Yasuyuki Ozeki - Academia.edu (original) (raw)

Papers by Yasuyuki Ozeki

The Journal of Physical Chemistry B, 1998

Excited states of free base chlorin (FBC), free base Bacteriochlorin (FBBC), pheophytin a (Pheo a... more Excited states of free base chlorin (FBC), free base Bacteriochlorin (FBBC), pheophytin a (Pheo a), and chlorophyll a (Chlo a), which are derivatives of free base porphine (FBP), were calculated by the SAC (symmetry adapted cluster)/SAC-CI (configuration interaction) method. The results reproduced well the experimentally determined excitation energies. The reduction of the outer double bonds in the porphine ring in the order of FBP, FBC, and FBBC causes a breakdown of the symmetry and a narrowing of the HOMO-LUMO gap, which result in a red shift of the Q x band and an increase of its intensity. In the change from Pheo a to Chlo a, the Mg coordination reduces the quasidegeneracy in the Q x state and then increases the spectral intensity. The disappearance of the Q y humps from the absorption spectrum of Pheo a, compared with that of Chlo a, is due to the red shift of the Q y state.

Conference on Lasers and Electro-Optics, 2019

We present a groundbreaking machine intelligence technology called "intelligent image-activated c... more We present a groundbreaking machine intelligence technology called "intelligent image-activated cell sorting" that achieves high-throughput image-triggered sorting of single cells by integrating high-speed fluorescence microscopy, cell focusing, cell sorting, and deep learning.

RSC Chemical Biology, 2020

Diphenyl acetylene derivatives containing various polymeric components have been designed as new ... more 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.

The Japan Society of Applied Physics, 2016

はじめに:ファイバレーザーを用いた光周波数コムは,小型・安定でほぼメンテナンスフリーと いった特徴を持つ実用的なコム光源として期待されている.しかし,通常の光ファイバは曲げや 捻り,温度変化など... more はじめに:ファイバレーザーを用いた光周波数コムは,小型・安定でほぼメンテナンスフリーと いった特徴を持つ実用的なコム光源として期待されている.しかし,通常の光ファイバは曲げや 捻り,温度変化などの外乱により伝搬する光の偏光状態が変化し,出力特性が不安定になるとい う問題がある.本研究では,安定・小型な実用的な光周波数コムを実現するため,単層カーボン ナノチューブ(SWNT)を用いた超短パルスファイバレー ザーをベースとした,全偏波保持型の光周波数コム光源 を開発し,光周波数コムの安定化制御に必要な fceo(carrier envelope offset frequency)の観測を行った. 実験と結果:本研究の実験系を Fig.1 に示す.ここで用い られている全てのファイバ,素子は偏波保持型のもので あり,空間に光を出さないために融着またはアダプタに より接続されている.はじめに,単層カーボンナノチュー ブ(SWNT)を用いた全偏波保持ファイバレーザーからの種光 をシミラリトン増幅した.次に,増幅されたパルスを偏波保 持シングルモードファイバ(PM-SMF)で分散補償することに より時間幅 41 fs,ピーク強度 71 kW の高強度超短パルスを 生成した(Fig.2).ここでは,台座成分が小さく,ピーク強度 が強くなるようファイバ長を最適化した.そして,生成した 高強度超短パルスを偏波保持高非線形分散シフトファイバ (PM-HN-DSF)に入射することにより波長 1.0~2.2 μm の 1 オ クターブ以上に広がるスーパーコンティニューム(SC)光を 生成した(Fig.3).この SC 光を導波路型の周期的分極反転ニ オブ酸リチウム(PPLN)結晶に入射することにより 2.13 μm の光に対する第二高調波を生成した.第二高調波と SC 光に 存在する 1.06 μm 成分を干渉させることで fceoを観測した. Fig.4 に観測した fceoの RF スペクトルを示す.励起強度を最 適化した際には,フリーラン時の fceo の線幅が 10 kHz とい う結果が得られた.fceo の周波数は時間的に安定な振る舞い を示した. Fig. 3 Spectrum of octave spanning SC. Fig. 1 Experimental set...

