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.

Vapor-Induced Conversion of a Centrosymmetric Organic-Inorganic Hybrid Crystal into a Proton-Conducting Second-Harmonic-Generation-Active Material.

Chemical responsivity in materials is essential to build systems with switchable functionalities. However, polarity-switchable materials are still rare because inducing a symmetry breaking of the crystal structure by adsorbing chemical species is difficult. In this study, we demonstrate that a molecular organic-inorganic hybrid crystal of (NEt)[MnN(CN)] () undergoes polarity switching induced by water vapor and transforms into a rare example of proton-conducting second-harmonic-generation-active material.

Label-free enzymatic reaction monitoring in water-in-oil microdroplets using ultra-broadband multiplex coherent anti-Stokes Raman scattering spectroscopy.

We propose a system for monitoring an enzymatic reaction, i.e., dehydrogenation of ethanol catalyzed by alcohol dehydrogenase, in microdroplets using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy.

Calibration for a count rate-dependent time correlation function and a random noise reduction in pulsed dynamic light scattering.

A pulsed dynamic light scattering (DLS) system, which would be potentially applied to nonlinear DLS with molecular selectivity, was developed by combining a sub-nanosecond pulsed laser with a software-based detection system. The distortion of the time correlation function due to the clipping effect in the photon counting module, and the resulting underestimation of the particle size, were successfully calibrated based on a theoretical simulation. The effective removal of random noises was also demonstrated via time gating synchronized to the laser pulses.

Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window.

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode was performed in a label-free manner.

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.

Optical coherence tomography-based tissue dynamics imaging for longitudinal and drug response evaluation of tumor spheroids.

We present optical coherence tomography (OCT)-based tissue dynamics imaging method to visualize and quantify tissue dynamics such as subcellular motion based on statistical analysis of rapid-time-sequence OCT signals at the same location. The analyses include logarithmic intensity variance (LIV) method and two types of OCT correlation decay speed analysis (OCDS). LIV is sensitive to the magnitude of the signal fluctuations, while OCDSs including early- and late-OCDS (OCDS and OCDS , respectively) are sensitive to the fast and slow tissue dynamics, respectively.

Identifying the molecular adsorption site of a single molecule junction through combined Raman and conductance studies.

Single-molecule junctions are ideal test beds for investigating the fundamentals of charge transport at the nanoscale. Conducting properties are strongly dependent on the metal-molecule interface geometry, which, however, is very poorly characterized due to numerous experimental challenges. We report on a new methodology for characterizing the adsorption site of single-molecule junctions through the combination of surface enhanced Raman scattering (SERS), current-voltage (-) curve measurements, and density functional theory simulations.

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.

Mechanism of co-aggregation in a protein mixture with small additives.

Co-aggregation plays an important role in processing protein-rich food materials under heterogeneous conditions. The main cause of co-aggregation is an electrostatic attraction between oppositely charged molecules. This study investigated thermal aggregation of β-lactoglobulin (BLG) (pI=5.

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

Purpose: In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas.

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.

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.

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.