Dual-Labeled Single Fluorescent Probes for the Simultaneous Two-Color Visualization of Dual Organelles and for Monitoring Cell Autophagy.

Dual-labeled single fluorescent probes are powerful tools for studying autophagy on the molecular scale, yet their development has been hampered by design complexity and a lack of valid strategies. Herein, for the first time, we introduce a combinatorial regulation strategy to fabricate dual-labeled probes for studying autophagy by integrating the specific organelle-targeting group and the functional fluorescence switch into a pentacyclic pyrylium scaffold (latent dual-target scaffold). For proof of concept, we prepared a range of dual-labeled probes ( TMOs ) that display different emission colors in duple organelles. In these probes, TMO1 and TMO2 enabled the simultaneous two-color visualization of the lysosomes and mitochondria. The other probes ( TMO3 and TMO4 ) discriminatively targeted lysosomes/nucleolus and lysosomes/lipid droplets (LDs) with dual-color emission characteristics, respectively. Intriguingly, by simply connecting the endoplasmic reticulum (ER) targeting group to the pentacyclic pyrylium scaffold, we created the first dual-labeled probe TMO5 for simultaneously labeling lysosomes/ER in distinctive fluorescent colors. Subsequently, using the dual-labeled probe TMO2 , drug-induced mitophagy was successfully recorded by evaluating the alterations of multiple mitophagy-related parameters, and the mitophagy defects in a cellular model of Parkinson's disease (PD) were also revealed by simultaneous dual-color/dual-organelle imaging. Further, the probe TMO4 can track the movement of lysosomes and LDs in real time and monitor the dynamic process of lipophagy. Therefore, this work not only presents attractive dual-labeled probes to promote the study of organelle interactions during autophagy but also provides a promising combinatorial regulation strategy that may be generalized for designing other dual-labeled probes with multiple organelle combinations.