The Japan Society of Applied Physics, 2016

近年、生体内のサブミリ秒オーダの高速なダイナミクスの観察や、多数の細胞集団を解析する フローサイトメトリへの応用などの要求に伴い、蛍光共焦点顕微鏡に周波数分割多重化の概念を 導入することで高速化... more 近年、生体内のサブミリ秒オーダの高速なダイナミクスの観察や、多数の細胞集団を解析する フローサイトメトリへの応用などの要求に伴い、蛍光共焦点顕微鏡に周波数分割多重化の概念を 導入することで高速化を狙った方式が複数提案されている。これらの方式は、互いに異なる周波 数で変調された多数のレーザ光を同時に試料に集光し、それらによる発生する蛍光を単一の検出 器(光電子増倍管など)で一括して検出したのち、周波数領域で各成分を分離、抽出して画像を 再構成する。並列化を行うためにスキャナの走査軸を 1 つ省略できることにより、従来よりも大 幅な高速化が可能である。特に、マルチビームの生成に音響光学ディフレクタ(AOD)を使用す る Fluorescence Imaging by Radiofrequency-tagged Emission (FIRE)法(図 1)が速度性能の点で優れて いるが、速度性能は上記 AODの変調帯域により制限され、さらなる高速化が望まれる[1]。 我々は上記の AOD による帯域制限を打破し、従来 FIRE 法に比べて 2 倍の速度性能を有する dual-AOD FIRE法を提案する。本方式では 2つの AODからそれぞれマルチビームを生成し、それ らを重ねることによって変調されたマルチビームを生成する(図 1)。これにより被測定信号の帯域 が AODの帯域に対して 2倍となるため、2倍高速な撮像が可能となる。本方式を用いてフレーム レート 16,000 フレーム/秒での蛍光画像の取得に成功している(図 2)。本講演では、dual-AOD FIRE 法において並列取得するピクセル数を増大した場合に隣接ピクセル間のクロストークによるアー ティファクトが発生しうることを示し、AODへの駆動信号を適切に設定することで本クロストー クを光学的にキャンセルする方法の実証について報告する。

ABSTRACTBy virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging f... more ABSTRACTBy virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. For example, at high flow speed (i.e., high throughput), the integration time of the image sensor becomes short, resulting in reduced sensitivity or pixel resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually “freezing” the motion of flowing cells on the image sensor to effectively achieve 1,000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells/s without sacrificing sensitivity and spatial resolution. T...

Cell, Jan 23, 2018

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly... more 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, resp...

IEEE Photonics Journal, 2017

Optical time-stretch microscopy has recently attracted intensive attention for its capability of ... more Optical time-stretch microscopy has recently attracted intensive attention for its capability of acquiring images at an ultrahigh frame rate. Unfortunately, its achievable frame rate is limited by the requirement of having no overlap between consecutive frames, which leads to a tradeoff between the frame rate (pulse repetition rate) and the amount of the temporal dispersion used for optical image serialization. In this paper, we demonstrate compressive sensing on the platform of optical time-stretch microscopy to overcome the tradeoff between frame rate and temporal dispersion (time stretch) and achieve 50 times higher frame rate than conventional optical time-stretch microscopy. Specifically, we computationally perform compressed optical time-stretch microscopy with an experimental dataset acquired by conventional optical time-stretch microscopy and demonstrate its effects in terms of spatial resolution and cell classification accuracy. Our results indicate that the spatial resolution and cell classification accuracy reach 780 nm and 95% at a line scan rate of 675 MHz and 6.75 GHz, respectively, which correspond to five times and 50 times higher frame rates than what conventional optical time-stretch microscopy can achieve with the same dispersion amount and digitizer sampling rate.

Seibutsu Butsuri, 2017

Recent technological advances have enabled microalgae-based engineering of bioproducts. Microalga... more Recent technological advances have enabled microalgae-based engineering of bioproducts. Microalgae have a higher photosynthetic efficiency than plants and can be cultivated in agriculturally unused land, which has helped us mass-produce microalgae such as Euglena gracilis. E. gracilis is a unicellular microalgal species found in freshwater and is an attractive species for producing lipids which can be converted into biofuels. However, its lipid production efficiency is low and unable to meet high demands from the competitive fuel market. In this article, we review a few innovative methods for developing super biofuels based on super microalgae that are anticipated to overcome the low lipid production efficiency.

Scientific Reports, 2017

In the last decade, high-content screening based on multivariate single-cell imaging has been pro... more In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological ...

