Coherent anti-Stokes Raman Scattering Signal Research Articles

Visualization of water concentration distribution in human skin by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) microscopy

Ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging was used to visualize the distribution of water concentration in human skin ex vivo. The CARS signal of the OH stretching vibrational mode of water was found to coexist with the signal of intercellular lipids such as ceramides, which were visualized by a sharp vibrational band at 2882 cm−1. Depth-resolved CARS spectroscopic imaging of a skin sample revealed that ceramides were localized in the stratum corneum. These findings demonstrate the powerful potential of CARS spectroscopic imaging for probing pathological changes caused by anomalous water concentration distribution in human skin.

Numerical simulation study of surface enhancement coherent anti-Stokes Raman scattering reinforced substrate

Plasma nanostructures are of particular significance for serving as a substrate for spectroscopic detection and identification of individual molecules. By combining the excitation wavelength of the molecule with the resonance wavelength of the nanostructure, the sensitive single-molecule Raman detection can be achieved. A high and stable plasma substrate for coherent anti-Stokes Raman scattering(CARS) is very useful for developing the surface-enhanced coherent anti-Stokes Raman scattering (SECARS). In the plasma nanostructures, the strong coupling of plasmonic nanoparticles with an inter-particle gap smaller than the diameter of the individual nanoparticles results in the hybridization of the optical properties of these individual nanoparticles. There are also the charge transfer plasmons(CTP) appearing in conductive bridging nanoparticles. Their unique properties make linked nanosystems a suitable candidate for building artificial molecules, nanomotors, sensors, and other optoelectronic devices. In this work, we, starting from reality, theoretically design a new linked nanosystem SECARS substrate where Fano resonance can be generated by the plasmon hybridization (PH) model resonance and the charge transfer plasmon resonance. The introduction of charge transfer plasma improves the tunability of structural resonance. By adjusting the conductivity of the conductive junction, the wavelength of the charge transfer plasma resonance can be easily adjusted to change the wavelength position of the Fano resonance. The data obtained by numerical simulation of the Raman mode at 1557 cm<sup>–1</sup> of L-tryptophan when a 1064 nm light source is used as the pump light show that this spatially symmetrical structure can generate multiple high-enhancement hot spots that do not depend on the polarization direction of the incident light. Ordinary CARS signal can generally be enhanced by 10<sup>12</sup>, and its maximum can reach 10<sup>14</sup>. Due to the ultrastrong field enhancement and insensitive-to-polarization, this method of using charge transfer plasma to design a substrate can be used in the practical substrate of SECARS and provide new ideas for designing other nonlinear optical processes such as four wave mixing and stimulated Raman scattering.

Three-beam rotational coherent anti-Stokes Raman spectroscopy thermometry in scattering environments.

Three-beam rotational coherent anti-Stokes Raman scattering (CARS) measurements performed in highly scattering environments are susceptible to contamination by two-beam CARS signals generated by the pump-probe and Stokes-probe interactions at the measurement volume. If this occurs, differences in the Raman excitation bandwidth between the two-beam and three-beam CARS signals can add significant errors to the spectral analysis. This interference to the best of our knowledge has not been acknowledged in previous three-beam rotational CARS experiments, but may introduce measurement errors up to 25% depending on the temperature, amount of scattering, and differences between the two-beam and three-beam Raman excitation bandwidths. In this work, the presence of two-beam CARS signal contamination was experimentally verified using a femtosecond-picosecond rotational CARS instrument in two scattering environments: (1) a fireball generated by a laboratory-scale explosion that contained particulate matter, metal fragments, and soot, and (2) a flow of air and small liquid droplets. A polarization scheme is presented to overcome this interference. By rotating the pump and Stokes polarizations +55∘ and -55∘ from the probe, respectively, the two-beam and three-beam CARS signals are orthogonally polarized and can be separated using a polarization analyzer. Using this polarization arrangement, the Raman-resonant three-beam CARS signal amplitude is reduced by a factor of 2.3 compared to the case where all polarizations are parallel. This method is successfully demonstrated in both scattering environments. A theoretical model is presented, and the temperature measurement error is studied for different experimental conditions. The criteria for when this interference may be present are discussed.

