コヒーレントラマン分光法は、高速かつ無標識に分子レベルの構造情報を得られる手法として近年注目を集めている。コヒーレントラマン分光法と顕微鏡を組み合わせた振動分光イメージングや、コヒーレントラマン分光法とフローサイトメトリーを組み合わせた大規模細胞解析法などがこれまでに実証されてきた。応募者らは超短パルスレーザーを用いて時間領域で分子振動を励起・検出するFourier-transform coherent Raman scattering (FT-CARS)法を用いて、高速（24,000 spectra/s）かつ広帯域（200 – 1600 cm-1）な振動分光計測を実現するとともに、様々な対象へとその応用を実証してきた。しかしながら、周波数領域で計測を行うCARS分光法やStimulated Raman scattering （SRS）法などと比較したときに、FT-CARS法では、CH伸縮振動が観測される高波数領域（2800 – 3400 cm-1）が計測できないといった欠点があった。

本研究では、これまでに開発してきたFT-CARS法に用いているチタンサファイアレーザーに同期して発振するYbファイバーレーザー（YbF）を併せて光源として用いることで、FT-CARS法でも高波数領域の計測が可能であることを実証した。これまでのFT-CARS計測では、パルス幅15 fs程度のチタンサファイアレーザー（TiS）からの出力(750 – 850 nm)をpump光とprobe光へと分割し、それらを様々なパルス間隔でサンプルへと照射することで、分子振動の時間発展を計測していた。今回開発した手法では、従来のFT-CARS分光計をベースとして、YbFからの出力（~1030 nm）を同時に照射することで、CH伸縮領域の計測を可能とした。TiSの出力の一部をYbFのキャビティ内へと導入し、相互位相変調によってYbFキャビティ内で2つのパルスが同じ群速度で伝搬しYbFとTiSの高精度な同期発振を実現した。

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.analchem.3c01958

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Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.algal.2023.102993

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：SPIE-Intl Soc Optical Eng  

DOI： 10.1117/1.apn.2.2.026008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The ability to perform sensitive, real-time, in situ, multiplex chemical analysis is indispensable for diverse applications such as human health monitoring, food safety testing, forensic analysis, environmental sensing, and homeland security. Surface-enhanced Raman spectroscopy (SERS) is an effective tool to offer the ability by virtue of its high sensitivity and rapid label-free signal detection as well as the availability of portable Raman spectrometers. Unfortunately, the practical utility of SERS is limited because it generally requires sample collection and preparation, namely, collecting a sample from an object of interest and placing the sample on top of a SERS substrate to perform a SERS measurement. In fact, not all analytes can satisfy this requirement because the sample collection and preparation process may be undesirable, laborious, difficult, dangerous, costly, or time-consuming. Here we introduce "Place & Play SERS" based on an ultrathin, flexible, stretchable, adhesive, biointegratable gold-deposited polyvinyl alcohol (PVA) nanomesh substrate that enables placing the substrate on top of an object of interest and performing a SERS measurement of the object by epi-excitation without the need for touching, destroying, and sampling it. Specifically, we characterized the sensitivity of the gold/PVA nanomesh substrate in the Place & Play SERS measurement scheme and then used the scheme to conduct SERS measurements of both wet and dry objects under nearly real-world conditions. To show the practical utility of Place & Play SERS, we demonstrated two examples of its application: food safety testing and forensic analysis. Our results firmly verified the new measurement scheme of SERS and are expected to extend the potential of SERS by opening up untapped applications of sensitive, real-time, in situ multiplex chemical analysis.

DOI： 10.1039/d2ay02090d

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)  

Flow cytometry is an indispensable tool in biology and medicine for counting and analyzing cells in large heterogeneous populations. It identifies multiple characteristics of every single cell, typically via fluorescent probes that specifically bind to target molecules on the cell surface or within the cell. However, flow cytometry has a critical limitation: the color barrier. The number of chemical traits that can be simultaneously resolved is typically limited to several due to the spectral overlap between fluorescence signals from different fluorescent probes. Here, we present color-scalable flow cytometry based on coherent Raman flow cytometry with Raman tags to break the color barrier. This is made possible by combining a broadband Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) flow cytometer, resonance-enhanced cyanine-based Raman tags, and Raman-active dots (Rdots). Specifically, we synthesized 20 cyanine-based Raman tags whose Raman spectra are linearly independent in the fingerprint region (400 to 1,600 cm-1). For highly sensitive detection, we produced Rdots composed of 12 different Raman tags in polymer nanoparticles whose detection limit was as low as 12 nM for a short FT-CARS signal integration time of 420 µs. We performed multiplex flow cytometry of MCF-7 breast cancer cells stained by 12 different Rdots with a high classification accuracy of 98%. Moreover, we demonstrated a large-scale time-course analysis of endocytosis via the multiplex Raman flow cytometer. Our method can theoretically achieve flow cytometry of live cells with >140 colors based on a single excitation laser and a single detector without increasing instrument size, cost, or complexity.