PloS one, 2016

The development of reliable, sustainable, and economical sources of alternative fuels is an impor... more The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a metho...

Cytometry. Part A : the journal of the International Society for Analytical Cytology, Jan 11, 2017

The development of reliable, sustainable, and economical sources of alternative fuels to petroleu... more The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is ...

Scientific reports, Jan 24, 2017

A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the ... more A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm(-1) was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the si...

Journal of biomedical optics, 2016

Visualization of epidermal cells is important because the differentiation patterns of keratinocyt... more Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

Biomedical Optics Express, 2016

We demonstrate high-throughput label-free single-cell image cytometry and image-based classificat... more We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our highthroughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-MHz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

2011 10th Euro-American Workshop on Information Optics, 2011

Huntington's disease, a major neurodegenerative disorder, involves expression and deposition of h... more Huntington's disease, a major neurodegenerative disorder, involves expression and deposition of highly aggregation-prone proteins with a long polyglutamine (polyQ) expansion. However, whether these intracellular aggregations are pathological or protective still remains elusive. Here, we propose stimulated Raman scattering (SRS) imaging of deuterium-labeled glutamine to investigate native polyQ aggregations in live cells with subcellular resolution. Technically, enrichment of deuterated glutamine in polyQ sequence enables sensitive and specific SRS imaging of carbon-deuterium bonds (C-D) from the aggregates without GFP labeling, as typically required for fluorescence microscopy. Biologically, we first reveal that these aggregates become 1.8 times denser without GFP, confirming its perturbation. Second, with SRS quantification, we discover a two-stage aggregation formation model, in which small aggregates are dominated by cytosolic proteins in the first stage, but then prefer to sequester polyQ proteins with size growth. This suggests that small aggregates are cytotoxic while large aggregates play a protective role. Third, we illustrate that polyQ aggregations are not a highly dense solid structure by calculating their absolute protein concentrations to be around millimolar range. Furthermore, hyperspectral SRS (hSRS) mapping of C-D vibrations reveals spectroscopic features corresponding to aggregation formation. This is largely absent in label-free SRS or SRS imaging of other non-glutamine amino acid probes. Applying hSRS to interrogating polyQ aggregations by inducing heat shock responses indicates a conformational intermediate with a changed hydrogen-bonding network. Altogether, our quantitative SRS imaging and spectroscopic analysis strategy offers new structural-functional insights for the molecular role of polyQ aggregates.

Journal of Laser Micro/Nanoengineering, 2012

This report describes fabrication of volume gratings inside various matrix polymers using femtose... more This report describes fabrication of volume gratings inside various matrix polymers using femtosecond laser irradiation at a wavelength of 400 nm. We investigated the diffraction efficiencies of volume gratings with a period of 10 μm by focusing 400-nm laser pulses with a 0.1-numerical aperture objective lens. We compared the polymers' diffraction efficiencies at 400 nm and 800 nm. The largest refractive index change was induced in polymers with low density. The large refractive index change was attributed to its volume contraction based on its low density.

JSAP-OSA Joint Symposia 2014 Abstracts, 2014

Stimulated Raman scattering (SRS) microscope has enabled label-free rapid Raman spectral imaging.... more Stimulated Raman scattering (SRS) microscope has enabled label-free rapid Raman spectral imaging. Recent studies have reported video-rate imaging with a pixel clock of over 10 MHz [1, 2]. However, these systems require expensive and bulky devices such as a solid-state laser or an optical parametric amplifier. On the other hand, fiber lasers are used for low-cost and compact systems although they are noisy compared with solid-state lasers. In order to reduce the noise, collinear balanced detection (CBD) has been proposed [3], although it has been performed at low data acquisition rate. Here, we demonstrate video-rate spectral imaging with a home-made Er-doped fiber laser (Er-FL) using the CBD technique.

Physical Review Letters, 2004

We show that modulational instability may arise even in the normal group-velocity dispersion regi... more We show that modulational instability may arise even in the normal group-velocity dispersion regime of an optical fiber when the fiber loss (gain) varies depending on the wavelength. A simple analytical expression for the instability gain is obtained, which reveals that the odd-order terms of the loss dispersion are responsible for this phenomenon. The instability gain is measured experimentally in an optical-parametric-amplification configuration. Large parametric gain is induced in a non-phasematched regime as we apply narrow band loss at the idler wavelength.

