A new near-field optical probe system designed for the measurements of intracellular free calcium concentrations in plants

一种新型近场光学探针系统，专为测量植物细胞内游离钙浓度而设计

基本信息

批准号：
07557144
负责人：
TAKAHASHI Tetsuo
金额：
$ 6.91万
依托单位：
Japan Advanced Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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 … More 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

为了测量高度光敏感植物的细胞内游离钙浓度，我们开发了一种新的近场扫描光学显微镜系统，该系统能够将钙敏感的荧光染料注入植物细胞中，并使用近场光点兴奋，均使用同一金属涂层的微型夹具。该系统在1）可避免的扰动中是有优势的显微镜。该系统将在植物生理学中有用，在植物生理学中，有时在加载荧光染料时报告了难度。近场光探针是用市售的微型移动物制成的：移液管……最初，更多的TE首先是用离子溅射的细铂涂层的，然后用铝果实真空。我们的目的是证明，通过相同的制造探针，近场兴奋和荧光染料的注射是可能的。设计了相对较大的扫描场，因为在我们的系统中不需要高空间分辨率。新的扫描设备由三个支持轻质扫描阶段的压电双线性组成。 A12*12MUM^2扫描图像1MUM荧光聚苯乙烯珠散布在盖玻片上证实了近场探测，使用相同的设备，我们成功地注入了小于1 PL的荧光染料溶液。尽管后者被刚性细胞壁包围，但可以将溶液注入溶液和Chara的炎性细胞中。我们得出的结论是，我们的系统通常适用于各种植物细胞，但光源的强度目前不足以监测细胞内游离钙的静息水平（-0.1MUM）。较少的