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.