Advances in ratiometric fluorescent probes for quantitative imaging of endogenous hydrogen sulfide.

Hydrogen sulfide (H₂S) has emerged as a crucial endogenous gaseous signaling molecule involved in diverse physiological and pathological processes, including vascular regulation, neuronal transmission, inflammation, and cellular redox homeostasis. The transient and highly reactive nature of endogenous H₂S necessitates the development of highly sensitive and selective analytical techniques capable of real-time detection in living systems. Among various sensing strategies, ratiometric fluorescent probes have attracted considerable attention due to their inherent ability to provide self-calibrated signals, thereby minimizing environmental interference and enabling quantitative imaging of H₂S in complex biological environments. Recent advances in probe design have introduced novel molecular scaffolds, organelle-targeted fluorophores, near-infrared emitters, and reaction-based sensing mechanisms that significantly enhance sensitivity, selectivity, and spatiotemporal resolution. These probes exploit diverse photophysical mechanisms including intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), excited-state intramolecular proton transfer (ESIPT), and photoinduced electron transfer (PET). In addition, nanomaterial-integrated probes and reversible sensing platforms have further improved quantitative detection and long-term monitoring of endogenous H₂S dynamics. Recent studies have demonstrated successful application of these probes in cellular systems, zebrafish, and mammalian disease models such as oxidative stress and acute liver injury. This review comprehensively summarizes the recent progress in ratiometric fluorescent probes for quantitative imaging of endogenous H₂S, focusing on molecular design strategies, sensing mechanisms, biological applications, and emerging technological trends. Furthermore, current challenges and future research directions are discussed to guide the development of next-generation H₂S imaging tools for biomedical and translational research.