DOI： 10.1093/pnasnexus/pgad001

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

Label-free imaging flow cytometry is a powerful tool for biological and medical research as it overcomes technical challenges in conventional fluorescence-based imaging flow cytometry that predominantly relies on fluorescent labeling. To date, two distinct types of label-free imaging flow cytometry have been developed, namely optofluidic time-stretch quantitative phase imaging flow cytometry and stimulated Raman scattering (SRS) imaging flow cytometry. Unfortunately, these two methods are incapable of probing some important molecules such as starch and collagen. Here, we present another type of label-free imaging flow cytometry, namely multiphoton imaging flow cytometry, for visualizing starch and collagen in live cells with high throughput. Our multiphoton imaging flow cytometer is based on nonlinear optical imaging whose image contrast is provided by two optical nonlinear effects: four-wave mixing (FWM) and second-harmonic generation (SHG). It is composed of a microfluidic chip with an acoustic focuser, a lab-made laser scanning SHG-FWM microscope, and a high-speed image acquisition circuit to simultaneously acquire FWM and SHG images of flowing cells. As a result, it acquires FWM and SHG images (100 × 100 pixels) with a spatial resolution of 500 nm and a field of view of 50 μm × 50 μm at a high event rate of four to five events per second, corresponding to a high throughput of 560-700 kb/s, where the event is defined by the passage of a cell or a cell-like particle. To show the utility of our multiphoton imaging flow cytometer, we used it to characterize Chromochloris zofingiensis (NIES-2175), a unicellular green alga that has recently attracted attention from the industrial sector for its ability to efficiently produce valuable materials for bioplastics, food, and biofuel. Our statistical image analysis found that starch was distributed at the center of the cells at the early cell cycle stage and became delocalized at the later stage. Multiphoton imaging flow cytometry is expected to be an effective tool for statistical high-content studies of biological functions and optimizing the evolution of highly productive cell strains.

DOI： 10.1002/cyto.a.24723

PubMed

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsphotonics.2c00768

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Language：Others   Publishing type：Research paper (scientific journal)  

The last two decades have witnessed a dramatic growth of wearable sensor technology, mainly represented by flexible, stretchable, on-skin electronic sensors that provide rich information of the wearer's health conditions and surroundings. A recent breakthrough in the field is the development of wearable chemical sensors based on surface-enhanced Raman spectroscopy (SERS) that can detect molecular fingerprints universally, sensitively, and noninvasively. However, while their sensing properties are excellent, these sensors are not scalable for widespread use beyond small-scale human health monitoring due to their cumbersome fabrication process and limited multifunctional sensing capabilities. Here, a highly scalable, wearable SERS sensor is demonstrated based on an easy-to-fabricate, low-cost, ultrathin, flexible, stretchable, adhesive, and biointegratable gold nanomesh. It can be fabricated in any shape and worn on virtually any surface for label-free, large-scale, in situ sensing of diverse analytes from low to high concentrations (10–106 × 10−9 m). To show the practical utility of the wearable SERS sensor, the sensor is tested for the detection of sweat biomarkers, drugs of abuse, and microplastics. This wearable SERS sensor represents a significant step toward the generalizability and practicality of wearable sensing technology.

DOI： 10.1002/adom.202200054

Scopus

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cyto.a.24664

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cyto.a.24664

Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/admt.202101567

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/admt.202101567

Language：Others   Publishing type：Research paper (scientific journal)  

Broadband Raman spectroscopy (detection bandwidth >1000 cm-1) is a valuable and widely used tool for understanding samples via label-free measurements of their molecular vibrations. Two important Raman spectral regions are the chemically specific "fingerprint"(200 to 1800 cm-1) and "low-frequency"or "terahertz"(THz) (<200 cm-1; <6 THz) regions, which mostly contain intramolecular and intermolecular vibrations, respectively. These two regions are highly complementary; broadband simultaneous measurement of both regions can provide a big picture comprising information about molecular structures and interactions. Although techniques for acquiring broadband Raman spectra covering both regions have been demonstrated, these methods tend to have spectral acquisition rates <10 spectra/s, prohibiting high-speed applications, such as Raman imaging or vibrational detection of transient phenomena. Here, we demonstrate a single-laser method for ultrafast (24,000 spectra/s) broadband Raman spectroscopy covering both THz and fingerprint regions. This is achieved by simultaneous detection of Sagnac-enhanced impulsive stimulated Raman scattering (SE-ISRS; THz-sensitive) and Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS; fingerprint-sensitive). With dual-detection impulsive vibrational spectroscopy, the SE-ISRS signal shows a >500 × enhancement of <6.5 THz sensitivity compared with that of FT-CARS, and the FT-CARS signal shows a >10 × enhancement of fingerprint sensitivity above 1000 cm-1 compared with that of SE-ISRS.

