Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe

Singlet oxygen ( 1 O 2 ) is a type of reactive oxygen species involved in numerous physiological activities. We previously reported that 1 O 2 -specific oxidation products are increased in patients with prediabetes, suggesting that measurement of 1 O 2 may be an important indicator of physiological and pathological conditions. The turnover in the generation and quenching of 1 O 2 is extremely rapid during biological activities owing to it high reactivity and short lifetime in solution. However, the dynamic changes in 1 O 2 generation in living cells have not been fully explored. In this study, we investigated whether the kinetics of 1 O 2 generation can be quantified using a far-red fluorescent probe for mitochondrial 1 O 2 , Si-DMA, following addition of the 1 O 2 generator, endoperoxide, to mammalian cells. The kinetics of Si-DMA fluorescence intensity dose-dependently increased following treatment of mammalian living cells with endoperoxide. Alternatively, treatment with 1 O 2 quenchers decreased the fluorescence intensities following endoperoxide treatment. Our results indicate that the kinetics of intracellular 1 O 2 can be readily obtained using Si-DMA and time-lapse imaging, which provides new insights into the mechanism of 1 O 2 generation in mammalian cells and the exploration of 1 O 2 generators and quenchers.