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

为了测量高度光敏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，该系统使用相同的金属包被微移管装置，将钙敏感荧光染料注射到植物细胞中，并用近场光斑激发这些染料。该系统的优势在于：1)避免了用于激发Ca^<2+>敏感荧光染料的光的可能扰动，2)排除了位于其他细胞间区室的染料的荧光，在这些区室中注入的染料可能被运输，3)避免了染料的漂白和/或通常采用外焦或共焦荧光显微镜的强烈激发对细胞造成的光损伤。该系统将在植物生理学中特别有用，在植物生理学中，有时在装载荧光染料方面有困难。近场光探针是由市售的微移液器制成的：首先通过离子溅射在微移液器上涂上一层薄薄的铂，然后真空沉积铝。我们的目的是证明近场激发和荧光染料的注入是可能的，通过同一制造探针。由于系统不需要高空间分辨率，所以设计了较大的扫描场。新的扫描装置由三个压电双晶片组成，支持轻质量扫描阶段。铺在盖盖上的1mum荧光聚苯乙烯珠的A12*12mum^2扫描图像证实了近场探测，并且使用相同的设备，我们成功地注入了不到1pl的荧光染料溶液。该溶液可以注射到真核细胞和结间细胞中，尽管后者被坚硬的细胞壁包围。我们的结论是，我们的系统一般适用于各种植物细胞，除了光源的强度目前不足以监测细胞内游离钙（-0.1muM）的静息水平。少

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