DOI： 10.1117/1.AP.4.1.016003

Scopus

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Language：Others   Publishing type：Research paper (scientific journal)  

A characteristic clinical feature of COVID-19 is the frequent incidence of microvascular thrombosis. In fact, COVID-19 autopsy reports have shown widespread thrombotic microangiopathy characterized by extensive diffuse microthrombi within peripheral capillaries and arterioles in lungs, hearts, and other organs, resulting in multiorgan failure. However, the underlying process of COVID-19-associated microvascular thrombosis remains elusive due to the lack of tools to statistically examine platelet aggregation (i.e., the initiation of microthrombus formation) in detail. Here we report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring of the blood of COVID-19 patients (n = 110). Surprisingly, our analysis of the big image data shows the anomalous presence of excessive platelet aggregates in nearly 90&#37; of all COVID-19 patients. Furthermore, results indicate strong links between the concentration of platelet aggregates and the severity, mortality, respiratory condition, and vascular endothelial dysfunction level of COVID-19 patients.

DOI： 10.1038/s41467-021-27378-2

Language：English   Publishing type：Research paper (scientific journal)  

We report highly sensitive Fourier-transform coherent anti-Stokes Raman scattering spectroscopy enabled by genetic algorithm (GA) pulse shaping for adaptive dispersion compensation. We show that the non-resonant four-wave mixing signal from water can be used as a fitness indicator for successful GA training. This method allows GA adaptation to sample measurement conditions and offers significantly improved performance compared to training using second-harmonic generation from a nonlinear crystal in place of the sample. Results include a 3× improvement to peak signal-to-noise ratio for 2-propanol measurement, as well as a 10× improvement to peak intensities from the high-throughput measurement of polystyrene microbeads under flow.

DOI： 10.1364/OL.434054

Language：English   Publishing type：Research paper (scientific journal)  

Fluorescence-encoded vibrational spectroscopy has become increasingly more popular by virtue of its high chemical specificity and sensitivity. However, current fluorescence-encoded vibrational spectroscopy methods lack sensitivity in the low-frequency region, which if addressed could further enhance their capabilities. Here, we present a method for highly sensitive low-frequency fluorescence-encoded vibrational spectroscopy, termed fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS). By first exciting molecules into vibrationally excited states and then promoting the vibrating molecules to electronic states at varying times, the molecular vibrations can be encoded onto the emitted time-domain fluorescence intensity. We demonstrate the sensitive low-frequency detection capability of FLETCHERS by measuring vibrational spectra in the lower fingerprint region of rhodamine 800 solutions as dilute as 250 nM, which is ∼1000 times more sensitive than conventional vibrational spectroscopy. These results, along with further improvement of the method, open up the prospect of performing single-molecule vibrational spectroscopy in the low-frequency region.

DOI： 10.1021/acs.jpclett.1c01741

Language：English   Publishing type：Research paper (scientific journal)  

In the past few decades, microalgae-based bioremediation methods for treating heavy metal (HM)-polluted wastewater have attracted much attention by virtue of their environment friendliness, cost efficiency, and sustainability. However, their HM removal efficiency is far from practical use. Directed evolution is expected to be effective for developing microalgae with a much higher HM removal efficiency, but there is no non-invasive or label-free indicator to identify them. Here, we present an intelligent cellular morphological indicator for identifying the HM removal efficiency of Euglena gracilis in a non-invasive and label-free manner. Specifically, we show a strong monotonic correlation (Spearman's ρ = -0.82, P = 2.1 × 10-5) between a morphological meta-feature recognized via our machine learning algorithms and the Cu2+ removal efficiency of 19 E. gracilis clones. Our findings firmly suggest that the morphology of E. gracilis cells can serve as an effective HM removal efficiency indicator and hence have great potential, when combined with a high-throughput image-activated cell sorter, for directed-evolution-based development of E. gracilis with an extremely high HM removal efficiency for practical wastewater treatment worldwide.

