An effective FRET-based two-photon ratiometric fluorescent probe with double well-resolved emission bands for lysosomal pH changes in living cells and zebrafish.

In cells, lysosome is an acidic organelle (approximately pH 4.5-5.5), whose pH changes plays a key role in mediating various biological processes. To address this issue, a lot of fluorescent probes have been developed and prepared for tracking lysosomal pH changes. However, few of these probes can realize the imaging of lysosomal pH changes in biosystems. Herein, a new two-photon (TP) ratiometric fluorescent probe (NpRhLys-pH) by adopting the fluorescence resonance energy transfer (FRET) strategy has been developed for imaging of lysosomal pH changes in living cells and zebrafish. In this probe NpRhLys-pH, constructed by conjugating a TP fluorophore (D-Π-A-structured naphthalimide derivative) with a rhodamine B fluorophore via a non-conjugated flexible linker, the morpholine moiety serves as a targeting unit for anchoring lysosomes, and the xanthane derivative shows a pH-modulated open/close form of the spirocycle. Such a scaffold affords the NpRhLys-pH is a reliable and specific probe for anchoring lysosomes in living cells and zebrafish with dual-channel emission peaks separated by 85 nm, and responds to lysosomal pH rapidly and reversibly with high selectivity and sensitivity, demonstrating it can be used as a powerful tool for the biological research of the relationship between physiology and pathology and lysosomal pH changes in biological systems.