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Single-fluorophore imaging under an epifluorescence microscope

Research Project

Project/Area Number 04508002
Research Category

Grant-in-Aid for Developmental Scientific Research (A)

Allocation TypeSingle-year Grants
Research Field 生物物性学
Research InstitutionKeio University

Principal Investigator

KINOSITA Kazuhiko  Keio University, Faculty of Science and Technology, Professor, 理工学部, 教授 (30124366)

Co-Investigator(Kenkyū-buntansha) MORI Hajime  Sigma Koki Co., Ltd.Managing Director, 技術部, 取締役部長
UETAKE Toshihumi  Orion Lens Design Co., Ltd.President, 代表取締役
HIRANO Ken'ichi  Hamamatsu Photonics K.K.Research Scientist, 筑波研究所, 研究員
MIYATA Hidetake  Keio University, Faculty of Science and Technology, Assistant Professor, 理工学部, 専任講師 (90229865)
Project Period (FY) 1992 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥59,200,000 (Direct Cost: ¥59,200,000)
Fiscal Year 1994: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1993: ¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 1992: ¥46,400,000 (Direct Cost: ¥46,400,000)
KeywordsFluorescence microscope / Background reduction / Fluorescence polarization / Myosin / Actin / Tetramethylrhodamine / Rotation imaging / Protein confomational changes / 1分子イメージング / テトラメチルロ-ダミン / 蛍光褪色 / 蛍光顕微鏡(超高感度) / 無蛍光対物レンズ / ローダミン / 核酸オリゴマー / 迷光 / 対物レンズ / 無蛍光材料 / エバネッセント波 / レーザー
Research Abstract

The purpose of this research was to develop an optical microscope that allows continuous observation of single fluorophores in aqueous solution. Real-time imaging of the behaviors of individual biological molecules, tagged with a fluorophore, in a living environment is the final goal.
For maximal flexibility, we modified a commercial epifluorescence microscope into a single-molecule imager. The high background luminescence inherent in the microscope could be reduced by two orders of magnitude by letting out the excitaion light that passed through the dichroic mirror and by confining the beam diameter of the excitation light. These modifications allowed real-time (30 frames/s) imaging of tetramethylrhodamine in an aqueous environment.
We also designed and constructed a non-fluorescent objective. Due to a difficulty in polishing the material we chose, we were unable to make an optically perfect objective. The one we made was gave smaller background compared to the best commercial objective developed recently, but the fluorescence throughput was not as high as expected. We therefore used the commercial objective of the latest design for single-molecule imaging.
Now we can split the fluorescence from a single fluorophore into vertically ahd horizonttaly polarized components. This allows real-time determination of the orientation of the fluorophore. Using this technique, we have been able to detect the axial rotation of an actin filament sliding over myosin. The successful imaging of rotation implies that we can now detect conformational changes (which necessarily involves orientational changes) in a single protein molecule during function.

Report

(4 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • 1992 Annual Research Report
  • Research Products

    (38 results)

All Other

All Publications (38 results)

  • [Publications] Hyuga,H.,et al.: "Steady-state deformation of a vesicle in alternating electric fields" Bioelectrochem.Bioenerg.32. 15-25 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hibino,M.,et al.: "Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential." Biophys.J.64. 1789-1800 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hakozaki,H.,et al.: "Sliding force between a small number of HMM molecules and a single actin filament under optical tweezers." J.Muscle Res.Cell Motility. 14. 358-358 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nishizaka,T.,et al.: "Measurement of sliding force generated on an actin filament under various concentrations of HMM in an in vitro motile system." J.Muscle Res.Cell Motility. 14. 357-358 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Miyata,H.,et al.: "Morphological change caused by sytochalasin D of liposomes encapsulating polymerized actin." J.Muscle Res.Cell Motility. 14. 367-368 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Itoh,H.,et al.: "Electroporation visualized under a multi-shot pulsed-laser fluorescence microscope system." Proc.SPIE. 2002. 118-125 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Kinosita,K.Jr.et al.: "Orientation of actin monomers in moving actin filaments." Plenum(New York), 9 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Hyuga: "Steady-state deformation of a vesicle in alternating electric fields." Bioelectrochem.Bioenerg.32. 15-25 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] M.Hibino: "Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential." Biophys.J.64. 1789-1800 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Hakozaki: "Sliding force between a small number of HMM molecules and a single actin filament under optical tweezers." J.Muscle Res.Cell Motility. 14. 358-358 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] T.Nishizaka: "Measurement of sliding force generated on an actin filament under various concentrations of HMM in an in vitro motile system." J.Muscle Res.Cell Motility. 14. 357-358 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Miyata: "Morphological change caused by sytochanlasin D of liposomes encapsulating polymerized actin." J.Muscle Res.Cell Motility. 14. 367-368 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H,Itoh: "Electroporation visualized under a multi-shot pulsed-laser fluorescence microscope system." Proc.SPIE. 2002. 118-125 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Miyata: "Stepwise motion of an actin filament pver a small number of heave meromyosin in in vitro motility assay." J.Biochem.115. 644-647 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Miyata: "Transformation of actin-encapsulating liposomes induced by cytochalasin D." Biophys.J.67. 922-928 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] K.Kinosita, Jr.: Orientation of actin monomers in moving actin filaments.Plenum (New York), 9 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Keisuke Suzuki,et al.,: "Spatiotemporal Relationships among Early Events of Fertilization in Sea Urchin Eggs Revealed by Multi-View Microscopy" J.Cell Biol.(in press).