DOI： 10.1021/acs.est.0c05278

Language：English   Publishing type：Research paper (scientific journal)  

Flow cytometry is a powerful tool with applications in diverse fields such as microbiology, immunology, virology, cancer biology, stem cell biology, and metabolic engineering. It rapidly counts and characterizes large heterogeneous populations of cells in suspension (e.g., blood cells, stem cells, cancer cells, and microorganisms) and dissociated solid tissues (e.g., lymph nodes, spleen, and solid tumors) with typical throughputs of 1,000-100,000 events per second (eps). By measuring cell size, cell granularity, and the expression of cell surface and intracellular molecules, it provides systematic insights into biological processes. Flow cytometers may also include cell sorting capabilities to enable subsequent additional analysis of the sorted sample (e.g., electron microscopy and DNA/RNA sequencing), cloning, and directed evolution. Unfortunately, traditional flow cytometry has several critical limitations as it mainly relies on fluorescent labeling for cellular phenotyping, which is an indirect measure of intracellular molecules and surface antigens. Furthermore, it often requires time-consuming preparation protocols and is incompatible with cell therapy. To overcome these difficulties, a different type of flow cytometry based on direct measurements of intracellular molecules by Raman spectroscopy, or "Raman flow cytometry" for short, has emerged. Raman flow cytometry obtains a chemical fingerprint of the cell in a nondestructive manner, allowing for single-cell metabolic phenotyping. However, its slow signal acquisition due to the weak light-molecule interaction of spontaneous Raman scattering prevents the throughput necessary to interrogate large cell populations in reasonable time frames, resulting in throughputs of about 1 eps. The remedy to this throughput limit lies in coherent Raman scattering methods such as stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS), which offer a significantly enhanced light-sample interaction and hence enable high-throughput Raman flow cytometry, Raman imaging flow cytometry, and even Raman image-activated cell sorting (RIACS). In this Account, we outline recent advances, technical challenges, and emerging opportunities of coherent Raman flow cytometry. First, we review the principles of various types of SRS and CARS and introduce several techniques of coherent Raman flow cytometry such as CARS, multiplex CARS, Fourier-transform CARS, SRS, SRS imaging flow cytometry, and RIACS. Next, we discuss a unique set of applications enabled by coherent Raman flow cytometry, from microbiology and lipid biology to cancer detection and cell therapy. Finally, we describe future opportunities and challenges of coherent Raman flow cytometry including increasing sensitivity and throughput, integration with droplet microfluidics, utilizing machine learning techniques, or achieving in vivo flow cytometry. This Account summarizes the growing field of high-throughput Raman flow cytometry and the bright future it can bring.

DOI： 10.1021/acs.accounts.1c00001

Language：English   Publishing type：Research paper (scientific journal)  

Raman optical activity (ROA) is effective for studying the conformational structure and behavior of chiral molecules in aqueous solutions and is advantageous over X-ray crystallography and nuclear magnetic resonance spectroscopy in sample preparation and cost performance. However, ROA signals are inherently minuscule; 3-5 orders of magnitude weaker than spontaneous Raman scattering due to the weak chiral light-matter interaction. Localized surface plasmon resonance on metallic nanoparticles has been employed to enhance ROA signals, but suffers from detrimental spectral artifacts due to its photothermal heat generation and inability to efficiently transfer and enhance optical chirality from the far field to the near field. Here we demonstrate all-dielectric chiral-field-enhanced ROA by devising a silicon nanodisk array and exploiting its dark mode to overcome these limitations. Specifically, we use it with pairs of chemical and biological enantiomers to show >100x enhanced chiral light-molecule interaction with negligible artifacts for ROA measurements.

DOI： 10.1038/s41467-021-23364-w

Language：English   Publishing type：Research paper (scientific journal)  

Dual-comb coherent Raman spectroscopy is a powerful tool for rapidly probing vibrational signatures of molecules in the fingerprint region. However, >99&#37; of its incident laser energy is unused and wasted since the duty cycle of its spectral acquisition is only less than 1&#37; due to the mismatch between the interval of the laser pulses (>1 ns) and the coherence lifetime of molecular vibrations (similar to 3 ps). Here we demonstrate similar to 100&#37; duty-cycle dual-comb coherent Raman spectroscopy with a "quasi"-dual-comb source. This is made possible by rapidly modulating the cavity length of one of the frequency combs and accurately measuring the group delay between pump and probe pulses by two-color interferometry for calibrating the phase of each Raman active mode in the sample. Specifically, we use the method to show a record high spectral acquisition rate of 100000 spectra/s with even higher sensitivity than conventional slower dual-comb coherent Raman spectroscopy.

DOI： 10.1021/acsphotonics.0c01656

Language：English   Publishing type：Research paper (scientific journal)  

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for vibrational spectroscopy as it provides several orders of magnitude higher sensitivity than inherently weak spontaneous Raman scattering by exciting localized surface plasmon resonance (LSPR) on metal substrates. However, SERS can be unreliable for biomedical use since it sacrifices reproducibility, uniformity, biocompatibility, and durability due to its strong dependence on "hot spots", large photothermal heat generation, and easy oxidization. Here, we demonstrate the design, fabrication, and use of a metal-free (i.e., LSPR-free), topologically tailored nanostructure composed of porous carbon nanowires in an array as a SERS substrate to overcome all these problems. Specifically, it offers not only high signal enhancement (~106) due to its strong broadband charge-transfer resonance, but also extraordinarily high reproducibility due to the absence of hot spots, high durability due to no oxidization, and high compatibility to biomolecules due to its fluorescence quenching capability.

