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Development of simultaneous ultra-fast 3D super resolution imaging and 3D single molecule tracking microscope system

Research Project

Project/Area Number 21K15058
Research Category

Grant-in-Aid for Early-Career Scientists

Allocation TypeMulti-year Fund
Review Section Basic Section 43040:Biophysics-related
Research InstitutionOkinawa Institute of Science and Technology Graduate University

Principal Investigator

Tsunoyama Takaaki  沖縄科学技術大学院大学, 膜協同性ユニット, スタッフサイエンティスト (90896862)

Project Period (FY) 2021-04-01 – 2024-03-31
Project Status Completed (Fiscal Year 2023)
Budget Amount *help
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2023: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2022: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2021: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Keywords超解像顕微鏡法 / 3次元観察 / 1分子蛍光追跡法 / 細胞膜 / 神経シナプス / 細胞膜受容体 / 1分子蛍光顕微鏡法 / 3次元超解像顕微鏡法 / 神経受容体 / 3次元蛍光1分子追跡 / シナプス / 群島構造
Outline of Research at the Start

神経細胞のシナプスでは受容体の分布が絶えず変化しており、信号伝達の調整メカニズムの解明のために活発な研究が行われている。しかし、神経シナプスは小さく、複雑な三次元構造を持っている故に顕微鏡観察による解析には限界があった。
本研究ではその限界を打ち破るため、超高速でシナプスの3次元超解像構造と受容体の蛍光1分子追跡を同時に行う方法を開発する。我々の作業仮設は、シナプス内の受容体は従来考えられていた巨大な塊として存在しているのでは無く、小さな群島の集まりとして存在しているというモデルである。新規に開発する顕微鏡技術によって、生きた神経細胞上での動的な構造変化を1分子レベルで検証する。

Outline of Final Research Achievements

It is a well-known theory that synaptic receptors are stably present (or anchored) on synapses. However, our recent findings suggest that receptors actually undergo frequent entering and departure movements, indicating a dynamic structure. In this study, we aimed to verify this hypothesis by developing a novel microscope system that simultaneously performs ultrafast 3D super-resolution observation and ultrafast 3D single-molecule fluorescence tracking.
We developed a novel microscope system characterized by high-speed Z-scanning using a liquid lens, enabling observations with a Z-directional resolution range of 2μm, which is more than twice the ordinary range, at a high speed of 250 Hz. Using this system, we were able to track the movement of receptors in and out of 3D super-resolved synapses in three dimensions.

Academic Significance and Societal Importance of the Research Achievements

超解像顕微鏡法や1分子蛍光観察法は今では広く用いられているが、3次元での観察となると応用例は少なく、ほとんどは固定細胞を用いたものである。特に神経細胞は複雑な3次元構造をもっており、3次元での生細胞を用いた動態の観察が重要であると考えられるが、限られた知見しか得られていない。
本研究で開発した液体レンズを用いる3次元顕微鏡システムはこの限界を打ち破るものであり、比較的安価に導入が可能で、その性能と安定性も十分なものであると証明ができた。このシステムは様々な観察系に導入することが可能で、3次元の高速観察、というニッチを補完するものであると確信している。

Report

(4 results)
  • 2023 Annual Research Report   Final Research Report ( PDF )
  • 2022 Research-status Report
  • 2021 Research-status Report
  • Research Products

    (6 results)

All 2023 2022

All Journal Article (2 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 2 results,  Open Access: 2 results) Presentation (4 results) (of which Int'l Joint Research: 4 results,  Invited: 4 results)

  • [Journal Article] Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics2023

    • Author(s)
      Fujiwara, T. K.Tsunoyama, T. A.Takeuchi, S.Kalay, Z.Nagai, Y.Kalkbrenner, T.Nemoto, Y. L.Chen, L. H.Shibata, A. C. E.Iwasawa, K.Ritchie, K. P.Suzuki, K. G. N.Kusumi, A.
    • Journal Title

      Journal of Cell Biology

      Volume: 222 Issue: 8

    • DOI

      10.1083/jcb.202110162

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology2023

    • Author(s)
      Fujiwara, T. K.Takeuchi, S.Kalay, Z.Nagai, Y.Tsunoyama, T. A.Kalkbrenner, T.Iwasawa, K.Ritchie, K. P.Suzuki, K. G. N.Kusumi, A.
    • Journal Title

      Journal of Cell Biology

      Volume: 222 Issue: 8

    • DOI

      10.1083/jcb.202110160

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Nano-liquid platform on the plasma membrane that integrates receptor signals for cancer promotion.2023

    • Author(s)
      4.Taka A. Tsunoyama, Christian Hoffmann, Daiki Sasaki, Bo Tang, Koichiro M. Hirosawa, Yuri L. Nemoto, Rinshi S. Kasai, Takahiro K. Fujiwara, Kenichi G.N. Suzuki, Hiroki Ishikawa, Dragomir Milovanovic, Akihiro Kusumi.
    • Organizer
      61st Annual Meeting of the Biophysical Society of Japan
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] iTRVZ: Liquid-like nanoscale signaling platform on the plasma membrane that integrates receptor signals leading to cancer promotion.2023

    • Author(s)
      4.Taka A. Tsunoyama, Christian Hoffmann, Daiki Sasaki, Bo Tang, Koichiro M. Hirosawa, Yuri L. Nemoto, Rinshi S. Kasai, Takahiro K. Fujiwara, Kenichi G.N. Suzuki, Hiroki Ishikawa, Dragomir Milovanovic, Akihiro Kusumi.
    • Organizer
      68th Annual Meeting of Biophysical Society
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Nanoscale condensed liquid platform on the plasma membrane for signal integration2022

    • Author(s)
      Tak-aki Tsunoyama
    • Organizer
      OIST-Kyoto University Joint Workshop
    • Related Report
      2022 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] Nanoscale LLPS-based liquid-like signaling platform that cooperatively integrates RTK, GPCR, and GPI-anchored receptor signals2022

    • Author(s)
      Tak-aki Tsunoyama
    • Organizer
      The 45th annual meeting of the molecular biology society of Japan
    • Related Report
      2022 Research-status Report
    • Int'l Joint Research / Invited

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Published: 2021-04-28   Modified: 2025-01-30  

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