    • Related Report
      1994 Annual Research Report
  • [Publications] Hidetake Miyata,et al.,: "Transformation of Actin-Encapsulating Liposomes Induced by Cytochalasin D" Biophys.J.67. 922-928 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Hidetake Miyata,et al.,: "Stepwise Motion of an Actin Filament over a Small Number of Heavy Meromyosin in in vitro Motility Assay" J.Biochem.(Tokyo). 115. 644-647 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Masahiro Hibino,et al.,: "Time Courses of Cell Electroporation As Revealed by Submicrosecond Imaging of Transmembrane Potential" Biophys.J.64. 1789-1800 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Hiroyuki Hyuga,et al.,: "Steady-State Deformation of a Vesicle in Alternating Electric Fields" Bioelectrochem.Bioenerg.32. 15-25 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Hiroyasu Itoh,et al.,: "Electroporation Visualized under a Multi-Shot Pulsed-Laser Fluoresce Microscope System" Proc.SPIE. 2002. 118-125 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kazuhiko Kinosita jr.,: "Orientation of Actin Monomers in Moving Actin Filaments" Plenum(New York), 9 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Hiroyuki HAKOZAKI,et al.,: "“Sliding Force between a Small Number of HMM Molecules and a Single Actin Filament under Optical Tweezers"" J.Muscle Res. Cell Motility,. 14. 358-358 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takayuki HISHIZAWA,et al.,: "Measurement of sliding Force Generated on an Actin Filament nuder Various Concentrations of HMM in an In Vitro Motile System"" J.Muscle Res. Cell Motility,. 14. 357-358 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroyasu ITOH,et al.,: "“Electroporation Visualized under a Multi‐Shot Pulsed‐Laser Fluorescence Microscope System" Proc.SPIE(The Interantional Society for Optical Engineering). 2002. 118-125 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroyuki HYUGA,et al.,: "“Steady‐State Deformation of a Vesicle in Alternating Electric Fields"" Bioelectrochem.Beoenerg.,. 32. 15-25 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Masahiro HIBINO,et al.,: "“Time Courses of Cell Electroporation As Revealed by Submicrosecond Imaging of Transmembrane Potential"" Biophys.J.64. 1789-1800 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hidetake MIYATA,et al.,: "“Transformation of Actin‐Encapsulating Liposomes Induced by CytochalasinD"" Biophys,J.,Submitted.

    • Related Report
      1993 Annual Research Report
  • [Publications] Kazuhiko KINOSITA,et al.,: "“Orientation of Actin Monomers in Moving Actin Filaments"" Plenum(New York), 9 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroyuki HAKOZAKI,et al.,: "¨Sliding Force between a Small number of HMM Molecules and a Single Actin Filament under Optical Tweeners¨" J.Muscle Res.Cell Motility,.

    • Related Report
      1992 Annual Research Report
  • [Publications] Takayuke NISHIZAKA,et al.,: "¨Measurement of Sliding Force Generated on an Actin Filament under Various Concentrations of HMM in an In Vitro Motile System¨" J.Muscle Res.Cell Motility,.

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuko KIMURA,et al.,: "¨Fluorescence Quenching Measurements of the Membrane Bound Lipid Haptens with Different Length Spacers¨" Biochim.Biophys.Acta,. 1104. 9-14 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Geraid MARRIOTT,et al.,: "¨Photomodulation of the Nucleatin Activity of a Photocleaveble Crosslinked Actin Dimer¨" Biochim.Intein.,. 26. 943-951 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Huroyuki HYUGA,et al.,: "¨Steady-State Deformation of a Vesicle in Alternatiyg Electric Fields¨" Bioelectrochem.Beoenerg.,.

    • Related Report
      1992 Annual Research Report
  • [Publications] Masahiro HIBTNO,et al.,: "¨Time Courses of Cell Electroporation As Revealed by Submicrosecond Imaging of Transmembrane Potential¨" Biophys.J.,.

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuheko KINISITA,Jr.,et al.,: "″Events of Membeane Electroporation Visualized on a Time Scale from Microsecond to Secinds″" Academic Press(New York), 18 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuhiko KINOSITA,Jr.,et al.,: "″Orientation of Actin Monomers in Moving Actin Filaments″" Plenum(New York),

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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