A dual-site modulated FRET-based two-photon ratiometric fluorescent probe for tracking lysosomal pH changes in living cells, tissues and zebrafish

Lysosomal pH plays an essential role in mediating various biological processes such as immunization, cell metabolism and enzyme activity. Herein, by utilizing the fluorescence resonance energy transfer (FRET) strategy, a two-photon (TP) ratiometric fluorescent probe (NpLys-pH) has been developed for tracking of lysosomal pH changes in living cells, tissues, and zebrafish. NpLys-pH was constructed by conjugating a pH turn-on TP fluorophore 1 (D-p-A-structured naphthalene derivative) with a pH turn-off naphthalimide fluorophore 2 via a non-conjugated linker. Meanwhile, NpLys-pH has two potential pH response sites that modulate the fluorescence signal by ICT and PET, respectively. The FRET process exists between the TP fluorophore 1 and naphthalimide fluorophore 2. In addition, NpLys-pH respond to pH rapidly and reversibly with high selectivity and sensitivity and has been applied for tracking lysosomal pH changes in living cells, tissues and zebrafish. We expect that the new probe present here can prompt the development of a wide variety of TP ratiometric fluorescent probes which can find application in detecting other important analytes in biological systems.