Hepatic ischemia–reperfusion injury (HIRI) is responsible for postoperative liver dysfunction and liver failure. Precise and rapid navigation of HIRI lesions is critical for early warning and timely development of pretreatment plans. Available methods for assaying liver injury fail to provide the exact location of lesions in real time intraoperatively. HIRI is intimately associated with oxidative stress which impairs lysosomal degradative function, leading to significant changes in lysosomal viscosity. Therefore, lysosomal viscosity is a potential biomarker for the precise targeting of HIRI. Hence, we developed a viscosity-activatable second near-infrared window fluorescent probe (NP-V) for the detection of lysosomal viscosity in hepatocytes and mice during HIRI. A reactive oxygen species–malondialdehyde–cathepsin B signaling pathway during HIRI was established. We further conducted high signal-to-background ratio NIR-II fluorescence imaging of HIRI mice. The contour and boundary of liver lesions were delineated, and as such the precise intraoperative resection of the lesion area was implemented. This research demonstrates the potential of NP-V as a dual-functional probe for the elucidation of HIRI pathogenesis and the direct navigation of HIRI lesions in clinical applications.

Dysregulated redox homeostasis under pathological conditions can eventually culminate in oxidative stress and associated disease damage. Spatial and temporal regulation of intracellular redox states involves two crucial parameters for elucidating oxidative stress-related molecular mechanisms. However, the lack of methods for real-time analysis of redox states is a considerable hurdle for the in-depth interpretation of pathogenic mechanisms. Herein, given the over-produced reactive oxygen species (ROS) and the translocation of redox-sensitive proteins as the potential biomarkers of oxidative stress, we developed a novel ROS-macromolecular protein synergistic imaging strategy that relied on a small-molecule fluorescent CPR-SK probe. The CPR-SK specifically activated the caffeic acid moieties or targeting peptides (EWWW) toward the biomarkers, including superoxide (O 2 •−) fluctuations and Keap1 translocation, achieving simultaneous real-time imaging of dual molecular events during oxidative stress. Importantly, in situ, CPR-SK exhibited both gentle elevation of O 2•− and subsequent migration of Keap1 from the cytoplasm to the nucleus, which were key indicators for determining slight injuries induced by hepatic ischemia-reperfusion. The results clearly demonstrated that this spatiotemporal imaging method was a reliable tool for analyzing dynamic intracellular changes of the redox state and elucidating the molecular mechanisms of oxidative stress-related diseases.