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Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer.

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter's small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Waseda University, National Institute for Japanese Language and Linguistics, 会議名: 言語資源活用ワークショップ2021, 開催地: オンライン, 会期: 2021年9月13日-14日, 主催: 国立国語研究所 コーパス開発センター, 近年, 音声の調音運動をMRI装置を用いて, リアルタイムに撮像することが可能になった.リアルタイムMRI(以下rtMRI)データは, 声道の正中矢状面全体の情報が含まれ, 調音音声学の再構築を促す可能性を秘めている. しかし, 収集されたデータに対する転記方法や分析方法は整備されているとは言いづらく, 単にデータを公開するだけではrtMRIデータに基づいた調音音声学研究は普及しにくいと予想される. そのため, 我々は過去にrtMRIデータ閲覧ツールとしてMRI Vuewerの設計と開発を行った. 上記ツールはrtMRIデータの音声的, 時間的側面の転記機能を有するものであったが, 画像的, 空間的側面の転記機能に不足があった. 本研究では近年行われたrtMRIデータを用いた研究から画像的, 空間的側面の転記に必要な機能を整理し, rtMRIデータ解析ツールとしてMRI Vuewerの再設計と再実装した結果を報告する.

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 ~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., Most current cell sorting methods are based on fluorescence detection with no imaging capability. Here the authors generate and use Raman image-activated cell sorting with a throughput of around 100 events per second, providing molecular images with no need for labeling.

Phototrophs assimilate CO2 into organic compounds that accumulate in storage organelles. Elucidation of the carbon dynamics of storage organelles could enhance the production efficiency of valuable compounds and facilitate the screening of strains with high photosynthetic activity. To comprehensively elucidate the carbon dynamics of these organelles, the intraorganellar distribution of the carbon atoms that accumulate at specific time periods should be probed. In this study, the biosynthesis of polysaccharides in storage organdies was spatiotemporally probed via stimulated Raman scattering (SRS) microscopy using a stable isotope (C-13) as the tracking probe. Paramylon granules (a storage organelle of beta-1,3-glucan) accumulated in a unicellular photosynthetic alga, Euglena gracilis, were investigated as a model organelle. The carbon source of the culture medium was switched from NaH12CO3 to NaH13CO3 during the production of the paramylon granules; this resulted in the distribution of the C-12 and C-13 constituents in the granules, so that the biosynthetic process could be tracked. Taking advantage of high-resolution SRS imaging and label switching, the localization of the C-12 and C-13 constituents inside a single paramylon granule could be visualized in three dimensions, thus revealing the growth process of paramylon granules. We propose that this method can be used for comprehensive elucidation of the dynamic activities of storage organelles.

National Institute for Japanese Language and Linguistics, Waseda University, ATR-Promotions, Chiba Institute of Technology, Konan University, Takushoku University, Picolab, 会議名: 言語資源活用ワークショップ2020, 開催地: オンライン, 会期: 2020年9月8日−9日, 主催: 国立国語研究所 コーパス開発センター, われわれは、日本語に関する調音音声学的研究の新しいインフラ提供をめざして、調音運動データベースの構築を進めてきている。声道全体の形状変化を毎秒14 ないし25 フレームのリアルタイムMRI 動画として記録したデータが、現時点で東京方言16 名、近畿方言5名分収録済である。1 名あたりの発話量は25〜30 分である。データには個々の発話の開始時刻と終了時刻のタグが付与されており、他に発話内容と話者に関するメタデータを検索に利用できる。

Diphenylacetylene derivatives containing different polymeric components, poly(l-lysine) (pLys) or tetra(ethylene glycol) (TEG) were designed as novel Raman imaging probes with high Raman sensitivity and low cytotoxicity in living plant cells. The pLys-conjugated probe is internalized via an endocytosis-dependent pathway, whereas TEG-conjugated probe most likely induces direct penetration into the plant cells., Diphenyl acetylene derivatives containing various polymeric components have been designed as new Raman imaging probes. These are taken up by plant cells via different pathways, and the internalization of exogenous molecules can be visualized.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae, Euglena gracilis, by semiclosed microchannels with liquid flow only. The isolated single cells were identified in isolating channels and continuously cultured to track, by Raman microscopy, for the formation of subcellular granules composed of polysaccharide paramylon, a unique metabolite of E. gracilis, generated through photosynthesis. Through low-temperature glass bonding, a thin glass interface was incorporated to the microfluidic device. Thus, the device could perform the direct measurements of cultured single cells at high magnification by Raman microscopy with low background noise. In this study, the first demonstration of sequential monitoring of paramylon biogenesis in a single identified E. gracilis cell is shown.