DOI： 10.1038/s41467-020-18590-7

Language：English   Publishing type：Research paper (scientific journal)  

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to similar to 100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

DOI： 10.1038/s41467-020-17285-3

Language：English   Publishing type：Research paper (scientific journal)  

The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of similar to 2000 events per second and a high sensitivity of similar to 50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

DOI： 10.1039/d0lc00080a

Language：English   Publishing type：Research paper (scientific journal)  

Cellular metabolites are valuable in a diverse range of applications. For example, the unicellular green algaHaematococcus lacustrisproduces as a secondary metabolite the carotenoid pigment astaxanthin (AXT), which is widely used in nutraceutical, cosmetic, and food industries due to its strong antioxidant activity. In order to enhance the productivity ofH. lacustris, spatial and temporal understanding of its metabolic dynamics is essential. Here we show spatiotemporal monitoring of AXT production inH. lacustriscells by resonance Raman microscopy combined with stable isotope labeling. Specifically, we incorporated carbon dioxide ((CO2)-C-13) labeled with a stable isotope (C-13) intoH. lacustriscells through carbon fixation and traced its conversion to(13)C-AXT using our resonance Raman microscope. We incubatedH. lacustriscells under various conditions by switching, pulsing, and replacing(13)CO(2)and(12)CO(2). By measurement of these cells we determined the fixation time of(13)C-carbon, visualized the intracellular localization of(13)C- and(12)C-AXTs, and revealed the dynamic consumption-production equilibrium of the accumulated AXT. This work is a valuable step in the development of effective screening criteria for high AXT-producingH. lacustriscells.

DOI： 10.1039/d0ra02803g

Language：English   Publishing type：Research paper (scientific journal)  

Large-scale label-free single-cell analysis of paramylon in Euglena gracilis by high-throughput broadband Raman flow cytometry.
Microalga-based biomaterial production has attracted attention as a new source of drugs, foods, and biofuels. For enhancing the production efficiency, it is essential to understand its differences between heterogeneous microalgal subpopulations. However, existing techniques are not adequate to address the need due to the lack of single-cell resolution or the inability to perform large-scale analysis and detect small molecules. Here we demonstrated large-scale single-cell analysis of Euglena gracilis (a unicellular microalgal species that produces paramylon as a potential drug for HIV and colon cancer) with our recently developed high-throughput broadband Raman flow cytometer at a throughput of >1,000 cells/s. Specifically, we characterized the intracellular content of paramylon from single-cell Raman spectra of 10,000 E. gracilis cells cultured under five different conditions and found that paramylon contents in E. gracilis cells cultured in an identical condition is given by a log-normal distribution, which is a good model for describing the number of chemicals in a reaction network. The capability of characterizing distribution functions in a label-free manner is an important basis for isolating specific cell populations for synthetic biology via directed evolution based on the intracellular content of metabolites.

DOI： 10.1364/BOE.382957

Language：English   Publishing type：Research paper (scientific journal)  

We experimentally demonstrate compressed time-domain coherent Raman spectroscopy with real-time random sampling. Specifically, we employ Fourier-transform coherent anti-Stokes Raman scattering as a platform for the spectroscopy and demonstrate reconstruction of Raman spectra from sparsely sampled time-domain inter-ferograms using the iterative shrinkage-thresholding algorithm. We show that Raman spectra reconstructed at compression ratios down to 0.3 retain the quality of a fully sampled Raman spectrum. We also verify this performance with multiple molecules to demonstrate its versatility. Advantages of our method include data volume reduction, signal acquisition time reduction, and alleviated requirements for measurement instruments. (C) 2019 Optical Society of America.

DOI： 10.1016/j.vibspec.2020.103042

Language：English   Publishing type：Research paper (scientific journal)  

Raman and fluorescence spectroscopies offer complementary approaches in bioanalytical chemistry, particularly in microbiological assays. The former method is used to detect lipids, metabolites, and nonspecific proteins and nucleic acids in a label-free manner, while the latter is used to investigate targeted proteins, nucleic acids, and their interactions via labeling or transfection. Despite their complementarity, these regimes are seldom used in conjunction due to fluorescent signals overwhelming inherently weak Raman signals by more than several orders of magnitude. Here we report a multimodal spectrometer that simultaneously performs Raman and fluorescence spectroscopies at high speed. It is made possible by Fourier-transform-coherent anti-Stokes Raman scattering (FT-CARS) and Fourier-transform-two-photon excitation (FT-TPE) measurements powered by a femtosecond pulse laser coupled to a homemade rapid-scan Michelson interferometer, operating at 24 000 spectra per second. As a proof-of-principle demonstration, we validate the ultrafast fluoRaman spectrometer by measuring coumarin dyes in organic solvents. To show its potential for applications that require rapid fluoRaman spectroscopy, we also demonstrate fluoRaman flow cytometry of Haematococcus pluvialis cells under varying culture conditions with a high throughput of similar to 10 events per second to perform large-scale single-cell analysis of their metabolic stress response.

