Unravelling Immune‐Inflammatory Responses and Lysosomal Adaptation: Insights from Two‐Photon Excited Delayed Fluorescence Imaging

Abstract Two‐photon excitation (TPE) microscopy with near‐infrared (NIR) emission has emerged as a promising technique for deep‐tissue optical imaging. Recent developments in fluorescence lifetime imaging with long‐lived emission probes have further enhanced the spatial resolution and precision of fluorescence imaging, especially in complex systems with short‐lived background signals. In this study, two innovative lysosome‐targeting probes, Cz‐NA and t Cz‐NA, are introduced. These probes offer a combination of advantages, including TPE ( λ ex = 880 nm), NIR emission ( λ em = 650 nm), and thermally activated delayed fluorescence (TADF) with long‐lived lifetimes (1.05 and 1.71 µs, respectively). These characteristics significantly improve the resolution and signal‐to‐noise ratio in deep‐tissue imaging. By integrating an acousto‐optic modulator (AOM) device with TPE microscopy, the authors successfully applied Cz‐NA in two‐photon excited delayed fluorescence (TPEDF) imaging to track lysosomal adaptation and immune responses to inflammation in mice. This study sheds light on the relationship between lysosome tubulation, innate immune responses, and inflammation in vivo, providing valuable insights for the development of autofluorescence‐free molecular probes in the future.