Mono o-nitrobenzyl dihydrofluorescein as a photoactivatable ROS sensor for oxidative stress in live cells

The confocal interpretation of organelle-selective fluorogenic chemosensors can be ambiguous because of inconsistency between the sensor localization and reaction site. To overcome this limitation, we implemented the photoactivation strategy which refers to light-controlled probe activation within cells at the desired time. In this study, we synthesized a photoactivatable dihydrofluorescein probe (1) and its cell-permeable derivative (3, acetoxymethyl ester of 1). We confirmed their reactivity to reactive oxygen species (ROS) and photoactivation in both solution and cells. The photoactivation of probe 3 facilitated temporal control between probe treatment and the occurrence of fluorogenic events. We conducted organelle colocalization studies and cellular ROS imaging, clearly showing that the probe primarily localized to early/late endosomes and reacted with intracellular ROS at these endosomes. These findings highlight the effectiveness of the photoactivation strategy for fluorogenic ROS probes in mitigating ambiguities associated with conventional fluorogenic ROS sensing approaches.