The Journal of Physical Chemistry B, 1998

Excited states of free base chlorin (FBC), free base Bacteriochlorin (FBBC), pheophytin a (Pheo a... more Excited states of free base chlorin (FBC), free base Bacteriochlorin (FBBC), pheophytin a (Pheo a), and chlorophyll a (Chlo a), which are derivatives of free base porphine (FBP), were calculated by the SAC (symmetry adapted cluster)/SAC-CI (configuration interaction) method. The results reproduced well the experimentally determined excitation energies. The reduction of the outer double bonds in the porphine ring in the order of FBP, FBC, and FBBC causes a breakdown of the symmetry and a narrowing of the HOMO-LUMO gap, which result in a red shift of the Q x band and an increase of its intensity. In the change from Pheo a to Chlo a, the Mg coordination reduces the quasidegeneracy in the Q x state and then increases the spectral intensity. The disappearance of the Q y humps from the absorption spectrum of Pheo a, compared with that of Chlo a, is due to the red shift of the Q y state.

Conference on Lasers and Electro-Optics, 2019

We present a groundbreaking machine intelligence technology called "intelligent image-activated c... more We present a groundbreaking machine intelligence technology called "intelligent image-activated cell sorting" that achieves high-throughput image-triggered sorting of single cells by integrating high-speed fluorescence microscopy, cell focusing, cell sorting, and deep learning.

RSC Chemical Biology, 2020

Diphenyl acetylene derivatives containing various polymeric components have been designed as new ... more 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.

The Japan Society of Applied Physics, 2016

はじめに:ファイバレーザーを用いた光周波数コムは,小型・安定でほぼメンテナンスフリーと いった特徴を持つ実用的なコム光源として期待されている.しかし,通常の光ファイバは曲げや 捻り,温度変化など... more はじめに:ファイバレーザーを用いた光周波数コムは,小型・安定でほぼメンテナンスフリーと いった特徴を持つ実用的なコム光源として期待されている.しかし,通常の光ファイバは曲げや 捻り,温度変化などの外乱により伝搬する光の偏光状態が変化し,出力特性が不安定になるとい う問題がある.本研究では,安定・小型な実用的な光周波数コムを実現するため,単層カーボン ナノチューブ(SWNT)を用いた超短パルスファイバレー ザーをベースとした,全偏波保持型の光周波数コム光源 を開発し,光周波数コムの安定化制御に必要な fceo(carrier envelope offset frequency)の観測を行った. 実験と結果:本研究の実験系を Fig.1 に示す.ここで用い られている全てのファイバ,素子は偏波保持型のもので あり,空間に光を出さないために融着またはアダプタに より接続されている.はじめに,単層カーボンナノチュー ブ(SWNT)を用いた全偏波保持ファイバレーザーからの種光 をシミラリトン増幅した.次に,増幅されたパルスを偏波保 持シングルモードファイバ(PM-SMF)で分散補償することに より時間幅 41 fs,ピーク強度 71 kW の高強度超短パルスを 生成した(Fig.2).ここでは,台座成分が小さく,ピーク強度 が強くなるようファイバ長を最適化した.そして,生成した 高強度超短パルスを偏波保持高非線形分散シフトファイバ (PM-HN-DSF)に入射することにより波長 1.0~2.2 μm の 1 オ クターブ以上に広がるスーパーコンティニューム(SC)光を 生成した(Fig.3).この SC 光を導波路型の周期的分極反転ニ オブ酸リチウム(PPLN)結晶に入射することにより 2.13 μm の光に対する第二高調波を生成した.第二高調波と SC 光に 存在する 1.06 μm 成分を干渉させることで fceoを観測した. Fig.4 に観測した fceoの RF スペクトルを示す.励起強度を最 適化した際には,フリーラン時の fceo の線幅が 10 kHz とい う結果が得られた.fceo の周波数は時間的に安定な振る舞い を示した. Fig. 3 Spectrum of octave spanning SC. Fig. 1 Experimental set...

