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

本研究的基本思路是在多光子激光扫描显微镜（以下简称MPLSM）中，激发光处于对植物有生理影响的波长范围之外。例如，在共聚焦激光扫描显微镜（以下简称CLSM）中，激发光的波长在很多情况下都在光合有效辐射的范围内，即观察本身的作用改变了被观察物体的状态。在叶绿体观测中，MPLSM观测几乎没有这种对被观测对象的“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MPLSM观察植物组织/细胞的例子，我们可以参考。例如，不知道CLSM观测中使用的荧光探针是否适用于MPLSM观测。我们进行了大量的试错。此外，我们不得不绕道去达到目的。例如，我们观察到没有叶绿体的根细胞在受到刺激时会发出荧光。因此，我们不得不花费大量的时间去发展观察方法，而不是观察本身，我们无法完成预期的目标：四维观察（空间三维+时间）。我们通过试错得到的主要结果如下：1。我们建立了以菠菜根为材料的pH观察方法，采用SNAR-4F作为探针，低温导入细胞的方法较为理想。我们可以根据周围溶液的pH变化来观察外质体的pH变化。为了观察拟南芥保护细胞中的Ca^<++>浓度，同时使用两种探针：Fra-red和fluo-3是我们所做的试验中最可取的。然而，为了进行准确的观测，需要对方法进行更多的改进。观察了蓝光照射下菠菜叶细胞原生质体叶绿体和液泡pH值的变化。液泡内pH值升高，而叶绿体内pH值基本保持不变。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