Fluorogenic Autophagic Vesicle-Engaging Probe Enables High-Contrast and Homogeneous Imaging of Autophagic Flux in Live Cells

Autophagic flux is a highly dynamic process essential for cellular homeostasis, yet its reliable visualization in live cells remains challenging due to the limitations of transfection-based LC3 reporters. Here, we present ATP3, a fluorogenic and ratiometric chemical probe that directly engages autophagic vesicles to enable the high-contrast and homogeneous imaging of autophagic flux without genetic manipulation. ATP3 constitutes a guanine targeting moiety and a smart fluorophore, with the former facilitating autophagic vesicle engagement, while the latter is intrinsically quenched and environment-responsive. ATP3 exhibits minimal background fluorescence and robust signal amplification upon autophagy-dependent engagement. Side-by-side comparisons demonstrate that ATP3 outperforms the conventional mRFP-GFP-LC3 assay in imaging contrast and staining uniformity. Furthermore, ATP3 enables dynamic monitoring of autophagic responses in an oxygen-glucose deprivation model, revealing progressive enhancement of autophagic flux under ischemic stress. Together, ATP3 provides a robust and broadly applicable chemical tool for visualizing autophagy dynamics in physiologically and disease-relevant contexts.