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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.


Average fluorescenceintensity in zebrafish intestinal bulb, normalizedto the mean of the buffer-gavaged set. Each dot represents one fish,each of which provided ∼107 intensity measurements.Boxes extend to the first and third quartile; whiskers enclose datawithin 1.5 times the interquartile range. Solid lines denote median,and dashed lines denote mean values. Shown are measurements for fishorally gavaged with 6.9 nL of buffered solutions (50 mM PIPES, pH7.4): buffer (N = 5), 5 μM MeRho-Az (N = 9), 5 μM MeRho-Az + 250μM DATS (N = 8), 5 μM MeRho-Az + 250 μM DATS + 250 μM GSH (N = 8),and 5 μM MeRho (N = 6).
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fig6: Average fluorescenceintensity in zebrafish intestinal bulb, normalizedto the mean of the buffer-gavaged set. Each dot represents one fish,each of which provided ∼107 intensity measurements.Boxes extend to the first and third quartile; whiskers enclose datawithin 1.5 times the interquartile range. Solid lines denote median,and dashed lines denote mean values. Shown are measurements for fishorally gavaged with 6.9 nL of buffered solutions (50 mM PIPES, pH7.4): buffer (N = 5), 5 μM MeRho-Az (N = 9), 5 μM MeRho-Az + 250μM DATS (N = 8), 5 μM MeRho-Az + 250 μM DATS + 250 μM GSH (N = 8),and 5 μM MeRho (N = 6).

Mentions: No difference in fluorescencewas observed when comparing the signalbetween a vehicle-gavaged control group and fish gavaged with either MeRho-Az alone or MeRho-Az + DATS, confirmingthat LSFM was not causing photoactivation of the azide and that H2S release from DATS was GSH-dependent (Figure 6).70 In contrast, fish gavagedwith both MeRho-Az and DATS/GSH were measurably brighterthan the vehicle or MeRho-Az alone (Figure 6), confirming that H2S was being captured by MeRho-Az and visualized using LSFM (Figure 5, see Supporting Information fora link to video compiling 2D image slices into a 3D representation).To compare the relative intensity of the fully activated probe, wealso gavaged fish with the fluorophore, MeRho, alone,which resulted in an identical intensity to that observed with MeRho-Az with DATS/GSH, which is consistent with efficientH2S-mediated activation of MeRho-Az in thezebrafish gut. To the best of our knowledge, these data demonstratethe first use of LSFM for live-animal imaging of analyte-responsivereaction-based probes, thus opening the door for new investigationsof whole-organism imaging in the context of reactive small moleculeanalytes. In a broader context, the three-dimensional imaging capabilityafforded by LSFM is crucial for accurately determining fluorescenceintensity in a whole organism due to the heterogeneity of basal autofluorescence,reflection, and absorption of various tissues and organs. Differentiationand separation of these different signals would not be possible withoutthe 3D intensity map afforded by LSFM imaging.


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)

Average fluorescenceintensity in zebrafish intestinal bulb, normalizedto the mean of the buffer-gavaged set. Each dot represents one fish,each of which provided ∼107 intensity measurements.Boxes extend to the first and third quartile; whiskers enclose datawithin 1.5 times the interquartile range. Solid lines denote median,and dashed lines denote mean values. Shown are measurements for fishorally gavaged with 6.9 nL of buffered solutions (50 mM PIPES, pH7.4): buffer (N = 5), 5 μM MeRho-Az (N = 9), 5 μM MeRho-Az + 250μM DATS (N = 8), 5 μM MeRho-Az + 250 μM DATS + 250 μM GSH (N = 8),and 5 μM MeRho (N = 6).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Average fluorescenceintensity in zebrafish intestinal bulb, normalizedto the mean of the buffer-gavaged set. Each dot represents one fish,each of which provided ∼107 intensity measurements.Boxes extend to the first and third quartile; whiskers enclose datawithin 1.5 times the interquartile range. Solid lines denote median,and dashed lines denote mean values. Shown are measurements for fishorally gavaged with 6.9 nL of buffered solutions (50 mM PIPES, pH7.4): buffer (N = 5), 5 μM MeRho-Az (N = 9), 5 μM MeRho-Az + 250μM DATS (N = 8), 5 μM MeRho-Az + 250 μM DATS + 250 μM GSH (N = 8),and 5 μM MeRho (N = 6).
Mentions: No difference in fluorescencewas observed when comparing the signalbetween a vehicle-gavaged control group and fish gavaged with either MeRho-Az alone or MeRho-Az + DATS, confirmingthat LSFM was not causing photoactivation of the azide and that H2S release from DATS was GSH-dependent (Figure 6).70 In contrast, fish gavagedwith both MeRho-Az and DATS/GSH were measurably brighterthan the vehicle or MeRho-Az alone (Figure 6), confirming that H2S was being captured by MeRho-Az and visualized using LSFM (Figure 5, see Supporting Information fora link to video compiling 2D image slices into a 3D representation).To compare the relative intensity of the fully activated probe, wealso gavaged fish with the fluorophore, MeRho, alone,which resulted in an identical intensity to that observed with MeRho-Az with DATS/GSH, which is consistent with efficientH2S-mediated activation of MeRho-Az in thezebrafish gut. To the best of our knowledge, these data demonstratethe first use of LSFM for live-animal imaging of analyte-responsivereaction-based probes, thus opening the door for new investigationsof whole-organism imaging in the context of reactive small moleculeanalytes. In a broader context, the three-dimensional imaging capabilityafforded by LSFM is crucial for accurately determining fluorescenceintensity in a whole organism due to the heterogeneity of basal autofluorescence,reflection, and absorption of various tissues and organs. Differentiationand separation of these different signals would not be possible withoutthe 3D intensity map afforded by LSFM imaging.

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.