Published papers

A multiplex CARS imaging system, equipped with an EM-CCD camera, was developed to improve the sensitivity of backward CARS imaging in biological analysis using an inverted microscope. The signal-to-noise ratio was improved by a factor of ca. 3 compared to a conventional CCD mode through the use of EM gain. When imaging epithelial cells in the backward CARS configuration, intracellular organelles such as lipid droplets and nuclei were spectroscopically identified with an exposure time of only 100 ms/pixel.

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. However, existing biomarkers have limitations of requiring fixation and labeling, or their molecular backgrounds are uncertain. Coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging is a novel option because it can assess and visualize molecular structures based on their molecular fingerprint. Here, we present a new label-free method to visualize cellular senescence using CARS imaging in nucleoli. We found the peak of the nucleolar amide I band shifted to a higher wavenumber in binuclear senescent cells, which reflects changes in the protein secondary structure from predominant α-helices to β-sheets originating from amyloid-like aggregates. Following this, we developed a procedure that can visualize the senescent cells by providing the ratios and subtractions of these two components. We also confirmed that the procedure can visualize nucleolar aggregates due to unfolded/misfolded proteins produced by proteasome inhibition. Finally, we found that this method can help visualize the nucleolar defects in naïve cells even before binucleation. Thus, our method is beneficial to evaluate ongoing cellular senescence through label-free imaging of nucleolar defects.

We analyzed the effects of a hypo-osmotic environment on rat Schwann cells, a type of glial cell surrounding neurons, using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. After hypo-osmotic treatment, we detected vacuole-like components in the cytoplasm using both bright-field and CARS spectroscopic imaging. An approach integrating both morphological examination and a spectroscopic analysis based on multiple vibrational bands revealed that these structures are predominantly water-filled, and their characteristics closely resembled those of the vacuoles observed in plant cells.

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. Graphical abstract: [Figure not available: see fulltext.].

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

We demonstrate how spatial beam self-cleaning and supercontinuum generation in graded-index multimode optical fibers can be directly applied in multiplex coherent anti-Stokes Raman Scattering (M-CARS) spectroscopy. Although supercontinuum generation causes pump depletion mainly in the center of the beam, the partial recovery of the pump brightness due to self-cleaning may enable self-referenced M-CARS, with no additional delay lines to synchronize pump and Stokes waves. As a proof-of-principle, we report examples of imaging of single chemical compounds and polystyrene beads. The new scheme paves the way towards simpler M-CARS systems based on multimode fiber sources.

Diffusion dynamics of polymers and colloidal dispersions at high concentrations have been widely studied using dynamic light scattering (DLS), a correlation spectroscopic method based on Rayleigh scattering. However, DLS has not been applied to complex systems such as biological cells because of its lack of molecular selectivity. Here, we propose dynamic Raman scattering (DRS), a novel molecular-selective correlation spectroscopic technique based on Raman scattering. We theoretically demonstrate that molecular-selective diffusion dynamics can be monitored by measuring the intensity fluctuation of coherent anti-Stokes Raman scattering in a phase-mismatching condition. We then experimentally prove the molecular selectivity of the DRS by extracting the polystyrene diffusion dynamics in a mixture of polystyrene and silica nanoparticles. We believe that DRS will be applicable for monitoring diffusion dynamics in complex dispersions without any pretreatment.

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. The reaction solution was encapsulated in water-in-oil microdroplets with diameters of 50 μm. The reaction was monitored by measuring the concentration of coenzymes from the CARS spectrum obtained in one-second exposure time. The results obtained using our system was consistent with those of the conventional fluorescence measurement system and indicate the potential of CARS spectroscopy for dropletbased high-throughput screening of enzymes.

Cellular responses to molecularly targeted agents (gefitinib and osimertinib) have been observed in cultured cancer cells using ultra-broadband multiplex coherent anti-Stokes Raman scattering and third harmonic generation microscopy. Lipid droplets (LDs) appeared in the cells 48 h after the exposure of cancer cells to gefitinib. This was also observed in the gefitinib-resistant strain, and the accumulation of LDs was significant. When exposed to osimertinib, LDs were not as pronounced as those with gefitinib. These agents act on cells through different mechanisms.

Multiplex coherent anti-stokes Raman scattering (M-CARS) and second harmonic generation (SHG) techniques are used to map the real part of the nonresonant third order nonlinear optical susceptibility and the second order nonlinear optical susceptibility of a thermally microimprinted niobium borophosphate glass. In particular, such bimodal nonlinear imaging is employed in order to precisely evaluate how thermal poling can modify the amplitude of the nonresonant third-order nonlinearity of the sample. A systematic decrease of the M-CARS intensity is found in the poled areas with respect to the unpoled ones, suggesting therefore a reduction of the real part of the nonresonant third order nonlinear susceptibility within these regions. Such a reduction in the M-CARS intensity can be explained by sodium depletion in the subanodic zone and is mostly linked to the reduction of both the matter density and the linear refractive index, in agreement with earlier works on thermal poling on the same family of glasses.

Opalescence of therapeutic antibody solutions is one of the concerns in drug formulation. However, the mechanistic insights into the opalescence of antibody solutions remain unclear. Here, we investigated the assembly states of antibody molecules as a function of antibody concentration. The solutions of bovine gamma globulin and human immunoglobulin G at around 100 mg/mL showed the formation of submicron-scale network assemblies. The network assembly resulted in the appearance of opalescence with a transparent blue color without the precipitates of antibodies. Furthermore, the addition of trehalose and arginine, previously known to act as protein stabilizers and protein aggregation suppressors, was able to suppress the opalescence arising from the network assembly. These results will provide an important information for evaluating and improving protein formulations.

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.

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. By combining SHG analysis with CARS, the two groups were identified as free fatty acids and wax esters, respectively.