A Molecular Engineering Strategy to Fine‐Tune Phototoxicity of AIE Probes for Super‐Resolution Imaging of Mitochondrial Cristae Dynamics

ABSTRACT Understanding mitochondrial morphology and function is critical for investigating mitochondrial diseases, yet fluorescent probes that label the inner mitochondrial membrane (IMM) and report dysfunction at high spatiotemporal resolution remain limited. Here, we rationally designed aggregation‐induced emission‐active probes with tunable alkyl chain lengths and carboxyl groups, allowing us to fine‑tune phototoxicity and achieve a controlled, mild level of reactive oxygen species (ROS) generation. Two probes, named OTS‐7C and OTS‐12C, enabled super‑resolution imaging of mitochondrial cristae using stimulated emission depletion microscopy (STED), while OTS‐12C further supported Hessian‐structured illumination microscopy (Hessian‐SIM) imaging in living cells. Importantly, the controllable ROS output of OTS‑12C supports prolonged time‐lapse imaging of mitochondrial stress responses, including swelling, cristae remodeling, and recovery. We further demonstrated that it could serve as a platform for evaluating antioxidant effectiveness, using Vitamin C and Astaxanthin as model antioxidants. Meanwhile, fluorescence lifetime imaging of OTS‐12C revealed that ROS‐induced oxidation of unsaturated lipids increased its fluorescence lifetime within the IMM, permitting real‐time monitoring of mitochondrial functional states. This work presents a simple yet effective strategy to fine‐tune the phototoxicity of AIE photosensitizers and provides OTS‐12C as a versatile fluorescent probe for high‐resolution visualization of mitochondrial ultrastructure, investigating mitochondrial stress management and evaluating drug antioxidant efficacy in living cells.