Highly Photostable Fluorescent Tracker with pH-Insensitivity for Long-Term Imaging of Lysosomal Dynamics in Live Cells

Visualizing and tracking lysosomal dynamic changes is crucially important in the fields of physiology and pathology. Most currently used pH-dependent small-molecule lysotrackers and sensors usually fail to visualize and track the changes due to (1) their leakage from lysosomes when the lysosomal pH increases and (2) their low photostability. Therefore, it is of significant interest to develop lysosomal probes for visualizing and tracking lysosomal dynamics independent of pH fluctuations and with high photostability. Herein, we found that the popular dicyanomethylene-4H-pyran (DCM) derivative DCM-NH2 can selectively target and label lysosomes with bright red fluorescence regardless of pH changes. The fluorescence enhancement in lysosomes has probably resulted from their microenvironment of polarity and viscosity. Compared with the commonly used commercial lysosomal molecular probes (LysoTracker Deep Red (LTDR) and LysoTracker Red DND-99), DCM-NH2 was demonstrated to exhibit a much stronger tolerance in lysosomes against various treatments and microenvironmental changes, and lysosomal membrane permeability could not cause DCM-NH2 to lose imaging of their targets as well. Moreover, DCM-NH2 exhibited a superior anti-photobleaching ability and low (photo-) cytotoxicity, which, along with pH-insensitivity, ensured its capability of long-term visualizing and tracking lysosomal dynamics. Lysosomal dynamic events such as the kiss-and-run process, fusion–fission, and mitophagy were successfully recorded with DCM-NH2. Our study thus confirms that DCM-NH2 is highly competitive for lysosomal imaging by overcoming the limitations of the commercial LysoTrackers and highlights the unexplored application of DCM-NH2 in bioimaging.