Post-detonation fireball thermometry via femtosecond-picosecond coherent anti-Stokes Raman Scattering (CARS)

Accurate knowledge of post-detonation fireball temperatures is important for understanding device performance and for validation of numerical models. Such measurements are difficult to make even under controlled laboratory conditions. In this work temperature measurements were performed in the fireball of a commercial detonator (RP-80, Teledyne RISI). The explosion and fragments were contained in a plastic enclosure with glass windows for optical access. A hybrid femtosecond-picosecond (fs-ps) rotational coherent anti-Stokes Raman scattering (CARS) instrument was used to perform gas-phase thermometry along a one-dimensional measurement volume in a single laser shot. The 13-mm-thick windows on the explosive-containment housing introduced significant nonlinear chirp on the fs lasers pulses, which reduced the Raman excitation bandwidth and did not allow for efficient excitation of high-J Raman transitions populated at flame temperatures. To overcome this, distinct pump and Stokes pulses were used in conjunction with spectral focusing, achieved by varying the relative timing between the pump and Stokes pulses to preferentially excite Raman transitions relevant to flame thermometry. Light scattering from particulate matter and solid fragments was a significant challenge and was mitigated using a new polarization scheme to isolate the CARS signal. Fireball temperatures were measured 35–40 mm above the detonator, 12–25 mm radially outward from the detonator centerline, and at 18 and 28 µs after initiation. At these locations and times, significant mixing between the detonation products and ambient air had occurred thus increasing the nitrogen-based CARS thermometry signal. Initial measurements show a distribution of fireball temperatures in the range 300–2000 K with higher temperatures occurring 28 µs after detonation.

Spatial frequency modulated imaging in coherent anti-Stokes Raman microscopy.

For sparse samples or in the presence of ambient light, the signal-to-noise ratio (SNR) performance of single-point-scanning coherent anti-Stokes Raman scattering (CARS) images is not optimized. As an improvement, we propose replacing the conventional CARS focus-point illumination with a periodically structured focus line while continuing to collect the transmitted CARS intensity on a single detector. The object information along the illuminated line is obtained by numerically processing the CARS signal recorded for various periods of the structured focus line. We demonstrate experimentally the feasibility of this spatial frequency modulated imaging (SPIFI) in CARS (SPIFI-CARS) and SHG (SPIFI-SHG) and identify situations where its SNR is better than that of the single-point-scanning approach.

An Efficient Switching-Off of Coherent Anti-Stokes Raman Scattering via Double Stimulated Raman Scattering Processes of Heteromolecular Vibrational Modes.

Recently, we introduced a new switching-off technique applicable to subdiffraction-limited coherent Raman imaging, where a coherent anti-Stokes Raman scattering (CARS) signal can be selectively suppressed via competitive stimulated Raman scattering (SRS) processes between vibrational modes of a single molecular species. Here, we show that such a three-beam CARS suppression can be made via double SRS processes between vibrational modes of heteromolecular species, a mixture of paraffin oil and benzene. We achieve more than 80% suppression of the pump-Stokes-beam CARS signal for the ring-breathing mode (target mode) of benzene when the C-H stretching mode (acceptor mode) of paraffin oil is used to deplete the pump photons via the pump-depletion-beam SRS process. The freedom in the choice of acceptor mode for depletion, which could be a critical advantage of the present switching-off scheme, can be of use for overcoming current challenges of depletion-based super-resolution coherent Raman imaging of biomolecules.

Theory of birefringence correction for polarization-controlled CARS.

Polarization-controlled coherent Raman spectroscopy is used as a high-throughput method to characterize the anisotropic nature of a molecular system, such as the molecular orientation distribution. However, optical birefringence originating from the molecular anisotropy can cause the observed Raman spectrum to be significantly distorted, making it extremely challenging to obtain quantitative information from polarization Raman measurements. Here, the birefringence effect on the signal intensity and the spectral shape of a polarization-controlled coherent anti-Stokes Raman scattering (CARS) is theoretically described using a uniaxially symmetrical model system. Due to the complexity, the effect of phase delay in the incident lights is not considered but only that of the generated CARS signal is considered. A new analytical method is presented to eliminate the birefringence contribution from polarization-controlled CARS data by analyzing polarization intensity profiles and retrieving the resonant Raman susceptibility spectra. This method is tested with two sets of polarization-controlled CARS data simulated with various combinations of symmetries of multiple underlying Raman modes. The analysis result clearly demonstrates that the effect of birefringence can be corrected for polarization-controlled CARS data and the symmetry tensor elements of all underlying Raman modes can be quantitatively characterized.

