pH-Dominated Selective Imaging of Lipid Droplets and Mitochondria via a Polarity-Reversible Ratiometric Fluorescent Probe

Elucidating the intrinsic relationship between mitochondrial pH (pH_m) fluctuation and lipid droplets (LDs) formation is vital in cell physiology. The development of small-molecular fluorescent probes for discrimination and simultaneous visualization of pH_m fluctuation toward LDs has not yet been reported. In this work, utilizing pH-driven polarity-reversible hemicyanine and rhodamine derivatives, a multifunctional fluorescent probe is developed for selectively identifying mitochondria and LDs under specific pH values via dual-emission channels. This rapid-response probe, Hcy-Rh, has two distinct chemical structures under acidic and alkaline circumstances. In acidic conditions, Hcy-Rh exhibits good hydrophilicity that can target mitochondria and display an intense red fluorescence. Conversely, the probe becomes lipophilic under weakly alkaline conditions and targets LDs, showing a strong blue emission. In this manner, Hcy-Rh can selectively label mitochondria and LDs, exhibiting red and blue fluorescence, respectively. Moreover, this ratiometric probe is applied to map pH_m changes in living cells under the stimulus with FCCP, NAC, and H₂O₂. The interplay of LD–mitochondria under oleic acid treatment and starvation-induced autophagy has been studied using this probe at different pH values. In a word, Hcy-Rh is a potential candidate for further exploring mitochondria–LD interaction mechanisms under pH_m fluctuation. Moreover, the polarity-dependent strategy is valuable for designing other functional biological probes in imaging multiple organelles.