1994 Fiscal Year Final Research Report Summary
Single-fluorophore imaging under an epifluorescence microscope
| Project/Area Number |
04508002
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Keio University |
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Principal Investigator |
KINOSITA Kazuhiko Keio University, Faculty of Science and Technology, Professor, 理工学部, 教授 (30124366)
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| 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)
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| Project Period (FY) |
1992 – 1994
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| Keywords | Fluorescence microscope / Background reduction / Fluorescence polarization / Myosin / Actin / Tetramethylrhodamine / Rotation imaging / Protein confomational changes |
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| 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.
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