| The redox state plays an important role in various cellular processes. In this study, we take advantage of a reduction-oxidation sensitive GFP (roGFP1) to real-timely image the mitochondrial redox state dynamics during the cisplatin-induced apoptosis. We also do pilot study on the multi-photo induced change of cellular redox state.The main contents and conclusions are as following:First, we optimize the imaging parameters for roGFP1 in FV1000 confocal microscope and analyze the impact of these parameters on the ratiometer of roGFP1. Concretely, we take time-lapse imaging for the mitochondrial targeted roGFP1 and we also study the impact of PMT voltage and pinhole on the ratiometer of roGFP1. We come to the conclusion that the concentration of roGFP1 protein is little related to the ratio value of roGFP1.Then, we investigate the response of roGFP1 to the exogenous membrane- permeant redox reagents H2O2 and DTT. The minimal H2O2 concentration that the probe roGFP1 can exhibit detectable change in fluorescence intensity ratio by our confocal system is 200μM. And the probe shows nice reversibility.As an application, we image the dynamical process of mitochondrial redox state in cisplatin induced apoptosis of Hela cells stable expressing roGFP1 gene. Our works also indicate that cells have a mechanism to antagonize the elevation of mitochondrial redox state. The ability of cells to check the increase of redox state in mitochondria is relevant to speed of advent of apoptosis.Last, using the roGFP1, we do pilot study on the multi-photo induced ROS production from cellular mitochondria. We encounter unexpected results. We found that multi-photo irradiation would change the spectrum of roGFP1. We next explore the movement of mitochondria based on this property of roGFP1.In summary, our work establishes a platform for real-time detecting the change of redox state, which has significant application to studying the role of redox state in various cellular processes.
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