Tuning Second Near-Infrared Fluorescence Activation by Regulating the Excited-State Charge Transfer Dynamics Change Ratio

Second near-infrared (NIR-II) fluorescence imaging holds great promise for studying biopathological processes with high spatial resolution. However, developing activatable NIR-II fluorescent probes (AFPs) remains challenging due to insufficient signal activation in response to biomarkers and labor-intensive probe optimization. Here, we identify the excited-state charge transfer dynamics change ratios (δ) as a critical determinant of the fluorescence "turn-on" ratio of AFPs. We design a series of AFPs and their uncaged counterparts (uAFPs) and systematically analyze their photophysical characteristics and responsiveness. Comprehensive analyses including computational calculations, femtosecond transient absorption spectroscopy, steady-state fluorescence spectra, and fluorescence titration experiments verify a strong correlation between the theoretical and experimental δ values and the fluorescence "turn-on" ratios of activated AFPs. As a proof of concept, the optimal probe AFP2 indicated by δ enables early diagnosis of drug-induced liver injury and ultrasensitive detection of tiny metastatic foci (<2 mm) in mouse models, demonstrating superior sensitivity outperforming conventional methods. This study highlights the potential of δ as a predictor of probe responsiveness, which can streamline and accelerate the development and optimization of NIR-II AFPs for broader preclinical and translational applications.