Impact of moderate pump–Stokes chirp on femtosecond coherent anti‐Stokes Raman scattering spectra

AbstractThe effects of inducing moderate chirp in the pump and Stokes beams in chirped‐probe‐pulse femtosecond coherent anti‐Stokes Raman scattering (CARS) spectroscopy are discussed. This is an important issue for measurements in high‐pressure systems where the CARS beams must be transmitted through glass of significant thickness to reach the probe volume of interest. The effects were investigated both experimentally, by inserting disks of SF‐11 glass into the pump and Stokes beam paths, and theoretically, by incorporating pulse chirp into our time‐dependent density matrix (TDDM) simulations. Experimentally, we found that inducing moderate pump and Stokes chirp produced significant spectral narrowing of the nonresonant four‐wave mixing contribution to the CARS signal, and this allowed us to control the resonant excitation bandwidth to eliminate interferences from neighboring species. The temperature measurement accuracy and precision were essentially unchanged with respect to the unchirped pump and Stokes case. The effect of moderate pump and Stokes chirp on the narrowing of the Raman excitation efficiency envelope was investigated by solving the full set of TDDM through direct numerical integration. The numerical efficiency of the solutions was enhanced dramatically by developing a parallel version of the TDDM code. The calculated Raman excitation efficiency profile was incorporated in our phenomenological spectral fitting codes.

Vibrational spectroscopy and imaging with non-resonant coherent anti-Stokes Raman scattering: double stimulated Raman scattering scheme.

We introduce a new coherent anti-Stokes Raman scattering (CARS) suppression scheme based on measuring a non-resonant CARS loss signal by three-beam (pump-Stokes-depletion) double stimulated Raman scattering (SRS) processes, which can be potentially of use for super-resolution Raman microscopy. In the converging configuration with employing both pump-depletion and Stokes-depletion SRS processes, we obtained approximately 94% suppression of non-resonant CARS signal, which is about 1.5 times more efficient than that with the parallel configuration with pump-Stokes and pump-depletion SRS processes. Such an enhanced suppression efficiency in the converging configuration results from a simultaneous loss of photons both in the pump and Stokes beams by double SRS processes, leading to an efficient suppression of the pump-Stokes-pump CARS signal. Based on the present method, we further propose two potential applications: (1) non-resonant background-free CARS imaging and (2) label-free super-resolution Raman imaging, and carry out simple numerical simulations to show their feasibility.

Ultra-multiplex CARS spectroscopic imaging with 1-millisecond pixel dwell time

We performed ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging by using a CCD camera with a fast readout time (<1 ms). The ultra-multiplex CARS signal of a polystyrene bead was detected in the range 600–3600 cm-1 with resolution <10 cm-1. The pixel dwell time was approximately 1 ms, which was limited by the readout time of the CCD camera rather than the exposure time. CARS images of 161 × 161 pixels were obtained of the polymer beads with a total data-acquisition time of approximately 28 s even with the use of a cost-effective microchip laser source. Label-free and ultra-multiplex (18 colors) imaging of living cells was also performed with an effective exposure time of 1.8 ms based on the molecular fingerprint as well as the C-H and O-H stretching vibrational modes using a master oscillator fiber amplifier laser source.

Characterization of Intra/Extracellular Water States Probed by Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopic Imaging.

