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

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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