Ratiometric Visualization of NO/H₂S Cross-Talk in Living Cells and Tissues Using a Nitroxyl-Responsive Two-Photon Fluorescence Probe

It is of scientific significance to explore the intricate relationship between two crucial gasotransmitters nitric oxide (NO) and hydrogen sulfide (H₂S) because they exert similar and interdependent biological actions within the living organisms. Nevertheless, visualization of the NO/H₂S crosstalk using effective molecular imaging tools remains challenging. To address this issue, and given that nitroxyl (HNO) has been implicated as the interdependent production of NO and H₂S via a network of cascading chemical reactions, we herein design a ratiometric two-photon fluorescent probe for HNO, termed TP-Rho-HNO, which consists of benzo­[h]­chromene-rhodol scaffold as two-photon energy transfer cassette with phosphine moiety as specific HNO recognition unit. The newly proposed probe has been successfully applied in ratiometric two-photon bioimaging of endogenous HNO derived from NO and H₂S interaction in the human umbilical vein cells (HUVECs) and as well as in rat brain tissues. Intriguingly, the imaging results consistently demonstrate that the mutually dependent upgeneration of H₂S and NO are present in living biosystems, indicating that this molecular probe would provide a powerful approach to elucidate the chemical foundation for the anfractuous cross-talk between the NO and H₂S signaling pathways in biology.