Rationally designed NIR-in/out fluorophore scaffold for a high-fidelity probe enabling deep-tissue H₂S imaging

Real-time, in situ deep-tissue bioimaging of living subjects imposes an ideal yet stringent requirement on fluorescent probes: their excitation and emission wavelengths should both reside within the "biological optical window" to minimize tissue absorption and scattering, thereby achieving a "near-infrared-in/near-infrared-out" (NIR-in/out) profile. For the most widely used single-photon microscopy, however, this ideal strategy has long been hampered by the "excitation wavelength bottleneck": most NIR probes still require visible-light excitation, severely limiting penetration depth and imaging fidelity. Existing NIR-in/out probes suffer from critical trade-offs, including small Stokes shifts, poor photostability, or inadequate water solubility.

To overcome this bottleneck, this study introduces a novel fluorophore platform, NAR, which successfully achieves NIR excitation/emission at 667/776 nm, a rare and exceptionally large Stokes shift of 109 nm, excellent water solubility, and outstanding photostability. To demonstrate its capabilities, we developed an H₂S probe, NAR-DBS, with a detection limit for H₂S as low as 38 nM. Notably, using only a conventional single-photon microscope, NAR-DBS achieved a deep-tissue imaging depth of approximately 120 μm in mouse liver sections, a performance that rivals or even surpasses that of many dedicated two-photon probes. Furthermore, when applied on a two-photon platform, the probe exhibited a remarkable imaging depth of ∼200 μm, significantly outperforming typical two-photon fluorescent probes.

This work not only provides a powerful tool for H₂S detection but also establishes an advanced fluorophore platform that both "democratizes" deep-tissue imaging by enabling superior single-photon performance with commonly available 640 nm or 660 nm lasers and sets a new benchmark for cutting-edge research with its state-of-the-art two-photon capabilities.