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

为了测量细胞内游离钙浓度的高感光性植物，我们开发了一种新的近场扫描光学显微镜系统，使钙敏感的荧光染料注入植物细胞和激发这些染料与近场光斑，都使用相同的金属涂层的微量移液器装置。该系统的优点在于：1）避免了用于激发Ca^2+敏感性荧光染料的光的可能扰动，2）排除了位于其他细胞间室中的染料的荧光，在这些细胞间室中注入的染料可能被转运，3）避免了通常在落射或共聚焦荧光显微镜中采用的强烈激发导致的染料漂白和/或细胞光损伤。该系统将是有用的，特别是在植物生理学中，有时报道的困难，在加载荧光染料。近场光探针是由商业上可获得的微量移液管：移液管 ...更多信息 首先通过离子溅射用铂薄层涂覆Te，然后用铝真空沉积。我们的目的是证明，近场激发和注入的荧光染料是可能的，通过同一制造的探针。由于本系统不需要很高的空间分辨率，所以设计了较大的扫描场。新的扫描设备包括三个压电双晶片，支持轻质量扫描阶段。一张12 * 12 μ m^2的1 μ m荧光聚苯乙烯微珠在盖玻片上的扫描图像证实了近场探测，并且使用相同的设备，我们成功地注入了少于1 pl的荧光染料溶液。注射的溶液是可能的眼虫细胞和节间细胞轮藻，虽然后者是由一个刚性的细胞壁包围。我们的结论是，我们的系统通常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的静息水平（~ 0.1 μ M）。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