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Analysis of biofunction by single molecule measurement

Research Project

Project/Area Number 11794015
Research Category

Grant-in-Aid for University and Society Collaboration

Allocation TypeSingle-year Grants
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, 教授 (50014246)

Co-Investigator(Kenkyū-buntansha) IAWATA Futoshi  Shizuoka University, Faculty of Engineering, Research Associate, 工学部, 助手 (30262794)
KAWATA Yoshimasa  Shizuoka University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (70221900)
SAKURAI Takashi  Hamamatsu University School of Medicine, Photon Medical Research Center, Research Associate, 光量子医学研究センター, 助手 (50283362)
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥3,000,000 (Direct Cost: ¥3,000,000)
Keywordsevanescence microscope / calcium transient / ultra high NA lens / neuronal cell death / single molecule fluorescence / single DNA / total reflection illumination / near field microscope / CCDカメラ / 高速度カメラ / 光電子増倍管 / マルチアノード / 膜電位色素 / 量子効率 / 緑色蛍光タンパク / 膜蛋白 / 緑色蛍光蛋白 / クロモグラニンB / 一分子ダイナミクス / DNA / フォトカウンティング / ケージトグルタミン酸
Research Abstract

Using our newly developed CCD camera, we observed rhodamine molecules dissolved into water under an evanescent field microscope equipped with an objective lens of NA=1. 65. The quantal bleach was captured at time resolution of 5 ms. A similar response of Rhod-2molecule was also detected. In this molecule, the brightness depended on the concentration of calcium ions in the medium. This opened a possibility that microenvironments can be monitored by a probe consisting of a single molecule. Hippocampal neurons loaded with Rhod-2 were observed at the bottom through the evanescent field microscope equipped with the new camera. We could detect Ca images at a 5-ms frame rate. Lowering the Rhod-2 concentration to one hundredths of the commonly used one, images became punctuated, and some spots of an extremely high signal/DC ratio were observed. These spotty responses could be Ca transients detected by a single Rhod-2 molecule and possibly caused by a single Ca channel. We also succeeded in observing endocytotic responses by using GFP-conjugated dynamin expressed in PC12 cells. Upon activation of the cell, dynamin translocated near to the cell membrane, and moved around beneath there as if to capture the membrane pits for endocytosis. From this result, we proposed a hypothesis, "sweeping model of dynamin," for regulation of endocytosis. We observed a shape of DNA molecules obtained from a few hippocampal neurons in culture facing an excitotoxic cell death due to glutamate application. The evanescent field microscopy reveled for the first time that the DNAs were fragmented in such an acute process of neuronal cell death. In order to overcome the problems due to the diffraction limit, we developed a new imaging technique by which an evanescent shadow is fixed on a thin film made of a photosensitive resin. Images of small vesicles in the cell could be captured.

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (34 results)

All Other

All Publications (34 results)

