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

Abstract

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 O-methylrhodol fluorophore, MeRho-Az, 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 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 H2S detection, we demonstrate the ability of MeRho-Az 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 MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az 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.

Dual‐Reactable Fluorescent Probes for Highly Selective and Sensitive Detection of Biological H2S

“…Such a fluorescent turn‐on response could be clearly visualized by the naked eye (Figure a). To our knowledge, the observed increase in fluorescence emission for 2 is the largest among the fluorescent probes reported so far for H 2 S …”

“…[6] Fluorescence-based methodsh ave recently emerged as au seful approach forH 2 Sd etection in biological systems. [7][8][9][10][11][12][13][14][15][16][17] An ideal intracellular H 2 Ss ensor should have al arge dynamic range for the selective detection of H 2 So ver higheri ntracellular concentrations of biothiols (such as millimolar concentrations of reduced glutathione,G SH). The reactivity of the probe toward H 2 Si sa lso essential forr eal-time detection.…”

Extended hypoxia‐mediated H2S production provides for long‐term oxygen sensing

“…Cells were transferred to 8 chamber mounted on #1.5 German borosilicate coverglass (Fisher) and grown to ~75% confluence. They were then treated with either the H 2 S‐specific fluorophore MeRho‐Az (10 μM), SSP4 (20 μM) or DCF and then exposed to 21% O 2 or 5% O 2 as described above. Cellular imaging was performed on a laser scanning confocal microscope (Zeiss LSM710 inverted microscope) using a Plan Apochromat 63x 1.4 NA oil objective.…”

A Benzooxazole‐Based Probe for the Sensitive Detection of Hydrogen Sulfide: Kinetic and Transition‐State Studies and In Vitro Application in HepG2 Cells

“…In recent times, numerous fluorescent probes have been developed for highly specific and sensitive in‐vivo imaging and in‐vitro detection of H 2 S in biological systems . These probes are normally designed on the basis of H 2 S‐persuaded highly specific reactions, like (i) reduction of azide and nitroso groups to amine,, (ii) thiolysis of dinitrophenyl ether or other leaving groups,, (iii) metal sulfide precipitation,, (iv) nucleophilic 1,4‐addition i. e., Michael‐type addition,, (v) a coordinative‐based approach, and (vi) cleavage of an S–O bond,, which are moderately slow and irreversible …”