Imaging of lysosomal oxidative stress during autophagy with a ratiometric probe featuring a large probe-product spectral separation

• The 3,6-bisamino-substituted anthracene reacts with highly reactive ROS to yield a NIR-emitting carbo-rhodamine. • The bridging group modification is a facile approach for ratiometric probes to yield large probe-product spectral separation. • The probe found use in monitoring the cellular ROS status during lysosomal autophagy process. Autophagy is a physiological process governing proteostasis and implicated in many diseases. Enhanced ROS levels was known to be associated with lysosomal autophagy and therefore a lyso-specific ROS probe could find applications in autophagy related studies. Following the probe-design principle of bridging-group modification, we previously developed a ROS probe capable of detect a broader range of reactive species than the existing probes. Additional merits of the probe include ratiometry-capability, minimal probe-product spectral crosstalk, resistance toward autoxidation at lysosomal acidity, and structural simplicity. It is the aim of this manuscript to render this probe lyso-specificity and examine its potentials to monitor autophagy-related ROS generation in HeLa cells treated by H 2 O 2 or 6-OHDA.