Coherent Raman Microspectroscopy Research Articles

Quantification of the nonlinear susceptibility of the hydrogen and deuterium stretch vibration for biomolecules in coherent Raman micro-spectroscopy.

Deuterium labelling is increasingly used in coherent Raman imaging of complex systems, such as biological cells and tissues, to improve chemical specificity. Nevertheless, quantitative coherent Raman susceptibility spectra for deuterated compounds have not been previously reported. Interestingly, it is expected theoretically that -D stretch vibrations have a Raman susceptibility lower than -H stretch vibrations, with the area of their imaginary part scaling with their wavenumber, which is shifted from around 2900 cm-1 for C-H into the silent region around 2100 cm-1 for C-D. Here, we report quantitative measurements of the nonlinear susceptibility of water, succinic acid, oleic acid, linoleic acid and deuterated isoforms. We show that the -D stretch vibration has indeed a lower area, consistent with the frequency reduction due to the doubling of atomic mass from hydrogen to deuterium. This finding elucidates an important trade-off between chemical specificity and signal strength in the adoption of deuterium labelling as an imaging strategy for coherent Raman microscopy.

Effective polarization suppression in two-beam 3-color broadband coherent Raman micro-spectroscopy (3CBCRS)

We report the extension of our formerly developed two-beam 3-color broadband coherent Raman methodology and the improvement of our tabletop system for fully polarization controlled low-frequency vibrational measurements. Each of the differently polarized partial signal in the coherent Raman scattering could be effectively eliminated at the theoretically predicted polarization analyzer orientations. The electronic contribution to the signal was suppressed without significantly reducing the vibrational contribution to the spectrum, which is a noteworthy advantage compared to Coherent anti-Stokes Raman Spectroscopy (CARS). The method was applied for well-characterized liquid CCl4 and CHCl3 samples for the first time. Coherent vibrational spectra were recorded simultaneously on the Stokes and anti-Stokes spectral-domain located near the zero frequency shift region. The methodology is easily extendable to measure vibrational or phonon spectra of anisotropic crystals.

Contribution to the development of low frequency terahertz coherent Raman micro-spectroscopy and microscopy

We report the construction and characterization of a coherent Raman tabletop system utilizing a novel astigmatic optical focusing geometry, a broadband nanosecond optical parametric oscillator and volumetric Bragg filters assisting 3CBCRS measuring system for the first time. In order to illustrate the versatility of the measurements and reveal the molecular information obtainable, two well-characterized chemicals were selected. Polarization sensitive epi-detected 3CBCRS spectra of liquid CCl4 and calcite crystal were recorded and analyzed. An unexpected polarization dependence of the signals of the lowest frequency modes of CCl4 was observed. The 1122 third order susceptibility component was phase flipped. The non-resonant susceptibility normalized 1122 component was found to be larger than the 1111 component for the lowest vibrational modes. This anomalous comportment was attributable to the anisotropy Raman tensor invariant in the third order nonlinear susceptibility tensor.

Direct Label-Free Measurement of the Distribution of Small Molecular Weight Compound Inside Thick Biological Tissue using Coherent Raman Microspectroscopy

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 /cm inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.Sci. Rep. 5, 13868; http://dx.doi.org/10.1038/srep13868 (2015).

Direct label-free measurement of the distribution of small molecular weight compound inside thick biological tissue using coherent Raman microspectroscopy.

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 cm−1 inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.

Chemical Analysis of Molecular Species through Turbid Medium

Subsurface analysis of chemical species is imperative for biomedical diagnostics and imaging, homeland security, and pharmaceutical and other industries; however, the access to the object of interest is often obscured by an optically scattering medium which limits the ability to inspect the chemical composition of the sample. In this report, we employ coherent Raman microspectroscopy in a combination with a hierarchical cluster analysis to mitigate the effect of scattering and demonstrate the identification of multiple chemical species.

Broadly wavelength- and pulse width-tunable high-repetition rate light pulses from soliton self-frequency shifting photonic crystal fiber integrated with a frequency doubling crystal

Soliton self-frequency shift (SSFS) in a photonic crystal fiber (PCF) pumped by a long-cavity mode-locked Cr:forsterite laser is integrated with second harmonic generation (SHG) in a nonlinear crystal to generate ultrashort light pulses tunable within the range of wavelengths from 680 to 1800 nm at a repetition rate of 20 MHz. The pulse width of the second harmonic output is tuned from 70 to 600 fs by varying the thickness of the nonlinear crystal, beam-focusing geometry, and the wavelength of the soliton PCF output. Wavelength-tunable pulses generated through a combination of SSFS and SHG are ideally suited for coherent Raman microspectroscopy at high repetition rates, as verified by experiments on synthetic diamond and polystyrene films.

Soliton dynamics in photonic-crystal fibers for coherent Raman microspectroscopy and microscopy

We describe various generation schemes that employ soliton dynamics in photonic-crystal fibers to generate a red-shifted femtosecond pulse with high efficiency. We then detail how to use this pulse along with the initial femtosecond laser pulse for coherent Raman experiments. We describe various measurement schemes that, though utilizing this pair of femtosecond pulses, mimick the preferred measurement scheme for coherent Raman microspectroscopy where one pulse is transform-limited and of picosecond duration; or for coherent Raman microscopy where both pulses are transform-limited and of picosecond duration. We finally discuss how to increase pulse energy and give some perspective on the realization of a coherent Raman endoscope based on soliton dynamics.

Detecting anthrax in the mail by coherent Raman microspectroscopy

In this report, we show the collection of spatial information through a turbid medium by coherent Raman microspectroscopic imaging. In particular, the technique is capable of identifying anthrax endospores inside a sealed paper envelope.

Hyperspectral coherent anti-Stokes Raman scattering microscopy imaging through turbid medium

Coherent Raman microspectroscopy imaging is an emerging technique for noninvasive, chemically specific optical imaging, which can be potentially used to analyze the chemical composition and its distribution in biological tissues. In this report, a hierarchical cluster analysis was applied to hyperspectral coherent anti-Stokes Raman imaging of different chemical species through a turbid medium. It was demonstrated that by using readily available commercial software (Cytospec, Inc.) and cluster analysis, distinct chemical species can be imaged and identified through a rather thick layer of scattering medium. Once the clusters of different chemical composition were distinguished, a phase retrieval algorithm was used to convert coherent anti-Stokes Raman spectra to Raman spectra, which were used for chemical identification of hidden microscopic objects. In particular, applications to remote optical sensing of potential biological threats and to imaging through a layer of skin tissue were successfully demonstrated.

Epi-detected hybrid coherent Raman micro-spectroscopy

We demonstrate efficient epi-detected coherent Raman micro-spectroscopy using a simple laser system based on a femtosecond mode-locked oscillator. In our experiment, the non-resonant background is eliminated by using a hybrid technique, which combines laser pulse shaping and timing with frequency-resolved detection, and is based on the different temporal behavior of parametric and nonparametric processes. We perform careful analysis to make sure the coherent Raman-resonant signal is generated in the backward direction, and not forward-generated and then back-scattered. This demonstration may have important implications for coherent Raman microscopy.

Molecular identification by generating coherence between molecular normal modes using stimulated Raman scattering

We propose a method of driving the vibrations of normal modes of a target molecule into coherence using stimulated Raman scattering. In concert many vibrations can produce a larger anti-Stokes signal than a single vibration. The same illumination does not drive other molecules to have coherent vibrations so that these molecules produce a weaker signal. We investigate how target and confounder molecules can be distinguished by pulses that drive many vibrations coherently, which has applications in coherent Raman microspectroscopy.