Coherent Raman microscopy visualizes ongoing cellular senescence through amide I peak shifts originating from β sheets in disordered nucleolar proteins.

Cellular senescence occurs through the accumulation of many kinds of stresses. Senescent cells in tissues also cause various age-related disorders. Therefore, detecting them without labeling is beneficial for medical research and developing diagnostic methods.

Molecular Fingerprinting of Mouse Brain Using Ultrabroadband Coherent Anti-Stokes Raman Scattering (CARS) Microspectroscopy Empowered by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS).

The Raman fingerprint spectral region provides abundant structural information on molecules. However, analyzing vibrational images within this region using coherent Raman imaging remains challenging due to the small Raman cross section and congested spectral features. In this study, we combined ultrabroadband coherent anti-Stokes Raman scattering (CARS) microspectroscopy across the spectral range of 500-4000 cm with multivariate curve resolution-alternating least-squares (MCR-ALS) to reveal hidden Raman bands in the fingerprint region.

Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging.

Visualization of intracellular lipid metabolism in brown adipocytes by time-lapse ultra-multiplex CARS microspectroscopy with an onstage incubator.

We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80 min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.

Visualizing intra-medulla lipids in human hair using ultra-multiplex CARS, SHG, and THG microscopy.

We performed label-free imaging of human-hair medulla using multi-modal nonlinear optical microscopy. Intra-medulla lipids (IMLs) were clearly visualized by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging. Two groups of IMLs were found: second harmonic generation (SHG) active and inactive.

Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB.

For many years, scientists have been looking for specific biomarkers associated with cancer cells for diagnosis purposes. These biomarkers mainly consist of proteins located at the cell surface (e.g.

Fast epi-detected broadband multiplex CARS and SHG imaging of mouse skull cells.

We present a bimodal imaging system able to obtain epi-detected mutiplex coherent anti-Stokes Raman scattering (M-CARS) and second harmonic generation (SHG) signals coming from biological samples. We studied a fragment of mouse parietal bone and could detect broadband anti-Stokes and SHG responses originating from bone cells and collagen respectively. In addition we compared two post-processing methods to retrieve the imaginary part of the third-order nonlinear susceptibility related to the spontaneous Raman scattering.

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation.

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining.

Spectro-temporal shaping of supercontinuum for subnanosecond time-coded M-CARS spectroscopy.

A supercontinuum laser source was designed for multiplex-coherent anti-Stokes Raman scattering spectroscopy. This source was based on the use of a germanium-doped standard optical fiber with a zero dispersion wavelength at 1600 nm and pumped at 1064 nm. We analyzed the nonlinear spectro-temporal interrelations of a subnanosecond pulse propagating in a normal dispersion regime in the presence of a multiple Raman cascading process and strong conversion.

Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source.

Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red(®) are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available.

Multimodal imaging of living cells with multiplex coherent anti-stokes raman scattering (CARS), third-order sum frequency generation (TSFG) and two-photon excitation fluorescence (TPEF) using a nanosecond white-light laser source.

The subnanosecond "white-light laser" source has been applied to multimodal, multiphoton, and multiplex spectroscopic imaging (M(3) spectroscopic imaging) with coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and two-photon excitation fluorescence (TPEF). As the proof-of-principle experiment, we performed simultaneous imaging of polystyrene beads with TSFG and TPEF. This technique is then applied to live cell imaging.

Near-infrared supercontinuum laser beam source in the second and third near-infrared optical windows used to image more deeply through thick tissue as compared with images from a lamp source.

With the use of longer near-infrared (NIR) wavelengths, image quality can be increased due to less scattering (described by the inverse wavelength power dependence 1/λ(n) where n ≥ 1 ) and minimal absorption from water molecules. Longer NIR windows, known as the second (1100 nm to 1350 nm) and third (1600 to 1870 nm) NIR windows are utilized to penetrate more deeply into tissue media and produce high-quality images. An NIR supercontinuum (SC) laser light source, with wavelengths in the second and third NIR optical windows to image tissue provides ballistic imaging of tissue.

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white-light laser source.

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously.

Electronically resonant third-order sum frequency generation spectroscopy using a nanosecond white-light supercontinuum.

Third-order sum frequency generation (TSFG) is one of the third-order nonlinear optical processes, and has the generation mechanism analogous to third harmonic generation (THG). By using a white-light supercontinuum, we can obtain broadband multiplex TSFG spectra. In the present study, we developed an electronically resonant TSFG spectrometer, and applied it to obtain TSFG spectra of hemoproteins.

Surfactant uptake dynamics in mammalian cells elucidated with quantitative coherent anti-stokes Raman scattering microspectroscopy.

The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%).

Time-frequency resolved analysis of a nanosecond supercontinuum source dedicated to multiplex CARS application.

In this paper, we describe and investigate the properties of a broadband source designed from a nanosecond microchip laser operating at high repetition rate and dedicated to multiplex-CARS application. We demonstrate that a strong reshaping of the initial pulse profile drastically affects the Stokes wave and therefore represents an important limitation in CARS experiment. In particular, we emphasize the saturation effect of the peak power of the Stokes wave resulting from supercontinuum generation.

Observation of Raman optical activity by heterodyne-detected polarization-resolved coherent anti-Stokes Raman scattering.

We report the first observation of Raman optical activity (ROA) by coherent anti-Stokes Raman scattering. Thanks to the more freedom of polarization configurations in coherent anti-Stokes Raman scattering than in spontaneous Raman spectroscopy, the contrast ratio of the chiral signal to the achiral background has been improved markedly. For (-)-β-pinene, it is 2 orders of magnitude better than that in the reported spontaneous ROA measurement.

Flow cytometer based on triggered supercontinuum laser illumination.

Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this multiparametric technique is currently used to differentiate blood cells subpopulations. The choice of excitation wavelengths matching fluorochrome spectra (it is currently the opposite) and the use of a broader range of fluorochromes can be made by taking advantage of a filtered supercontinuum white light source.

Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation).

We have developed a new multimodal molecular imaging system that combines CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), THG (third harmonic generation) and multiplex TSFG (third-order sum frequency generation) using a subnanosecond white-light laser source. Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG. A correlation image of CARS and TSF reveals that the TSF signal is generated predominantly from lipid droplets inside a cell as well as the peripheral cell wall.

Protein secondary structure imaging with ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy.

Protein secondary structures in human hair have been studied with ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The CARS peak-shift mapping method has been developed and applied to hair samples with and without treatments by chemical reduction and mechanical extension. It clearly visualizes the treatment induced changes in protein secondary structures and their spatial distributions.