The Japan Society of Applied Physics, 2016

近年、生体内のサブミリ秒オーダの高速なダイナミクスの観察や、多数の細胞集団を解析する フローサイトメトリへの応用などの要求に伴い、蛍光共焦点顕微鏡に周波数分割多重化の概念を 導入することで高速化... more 近年、生体内のサブミリ秒オーダの高速なダイナミクスの観察や、多数の細胞集団を解析する フローサイトメトリへの応用などの要求に伴い、蛍光共焦点顕微鏡に周波数分割多重化の概念を 導入することで高速化を狙った方式が複数提案されている。これらの方式は、互いに異なる周波 数で変調された多数のレーザ光を同時に試料に集光し、それらによる発生する蛍光を単一の検出 器(光電子増倍管など)で一括して検出したのち、周波数領域で各成分を分離、抽出して画像を 再構成する。並列化を行うためにスキャナの走査軸を 1 つ省略できることにより、従来よりも大 幅な高速化が可能である。特に、マルチビームの生成に音響光学ディフレクタ(AOD)を使用す る Fluorescence Imaging by Radiofrequency-tagged Emission (FIRE)法(図 1)が速度性能の点で優れて いるが、速度性能は上記 AODの変調帯域により制限され、さらなる高速化が望まれる[1]。 我々は上記の AOD による帯域制限を打破し、従来 FIRE 法に比べて 2 倍の速度性能を有する dual-AOD FIRE法を提案する。本方式では 2つの AODからそれぞれマルチビームを生成し、それ らを重ねることによって変調されたマルチビームを生成する(図 1)。これにより被測定信号の帯域 が AODの帯域に対して 2倍となるため、2倍高速な撮像が可能となる。本方式を用いてフレーム レート 16,000 フレーム/秒での蛍光画像の取得に成功している(図 2)。本講演では、dual-AOD FIRE 法において並列取得するピクセル数を増大した場合に隣接ピクセル間のクロストークによるアー ティファクトが発生しうることを示し、AODへの駆動信号を適切に設定することで本クロストー クを光学的にキャンセルする方法の実証について報告する。

ABSTRACTBy virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging f... more ABSTRACTBy virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. For example, at high flow speed (i.e., high throughput), the integration time of the image sensor becomes short, resulting in reduced sensitivity or pixel resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually “freezing” the motion of flowing cells on the image sensor to effectively achieve 1,000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells/s without sacrificing sensitivity and spatial resolution. T...

Cell, Jan 23, 2018

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly... more 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, resp...

IEEE Photonics Journal, 2017

Optical time-stretch microscopy has recently attracted intensive attention for its capability of ... more Optical time-stretch microscopy has recently attracted intensive attention for its capability of acquiring images at an ultrahigh frame rate. Unfortunately, its achievable frame rate is limited by the requirement of having no overlap between consecutive frames, which leads to a tradeoff between the frame rate (pulse repetition rate) and the amount of the temporal dispersion used for optical image serialization. In this paper, we demonstrate compressive sensing on the platform of optical time-stretch microscopy to overcome the tradeoff between frame rate and temporal dispersion (time stretch) and achieve 50 times higher frame rate than conventional optical time-stretch microscopy. Specifically, we computationally perform compressed optical time-stretch microscopy with an experimental dataset acquired by conventional optical time-stretch microscopy and demonstrate its effects in terms of spatial resolution and cell classification accuracy. Our results indicate that the spatial resolution and cell classification accuracy reach 780 nm and 95% at a line scan rate of 675 MHz and 6.75 GHz, respectively, which correspond to five times and 50 times higher frame rates than what conventional optical time-stretch microscopy can achieve with the same dispersion amount and digitizer sampling rate.

Seibutsu Butsuri, 2017

Recent technological advances have enabled microalgae-based engineering of bioproducts. Microalga... more Recent technological advances have enabled microalgae-based engineering of bioproducts. Microalgae have a higher photosynthetic efficiency than plants and can be cultivated in agriculturally unused land, which has helped us mass-produce microalgae such as Euglena gracilis. E. gracilis is a unicellular microalgal species found in freshwater and is an attractive species for producing lipids which can be converted into biofuels. However, its lipid production efficiency is low and unable to meet high demands from the competitive fuel market. In this article, we review a few innovative methods for developing super biofuels based on super microalgae that are anticipated to overcome the low lipid production efficiency.

Scientific Reports, 2017

In the last decade, high-content screening based on multivariate single-cell imaging has been pro... more In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological ...

