretching modes evidently becomes slower compared with that at room temperature. These temperature‐dependent measurements enable us to discriminate the contribution of pure dephasing mechanism, due to phonons and two‐level systems in polymer, from the contribution of lifetime of the vibrational excited state to the coherent vibrational relaxation of CH₂ stretching modes. We conclude that the coherent vibrational relaxation of symmetric CH₂ stretching modes at room temperature consists of the contribution of lifetime and approximately 1.5 times larger contribution of pure dephasing. Copyright © 2015 John Wiley & Sons, Ltd.     

Visualization of β‐carotene and starch granules in plant cells using CARS and SHG microscopy

Information on the content and bioavailability of provitamin A carotenoids, such as β‐carotene, in plant foods is of great interest due to the widespread vitamin A deficiency in developing countries. While the amount of β‐carotene can readily be quantified with analytical techniques, there is limited information on β‐carotene morphology in native plant materials. Here, we introduce nonlinear microscopy for three‐dimensional, label‐free imaging of carotenoids in fresh and thermally treated plant tissues, providing quantitative information at single‐aggregate level and detailed insight into their distribution. Carotenoids in orange‐fleshed sweet potato (OFSP), carrot, and mango were visualized by coherent anti‐Stokes Raman scattering (CARS) microscopy and, in the case of OFSP, related to the plant‐matrix morphology by simultaneous second‐harmonic generation (SHG) microscopy of starch granules. Sizes, shapes, densities, and location of different types of carotenoid bodies were quantified. While OFSP and carrot showed heterogeneous rod‐shaped bodies with high carotenoid densities indicated by higher CARS signals, the carotenoid‐filled lipid droplets in mango appeared as homogeneous low‐density aggregates of rounded shape. In addition, β‐carotene densities and morphologies in OFSP were studied after thermal processing, showing that the bodies remain intact despite significant changes of the surrounding starch granules. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy

Broadband multiplex coherent anti‐Stokes Raman scattering (MCARS) microscopy allows the rapid chemical mapping and molecular imaging of untreated material samples with three‐dimensional sectioning capabilities. It can be realized with a single laser in a simple and robust setup using supercontinuum generation in a microstructured fiber. The successful implementation of a MCARS microscope is discussed in detail, its parameters are characterized, and applications are shown for the identification and mapping of polymer blends. An evolutionary fitting routine is presented, which allows a fully quantitative analysis of the MCARS information resulting in high‐contrast chemical maps. The established setup enables reliable day‐to‐day operation as a valuable tool for rapid material characterization. Copyright © 2007 John Wiley & Sons, Ltd.     

On the sensitivity of rotational CARS N2 thermometry to the Herman–Wallis factor

Purely rotational spectral signals of coherent anti‐Stokes Raman scattering (CARS) from nitrogen molecules are studied as a function of the vibration–rotation interaction that weakens the rigid rotor approximation under which the dominant terms of the Raman cross section are calculated. The effect of the vibration–rotation interaction is quantified by means of the Herman–Wallis (HW) factor, and different approaches to its determination are evaluated in terms of their relative contribution to the CARS intensity and thermometric measurements made in a fuel‐rich hydrocarbon flame. Known HW factors are contrasted with more complete expressions of recent derivation, and it is found that relative line strength adjustments amount to about a few percent. Such differences result in temperature corrections of less than 1%. This value should be considered for the definition of the ideal thermometric accuracy of the technique but it is of minor importance in comparison with other sources of uncertainty (e.g. Raman line widths) that emerge from the complexity typical of reactive gas mixtures. Copyright © 2011 John Wiley & Sons, Ltd.     

Quadruplex CARS micro‐spectroscopy

We demonstrate a technique for simultaneous detection of coherent anti‐Stokes Raman scattering (CARS) at four vibrational frequencies, using simple passive optical elements and without spectrally resolved detection. The technique is based on pump and Stokes femtosecond pulses selectively exciting vibrational resonances through spectral focusing. By replicating the pump and Stokes pair into four pairs, each traveling through appropriate glass elements, we simultaneously excite four different vibrational frequencies. The resulting CARS is a periodic train of intensities detected by a single photomultiplier and frequency analyzed to retrieve its Fourier coefficients. We demonstrate detection of methanol and ethanol mixtures in water and quantitative determination of their concentration owing to the improved chemical selectivity of this quadruplex CARS scheme. Copyright © 2012 John Wiley & Sons, Ltd.     

Measurement and calculation of the Q‐branch spectrum of nitrogen using inverse Raman spectroscopy and cw Raman‐induced polarization spectroscopy

The techniques of inverse Raman spectroscopy, Raman‐induced polarization spectroscopy (RIPS), and optical heterodyne RIPS (OHD‐RIPS) are compared by probing the Q‐branch of the nitrogen molecule. The signal is measured employing either a photomultiplier tube (low background level–RIPS) or a photodetector (high background level–IRS and OHD‐RIPS). The measurements are performed using atmospheric mixtures of N₂ Ar with concentrations varying from 0 to 79% N₂. This strategy permits estimation of detection limits using the different techniques. Pump and probe energy levels are varied independently to study signal dependence on laser irradiance. A theoretical treatment is presented on the basis of the Raman susceptibility equations, which permits the calculation of spectra for all three techniques. Calculated Q‐branch spectra are compared with the measured spectra for the interactions of a linearly polarized probe beam with a linearly or circularly polarized pump beam. The polarizer angle in the detection path for OHD‐RIPS has a dramatic effect on the shape of the spectrum. The calculated and experimental OHD‐RIPS spectra are in good agreement over the entire range of investigated polarizer angles. Detection limits using these techniques are analyzed to suggest their applicability for measuring other species of importance in combustion and plasma systems. Copyright © 2007 John Wiley & Sons, Ltd.     

