A Bright Fluorescent Probe for H(2)S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

[Image: see text] Hydrogen sulfide (H(2)S) 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 H(2)S 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 O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H(2)S, is selective for H(2)S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az 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 H(2)S detection, we demonstrate the ability of MeRho-Az to detect differences in H(2)S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H(2)S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H(2)S 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.