Abstract
Intracellular pH is an important measurement of cell activity. Among pH measurements, fluorescence measurement is generally used because of the advantage of non-contact, real-time and two-dimensional distribution. The other hand, this measurement itself is very delicate and tough because fluorescence is essentially the light with extremely weak intensity. In this measurement, to avoid the influence of fade on measuring accuracy, fluorescence pH ratiometry was proposed and frequently used. However, in applying this method to plant cell in accordance with the protocol of reagent company, the pH value varied widely even in same cell in the stage of calibration between pH and intensity ratio. This research was carried out to establish the practical procedure for obtaining the result with the higher reliability. Here, Allium cepa was used for plant tissue. BCECF-AM was used for pH-indicator. The experiment was made using the cryomicroscopy having cooled CCD camera. First, to decide the conditions of obtaining sufficient image to be analysed, the calibration was made many times with varying the reagent concentration, incubation-time in addition to pH concentration. Next, we examined the distributions of luminance and fluorescence intensity ratio in detail using the good images to understand the causes of scattering of value respectively. Through above discussions, the procedure to make the smallest scatter of data was found out. Further, we applied this established procedure to the plant tissue which was in cooling process to understand the difference in pH-change due to cooling rate.