Recent development of stimulated Raman scattering (SRS) microscopy allows for label-free biological imaging with chemical specificity based on molecular-vibrational signatures. In particular, hyperspectral SRS imaging can acquire a molecular-vibrational spectrum at each pixel, allowing us not only to investigate the spectral difference of various biological molecules but also to discriminate different constituents based on their spectral difference. However, the number of constituents discriminated in previous label-free SRS imaging was limited to four because of the subtleness of spectral difference. Here, we report hyperspectral SRS imaging of plant tissues including leaves of Camellia japonica, roots of Arabidopsis thaliana, and thalli of a liverwort Marchantia polymorpha L. We show that SRS can discriminate as many as six components in Marchantia polymorpha L. without labeling. Our results demonstrate the effectiveness of hyperspectral SRS imaging as a tool for label-free multicolour imaging analysis of various biomolecules in plant tissues.

Imaging of biological cells and tissues with subcellular spatial resolution is important in biology and medicine because it allows us to explore the dynamics of cells and to diagnose the structure of tissues. Among various optical imaging modalities, laser microscopy with fluorescent staining is a powerful method for this purpose. However, it still suffers from the limited applicability and cytotoxicity of a staining process. Stimulated Raman scattering (SRS) microscopy is an emerging technique of biological imaging based on molecular vibrational contrast, offering new opportunities of biomedical microscopy. In this paper, we introduce the principle and applications of SRS microscopy and discuss various methods of spectral imaging with SRS microscopy. Then, we present our multicolor SRS microscope, which employs a fast wavelength-tunable picosecond Yb fiber laser and a picosecond Ti:sapphire laser. In particular, we describe the technical details of the Yb fiber oscillator, synchronization electronics, high-speed wavelength-tunable filter, video-rate microscope setup and signal detection electronics including the photodetector and the lock-in amplifier. We hope that this paper will be informative for those interested in the application of ultrafast lasers to coherent Raman microscopy including SRS microscopy.

Epidermal keratinocyte (KC) differentiation, which involves the process from proliferation to cell death for shedding the outermost layer of skin, is crucial for the barrier function of skin. Therefore, in dermatology, it is important to elucidate the epidermal KC differentiation process to evaluate the symptom level of diseases and skin conditions. Previous dermatological studies used staining or labelling techniques for this purpose, but they have technological limitations for revealing the entire process of epidermal KC differentiation, especially when applied to humans. Here, we demonstrate label-free visualization of three-dimensional (3D) intracellular morphological changes of ex vivo human epidermis during epidermal KC differentiation using stimulated Raman scattering (SRS) microscopy. Specifically, we observed changes in nuclei during the initial enucleation process in which the nucleus is digested prior to flattening. Furthermore, we found holes left behind by improperly digested nuclei in the stratum corneum, suggesting abnormal differentiation. Our findings indicate the great potential of SRS microscopy for discrimination of the degree of epidermal KC differentiation.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae

会議名: 言語資源活用ワークショップ2016, 開催地: 国立国語研究所, 会期: 2017年3月7日-8日, 主催: 国立国語研究所 コーパス開発センター, 喉頭音源由来の声質の違いは，話者のパラ言語メッセージならびに心的・認知的状態を伝えるシグナルであり，自発音声コーパスに求められる重要な情報であるが，そのアノテーションは音声学の専門家でなければ難しくコストが大きい。本研究は，機械学習による声質の自動アノテーションの可能性を探ることを目的とする。本研究では，非流暢性にも関連する従来よく用いられてきた発見的な音響特徴量に加え，近年音声からの感情認識で広く用いられるようになった大規模な特徴量セットの効果を検証した結果を報告する。

We have developed a two-way multi-view 2-D/3-D display combining a liquid crystal lens and horizontally and vertically x times-density pixels (HVxDP) arrangement. The two-way multi-view display features the same display resolution in 2-D and 3-D modes and a quite small color moire for landscape and portrait, respectively, when using the HVxDP arrangement. In this paper, we realized suitable 3-D properties for achieving a good balance between 3-D moire and 3-D crosstalk for landscape and portrait by a two-way liquid crystal lens with two kinds of focal lengths for the edge part and the center part of the lens.

