Near‐Infrared II Nanohybrid Enables Single‐Excitation, Fully Ratiometric Imaging of Hydrogen Sulfide In Vivo

ABSTRACT As a crucial endogenous gasotransmitter, the precise in vivo monitoring of hydrogen sulfide (H 2 S) is of fundamental importance for elucidating its associated physiological and pathological processes. However, current fluorescence imaging systems for H 2 S critically lack ratiometric probes that simultaneously feature a single excitation wavelength alongside both working and reference emission bands operating entirely within the second near‐infrared (NIR‐II) window. To address this gap, we present a NIR‐II ratiometric nanohybrid, ErNPs@mSiO 2 ‐Cy‐NO 2 , that integrates Er 3+ ‐doped lanthanide downconversion nanoparticles encapsulated by mesoporous silica loading with NIR II cyanine dye (Cy‐NO 2 ). In the presence of H 2 S, Cy‐NO 2 is converted to Cy‐NH 2 , regulating an intramolecular photoinduced electron‐transfer (PET) process. Under single 980 nm excitation, this reaction selectively modulates the ∼1026 nm emission (located in NIR II), while the Er 3+ emission at 1550 nm (located in NIR II) remains invariant and serves as an internal reference. The resulting fully NIR II ratiometric readout (I 1026 /I 1550 ) enables real‐time visualization of H 2 S in vitro and in vivo. Overall, ErNPs@mSiO 2 ‐Cy‐NO 2 establishes a robust platform for monitoring of H 2 S and holds broad potential for advanced biosensing and diagnostic applications.