A molecular engineering platform for enhanced Stokes shift NIR-II fluorophores enabling high-fidelity 1400 nm in vivo imaging

Fluorescence imaging in the second near-infrared window (NIR-II, 900-1700 nm) offers superior spatial resolution and penetration depth for in vivo visualization due to reduced tissue scattering and autofluorescence. However, the advancement of this modality is often constrained by the availability of organic fluorophores that combine straightforward synthesis, a large Stokes shift to minimize self-absorption, and high stability under physiological conditions. To address this challenge, we developed a simple yet effective molecular design strategy through the synergistic enhancement of π-conjugation via benzannulation and terminal donor engineering. This approach facilely yields a series of novel asymmetric xanthene dyes (NIR-820, NIR-842, and NIR-864) exhibiting tunable deep-NIR emission, enhanced Stokes shifts (∼54 nm), and remarkable photo/chemo-stability. The representative NIR-842 nanoparticles enabled high-contrast visualization of vasculature networks. Impressively, leveraging its extended emission tail beyond 1400 nm, high-fidelity real-time angiography with an outstanding signal-to-background ratio was achieved under 1400 nm long-pass filtration. This work not only presents a robust fluorophore for high-quality bioimaging, but also establishes a versatile molecular platform for the future development of high-performance NIR-II probes.