DOI： 10.1021/acs.analchem.9b03563

Language：English   Publishing type：Research paper (scientific journal)  

The "fingerprint" (500-1800 cm(-1)) and "high-frequency" (2400-4000 cm(-1)) regions in Raman spectroscopy are commonly used for label-free chemical analysis, while the "low-frequency" region (<200 cm(-1)) is often overlooked, despite containing rich information. This is largely due to the challenge of measuring weak Raman signals that are obscured by strong Rayleigh scattering. Here we propose and experimentally demonstrate Sagnac-enhanced impulsive stimulated Raman scattering (SE-ISRS), a filter-free method for time-domain Raman spectroscopy that overcomes the challenge and provides low-frequency Raman spectra at all probe frequencies. Using SE-ISRS for simultaneous low-frequency and fingerprint region measurements, we demonstrate a >5x enhancement of the signal-to-noise ratio compared to conventional ISRS spectroscopy. (C) 2019 Optical Society of America

DOI： 10.1364/OL.44.005282

Language：English  

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DOI： 10.1038/s41596-019-0252-5

Language：English   Publishing type：Research paper (scientific journal)  

We demonstrate broadband Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectral microscopy with a pixel dwell time of 42 mu s, which is ~50 times shorter than the shortest-to-date pixel dwell time for CARS spectral microscopy. Our broadband FT-CARS spectral microscope is composed of an FT-CARS spectrometer, a rapid galvanometric scanner, and a high-speed image acquisition circuit, enabling a frame rate of 2.4 fps with a pixel resolution of 100 x 100 pixels, a bandwidth of 600-1,200 cm(-1), a spatial resolution of 0.95 mu m, and a spectral resolution of 37 cm(-1). As a proof-of-principle demonstration, we used the high-speed FT-CARS spectral microscope to perform CARS imaging of polymer beads and Haematococcus lacustris cells. Our high-speed broadband CARS spectral microscope holds promise for studying rapid cellular dynamics, such as signaling, cell-to-cell communication, and molecular transport in a label-free manner.

DOI： 10.1002/jrs.5630

Language：English   Publishing type：Research paper (scientific journal)  

Intelligent image-activated cell sorting (iIACS) is a machine-intelligence technology that performs real-time intelligent image-based sorting of single cells with high throughput. iIACS extends beyond the capabilities of fluorescence-activated cell sorting (FACS) from fluorescence intensity profiles of cells to multidimensional images, thereby enabling high-content sorting of cells or cell clusters with unique spatial chemical and morphological traits. Therefore, iIACS serves as an integral part of holistic single-cell analysis by enabling direct links between population-level analysis (flow cytometry), cell-level analysis (microscopy), and gene-level analysis (sequencing). Specifically, iIACS is based on a seamless integration of high-throughput cell microscopy (e.g., multicolor fluorescence imaging, bright-field imaging), cell focusing, cell sorting, and deep learning on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, intelligent decision making, and actuation. Here, we provide a practical guide to iIACS that describes how to design, build, characterize, and use an iIACS machine. The guide includes the consideration of several important design parameters, such as throughput, sensitivity, dynamic range, image quality, sort purity, and sort yield; the development and integration of optical, microfluidic, electrical, computational, and mechanical components; and the characterization and practical usage of the integrated system. Assuming that all components are readily available, a team of several researchers experienced in optics, electronics, digital signal processing, microfluidics, mechatronics, and flow cytometry can complete this protocol in similar to 3 months.

DOI： 10.1038/s41596-019-0183-1

Language：English   Publishing type：Research paper (scientific journal)  

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm(-1)) with a record--high throughput of similar to 2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.

