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Development for single-molecule deep imaging method of biological sample under cryogenic conditions

Research Project

Project/Area Number 19K22162
Research Category

Grant-in-Aid for Challenging Research (Exploratory)

Allocation TypeMulti-year Fund
Review Section Medium-sized Section 32:Physical chemistry, functional solid state chemistry, and related fields
Research InstitutionTokyo Institute of Technology

Principal Investigator

Fujiyoshi Satoru  東京工業大学, 理学院, 助教 (70371705)

Project Period (FY) 2019-06-28 – 2021-03-31
Project Status Completed (Fiscal Year 2020)
Budget Amount *help
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2020: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Fiscal Year 2019: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Keywords1分子観察 / 可変浸レンズ / 蛍光顕微鏡 / 三次元イメージング / 生体試料 / 1分子分光 / 深部観察 / 顕微鏡
Outline of Research at the Start

蛍光顕微鏡で観察する試料の表面には、空気/水(または油/水などの)界面が存在する。試料表面は平らな場合が多く、平らな界面で光が屈折すると収差が産まれ、顕微鏡の分解能と検出効率が大きく低下する このため、蛍光顕微鏡でも、細胞や組織のような厚みのある試料全体を正しく画像化することは難しい。そこで、当該研究では、界面屈折の影響をゼロにできるWILを開発する。

Outline of Final Research Achievements

Three-dimensional optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. In this project, we have developed a variable immersion lens (VIL) for deep fluorescence imaging A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror). VIL microscope enables diffraction-limited 1.2-NA imaging in water (refractive index of 1.34) at a depth of 0.3 mm by minimizing aberrations due to refraction of a sample interface. Another aberration due to refractive index mismatching between a mounting medium and an object can be also corrected by the VIL system because various fluids with different refractive indexes can be used as mounting media for the VIL. As a result, we have demonstrated that a cell spheroid can be imaged at a true dimension.

Academic Significance and Societal Importance of the Research Achievements

これまで細胞内の小器官を測定する場合、培養細胞ぐらいの厚さ(10ミクロン)がせいぜいで組織レベルの試料になると細胞ごとの分解能が限界であった。我々の提案する方法は生体組織の中の小器官を鮮明に取得できる方法であり、とても大きな意義があると考えている。この技術は医学、薬学にもすぐに応用できる方法であり、こちらの方向を伸ばすことで社会に貢献したい。

Report

(3 results)
  • 2020 Annual Research Report   Final Research Report ( PDF )
  • 2019 Research-status Report
  • Research Products

    (18 results)

All 2021 2020 2019

All Journal Article (5 results) (of which Peer Reviewed: 5 results,  Open Access: 1 results) Presentation (12 results) (of which Invited: 3 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] Variable immersion microscopy with a high numerical aperture2021

    • Author(s)
      Keita Ishida, Kanta Naruse, Yuta Mizouchi, Yoshihiro Ogawa, Michio Matsushita, Takeshi Shimi, Hiroshi Kimura, Satoru Fujiyoshi
    • Journal Title

      Optics Letters

      Volume: 46 Issue: 4 Pages: 856-859

    • DOI

      10.1364/ol.416006

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cryogenic Single-Molecule Spectroscopy of the Primary Electron Acceptor in Photosynthetic Reaction Center2020

    • Author(s)
      T. Kondo, R. Mutoh, H. Tabe, G. Kurisu, H. Oh-oka, S. Fujiyoshi, and M. Matsushita
    • Journal Title

      J. Phys. Chem. Lett.

      Volume: - Issue: 10 Pages: 3980-3986

    • DOI

      10.1021/acs.jpclett.0c00891

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami2020

    • Author(s)
      Furubayashi Taku、Ishida Keita、Nakata Eiji、Morii Takashi、Naruse Kanta、Matsushita Michio、Fujiyoshi Satoru
    • Journal Title

      The Journal of Physical Chemistry B

      Volume: 124 Issue: 35 Pages: 7525-7536

    • DOI

      10.1021/acs.jpcb.0c04721

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Aberration-corrected cryogenic objective mirror with a 0.93 numerical aperture2019

    • Author(s)
      Fujiwara Masanori、Ishii Takaki、Ishida Keita、Toratani Yasuharu、Furubayashi Taku、Matsushita Michio、Fujiyoshi Satoru
    • Journal Title

