A Bright Fluorescent Probe for H₂S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

Hydrogen sulfide (H₂S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H₂S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized <i>O</i>-methylrhodol fluorophore, <b>MeRho-Az</b>, which exhibits a rapid >1000-fold fluorescence response when treated with H₂S, is selective for H₂S over other biological analytes, and has a detection limit of 86 nM. Additionally, the <b>MeRho-Az</b> scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H₂S detection, we demonstrate the ability of <b>MeRho-Az</b> to detect differences in H₂S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H₂S synthesis. Expanding the use of <b>MeRho-Az</b> to complex and heterogeneous biological settings, we used <b>MeRho-Az</b> in combination with light sheet fluorescence microscopy (LSFM) to visualize H₂S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems