Development of Wide-area Fluorescent Image Observing System for Analysis of the Relationship between Function and High-dimension Structure in Biological System.

开发广域荧光图像观测系统，用于分析生物系统功能与高维结构的关系。

• 批准号: 05554028
• 负责人: MIMURA Tetsuro
• 金额: $ 6.02万
• 依托单位: Hitotsubashi University (1994)Himeji Institute of Technology (1993)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05554028/
• 关键词: Image analysis; Chlorophyll Fluorescence; Fluorescence measurement; Photosynthesis; Intracellular pH; Membrane potential; Stereoscopic microscope; Plant tissue

Recent fluorescent microscopy is working as an extremely powerful tool in biological science. Combining the newly developed fluorescent probes with an improved fluorescent microscope, we are now able to observe not only morphological structure, but also biophysical situation like intracellular distribution of Ca^<2+> and pH,etc. Furthermore, in plant cells, auto fluorescence of chlorophyll has been used to understand the various photosynthetic phenomena.In the present study, we planed to develop a new-type fluorescence observing system. This is an apparatus that equips a computer-controlled wide area irradiation system and high-sensitive video camera. Using this system, we will be able to observc a fluorescent imagc of wide area like a stereoscopic microscope.If we use newly developed fluorescent probes (such as BCECF,Fura 2, etcf.) with a wide-area fluorescence viewing systcm, we will be able to detect an interaction between cells in a tissue besides intracellular reactions in the individual cell.We basically planned the system and constructed the irradiation system and the fluorescent observing system in 1993. In 1994 and 1995, we improved the following points.(1) To detect the auto fluorescence of chlorophyll under saturated irradiation, the irradiation system with Xenon lump was improved. Furthermore, a halogen irradiation system was added to illuminate the sample.(2) Computer-controlled exchange system of filters was added to the irradiation system.(3) Video camera controlling system was improved to expand the detection range.(4) Computer program was developed for the fluorescent image analysis.(5) The irradiation system was improved to be applicable for the ordinal fluorescence microscope.(6) As the preliminary experiments, a fluorescent induction fo chlorophyll and non-photo chemical quenching was observed using this system.This apparatus is still under construction. We need further improvements of both hard and soft wares.

近年来，荧光显微镜在生物科学中成为一种非常有力的工具。将新开发的荧光探针与改进的荧光显微镜相结合，我们现在不仅可以观察到形态结构，还可以观察到细胞内Ca^<2+>和pH等生物物理情况。此外，在植物细胞中，叶绿素的自荧光已被用来了解各种光合现象。在本研究中，我们计划开发一种新型荧光观测系统。这是一种配备电脑控制的广域辐射系统和高灵敏度摄像机的设备。使用该系统，我们将能够像立体显微镜一样观察到大面积的荧光图像。如果我们使用新开发的荧光探针（如BCECF，Fura 2等）和广域荧光观察系统，除了单个细胞内的细胞内反应外，我们将能够检测组织中细胞之间的相互作用。1993年，我们对系统进行了基本规划，建成了辐照系统和荧光观测系统。在1994年和1995年，我们改进了以下几点：(1)为了检测饱和辐照下叶绿素的自荧光，对氙块辐照系统进行了改进。此外，还增加了卤素照射系统来照射样品。(2)在辐照系统中增加了计算机控制的过滤器交换系统。(3)改进了摄像机控制系统，扩大了探测范围。(4)开发了荧光图像分析的计算机程序。(5)改进了辐照系统，使其适用于有序荧光显微镜。(6)初步观察到该体系对叶绿素的荧光诱导和非光化学猝灭。这台设备仍在建造中。我们在硬件和软件方面都需要进一步改进。

项目成果

Shimmen, T., Mimura T., Kikuyama M., Tazawa M.: "Characean cells as a tool for studying electro-physiological characteristics of plant cells." Cell Struct. & Funct.19. 263-278 (1994)

Shimmen, T.、Mimura T.、Kikuyama M.、Tazawa M.：“Characean 细胞作为研究植物细胞电生理特征的工具。”

Shimmen, T., Hamatani M., Saito S., Yokota E., Mimura T., Fusetani N., Karaki H.: "Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells of Hydrocharis." Protoplasma. (in press).

Shimmen, T.、Hamatani M.、Saito S.、Yokota E.、Mimura T.、Fusetani N.、Karaki H.：“肌动蛋白丝在 Hydrocharis 根毛细胞的细胞质流动和跨液泡链组织中的作用。”

Beilby, M.J.: "The proton pump,high pH channels, and excitation: voltage clamp studies of intact and perfused cells on Nitellopsis obtusa." Protoplasma. 175. 144-152 (1993)

Beilby, M.J.：“质子泵、高 pH 通道和激发：对 Nitellopsis obtusa 上完整和灌注细胞的电压钳研究。”

Shimmen, T.: "Characean cells as a tool for studying electrophysiogical characteristics 0f plant cells." Cell Struct. &Funct.19. 263-278 (1994)

Shimmen, T.：“Characean 细胞作为研究植物细胞电生理特性的工具。”

Mimura, T., Shimmen, T.: "Characterization of the Ca^<2+>-dependent C1^- efflux in perfused Chara cells" Plant & Cell Physiol. 35. 793-800 (1994)

Mimura, T.，Shimmen, T.：“灌注轮藻细胞中 Ca^2 依赖性 C1^- 流出的表征” Plant