Visual monitoring of the lysosomal pH changes during autophagy with a red-emission fluorescent probe

Autophagy plays crucial roles in maintaining normal intracellular homeostasis. Molecular probes capable of monitoring lysosomal pH changes during autophagy are still highly required yet challenging to develop. Here, a lysosome-targeting fluorescent pH probe, RML, is presented by introducing a methylcarbitol unit as the lysosome-targeting group to rhodamine B, which is highly sensitive to pH changes. RML exhibits remarkable pH-dependent behavior at 583 nm with a fluorescent enhancement of more than 148-fold. The pK_a value is determined as 4.96, and the linear response with pH changes from 4.50-5.70, which is favorable for lysosomal pH imaging. We also confirm that RML diffuses selectively into lysosomes using confocal fluorescence microscopy. Using RML, we have successfully visualized autophagy by monitoring the lysosomal pH changes.