Rational linker engineering enables near-infrared visualization of cysteine fluctuations in vivo.

Cysteine (Cys) dynamics govern cellular redox homeostasis, yet existing fluorescent probes operating in the visible region suffer from limited tissue penetration due to strong light scattering and autofluorescence. Herein, we report a rational linker engineering strategy to develop a near-infrared (NIR) fluorescent probe for real-time Cys imaging in vivo. By systematically varying the π-conjugated bridge (furan, thiophene, EDOT, and benzene) within a dicyanoisophorone-based donor-π-acceptor scaffold, we identified furan as the optimal linker that maximizes intramolecular charge-transfer (ICT) efficiency. The optimized probe, FTCN-CA, exhibits NIR emission at 720 nm with a large Stokes shift of 250 nm, detects Cys with a detection limit of 8.7 × 10-4 μM (DMSO-PBS, 1:1, v/v, pH 7.4), and discriminates Cys from other biothiols with high selectivity. Importantly, FTCN-CA enables visualization of endogenous and exogenous Cys fluctuations in live HeLa cells and in living mice under both physiological and oxidative stress conditions. In a renal ischemia-reperfusion injury model, FTCN-CA reveals elevated Cys levels in pathological kidney tissue, providing direct visual evidence of a compensatory antioxidant response. This work establishes a generalizable linker-engineering strategy for NIR probe development and provides a robust tool for investigating Cys-associated redox biology.