The management of oily wastewater produced in the petrochemical industries poses an immense challenge from both practical and environmental points of view. Filtration using ceramic membranes has been considered a promising technology to remove stable emulsions from oily wastewater; however, the separation performances are severely limited by membrane fouling. In this study, we evaluated the filtration performance of porous ceramic membranes for the treatment of oil/water emulsions. Porous ceramic membrane, having different pore sizes (0.1–1.4 μm) and made from various materials (Al2O3, TiO2–ZrO2, TiO2, and SiC), were tested in a crossflow microfiltration system using oil/water emulsions. The emulsions were stabilized by anionic and cationic surfactants. The fouling caused by filtration is discussed in terms if membrane and emulsion properties. Pore size and surface charge were the two most important prominent fouling factors during the oil/water emulsion filtrations. For membranes with small pores, serious fouling was observed, irrespective of the type of surfactant; however, these membranes exhibited excellent oil rejection. The fouling was caused by the accumulation of oil droplets on the membrane surface, which subsequently led to the formation of a cake layer and/or a continuous oil layer. The effect of surface charge became significant regarding membranes of larger pore sizes, especially when membrane pore sizes were comparable to that of the droplet sizes. When the membrane and oil droplets had the same charge, electrostatic repulsion occurred, preventing the penetration of oil droplets into the pores. Although some of the small oil droplets could enter the pores, they would be trapped at the narrowing and/or flexing inside the pores because droplet deformation was inhibited by the electrostatic repulsion. Therefore, fouling occurred on the membrane surface and inside. As a result, a severe decline in water permeation was observed, together with a moderate oil rejection. By contrast, when the membrane and oil droplets were oppositely charged, oil droplets were deformed and entered the pores, resulting in permeation through the membrane. Therefore, water permeation was high, but oil rejection was sacrificed. These results indicate that both pore size and surface charge of membranes should be optimized to achieve better filtration performance.

We present an environmentally stable, high-speed wavelength-tunable laser system with a tunability of >280 cm−1. This laser is synchronized to a picosecond Ti:sapphire laser to demonstrate video-rate hyperspectral stimulated Raman imaging of polymer beads.

To maximize excellent property of hollow nanoparticles, it is important to determine physical property of shell because it directly affects functionality of the final product. In this study, hollow silica nanoparticles (HSNPs) with different shell structure were prepared based on sol–gel reaction using polystyrene (PS) beads template. As physical property, apparent shell density was determined using specific surface area and shell thickness. The obtained HSNPs were categorized into three types of (A) thickness of silica shell is relatively thin and spherical shape of the stacked particulates is clearly identified in the shell, (B) thickness of silica shell is similarly thin to type A, but shape of the stacked particulates is identified as half-round shape, and (C) thickness of silica shell is thick and the stacked particulates are not recognized in the shell, which were controlled by suspension pH and reaction time. Apparent shell density of types A, B, C appeared in the 1.4–2.2 g/cm 3 and it increased with increase in pH and/or reaction time. This must be useful technique to design hollow silica nanoparticles.

We present the temperature--pressure--magnetic-field phase diagram of the itinerant-electron metamagnet UCoAl determined from the ac susceptibility measurement. The quantum critical point (QCP) was found to be located at $2.9\ifmmode\pm\else\textpm\fi{}0.2$ GPa ($\ensuremath{\equiv}{P}_{\mathrm{QCP}}$) and $130\ifmmode\pm\else\textpm\fi{}5$ kOe. In addition to the determination of the phase diagram, we report magnetic and transport properties in the paramagnetic state and around the phase boundary. The critical divergence of the magnetic susceptibility is suppressed toward the QCP. From the longitudinal magnetoresistance and the resistivity under magnetic fields, enhancement of the resistivity is observed in the so-called supercritical region. In the paramagnetic region, the magnetoresistance and temperature dependence of the resistivity exhibit complex behaviors, especially in the vicinity of 1.5 GPa and ${P}_{\mathrm{QCP}}$.

We have performed the de Haas–van Alphen (dHvA) experiment to elucidate the Fermi surface (FS) property of the typical localized 5 f -electron system of UPd 3 using a high-quality sample (RRR = 100). Many dHvA branches are newly observed. The FS topology is discussed comparing the angular dependence of the dHvA frequency and the two different band models. An anomaly due to the quadrupolar or magnetic interaction can not be found in the observed dHvA signal as well as the ac susceptibility up to 17.5 T.

We have studied the effect of an alternating magnetic field on the growth rate of primary roots in Zea mays. Corn seedlings were grown in the dark, under constant conditions of temperature (25°C) and humidity (100%). They were also subjected to a 5 mT magnetic field, alternating at frequencies of either 5, 10, 20 or 40 Hz. The rate of primary root growth, in plants grown at each frequency, was measured and compared to a control group. Control plants were grown under similar conditions, in the absence of the magnetic field. The growth rate of primary roots in seedlings grown at each of the magnetic field frequencies used, was increased compared to the control group. The highest growth rate was seen in seedlings exposed to a magnetic field alternating at 10 Hz.