Detailed knowledge of the water status in living organisms is crucial for understanding their physiology and pathophysiology. Here, we developed a technique to spectroscopically image water at high resolution using ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy equipped with a supercontinuum light source. This system allows for the visualization of a wide spectrum of CARS signals from the fingerprint to the end of O-H stretching at a spectral resolution of ∼10 cm-1. Application of the system to living mammalian cells revealed a spectral red shift of the O-H stretching vibrational band inside compared to outside the cells, suggesting the existence of stronger hydrogen bonds inside the cells. Furthermore, potential changes in spectra were examined by adding mannitol to the extracellular solution, which increases the osmolality outside the cells and thereby induces dehydration of the cells. Under this treatment, the red shift of the O-H stretching band was further enhanced, revealing the effects of mannitol on water states inside the cells. The methodology developed here should serve as a powerful tool for the chemical imaging of water in living cells in various biological and medical contexts.

Selective suppression of CARS signal with two competing stimulated Raman scattering processes.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy have been used in studying the structure and dynamics of a wide range of chemical and biological systems. However, the spatial resolution of CARS microscopy is still limited by the diffraction barrier, and hence a suitable scheme to selectively switch off the CARS imaging signal is essential for super-resolution CARS microscopy. Here, we present theoretical descriptions about three different ways to selectively suppress the pump-Stokes-pump two-beam CARS signal by employing three-beam double stimulated Raman scattering (SRS) schemes. Using a semiclassical theory for the interaction of radiation with the Raman-active molecule, we obtain coupled differential equations for the intensities of the pump, Stokes, depletion, and the generated CARS signal fields. We find approximate solutions of these coupled differential equations. They are then used to show that the pump-Stokes-pump CARS signal can be selectively suppressed by increasing the added depletion beam intensity, when the three injected beam frequencies are tuned in such a way that they can induce two SRS processes simultaneously. To show that these switching-off methods can be used to develop super-resolution CARS imaging techniques, we numerically calculate the full-width-at-half-maximum of the CARS imaging point spread function assuming that the spatial profiles of the pump and Stokes beams are Gaussian functions and that the spatial profile of the depletion beam is doughnut-shaped. We anticipate that the proposed selective CARS suppression schemes will be of use in developing super-resolution, label-free CARS microscopy.

Cell-Nanoparticle Interactions at (Sub)-Nanometer Resolution Analyzed by Electron Microscopy and Correlative Coherent Anti-Stokes Raman Scattering.

A wide variety of nanoparticles are playing an increasingly important role in drug delivery. Label-free imaging techniques are especially desirable to follow the cellular uptake and intracellular fate of nanoparticles. The combined correlative use of different techniques, each with unique advantages, facilitates more detailed investigation about such interactions. The synergistic use of correlative coherent anti-Stokes Raman scattering and electron microscopy (C-CARS-EM) imaging offers label-free, chemically-specific, and (sub)-nanometer spatial resolution for studying nanoparticle uptake into cells as demonstrated in the current study. Coherent anti-Stokes Raman scattering (CARS) microscopy offers chemically-specific (sub)micron spatial resolution imaging without fluorescent labels while transmission electron microscopy (TEM) offers (sub)-nanometer scale spatial resolution and thus visualization of precise nanoparticle localization at the sub-cellular level. This proof-of-concept imaging platform with unlabeled drug nanocrystals and macrophage cells revealed good colocalization between the CARS signal and electron dense nanocrystals in TEM images. The correlative TEM images revealed subcellular localization of nanocrystals inside membrane bound vesicles, showing multivesicular body (MVB)-like morphology typical for late endosomes (LEs), endolysosomes, and phagolysosomes. C-CARS-EM imaging has much potential to study the interactions between a wide range of nanoparticles and cells with high precision and confidence.

Interference-free hybrid fs/ps vibrational CARS thermometry in high-pressure flames.

Interference-free hybrid femtosecond/picosecond vibrational coherent anti-Stokes Raman scattering (CARS) of nitrogen is reported for temperature measurements of 1300-2300K in high-pressure, laminar H2-air and CH4-air diffusion flames up to 10bar. Following coherent Raman excitation by 100fs duration pump and Stokes pulses, a time-asymmetric probe pulse is used for the detection of spectrally resolved N2 CARS signals at probe delays as early as ∼200-300 fs. This allows for full rejection of nonresonant contributions while being independent of collisions for single-shot precision of ±2% at elevated pressures. The effects of collisions at longer probe-pulse delays are also investigated to determine the feasibility of varying the detection timing from 200fs to 100ps.

