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

Hammers MD, Taormina MJ, Cerda MM, Montoya LA, Seidenkranz DT, Parthasarathy R, Pluth MD - J. Am. Chem. Soc. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: †Department of Chemistry and Biochemistry, ‡Department of Physics, §Institute of Molecular Biology, ∥Materials Science Institute. University of Oregon, Eugene, Oregon 97403-1253, United States.

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.

No MeSH data available.


Fluorescence imaging of H2S in C6 cells. Cells wereimaged after incubation with 5 μM MeRho-Az for45 min after pretreatment with (a) no pretreatment, (b) 100 nM AP39for 60 min, or (c) 20 μM AOAA for 45 min. Scale bars = 5 μm.(d) Quantified cellular fluorescence after reaction of MeRho-Az with endogenous H2S (MeRho-Az, N = 24 cells), after addition of exogenous H2S (AP39, N = 24 cells), and after inhibition of enzymatic H2S production (AOAA, N = 24 cells).
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fig4: Fluorescence imaging of H2S in C6 cells. Cells wereimaged after incubation with 5 μM MeRho-Az for45 min after pretreatment with (a) no pretreatment, (b) 100 nM AP39for 60 min, or (c) 20 μM AOAA for 45 min. Scale bars = 5 μm.(d) Quantified cellular fluorescence after reaction of MeRho-Az with endogenous H2S (MeRho-Az, N = 24 cells), after addition of exogenous H2S (AP39, N = 24 cells), and after inhibition of enzymatic H2S production (AOAA, N = 24 cells).

Mentions: On the basis of the excellent H2S sensingpropertiesof MeRho-Az, we sought to establish the efficacy of MeRho-Az for detecting endogenously produced H2S in cells. After incubation of C6 rat glial cells, which expressthe H2S-producing CBS enzyme, with 5 μM MeRho-Az for 45 min, the cells were fixed and imaged using a fluorescencemicroscope (Figure 4). We then compared thisfluorescence response with cells that had been pretreated with eithera slow-releasing H2S donor (AP39, 100 nM)66 or a common CBS inhibitor (aminooxyacetic acid, AOAA, 20μM).67 We observed a significantreduction in fluorescence in cells treated with AOAA by contrast tountreated cells, suggesting that MeRho-Az is sufficientlysensitive to detect endogenous levels of enzymatically produced H2S. Furthermore, cells treated with low concentrations of AP39showed enhanced fluorescence, highlighting the sensitivity of thesystem. These results demonstrate the applicability of the MeRho-Az platform in cellular environments, which can likely be extendedto assays involving biological fluids such as serum, blood, or tissuehomogenates.


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

Hammers MD, Taormina MJ, Cerda MM, Montoya LA, Seidenkranz DT, Parthasarathy R, Pluth MD - J. Am. Chem. Soc. (2015)

Fluorescence imaging of H2S in C6 cells. Cells wereimaged after incubation with 5 μM MeRho-Az for45 min after pretreatment with (a) no pretreatment, (b) 100 nM AP39for 60 min, or (c) 20 μM AOAA for 45 min. Scale bars = 5 μm.(d) Quantified cellular fluorescence after reaction of MeRho-Az with endogenous H2S (MeRho-Az, N = 24 cells), after addition of exogenous H2S (AP39, N = 24 cells), and after inhibition of enzymatic H2S production (AOAA, N = 24 cells).
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Related In: Results  -  Collection

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fig4: Fluorescence imaging of H2S in C6 cells. Cells wereimaged after incubation with 5 μM MeRho-Az for45 min after pretreatment with (a) no pretreatment, (b) 100 nM AP39for 60 min, or (c) 20 μM AOAA for 45 min. Scale bars = 5 μm.(d) Quantified cellular fluorescence after reaction of MeRho-Az with endogenous H2S (MeRho-Az, N = 24 cells), after addition of exogenous H2S (AP39, N = 24 cells), and after inhibition of enzymatic H2S production (AOAA, N = 24 cells).
Mentions: On the basis of the excellent H2S sensingpropertiesof MeRho-Az, we sought to establish the efficacy of MeRho-Az for detecting endogenously produced H2S in cells. After incubation of C6 rat glial cells, which expressthe H2S-producing CBS enzyme, with 5 μM MeRho-Az for 45 min, the cells were fixed and imaged using a fluorescencemicroscope (Figure 4). We then compared thisfluorescence response with cells that had been pretreated with eithera slow-releasing H2S donor (AP39, 100 nM)66 or a common CBS inhibitor (aminooxyacetic acid, AOAA, 20μM).67 We observed a significantreduction in fluorescence in cells treated with AOAA by contrast tountreated cells, suggesting that MeRho-Az is sufficientlysensitive to detect endogenous levels of enzymatically produced H2S. Furthermore, cells treated with low concentrations of AP39showed enhanced fluorescence, highlighting the sensitivity of thesystem. These results demonstrate the applicability of the MeRho-Az platform in cellular environments, which can likely be extendedto assays involving biological fluids such as serum, blood, or tissuehomogenates.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: †Department of Chemistry and Biochemistry, ‡Department of Physics, §Institute of Molecular Biology, ∥Materials Science Institute. University of Oregon, Eugene, Oregon 97403-1253, United States.

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.

No MeSH data available.