Rhodamine‐Based Fluorescence Probe for Monitoring of Lysosomal pH: Spectroscopic Insights and Cellular Applications

Abstract Lysosomes play a pivotal role in various cellular processes, and their dysregulation can lead to severe health conditions. Monitoring lysosomal pH changes in real time is crucial for understanding and controlling lysosome‐related disorders. Herein, we report the development of a rhodamine‐based fluorescence probe, Rh‐Mo, designed for lysosomal pH sensing and lysosome‐specific imaging of human cells. Spectroscopic studies reveal that Rh‐Mo undergoes a color change and fluorescence enhancement in acidic conditions. The probe exhibits a high fluorescence quantum yield at acidic pH (0.84 at pH = 4.0) and a p K a value of 5.85, enabling sensitive detection of lysosomal pH changes. Theoretical calculations support the mechanism of protonation‐induced ring opening, elucidating the spectroscopic behavior of Rh‐Mo. In cellular studies, Rh‐Mo exhibits low cytotoxicity and efficient uptake and colocalization with lysosomes in MCF‐7 cells, highlighting its potential for bioimaging applications.