A pH-insensitive near-infrared fluorescent probe for wash-free lysosome-specific tracking with long time during physiological and pathological processes

Abstract A novel dicyanomethylene-4H-pyran based lysosome-specific near-infrared (NIR) probe, DCM-ML , is reported. It exhibits very weak fluorescence (Ф=0.007) in aqueous solution due to the energy relaxation from intramolecular rotation; but a large "off-on" NIR emission (20-folds) after specifically labeling lysosomes due to the local high viscosity instead of the local pH by both one- and two-photon excitation microscopy. Therefore, the imaging ability of DCM-ML will not confront the problems suffered by the currently used and reported pH-dependent lysosome probes (e.g., once lysosomal pH increases, their fluorescence gets quenched); The pH-independent feature of DCM-ML is further demonstrated to be a great advantage for tracing lysosomes’movements in a relatively long time, even during cellular processes when lysosomes are suffered from external stimulations that induce pH increase and apoptosis. In addition to the characteristics of pH-insensitivity and "off-on" NIR emission, DCM-ML also possesses characteristics of excellent membrane permeability, high selectivity, low cytotoxicity, good photostability and large Stokes shift (>200 nm), which qualify it as a superior wash-free NIR probe for tracking dynamic changes of lysosomes under both physiological and toxicological conditions. Hence, we believe DCM-ML may find a great potential of application in the future.