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

Elucidating the intrinsic relationship between mitochondrial pH (pHm) fluctuation and lipid droplets (LDs) formation is vital in cell physiology. The development of small-molecular fluorescent probes for discrimination and simultaneous visualization of pHm 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 pHm 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 pHm fluctuation. Moreover, the polarity-dependent strategy is valuable for designing other functional biological probes in imaging multiple organelles.