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      Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering

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      a , a , b , a , a , c , b , d , c , a , a , b , b , e , f , e , e , g , g , g , b , b , h , b , i , j , k , k , l , l , l , m , m , b , h , b , h , b , d , h , n , a , 1
      Proceedings of the National Academy of Sciences of the United States of America
      National Academy of Sciences
      imaging flow cytometry, stimulated Raman scattering, metabolite imaging, microalgae, cancer cells

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          Significance

          Imaging flow cytometry is a powerful tool for analyzing every single cell in a large heterogeneous population but relies on fluorescent labeling, which comes with cytotoxicity, nonspecific binding, and interference with natural cellular functions. This paper presents label-free multicolor chemical imaging flow cytometry based on stimulated Raman scattering (SRS), a highly sensitive method of molecular vibrational spectroscopy. With the help of deep learning, it demonstrates high-precision characterization and classification of microalgal cells and cancer cells without the need for fluorescent labeling.

          Abstract

          Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

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          Most cited references36

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          A functional perspective on phenotypic heterogeneity in microorganisms.

          Most microbial communities consist of a genetically diverse assembly of different organisms, and the level of genetic diversity plays an important part in community properties and functions. However, biological diversity also arises at a lower level of biological organization, between genetically identical cells that reside in the same microenvironment. In this Review, I outline the molecular mechanisms responsible for phenotypic heterogeneity and discuss how phenotypic heterogeneity allows genotypes to persist in fluctuating environments. I also describe how it promotes interactions between phenotypic subpopulations in clonal groups, providing microbial groups with new functionality.
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            Biological imaging software tools.

            Few technologies are more widespread in modern biological laboratories than imaging. Recent advances in optical technologies and instrumentation are providing hitherto unimagined capabilities. Almost all these advances have required the development of software to enable the acquisition, management, analysis and visualization of the imaging data. We review each computational step that biologists encounter when dealing with digital images, the inherent challenges and the overall status of available software for bioimage informatics, focusing on open-source options.
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              Exploiting diversity and synthetic biology for the production of algal biofuels.

              Modern life is intimately linked to the availability of fossil fuels, which continue to meet the world's growing energy needs even though their use drives climate change, exhausts finite reserves and contributes to global political strife. Biofuels made from renewable resources could be a more sustainable alternative, particularly if sourced from organisms, such as algae, that can be farmed without using valuable arable land. Strain development and process engineering are needed to make algal biofuels practical and economically viable.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                6 August 2019
                19 July 2019
                19 July 2019
                : 116
                : 32
                : 15842-15848
                Affiliations
                [1] aDepartment of Electrical Engineering and Information Systems, The University of Tokyo , 113-8656 Tokyo, Japan;
                [2] bDepartment of Chemistry, The University of Tokyo , 113-0033 Tokyo, Japan;
                [3] cDepartment of Photonics, National Chiao Tung University , 300 Hsinchu, Taiwan;
                [4] dInstitute of Technological Sciences, Wuhan University , 430072 Wuhan, China;
                [5] eDepartment of Micro-Nano Mechanical Science and Engineering, Nagoya University , 464-8601 Nagoya, Japan;
                [6] fDepartment of Precision Mechanics, Chuo University , 112-8551 Tokyo, Japan;
                [7] gDepartment of Creative Informatics, The University of Tokyo , 113-0033 Tokyo, Japan;
                [8] hJapan Science and Technology Agency , 332-0012 Saitama, Japan;
                [9] iDepartment of Gastroenterology, Cancer Institute Hospital, Japanese Foundation for Cancer Research , 135-8550 Tokyo, Japan;
                [10] jClinical Research and Regional Innovation, Faculty of Medicine, University of Tsukuba , 305-8575 Ibaraki, Japan;
                [11] kDivision of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research , 135-8550 Tokyo, Japan;
                [12] lDepartment of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo , 113-0033 Tokyo, Japan;
                [13] mCenter for Biosystems Dynamics Research, RIKEN , 565-0871 Osaka, Japan;
                [14] nDepartment of Bioengineering, University of California, Los Angeles , CA 90095
                Author notes
                1To whom correspondence may be addressed. Email: ozeki@ 123456ee.t.u-tokyo.ac.jp .

                Edited by Björn F. Lillemeier, Salk Institute, La Jolla, CA, and accepted by Editorial Board Member John W. Sedat June 14, 2019 (received for review February 8, 2019)

                Author contributions: Y.S., K.G., and Y.O. designed research; Y.S., K. Kobayashi, Y.W., D.D., S.T., S.L., A.I., Y.K., T.H., S.S., F.A., K. Koizumi, H.T., M.I., K.H., Y. Yalikun, Y.T., T.S., and N.N. performed research; T.I., M.H., S.M., K. Shiba, K. Suga, M.N., M.J., and Y. Yatomi contributed new reagents/analytic tools; Y.S., C.-J.H., C.L., C.-W.S., H.L., and Y.F. analyzed data; and Y.S., K.G., and Y.O. wrote the paper.

                Author information
                http://orcid.org/0000-0003-0391-9832
                http://orcid.org/0000-0002-4145-7264
                http://orcid.org/0000-0001-6459-0204
                http://orcid.org/0000-0003-1034-5718
                http://orcid.org/0000-0002-9004-0799
                Article
                201902322
                10.1073/pnas.1902322116
                6690022
                31324741
                33753269-8166-4fb8-bb89-1ee477683539
                Copyright © 2019 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 7
                Funding
                Funded by: Cabinet Office, Government of Japan 501100002770
                Award ID: 2014-PM02
                Award Recipient : Yuta Suzuki Award Recipient : Koya Kobayashi Award Recipient : Yoshifumi Wakisaka Award Recipient : Dinghuan Deng Award Recipient : Shunji Tanaka Award Recipient : Chun-Jung Huang Award Recipient : Cheng Lei Award Recipient : Hanqin Liu Award Recipient : Yasuhiro Fujiwaki Award Recipient : Sangwook Lee Award Recipient : Akihiro Isozaki Award Recipient : Yusuke Kasai Award Recipient : Takeshi Hayakawa Award Recipient : Shinya Sakuma Award Recipient : Fumihito Arai Award Recipient : Kenichi Koizumi Award Recipient : Hiroshi Tezuka Award Recipient : Mary Inaba Award Recipient : Kei Hiraki Award Recipient : Takuro Ito Award Recipient : Misa Hase Award Recipient : Satoshi Matsusaka Award Recipient : Kiyotaka Shiba Award Recipient : Kanako Suga Award Recipient : Masako Nishikawa Award Recipient : Masahiro Jona Award Recipient : Yutaka Yatomi Award Recipient : Yalikun Yaxiaer Award Recipient : Yo Tanaka Award Recipient : Takeaki Sugimura Award Recipient : Nao Nitta Award Recipient : Keisuke Goda Award Recipient : Yasuyuki Ozeki
                Categories
                PNAS Plus
                Physical Sciences
                Engineering
                PNAS Plus

                imaging flow cytometry,stimulated raman scattering,metabolite imaging,microalgae,cancer cells

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