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In order to understand the physiological functions of reactive oxygen species (ROS) generated in leaves, their direct measurement in vivo is of special importance. Here we report experiments with two dansyl-based ROS sensors, the singlet oxygen specific DanePy and HO-1889NH, which is reactive to both singlet oxygen and superoxide radicals. Here we report in vivo detection of ¹O₂ and O₂^–• by fluorescence quenching of two dansyl-based ROS sensors, the ¹O₂ specific DanePy and HO-1889NH, which was reactive with both ¹O₂ and O₂^–•. The ROS sensors were administered to spinach leaves through a pinhole, and then the leaves were exposed to either excess photosynthetically active radiation or UV (280–360 nm) radiation. Microlocalization of the sensors’ fluorescence and its ROS-induced quenching was followed with confocal laser scanning microscopy and with fluorescence imaging. These …