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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.


2D slices of LSFM images of live zebrafish 60 min after gavage.(a) Larval zebrafish (7 dpf) gavaged with phenol red to highlightthe intestine (scale bar = 1 mm). The boxed region corresponds tothe intestinal bulb expanded below in (b,c). Zebrafish gavaged with(b) 5 μM MeRho-Az + 250 μM DATS + 250 μMGSH or (c) 5 μM MeRho-Az. Scale bar in (b) and(c) = 10 μm.
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fig5: 2D slices of LSFM images of live zebrafish 60 min after gavage.(a) Larval zebrafish (7 dpf) gavaged with phenol red to highlightthe intestine (scale bar = 1 mm). The boxed region corresponds tothe intestinal bulb expanded below in (b,c). Zebrafish gavaged with(b) 5 μM MeRho-Az + 250 μM DATS + 250 μMGSH or (c) 5 μM MeRho-Az. Scale bar in (b) and(c) = 10 μm.

Mentions: To further establish MeRho-Az as anin vivo H2S reporter, we next examined its biocompatibilityusing LSFM.Because little is known about endogenous sulfide dynamics in developingzebrafish, we focused our initial efforts on H2S releasefrom a commonly used slow-releasing H2S donor, diallyltrisulfide (DATS). To confirm, as previously reported, that a thiolsuch as GSH is required to achieve H2S release from DATS,68 we used MeRho-Az with DATS to detectliberated H2S and observed a dose-dependent release ofH2S in response to [GSH] (Figure S3). To expand on the use of MeRho-Az and to establishits validity for use with LSFM in live organisms, we chose to uselarval (7 days post fertilization) zebrafish for imaging studies.At this stage in their development, zebrafish are approximately 3mm in length and maintain a high level of transparency. Also, a keybenefit of LSFM is that the collection of illuminated sheets thatmake up the three-dimensional images is obtained on a time scale (∼10s in total) significantly faster than the time scale of gut peristalsis(∼1 min), which allows for direct analysis of the actual gutvolume with minimal artifacts from translational movement. We firsttested the toxicity of MeRho-Az in larval zebrafish byorally gavaging69 7 nL of buffered solutions(50 mM PIPES, 100 mM KCl, pH 7.4) containing 5 μM MeRho-Az and monitored the fish over time. No toxicity was observed for 20h, and although this cannot completely rule out unwanted biologicaleffects in longer term experiments, it suggests the safe use of MeRho-Az as a viable in vivo fluorescent reporter over thetime scale of hours. To test the ability of MeRho-Az toprovide an analyte-responsive signal toward H2S, larvalzebrafish were orally gavaged with buffered solutions containing:buffer only, 5 μM MeRho-Az, 5 μM MeRho-Az + 250 μM DATS, 5 μM MeRho-Az + 250 μMDATS + 250 μM GSH, or 5 μM MeRho. After 60min of recovery time, a three-dimensional image of the intestinalbulb for each fish was acquired using LSFM (Figure 5).


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)

2D slices of LSFM images of live zebrafish 60 min after gavage.(a) Larval zebrafish (7 dpf) gavaged with phenol red to highlightthe intestine (scale bar = 1 mm). The boxed region corresponds tothe intestinal bulb expanded below in (b,c). Zebrafish gavaged with(b) 5 μM MeRho-Az + 250 μM DATS + 250 μMGSH or (c) 5 μM MeRho-Az. Scale bar in (b) and(c) = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543995&req=5

fig5: 2D slices of LSFM images of live zebrafish 60 min after gavage.(a) Larval zebrafish (7 dpf) gavaged with phenol red to highlightthe intestine (scale bar = 1 mm). The boxed region corresponds tothe intestinal bulb expanded below in (b,c). Zebrafish gavaged with(b) 5 μM MeRho-Az + 250 μM DATS + 250 μMGSH or (c) 5 μM MeRho-Az. Scale bar in (b) and(c) = 10 μm.
Mentions: To further establish MeRho-Az as anin vivo H2S reporter, we next examined its biocompatibilityusing LSFM.Because little is known about endogenous sulfide dynamics in developingzebrafish, we focused our initial efforts on H2S releasefrom a commonly used slow-releasing H2S donor, diallyltrisulfide (DATS). To confirm, as previously reported, that a thiolsuch as GSH is required to achieve H2S release from DATS,68 we used MeRho-Az with DATS to detectliberated H2S and observed a dose-dependent release ofH2S in response to [GSH] (Figure S3). To expand on the use of MeRho-Az and to establishits validity for use with LSFM in live organisms, we chose to uselarval (7 days post fertilization) zebrafish for imaging studies.At this stage in their development, zebrafish are approximately 3mm in length and maintain a high level of transparency. Also, a keybenefit of LSFM is that the collection of illuminated sheets thatmake up the three-dimensional images is obtained on a time scale (∼10s in total) significantly faster than the time scale of gut peristalsis(∼1 min), which allows for direct analysis of the actual gutvolume with minimal artifacts from translational movement. We firsttested the toxicity of MeRho-Az in larval zebrafish byorally gavaging69 7 nL of buffered solutions(50 mM PIPES, 100 mM KCl, pH 7.4) containing 5 μM MeRho-Az and monitored the fish over time. No toxicity was observed for 20h, and although this cannot completely rule out unwanted biologicaleffects in longer term experiments, it suggests the safe use of MeRho-Az as a viable in vivo fluorescent reporter over thetime scale of hours. To test the ability of MeRho-Az toprovide an analyte-responsive signal toward H2S, larvalzebrafish were orally gavaged with buffered solutions containing:buffer only, 5 μM MeRho-Az, 5 μM MeRho-Az + 250 μM DATS, 5 μM MeRho-Az + 250 μMDATS + 250 μM GSH, or 5 μM MeRho. After 60min of recovery time, a three-dimensional image of the intestinalbulb for each fish was acquired using LSFM (Figure 5).

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.