A two-photon ratiometric fluorescent probe for effective monitoring of lysosomal pH in live cells and cancer tissues

Lysosomes are ubiquitous acidic organelles responsible for breaking down of biological substances. Acidic pH is a characteristic feature of lysosomes whereas aberrant lysosomal pH has been manifested in myriad diseases. Thus, being able to perform real-time monitoring of lysosomal pH is crucial to understand and investigate pathological and physiological processes. Herein, we report a novel and efficient carbazole-benzimidazole-based two-photon ratiometric fluorescence probe, namely BIMC for fast-response lysosomal pH detection and imaging. In this probe, the methylcarbitol substituent serves as a targeting unit for lysosomes and the benzimidazole moiety acts as a proton reaction site. BIMC exhibits an ideal pK_a value of 4.46, which is found useful for quantitative detection and imaging of lysosomal pH change in the linearly proportional range of 3.8–5.0. Two-photon ratiometric imaging studies shows that BIMC can selectively monitor pH with excellent photostability and low cytotoxicity in the lysosomes of live cells and in living cancer tissues. All these results demonstrate that BIMC is a promising two-photon fluorescent probe for a practical use in investigating lysosome-associated pathological and physiological processes.