Construction of single fluorescent probes for separately visualizing duple organelles in different emission colors

• A fluorophore with D-π-A and rigid structure was constructed with high sensitivity to polarity. • A robust probe HFM was fabricated to image LDs and ER in dual emission colors. • A novel probe HFM-M was delivered to visualize LDs and lysosomes in dual emission colors. • Changes in LDs/ER or LDs/lysosomes were simultaneously visualized during external stimuli. • Depletion of LDs and ER during alcohol stimuli was successfully revealed with HFM . Fluorescent probes separately labelling duple organelles in different colors are powerful molecular tools to investigate the interplay between different organelles. In this work, we have presented a fluorescent dye with high sensitivity to environmental polarity, and prepared two fluorescent probes to visualize duple organelles in dual emission colors based on the platform. Probe HFM simultaneously targeted lipid droplets (LDs) and endoplasmic reticulum (ER) in living cells, and imaged the two organelles in blue and green emission, respectively. Meanwhile, HFM-M enabled visualizing LDs and lysosomes in blue and green fluorescence, respectively. Using the two newly designed fluorescent probe, the changes in LDs/ER or LDs/lysosomes during oleic acid and starvation stimuli have been visualized. We have successfully revealed that excessive alcohol can lead to the depletion of LDs and ER in living cells. We believe that the unique fluorescent dye highly sensitive to environmental polarity can serve as desirable platform for probes design. The probes HFM and HFM-M are valuable molecular tools to investigate the interplay between LDs/ER or LDs/lysosome, and facilitate the researches on relative biological fields.