DOI： 10.1126/sciadv.aau0241

Language：Japanese   Publishing type：Research paper (scientific journal)  

Ultrafast chiroptical spectroscopy

Language：English   Publishing type：Research paper (scientific journal)  

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

DOI： 10.1016/j.cell.2018.08.028

Language：English   Publishing type：Research paper (scientific journal)  

Label-free particle analysis is a powerful tool in chemical, pharmaceutical, and cosmetic industries as well as in basic sciences, but its throughput is significantly lower than that of fluorescence-based counterparts. Here we present a label-free single-particle analyzer based on broadband dual-comb coherent Raman scattering spectroscopy operating at a spectroscopic scan rate of 10 kHz. As a proof-of-concept demonstration, we perform broadband coherent anti-Stokes Raman scattering measurements of polystyrene microparticles flowing on an acoustofluidic chip at a high throughput of >1000 particles per second. This high-throughput label-free particle analyzer has the potential for high-precision statistical analysis of a large number of microparticles including biological cells. (C) 2018 Optical Society of America

DOI： 10.1364/OL.43.004057

Language：English   Publishing type：Research paper (scientific journal)  

In this study, we developed a hyper-Raman (HR) microscopic system using an optical parametric oscillator (OPO) pumped by a picosecond mode-locked Nd:YVO4 laser source. Owing to the wavelength tunability of OPO, the two-photon electronic resonance effect can be investigated in a broad spectral range of 345-495 nm. As a proof-of-principle experiment, we performed HR imaging of TiO2 single crystals. In addition to HR, second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) were observed simultaneously depending on the excitation wavelength.

DOI： 10.1246/cl.180077

Language：English   Publishing type：Research paper (scientific journal)  

High-speed Raman spectroscopy has become increasingly important for analyzing chemical dynamics in real time. To address the need, rapid-scan Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy has been developed to realize broadband CARS measurements at a scan rate of more than 20,000 scans/s. However, the detection sensitivity of FT-CARS spectroscopy is inherently low due to the limited number of photons detected during each scan. In this Letter, we show our experimental demonstration of enhanced sensitivity in rapid-scan FT-CARS spectroscopy by heterodyne detection. Specifically, we implemented heterodyne detection by superposing the CARS electric field with an external local oscillator (LO) for their interference. The CARS signal was amplified by simply increasing the power of the LO without the need for increasing the incident power onto the sample. Consequently, we achieved enhancement in signal intensity and the signal-to-noise ratio by factors of 39 and 5, respectively, compared to FT-CARS spectroscopy with homodyne detection. The sensitivity-improved rapid-scan FT-CARS spectroscopy is expected to enable the sensitive real-time observation of chemical dynamics in a broad range of settings, such as combustion engines and live biological cells. (C) 2017 Optical Society of America

DOI： 10.1364/OL.42.004335

Language：English   Publishing type：Research paper (scientific journal)  

We developed a Raman optical activity (ROA) spectroscopic system with visible-excited coherent anti-Stokes Raman scattering (CARS). A supercontinuum within the visible region was generated with a photonic crystal fiber pumped with both 532 and 1064 nm excitation, generating a multiplexed CARS-ROA spectrum covering the whole fingerprint region. In visible excitation, the CARS-ROA spectrum of (-)-beta-pinene shows a higher contrast ratio of the chirality-induced signal to the achiral background than that of the previously reported near-infrared CARS-ROA spectrum. (C) 2015 Optical Society of America

DOI： 10.1364/OL.40.004170

Language：English   Publishing type：Research paper (scientific journal)  

We report the development of broadband and sensitive time-resolved circular dichroism (TRCD) spectroscopy by exploiting optical heterodyne detection. Using this method, transient CD signals of submillidegree level can be detected over the spectral range of 415-730 nm. We also demonstrate that the broadband measurement with the aid of singular value decomposition enables the discrimination of genuine TRCD signals from artificial optical-anisotropy, such as linear birefringence and linear dichroism, induced by photoexcitation. (C) 2015 AIP Publishing LLC.

DOI： 10.1063/1.4932229

Language：English   Publishing type：Research paper (scientific journal)  

We demonstrate a method to measure Raman optical activity (ROA) by using coherent anti-Stokes Raman scattering (CARS) spectral interferometry. An extremely weak chirality-induced CARS field is amplified through the interference with a strong CARS field generated from an external reference and is extracted by the Fourier transformation. In this interferometric coherent Raman optical activity (iCROA), both the sign and the magnitude of optical active non-resonant background susceptibility can be directly determined. Measurement of a CARS-ROA spectrum with less artifact is obtained because a broad offset artifact due to optical rotatory dispersion is clearly distinguished in iCROA. (C) 2013 Optical Society of America

DOI： 10.1364/OE.21.013515

Language：English   Publishing type：Research paper (scientific journal)  

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 (-)-beta-pinene, it is 2 orders of magnitude better than that in the reported spontaneous ROA measurement. This is also the first measurement of ROA signal using a pulsed laser source.