The impact of cell fixation on coherent anti-stokes Raman scattering signal intensity in neuronal and glial cell lines.

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid-related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti-stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.

Invited Article: CARS molecular fingerprinting using sub-100-ps microchip laser source with fiber amplifier

We have developed an ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopic system using a supercontinuum (SC) seeded by sub-100-ps (85 ps) laser pulses with a sub-MHz (0.82 MHz) repetition rate. Because of the high peak power and ultrabroadband spectral profile of the SC, we can efficiently generate multiplex CARS signals in the spectral range of 600–3600 cm−1, which covers the entire molecular fingerprint region, as well as the C—H and O—H stretching regions. Due to the high peak power of the new laser source, the exposure time (pixel dwell time) for CARS imaging of polymer beads was reduced to less than 1 ms (0.8 ms), which was limited by the readout time of a CCD camera. Owing to the improvement in CARS spectral quality, clear molecular fingerprinting was achieved for living HeLa cells at different phases in the cell cycle.

Invited Article: Label-free nerve imaging with a coherent anti-Stokes Raman scattering rigid endoscope using two optical fibers for laser delivery

A coherent anti-Stokes Raman scattering (CARS) rigid endoscope using two optical fibers to deliver excitation beams individually is developed. The use of two optical fibers allows the correction of longitudinal chromatic aberration and enhances the CARS signal by a factor of 2.59. The endoscope is used to image rat sciatic nerves with an imaging time of 10 s. Imaging of the rabbit prostatic fascia without sample slicing is also demonstrated, which reveals the potential for the application of the CARS endoscope to robot-assisted surgery.

Ultrabroadband Multiplex Coherent anti-Stokes Raman Scattering (CARS) Microspectroscopy Using a CCD Camera with an InGaAs Image Intensifier

We developed a new microspectroscopic system based on the ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) process using a CCD camera with an InGaAs image intensifier. Owing to the gated amplification of the CARS signal by the InGaAs Image intensifier, the CARS signal was detected with less than 10 laser shots. As a proof-of-principle study, we applied our system to visualizing the heterogeneous structure of a blended polymer consisting of cellulose acetate and cellulose tris(phenylcarbamate). The clear vibrational contrast was observed under weak excitation conditions.

Six-wave mixing coherent anti-Stokes Raman scattering microscopy.

Acquiring images of biological tissues and cells without the assistance of exogenous labels with a fast repetition rate and chemical specificity is what coherent anti-Stokes Raman Scattering (CARS) imaging offers. Nonresonant background (NRB) is one of the main drawbacks of the CARS microscopy technique because it limits the detection of weak Raman lines and the detection of low-concentration molecules. We show that a six-wave mixing process with two beams, which is a cascade effect of CARS, show better signal/NRB ratio and can be utilized for biological tissues imaging. The cascade CARS (CCARS) depends on chi-3 to the fourth power, instead of chi-3 squared as in the usual CARS signal; therefore, the contrast ratio with NRB is higher for CCARS than for CARS. We present analytic calculations showing that CCARS have better contrast over CARS in any situation. Comparison of the signals of both techniques generated on water-ethanol solutions confirm these results. Finally, we acquired CCARS images of fresh biological tissues, attesting that it is a useful tool for biological studies.

Simple model of coherent anti‐Stokes Raman scattering signals generated by means of linearly chirped ultrashort Gaussian laser pulses

AbstractTime and frequency dependences of coherent anti‐Stokes Raman scattering (CARS) are here found analytically for the increasingly popular experimental approach where the Raman preparation and probing are realized by means of very short linearly chirped laser pulses. These are chosen with Gaussian lineshapes that, besides being rather common in ultrashort laser physics, guarantee full solution to the nonlinear scattering problem at hand. The solution appears rather simple in spite of the complications arising from the treatment of optical nonlinearities of different nature (dielectric susceptibility of the third order for the Raman medium and quadratic phases of three laser fields). The simplicity of the solution allows versatile applications that are here shown for different chirp effects in vibrational and purely rotational N2 CARS whose spectral complexity provides an excellent ground to test the model. The comparison with known experiments shows a good agreement except for some limitations imposed by deviations of the nonresonant CARS signal from the Gaussian structure assumed in the model.