PloS one, 2016

The development of reliable, sustainable, and economical sources of alternative fuels is an impor... more The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a metho...

Cytometry. Part A : the journal of the International Society for Analytical Cytology, Jan 11, 2017

The development of reliable, sustainable, and economical sources of alternative fuels to petroleu... more The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is ...

Scientific reports, Jan 24, 2017

A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the ... more A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm(-1) was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the si...

Journal of biomedical optics, 2016

Visualization of epidermal cells is important because the differentiation patterns of keratinocyt... more Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

Biomedical Optics Express, 2016

We demonstrate high-throughput label-free single-cell image cytometry and image-based classificat... more We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our highthroughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-MHz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

2011 10th Euro-American Workshop on Information Optics, 2011

Huntington's disease, a major neurodegenerative disorder, involves expression and deposition of h... more Huntington's disease, a major neurodegenerative disorder, involves expression and deposition of highly aggregation-prone proteins with a long polyglutamine (polyQ) expansion. However, whether these intracellular aggregations are pathological or protective still remains elusive. Here, we propose stimulated Raman scattering (SRS) imaging of deuterium-labeled glutamine to investigate native polyQ aggregations in live cells with subcellular resolution. Technically, enrichment of deuterated glutamine in polyQ sequence enables sensitive and specific SRS imaging of carbon-deuterium bonds (C-D) from the aggregates without GFP labeling, as typically required for fluorescence microscopy. Biologically, we first reveal that these aggregates become 1.8 times denser without GFP, confirming its perturbation. Second, with SRS quantification, we discover a two-stage aggregation formation model, in which small aggregates are dominated by cytosolic proteins in the first stage, but then prefer to sequester polyQ proteins with size growth. This suggests that small aggregates are cytotoxic while large aggregates play a protective role. Third, we illustrate that polyQ aggregations are not a highly dense solid structure by calculating their absolute protein concentrations to be around millimolar range. Furthermore, hyperspectral SRS (hSRS) mapping of C-D vibrations reveals spectroscopic features corresponding to aggregation formation. This is largely absent in label-free SRS or SRS imaging of other non-glutamine amino acid probes. Applying hSRS to interrogating polyQ aggregations by inducing heat shock responses indicates a conformational intermediate with a changed hydrogen-bonding network. Altogether, our quantitative SRS imaging and spectroscopic analysis strategy offers new structural-functional insights for the molecular role of polyQ aggregates.

Journal of Laser Micro/Nanoengineering, 2012

This report describes fabrication of volume gratings inside various matrix polymers using femtose... more This report describes fabrication of volume gratings inside various matrix polymers using femtosecond laser irradiation at a wavelength of 400 nm. We investigated the diffraction efficiencies of volume gratings with a period of 10 μm by focusing 400-nm laser pulses with a 0.1-numerical aperture objective lens. We compared the polymers' diffraction efficiencies at 400 nm and 800 nm. The largest refractive index change was induced in polymers with low density. The large refractive index change was attributed to its volume contraction based on its low density.

JSAP-OSA Joint Symposia 2014 Abstracts, 2014

Stimulated Raman scattering (SRS) microscope has enabled label-free rapid Raman spectral imaging.... more Stimulated Raman scattering (SRS) microscope has enabled label-free rapid Raman spectral imaging. Recent studies have reported video-rate imaging with a pixel clock of over 10 MHz [1, 2]. However, these systems require expensive and bulky devices such as a solid-state laser or an optical parametric amplifier. On the other hand, fiber lasers are used for low-cost and compact systems although they are noisy compared with solid-state lasers. In order to reduce the noise, collinear balanced detection (CBD) has been proposed [3], although it has been performed at low data acquisition rate. Here, we demonstrate video-rate spectral imaging with a home-made Er-doped fiber laser (Er-FL) using the CBD technique.

Physical Review Letters, 2004

We show that modulational instability may arise even in the normal group-velocity dispersion regi... more We show that modulational instability may arise even in the normal group-velocity dispersion regime of an optical fiber when the fiber loss (gain) varies depending on the wavelength. A simple analytical expression for the instability gain is obtained, which reveals that the odd-order terms of the loss dispersion are responsible for this phenomenon. The instability gain is measured experimentally in an optical-parametric-amplification configuration. Large parametric gain is induced in a non-phasematched regime as we apply narrow band loss at the idler wavelength.