DOI： 10.1103/PhysRevLett.109.083901

Event date： 2023.6

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Broadband fluorescence-encoded vibrational imaging

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High-speed FT-CARS spectroscopy for high-throughput phenotyping

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Event date： 2022.9

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Fluorescence-Encoded Time-Domain Coherent Raman Spectroscopy

Event date： 2022.8

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Super-multiplex flow cytometry by cyanine-based Raman tags

Event date： 2022.8

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Label-free characterization of rare-cell populations by high-throughput Raman flow cytometry

Event date： 2022.8

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Gold nanomesh for wearable SERS

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Gold nanomesh for wearable SERS

Event date： 2022.8

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Event date： 2022.6

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Unconventional modalities in flow cytometry FLIM, Raman, SHG, etc.

Event date： 2022.6

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Observation of Raman optical activity with polarization-resolved heterodyne coherent anti-Stokes Raman scattering

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Observation of molecular chirality by CARS-ROA

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Shot-noise limited detection of CARS-ROA

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Raman optical activity measured by CARS spectral interferometry

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Raman optical activity measured by nonlinear Raman scattering

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Raman optical activity measured by coherent anti-Stokes Raman scattering

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Development of Raman optical activity spectroscopy with cohernet Raman scattering

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Molecular chirality probed by coherent Raman scattering

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Coherent Raman optical activity spectroscopy

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Development of achiral-background-free chiroptical spectroscopies with heterodyne detection

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Development of broadband and sensitive femtosecond time-resolved circular dichroism spectroscopy

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Development of broadband and sensitive femtosecond time-resolved circular dichroism spectroscopy by using spectral interferometry

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Visible-excited CARS-ROA spectroscopy

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Broadband and ultrasensitive femtosecond time-resolved circular dichroism spectroscopy using spectral interferometry

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Excited-state dynamics of biomolecules studied by time-resolved circular dichroism spectroscopy

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Rapid-Scan Fourier-Transform CARS Spectroscopy with Heterodyne Detection

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Development and application of ultrafast chiroptical spectroscopy

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Rapid Fourier-transform CARS microscopy

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Coherent ROA spectroscopy

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High-speed broadband Fourier-transform CARS microscopy

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Heterodyne-detected rapid-scan Fourier-transform CARS spectroscopy

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High-speed Fourier-transform coherent anti-Stokes Raman scattering spectroscopy with heterodyne detection

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Simultaneous FT-CARS and fluorescence spectroscopy at 24,000 spectra per second

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Time-domain Raman spectroscopy and its application to biological research

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Microfluidic particle analysis with dual-comb coherent Raman spectroscopy

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High-speed simultaneous Raman-fluorescence spectrometer

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High-throughput broadband CARS flow cytometry at >2,000 cells/s

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High-speed simultaneous CARS and fluorescence spectroscopies

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Coherent ROA spectroscopy

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Intelligent Image-Activated Cell Sorting: Principles and Applications

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High-throughput broadband Raman flow cytometry

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Label-free molecularly specific flow cytometry and beyond

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High-throughput Raman spectroscopic flow cytometry"

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Coherent Raman spectroscopic flow cytometry for large-scale single cell analysis

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Simultaneous Raman-Fluorescence Flow Cytometry

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Large-scale single-cell analysis by high-speed Raman spectroscopy

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Country：China  

Large-scale single-cell analysis by ultra-rapid time-domain Raman spectroscopy

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High-throughput Raman flow cytometry

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High-throughput Broadband Vibrational Flow Cytometry

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Multiplex spectroscopy for large-scale single-cell analysis

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High-throughput vibrational spectroscopic flow cytometry

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Highly sensitive low-frequency Raman spectroscopy enabled by Sagnac-enhanced impulsive stimulated Raman scattering

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High-throughput vibrational flow cytometry

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High-throughput multimodal Raman-fluorescence flow cytometry

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High-speed low-frequency vibrational spectroscopy using a Sagnac interferometer

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Fluorescence-encoded time-domain coherent Raman spectroscopy through single-photon counting

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Fast, sensitive dual-comb CARS spectroscopy with a quasi-dual-comb laser

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Raman spectroscopy and imaging of single cells in flow

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Fluorescence-Encoded Time-Domain Coherent Raman Spectroscopy

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Multi-cell analysis by high-throughput Raman flow cytometry

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Super-sensitive Raman spectroscopy by fluorescence encoding

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THz-fingerprint coherent Raman spectroscopy at over 20,000 spectra/sec

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Raman image-activated cell sorting and beyond

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Raman flow cytometry on a chip

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Porous carbon nanowires for plasmon-free SERS

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High-speed Fourier-transform spectroscopy by non-mechanical cavity modulation

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Chiral-field-enhanced Raman optical activity by a silicon nanodisk array

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Ultra-broadband time-domain Raman spectroscopy using synchronized mode-locked lasers

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Language：English   Presentation type：Oral presentation (invited, special)  

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Authorship：Lead author,　Corresponding author  

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CiNii Research

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Type：Peer review 

Number of peer-reviewed articles in foreign language journals：4

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：6

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：7

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：4

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1