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.