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

本研究的基本思想是，在多光子激光扫描显微镜（以下简称MPLSM）的激发光的波长范围内，有生理效应的植物。例如，在共焦激光扫描显微镜（下文中，CLSM）的情况下，激发光的波长在许多情况下在光合有效辐射的范围内，即，观察动作本身改变了被观察对象的状态。MPLSM观察几乎没有叶绿体观察中观察对象的这种“破坏”。然而，我们在研究过程中遇到了许多意想不到的困难，因为在我们开始这项研究的时候，几乎没有使用我们可以参考的MPLSM观察植物组织/细胞的例子。例如，不知道CLSM观测中使用的哪些荧光探针或是否适用于MPLSM观测。我们做了很多尝试和错误。F ...更多信息 我们不得不紧急绕道到达目的地。例如，我们观察到没有叶绿体的根细胞在受到激发时发出荧光。因此，我们不得不花费大量的时间来开发观察方法，而不是观察本身，我们无法实现预期的目标：四维观察（空间三维+时间）。通过试验和误差分析，我们得到了以下主要结果：1.建立了菠菜根pH值的检测方法：以SNAR-4F为探针，采用低温导入法较好。根据周围溶液pH值的变化，可以观察质外体pH值的变化.对于拟南芥保卫细胞中Ca^++浓度的观察，在我们所做的试验中，同时使用两种探针：Fra-red和fluo-3是最好的。然而，需要对方法进行更多的改进以进行精确的观测。我们观察了蓝光照射下菠菜叶细胞原生质体中叶绿体和液泡的pH值变化。液泡中的pH值升高，但叶绿体中的pH值几乎保持不变。少

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