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Visulization of a single molecule in a living cell

Research Project

Project/Area Number 08557003
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section展開研究
Research Field General physiology
Research InstitutionHamamatsu University School of Medicine

Principal Investigator

TERAKAWA Susumu  Hamamatsu University School of Medicine, Photon Medical Research Center, Professor, 光量子医学研究センター, 文部教官教授 (50014246)

Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 1997: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1996: ¥5,400,000 (Direct Cost: ¥5,400,000)
Keywordsevanescence / exocytosis / confocal microscope / chromaffin cell / K-channel / gating / single molecule fluorescence / エバネッセント光 / エキソサイトーシス / 量子放出 / 一分化可視化 / イメージング / 分泌顆粒 / キナクリン / レーザー
Research Abstract

The purpose of this project is to visualize real-time images of molecules in living cells. To test this, I employed the evanescence microscopy and the confocal microscopy with a microlens-attached Nipkow disk scanner. By these techniques, catecholamine release from chromaffin vesicles and gating activity of K-cahnnels expressed in Xenopus oocytes were captured as dynamic 2-dimesnional images.
Exocytotic release of catecholamine was visualized by first loading a fluorescent dye, quinacrine, into chromaffin vesicles, and then by observing the fluorescence of the dye with the confocal microscope equipped with an objective lens of a high numerical aperture. It was found that some vesicles release their contents only partially and recycle without a full release. This findings do not support the widely accepted hypothesis of quantal release of tranamitters.
Messenger RNA for K-channel was modified at 351 position, so that K-channel expressed in Xenpus oocytes injected with the mRNA can be specifically labeled with tetramthyl rhodamine maleimide (TMRM). Under the voltage clamp condition, the fluorescence of TMRM on the oocyte membrane was examined with the evanescence microscope. Many fluorescent spots showed voltage dependent changes in brightness (50% in amplitude with 100mV depolarization) that reflects the gating process of the ion channel. Moreover, with the time of illumination these spots disappeared abruptly in a mode of quantal bleaching, as expected for a single fluorescent molecule. This is evidence for the first observation of single molecules on the plasma membrane in a livinf cell.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] 寺川 進: "ビデオ強化顕微鏡による生体可視化技術" 応用物理. 66(5). 444-449 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 寺川 進: "開口分泌現象のイメージング-コンピュータ画像解析" 生体の科学. 48. 205-211 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 寺川 進: "微分干渉顕微鏡" Medical Imaging Technology. 15. 702-708 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] E.Suzaki: "Video-rate dynamics of exocytotic event associated with phagocytosis" Cell Motility and Cytoskeletons. 38. 215-228 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.Terakawa: "Dynamics of exocytotic release of catecholamine,in:Adrenal Chromattin Cell" Hokkaido University Press(ed.by T.Kanno)(印刷中), (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 寺川 進: "エキソサイトーシス(標準分子医化学)" 医学書院(藤田道也編), 分担4ページ (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.Terakawa: "Exocytosis of adrenal chromaffin cells observed with a video-enhanced differential interference contrast microscope and a fluorescence microscope" Adrenal Chromaffin Cell, Hokkaido University Press. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.Terakawa: "Differential interference contrast microscope" Medical Imaging Technology. 15. 702-708 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] E.Suzaki: "Video-rate dynamics of exocytotic events associated with phagocytosis" Cell Motility and Cytoskeletons. 38. 215-228 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.Terakawa, S.Sakurai, T.: "Dynamics of exocytotic release of catecholamine measured simultaneously by VEC-DIC microscopy and DC-amperometry." Jpn.J.Physiol.46 (suppl). S7 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 寺川 進: "ビデオ強化顕微鏡による生体可視化技術" 応用物理. 66(5). 444-449 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 寺川 進: "開口分泌現象のイメージング-コンピュータ画像解析" 生体の科学. 48. 205-211 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 寺川 進: "微分干渉顕微鏡" Medical Imaging Technology. 15. 702-708 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] E.Suzaki: "Video-rate dynamics of exocytotic events associated with phagocytosis" Cell Motility and Cytoskeletons. 38. 215-228 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] S.TERAKAWA: "Adrenal Chromattin Cell:archetype and exemplar of cellular signalling in secretory control." Hokkaido-University Press(ed.T.Kanno)(印刷中), (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 寺川 進: "エキソサイトーシス(標準分子医化学" 医学書院(藤田道也 編), 4 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Pozzi,G.: "Single-chain polyprenyl phosphates form "primitive" membranes" Angewande Chemie. 35. 177-180 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Igarashi,M.: "Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1." Journal of Cell Biology. 134. 205-215 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 寺川進: "神経細胞動態のビデオマイクロスコピー" 日本神経精神薬理学雑誌. 16. 59-64 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] TERAKAWA,S.: "Dynamics of exocytotic release of catecholamine measured simultaneously by VEC-DIC microscopy and DC amperometry" Japanese Journal of Physiology. 46. S7- (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 寺川進: "ビデオ強化顕微鏡-分解能の限界を超えて「バイオイメージング」" 共立出版(印刷中),

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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