Four-dimensional observation of chloroplast reactions to environmental variations using a multi-photon laser scanning florescence microscope.

使用多光子激光扫描荧光显微镜四维观察叶绿体对环境变化的反应。

基本信息

批准号：
15380171
负责人：
KURATA Kenji
金额：
$ 8.9万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

The basic idea of this research was that in a multiphoton laser scanning microscope (hereafter, MPLSM) the excitation light is out of the range of the wavelength that has physiological effects to the plants. For example, in the case of confocal laser scanning microscope (hereafter, CLSM), the wavelenghths of the excitation light are in many cases in the range photosynthetically active radiation, i.e., action of observation itself changes the status of the observed objects. MPLSM observation is almost free from such 'destruction' of the observed objects in the case of chloroplast observation. However, we confronted much of unexpected difficulties during the research, because at the time when we began this research there had been almost no examples of plant tissue/cells observations using a MPLSM to which we could refer. For example, it was unknown which of or whether the fluorescent probes used in CLSM observations are applicable to MPLSM observations. We made much of trial and error. F … More urthermore, we were obliged to make a detour to reach the goal. For example, we observed roots cells which are free from chloroplasts that emit florescence when excited. For this reason, we were obliged to spend much of time for developing observation methods, not for observation itself and we could not accomplish the expected object : four dimensional observation (spatial three dimensions + time). The main results we obtained through trial and error are the followings :1. We established the pH observation method using a spinach roots : use of SNAR-4F as a probe and its low temperature introduction method into cells were preferable. We could observe the change in pH in apoplasts according to the change in pH of the surrounding solution.2. For the observation of Ca^<++> concentrations in guard cells of Arabidopsis, simultaneous use of two probes : Fra-red and fluo-3 was most preferable among trials we made. However, more improvements in the methods were required to make accurate observations.3. We observed the change in pH in chloroplasts and vacuoles of spinach leaf cell protoplasts when irradiated by blue light. The pH in vacuoles increased but pH in chloroplasts remained almost constant. Less

这项研究的基本思想是，在多光子激光扫描显微镜（以下是MPLSM）中，兴奋光不超出对植物具有物理作用的波长范围。例如，在共聚焦激光扫描显微镜（以下简称CLSM）的情况下，在许多情况下，兴奋光的葡萄酒光在照片同步活动辐射范围内，即观察的作用本身会改变观察到的对象的状态。在叶绿体观察的情况下，MPLSM观察几乎没有这种观察到的物体的“破坏”。但是，我们在研究期间面临着许多意外的困难，因为在我们开始研究时，几乎没有使用MPLSM观察植物组织/细胞观察的例子。例如，未知CLSM观测中使用的荧光探针是否适用于MPLSM观测值。我们做了很多反复试验。 f…更urthermore，我们有义务绕行目标。例如，我们观察到根细胞，这些细胞不含叶绿体，这些细胞在激发时发出荧光。因此，我们必须花费大量时间来开发观察方法，而不是进行观察本身，而我们无法完成预期的对象：四维观察（空间三维 +时间）。我们通过反复试验获得的主要结果是以下内容：1。我们使用菠菜根建立了pH观测方法：使用SNAR-4F作为探针，其低温介绍方法是首选。我们可以根据周围溶液的pH变化观察到pH的变化2。为了观察拟南芥警卫细胞中Ca^<++>的浓度，简单地使用了两个问题：在我们进行的试验中，最优选的是FRA-RED和FLUO-3。但是，需要对这些方法进行更多改进以进行准确的观察。3。当被蓝光辐射时，我们观察到质子成形片和菠菜叶细胞原生质体的真空的pH变化。真空吸尘器中的pH值增加，但叶绿体中的pH值几乎保持恒定。较少的