      Applied Physics Letters

      Volume: 115 Issue: 3 Pages: 033701-033701

    • DOI

      10.1063/1.5110546

    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Journal Article] Nanometer Accuracy in Cryogenic Far-Field Localization Microscopy of Individual Molecules2019

    • Author(s)
      Furubayashi Taku、Ishida Keita、Kashida Hiromu、Nakata Eiji、Morii Takashi、Matsushita Michio、Fujiyoshi Satoru
    • Journal Title

      The Journal of Physical Chemistry Letters

      Volume: 10 Issue: 19 Pages: 5841-5846

    • DOI

      10.1021/acs.jpclett.9b02184

    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Presentation] 超流動ヘリウム1分子分光で見たタンパク質の中の水素結合2020

    • Author(s)
      藤芳 暁
    • Organizer
      量子生命科学会第二回大会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] Single-molecule nanoscopy by using cryogenic fluorescence microscopy2020

    • Author(s)
      藤芳 暁
    • Organizer
      日本顕微鏡学会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 可変浸レンズ:実験とシミュレーション2020

    • Author(s)
      成瀬寛太、石田啓太、溝内雄太、松下道雄、志見剛、木村宏、藤芳暁
    • Organizer
      分子科学会 オンライン討論
    • Related Report
      2020 Annual Research Report
  • [Presentation] 反応性FRETペアによる細胞内標識2020

    • Author(s)
      溝内雄太、成瀬寛太、松下道雄、工藤史貴、佐藤優子、木村宏、藤芳暁
    • Organizer
      分子科学会 オンライン討論
    • Related Report
      2020 Annual Research Report
  • [Presentation] 液中の深いところの顕微観察を可能にする可変浸レンズ2020

    • Author(s)
      成瀬寛太、石田啓太、溝内雄太、松下道雄、志見剛、木村宏、藤芳暁
    • Organizer
      日本物理学会年次大会
    • Related Report
      2020 Annual Research Report
  • [Presentation] 三次元カメラ共焦点クライオ(C3)蛍光顕微鏡2019

    • Author(s)
      藤芳 暁
    • Organizer
      放射線影響学会
    • Related Report
      2019 Research-status Report
    • Invited
  • [Presentation] 開口数0.93の収差補正クライオ対物鏡の開発2019

    • Author(s)
      石井啓暉, 虎谷泰靖, 藤原正規, 石田啓太, 藤芳暁, 松下道雄
    • Organizer
      日本物理学会秋季大会
    • Related Report
      2019 Research-status Report
  • [Presentation] 高開口数蛍光顕微鏡の界面屈折に由来する収差の研究:メニスカスレンズによる収差補正2019

    • Author(s)
      石田啓太, 藤芳暁, 松下道雄
    • Organizer
      日本物理学会秋季大会
    • Related Report
      2019 Research-status Report
  • [Presentation] 温度安定化循環水によるクライオ蛍光顕微鏡のナノレベル安定化2019

    • Author(s)
      滝島研人, 古林琢, 松下道雄, 藤芳暁
    • Organizer
      日本物理学会秋季大会
    • Related Report
      2019 Research-status Report
  • [Presentation] クライオ蛍光顕微鏡による分子確度イメージングの実現2019

    • Author(s)
      古林 琢、中田栄司、森井孝、藤芳暁、松下道雄
    • Organizer
      分子科学討論会
    • Related Report
      2019 Research-status Report
  • [Presentation] 三次元カメラ共焦点顕微鏡によるクライオ1分子イメージング2019

    • Author(s)
      松田剛、古林 琢、松下道雄、藤芳暁
    • Organizer
      分子科学討論会
    • Related Report
      2019 Research-status Report
  • [Presentation] DNAオリガミを用いたクライオ超解像蛍光イメージング用色素の探索2019

    • Author(s)
      溝内雄太、石井啓暉、中田栄司、森井孝、藤芳暁、松下道雄
    • Organizer
      分子科学討論会
    • Related Report
      2019 Research-status Report
  • [Patent(Industrial Property Rights)] 顕微鏡2020

    • Inventor(s)
      藤芳暁、石田啓太
    • Industrial Property Rights Holder
      藤芳暁、石田啓太
    • Industrial Property Rights Type
      特許
    • Filing Date
      2020
    • Related Report
      2019 Research-status Report

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Published: 2019-07-04   Modified: 2022-12-28  

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