All-in-One Fluorescent Probe: Enabling Simultaneous Tracking of Multiple Dynamic Organellar Perturbations during...

Ferroptosis is a regulated form of cell death driven by lipid peroxidation, involving intricate crosstalk between subcellular organelles and dynamic microenvironmental perturbations. However, this mechanism remains incompletely elucidated due to the lack of versatile tools enabling the simultaneous monitoring of multiple organelles and their microenvironments. Herein, we report a novel single fluorescent probe (ATBI) that allows concurrent discrimination and visualization of three key subcellular organelles: mitochondria, lysosomes, and lipid droplets (LDs). ATBI lights up mitochondria/lysosomes (∼719 nm) and LDs (∼425 nm) simultaneously with high fidelity at distinctly separated emission wavelengths. Notably, mitochondria and lysosomes can be effectively distinguished by their distinct morphological features and fluorescence lifetimes, while changes in viscosity within mitochondria/lysosomes can be further quantified based on lifetime variations. Using this multifunctional probe, we visualized the dynamic process of Erastin-induced ferroptosis: LD accumulation, increased lysosomal viscosity, and decreased mitochondrial viscosity. Furthermore, we demonstrated that SLC7A11, a key regulatory factor of ferroptosis, restores the normal morphology and viscosity homeostasis of these organelles, highlighting the critical role of maintaining subcellular organellar morphology and microenvironmental stability in resisting ferroptosis.