Analyzing ultrafast multiplex coherent anti‐Stokes Raman spectra with picosecond probing

This article reports an efficient method to simulate time and frequency resolved coherent anti‐Stokes Raman scattering spectra measured with picosecond pump and probe fields and ultrashort Stokes pulses. A systematic comparison of measured and simulated time and frequency dependent data is presented for carbon tetrachloride, chloroform, cyclohexane, octane, and poly(methyl methacrylate). While the first compound exhibits no Raman active modes in the considered spectral region of the CH‐stretch vibrations, the other ones show Raman spectra of increasing complexity. Vibrational frequencies and homogeneous dephasing rates are extracted by fitting explicit analytical formulas to the recorded data. Interference between nonresonant and resonant contributions to the nonlinear polarization is taken fully into account. The ability to measure the influence of inhomogeneous broadening is discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Fiber‐based light sources for biomedical applications of coherent anti‐Stokes Raman scattering microscopy

During the past decade coherent anti‐Stokes Raman scattering (CARS) microscopy has evolved to one of the most powerful imaging techniques in the biomedical sciences, enabling the label‐free visualization of the chemical composition of tissue in vivo in real time. While the acquisition of high‐contrast images of single cells up to large tissue sections enables a wide range of medical applications from routine diagnostics to surgical guidance, to date CARS imaging is employed in fundamental research only, essentially because the synchronized multiple wavelength pulsed laser sources required for CARS microscopy are large, expensive and require regular maintenance. Laser sources based on optical fibers can overcome these limitations combining highest efficiency and peak powers with an excellent spatial beam profile and thermal stability. In this review we summarize the different fiber‐based approaches for laser sources dedicated to coherent Raman imaging, in particular active fiber technology and passive fiber‐based frequency conversion processes, i.e. supercontinuum generation, soliton self‐frequency shift and four‐wave mixing. We re‐evaluate the ideal laser parameters for CARS imaging and discuss the suitability of different laser concepts for turn‐key operation required for routine application in clinics.

     

General criterion to discern between coherent and incoherent synthesis of broadband coherent anti‐Stokes Raman spectra

Recently, the ordinary qualitative criterion on how to distinguish between coherent and incoherent convolutions of broadband coherent anti‐Stokes Raman (CARS) signals generated by degenerate pump lasers has been revised in view of a quantitative analysis. The revision has established that incoherent CARS approach can be justified as unitary limit of the function ] erfc(Γ/σ₁)/σ₁, where Γ and σ₁ are respectively the spectral widths of the Raman line and the degenerate pump lasers. The result was, however, limited to nonoverlapping Raman lines. In this work, the extension to a more common situation of closely spaced Raman transitions is considered. For large overlap between adjacent Raman lines, the new analysis suggests significant deviations from the previous result. Weak line mixing is also taken into consideration. Nonetheless, all types of deviations are characterized by a common tendency toward the incoherent limit. Copyright © 2007 John Wiley & Sons, Ltd.     

Polarization‐sensitive CARS spectroscopy on free‐base porphyrins: coproporphyrin I tetramethyl ester

The application of polarization‐sensitive (PS) coherent anti‐Stokes Raman scattering (CARS) spectroscopy for the investigation of highly luminescent free‐base porphyrins under Q_x band resonance is discussed. For coproporphyrin I tetramethyl ester (CP‐I‐TME), PS CARS spectra involving resonances with the electronic Q_x absorption band as well as polarized spontaneous Raman spectra involving B band resonance are presented. A quantitative evaluation of the CP‐I‐TME spectra is performed and the results are compared to our previously presented data on free‐base octaethylporphine (OEP) and mesoporphyrin IX dimethyl ester (MP‐IX‐DME), which were obtained under identical excitation conditions. This comprehensive analysis reveals several spectral differences that can be attributed to the different β–substitution pattern of the porphyrin macrocycle. Additionally, the strong resonance enhancement of totally symmetric modes under Q_x band excitation is identified as a common property for OEP, CP‐I‐TME, and MP‐IX‐DME; this enhancement selectivity distinguishes the investigated substances from what is generally observed for metallo porphyrins. Copyright © 2008 John Wiley & Sons, Ltd.     

Raman spectroscopy and microscopy of individual cells and cellular components

Raman spectroscopy provides the unique opportunity to nondestructively analyze chemical concentrations in individual cells on the submicrometer length scale without the need for optical labels. This enables the rapid assessment of cellular biochemistry inside living cells, and it allows for their continued analysis. Here, we review recent developments in the analysis of single cells, subcellular compartments, and chemical imaging based on Raman spectroscopy. Spontaneous Raman spectroscopy provides for the full spectral assessment of cellular biochemistry, while coherent Raman techniques, such as coherent anti‐Stokes Raman scattering is primarily used as an imaging tool comparable to confocal fluorescence microscopy. These techniques are complemented by surface‐enhanced Raman spectroscopy, which provides higher sensitivity and local specificity, and also extends the techniques to chemical indicators, i.e. pH sensing. We review the strengths and weaknesses of each technique, demonstrate some of their applications and discuss their potential for future research in cell biology and biomedicine.