  • [Publications] Tsuboi T: "Sweeping model of dynamin activity"J.Biol.Chem.. Feb.20(JBC On line). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Scalettar BA: "Neuronal calcium sensor-1 binds selectively to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells"J.Cell Sci.. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuboi T: "Water secretion associated with exocytosis in endocrine cells revealed by micro forcemetry and evanescent wave microscopy"Biophys.J.. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Terakawa S: "Fluorescence micro-imaging of living cells and biomolecules with ultra high NA objectives"Proc.SPIE. 4597. 121-127 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuboi T: "Supply of secretory granule enhanced by protein kinase C in the bovine chromaffin cell"Biochem Biophys Res Commun.. 282. 621-628 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 寺川 進: "光マイクロイメージング法 -医学生物学への応用展開"Otol.Jpn.. 11. 1-5 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Terakawa S: "Modern Problems of Cellular and Molecular Biophysics(DIC Microscopy : Characteristics and Application for Studies of Living Cells)"Noyan Tapan. 155-166 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuboi T, Sakurai T, Kimura T, Terakawa S: "Sweeping model of dynamin activity."J. Biol. Chem.. Feb. 28, JBC on line. (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Scaletter BA, Rosa P, Taverna E, et al.: "Neuronal calcium sensor-1 bidns selectively to Regulated secretory organelles and functions in basal And stimulated exocytosis in PC12 cells."J. Cell Sci.. (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuboi T, Kikuta T, Sakurai S, Terakawa S: "Water secretion associated with exocytosis in Endocrine cells revealed by micro focremetry and Evanescent wave microscopy"Biophys. J.. (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Iwata F, Kobyashi K, Sasaki A, et al.: "Nanometer-scale modification of a uethane-urea copolymer film using local field enhancmenent at an apex of a metal coated probe"Nanotechnol.. 13. 138-142 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Terakawa S, Tsuboi T, Sakurai T, et al.: "Fluorescence micro-imaging of living cells and biomoleuces with ultra high NA objective."Proc. SPIE. 4597. 121-127 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuboi T, Kikuta T, Warashian A, Terakawa S: "Supply of secretory granule enhanced by protein kinase C in the bovine chromaffin cell"Biochem. Biophys. Res. Commun.. 282. 621-628 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Terakawa S: "DIC microscopyc and application for studies of living cells."Modern Problems of Cellular and Molecular Biophysics, Noyan Tapan. 155-166 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Murakmi M, Tsuboi T, Kawata Y, Terakawa S, et al.: "High-resolution imaging of living biological specimens"CLEO Pac. Rim Tech. Dig.. 1184-1185 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Inami W, Kawata Y: "Analysis of the scattering light distribution of a Tightly focused laser beam by a particle near a substrate"J. Appl. Phys.. 89. 5876-5880 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sakaguchi H, Hirai A, Iwata F, et al.: "Determination of performance on tunnel conduction through molecular wire using a conductive atomic force microscope"Appl. Phys. Lett.. 79. 3708-3710 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Iwata F, Someya D, Sakaguchi H, et al.: "Photoconductive imaging in a photon scanning tunneling microscope capable of point-contact current sensing using a conductive fiber"J. Microscopy. 202. 188-192 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tsuboi T: "Sweeping model of dynamin activity"J. Biol. Chem.. Feb.20(JBC On line). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Scalettar BA: "Neuronal calcium sensor-l binds selectively to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells"J. Cell Sci.. (in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Tsuboi T: "Water secretion associated with exocytosis in endocrine cells revealed by micro forcemetry and evanescent wave microscopy"Biophys. J.. (in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Terakawa S: "Fluorescence micro-imaging of living cells and biomolecules with ultra high NA objectives"Proc. SPIE. 4597. 121-127 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Tsuboi T: "Supply of secretory granule enhanced by protein kinase C in the bovine chromaffin cell"Biochem Biophys Res Commun. 282. 621-628 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 寺川進: "光マイクロイメージング法-医学生物学への応用展開"Otol.Jpn.. 11. 1-5 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Terakawa S: "Modern Problems of Cellular and Molecular Biophysics(DIC Microscopy : Characteristics and Application for Studies of Living Cells)"Noyan Tapan. 155-166 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Tsuboi T: "Supply of secretory granule enhanced by protein kinase C in the bovine chromaffin cell."Biochemistry and Biophyscs Reasearch Commununications.. (印刷中). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kawata Y: "Non-optically probing near-field microscopy for the observation of biological living specimens,".(in press)."Applied Physics Letter. (印刷中). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kawano Y: "High numerical aperture objective lenses and optical system improved objective type total internal reflection fluorescence microscopy."Procedings of SPIE. 4098. 142-151 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tsuboi T: "Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event."Current Biology. 10. 1307-1310 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ishihara Y: "Exocytosis in the dissociated pancreatic acinar cells of the guinea pig directly visualized by VEC-DIC microscopy."Biochemistry and Biophyscs Research. 277. 134-137 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ishihara Y: "Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc."American Journal of Physiology. 279. C1177-C1188 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Sakurai T: "Superoxide production in the islet of Langerhans In : Ultrastructure and molecular biology protocols for oxidants and antioxidants (ed.D.Armstrong)"Academic Press印刷中. (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] 坪井貴司: "光ピンセット法とその応用"日本レーザー医学会誌. 20巻・4号. 367-372 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 櫻井孝司: "ケイジド化合物の三次元マイクロフォトリシス(バイオイメージングの最先端)"先端医療技術研究所(寺田弘司). 182-187(324) (1999)

    • Related Report
      1999 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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