2001 Fiscal Year Final Research Report Summary
Development of an in vivo single-molecule fluorescence microscope and its application to the studies of cell signaling
| Project/Area Number |
12558082
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
SAKO Yasushi Graduate School of Medicine, Osaka University, Associate Professor, 医学系研究科, 助教授 (20215700)
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| Co-Investigator(Kenkyū-buntansha) |
SASE Ichiro Nikon Microscience Co., Researcher, 研究員
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| Project Period (FY) |
2000 – 2001
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| Keywords | total internal reflection / fluorescence / calcium / photobleach / multicolor microscopy / EGF / Ras |
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| Research Abstract |
In this project, we developed an "in vivo single-molecule fluorescence microscope", which can observe single particles of bio-molecules and complexes of bio-molecules in living cells. Various kinds of optical manipulation of cells can be executable simultaneously of the single molecule observation. The specifications of this microscope are as follows.
(1) Single molecule observation of fluorescent molecules can be achieved using the objective-type total internal reflection (TIR) optics.
(2) An Kr-Ar ion laser (488, 568, 647 nm) and a Nd/YAG laser (532 nm) are equipped as the light sources.
(3) Dual-view optics are equipped before the detector for the simultaneous dual color observation.
(4) Epi-illumination optics with a halogen arc lamp can be used simultaneously with the illumination using TIR. The epi-illumination can be used for the measurements with various intracellular fluorescence indicators and photochemical reactions of caged compounds.
(5) TIR photobleaching experiment can be don
… More
e in a very limited excitation volume (1 μm^2 x 150 nm).
Performance of the prototype of this microscope was improved through an application that is an analysis of intracellular signaling events induced by epidermal growth factor (EGF). First, we examined the number of EGF-binding on the cell surface that is enough to induce intracellular calcium response. A pulse of EGF labeled with a fluorophore Cy3 was added to cells loaded with a fluorescence calcium indicator Fluo-4. In the same cells, the number of EGF-binding was counted using TIR microscopy and the intracellular calcium concentrations were measured using Fluo-4 excited by epi-illumination. We have found that the binding of 150 molecules of Cy3-EGF is enough to induce calcium response in every cell. Magnitude of the response was not related to the number of EGF-binding. Cells has several tens thousands of EGFRs on the cell surface. Therefore, activation of less than 1 % of receptors was enough to induce cellular response. Now, we are studying the lateral movement of cell signaling proteins Ras and Raf1 under the plasma membrane. In this study, TIR photobleaching technique will be used. Less
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[Publications] Sako Y., Hibino, K., Miyauchi, T., Miyamoto, Y., Ueda, M., and Yanagida, T.: "Single-molecule imaging of signaling molecules in living cells"Single Mol.. 1. 151-155 (2000)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Yanagida, T., Tanaka, H., Kitamura, K., Wazawa, T., Nishiyama, M., Esaki, S., Sako, Y., Ide, T., Iwane, A. H-, Ishii, Y.: "Single molecule techniques in biophysics""Na/K-ATPase and related ATPases" Taaniguchi, K. and Kaya, S. ed.. 71-85 (2000)
Description
「研究成果報告書概要(欧文)」より
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