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

最近的荧光显微镜在生物科学中发挥着极其强大的作用。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形态结构，还可以观察生物物理情况，如细胞内钙离子的分布和pH等。此外，在植物细胞中，叶绿素的自动荧光已经被用来了解各种光合作用现象。在本研究中，我们计划开发一种新型的荧光观测系统。这是一种配备了计算机控制的广域照射系统和高灵敏度摄像机的装置。使用这个系统，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)。有了广域荧光观察系统，我们将能够探测到组织中细胞之间的相互作用，除了单个细胞的细胞内反应。我们基本规划了该系统，并在1993年建立了照射系统和荧光观察系统。1994年和1995年，我们对以下几个方面进行了改进：(1)改进了氙块辐照系统，以检测饱和辐照下的叶绿素自体荧光。此外，还增加了卤素辐照系统来照亮样品。(2)在辐照系统中增加了滤光片的计算机控制交换系统。(3)改进了摄像机控制系统，扩大了检测范围。(4)开发了荧光图像分析的计算机程序。(5)改进了辐照系统，使其适用于常规荧光显微镜。(6)作为初步实验，利用该系统观察到了对叶绿素和非光化学猝灭的荧光诱导。该装置仍在建造中。我们在硬件和软件方面都需要进一步改进。

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.