| Project/Area Number |
07557144
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Physical pharmacy
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| Research Institution | Japan Advanced Institute of Science and Technology |
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Principal Investigator |
TAKAHASHI Tetsuo JAIST,School of Materials Sci.Associate Professor, 材料科学研究科, 助教授 (90133769)
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| Co-Investigator(Kenkyū-buntansha) |
TSUJIMOTO Kazuo JAIST,School of Materials Sci.Professor, 材料科学研究科, 教授 (50017414)
KATAOKA Hironao Tohoku Univ.Inst.Genetic Ecology Associate Professor, 遺伝生態研究センター, 助教授 (30108568)
NAGAMURA Toshihiko Unisoku Co.Ltd., Research Center, Director, 開発研究所, 所長(研究職)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1995: ¥9,400,000 (Direct Cost: ¥9,400,000)
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| Keywords | Near-field / fluorescent dyes / plant cells / microinjection / microloading / scanning microscope / sub-picoliters / calcium / カルシウムイオン / 走査型光学顕微鏡 / マイクロインジェクション / 蛍光色素 / カルシウム / 蛍光プローブ / ナノスケール / マニピュレート顕微鏡 / マイクロキャピラリー |
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| Research Abstract |
To measure intracellular free calcium concentrations of highly photosensitive plants, we developed a new near-field scanning optical microscope system that enabled injection of calcium-sensitive fluorescent dyes into plant cells and the excitation of these dyes with a near-field light spot, both using the same metal-coated micropipette apparatus. This system is advantageous in 1) avoiding possible perturbation from light used for the excitation of Ca^<2+> sensitive fluorescent dyes, 2) excluding fluorescence from dyes located in other intercellular compartments where the injected dyes may be transported, 3) circumventing bleaching of dyes and/or photodamages to the cells resulting from intense excitation normally adopted in epi-or conforcal fluorescent microscopy. This system will be useful especially in plant physiology where the difficulty is sometimes reported in loading fluorescent dyes.
The near-field light probes were fabricated from commercially available micropipette : the pipet
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te was first coated with a thin layr of platinum through ion sputtering and then vacuum deposited with aluminum. Our aim is to demonstrate that the near-field excitation and the injection of the fluorescent dyes is possible through the same fabricated probe. Relatively large scanning field was designed because high spatial resolution was not necessary in our system. The new scanning device consists of three piezo bimorphs that support a light-mass scanning stage. A12*12mum^2 scanning image of 1mum fluorescent polystirene beads spread on a coverslip confirmed the near-field probing, and using the same apparatus, we succeeded in injecting less than 1 pl of a fluorescent dye solution. Injection of a solution was possible into both Euglena cells and internodal cells of Chara, although the latter were surrounded by a rigid cell wall. We concluded that our system is generally applicable to various plant cells, except that the intensity of the light source is currently not enough to monitor resting levels of intracellular free calcium (-0.1muM). Less
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