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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.


Related in: MedlinePlus

(a) Uncorrected fluorescentresponse of MeRho-Az toNaHS treatment over 60 min. Conditions: 5 μM MeRho-Az, 250 μM NaHS, PIPES buffer (50 mM, 100 mM KCl, pH 7.4), λex = 476 nm, λem = 480–650 nm, 37 °C.(b) Concentration-dependent fluorescence of MeRho-Az whentreated with 0.10, 2.5, 5.0, 7.5, and 15 μM NaHS and incubationfor 90 min at 37 °C. Each data point represents the average ofat least three trials. Error bars were calculated as standard deviation.(c) Fluorescence photoactivation response of HSN2 (λex = 432 nm, λem = 542 nm), DNS-N3 (λex = 340 nm, λem = 550 nm), C7-Az (λex = 340nm, λem = 445 nm), and MeRho-Az (λex = 476 nm, λem = 516 nm). Excitation slits:2.6 nm. Data measured at 4 s–1.
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fig2: (a) Uncorrected fluorescentresponse of MeRho-Az toNaHS treatment over 60 min. Conditions: 5 μM MeRho-Az, 250 μM NaHS, PIPES buffer (50 mM, 100 mM KCl, pH 7.4), λex = 476 nm, λem = 480–650 nm, 37 °C.(b) Concentration-dependent fluorescence of MeRho-Az whentreated with 0.10, 2.5, 5.0, 7.5, and 15 μM NaHS and incubationfor 90 min at 37 °C. Each data point represents the average ofat least three trials. Error bars were calculated as standard deviation.(c) Fluorescence photoactivation response of HSN2 (λex = 432 nm, λem = 542 nm), DNS-N3 (λex = 340 nm, λem = 550 nm), C7-Az (λex = 340nm, λem = 445 nm), and MeRho-Az (λex = 476 nm, λem = 516 nm). Excitation slits:2.6 nm. Data measured at 4 s–1.

Mentions: Having established that MeRho offers a bright,biocompatiblefluorophore platform, we next investigated the viability of MeRho-Az as a fluorescent H2S sensor. MeRho-Az (5 μM) exhibits a rapid increase in fluorescence when treatedwith 50 equiv of NaHS (250 μM) in aqueous PIPES buffer (50 mM,100 mM KCl, pH 7.4). Owing to the stark contrast in brightness betweenthe azide- and amine-functionalized rhodol scaffolds, reduction of MeRho-Az to the parent amine produces a 1200-fold fluorescenceturn on (F/F0) over 60min (440-fold without any background correction) (Figure 2a). This represents one of the strongest fluorescentresponses from H2S detection recorded to date. While thereaction of some probes with H2S may reach completion morequickly, the magnitude of response with MeRho-Az afteronly 5 min is significant. Furthermore, the fluorescence turn-on characteristicsof MeRho-Az are faster and stronger than a recently reportednitro-reduction rhodol platform,64 whichis consistent with previous findings from our group in which azidereduction on a naphthalimide scaffold proceeds faster and has a strongerturn-on than the corresponding nitro-functionalized analogue.65 After determining that MeRho-Az effectively reports on H2S, the sensitivity and detectionlimit of the probe was examined. A linear, concentration-dependentfluorescence relationship was observed between MeRho-Az fluorescence and increasing H2S concentrations (Figure 2b, Table S1). The detectionlimit was calculated to be the concentration at which the fluorescenceequals that of [blank + 3σ] according to a linear regressionfit of the data and determined to be 86 ± 7 nM. Supporting thevalidity of this detection limit, the MeRho-Az probecan differentiate between 1.0 and 0.10 μM H2S witha p value <0.01. Finally, to test the photostabilityof MeRho-Az, we prepared three common azide-based H2S detection probes HSN2, DNS-N3, and C7-Az,31,40,65 which are based on naphthalimide,dansyl, and coumarin fluorophores, respectively, and compared thephotoactivation of each azide under identical conditions in the absenceof H2S. As expected, the rhodol system in MeRho-Az exhibits significantly less photoactivation than the other azide-basedsystems (Figure 3c). Taken together, thesedata demonstrate the reactivity of MeRho-Az with H2S and highlights its sensitivity and potential for use inbiological applications.


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)

(a) Uncorrected fluorescentresponse of MeRho-Az toNaHS treatment over 60 min. Conditions: 5 μM MeRho-Az, 250 μM NaHS, PIPES buffer (50 mM, 100 mM KCl, pH 7.4), λex = 476 nm, λem = 480–650 nm, 37 °C.(b) Concentration-dependent fluorescence of MeRho-Az whentreated with 0.10, 2.5, 5.0, 7.5, and 15 μM NaHS and incubationfor 90 min at 37 °C. Each data point represents the average ofat least three trials. Error bars were calculated as standard deviation.(c) Fluorescence photoactivation response of HSN2 (λex = 432 nm, λem = 542 nm), DNS-N3 (λex = 340 nm, λem = 550 nm), C7-Az (λex = 340nm, λem = 445 nm), and MeRho-Az (λex = 476 nm, λem = 516 nm). Excitation slits:2.6 nm. Data measured at 4 s–1.
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fig2: (a) Uncorrected fluorescentresponse of MeRho-Az toNaHS treatment over 60 min. Conditions: 5 μM MeRho-Az, 250 μM NaHS, PIPES buffer (50 mM, 100 mM KCl, pH 7.4), λex = 476 nm, λem = 480–650 nm, 37 °C.(b) Concentration-dependent fluorescence of MeRho-Az whentreated with 0.10, 2.5, 5.0, 7.5, and 15 μM NaHS and incubationfor 90 min at 37 °C. Each data point represents the average ofat least three trials. Error bars were calculated as standard deviation.(c) Fluorescence photoactivation response of HSN2 (λex = 432 nm, λem = 542 nm), DNS-N3 (λex = 340 nm, λem = 550 nm), C7-Az (λex = 340nm, λem = 445 nm), and MeRho-Az (λex = 476 nm, λem = 516 nm). Excitation slits:2.6 nm. Data measured at 4 s–1.
Mentions: Having established that MeRho offers a bright,biocompatiblefluorophore platform, we next investigated the viability of MeRho-Az as a fluorescent H2S sensor. MeRho-Az (5 μM) exhibits a rapid increase in fluorescence when treatedwith 50 equiv of NaHS (250 μM) in aqueous PIPES buffer (50 mM,100 mM KCl, pH 7.4). Owing to the stark contrast in brightness betweenthe azide- and amine-functionalized rhodol scaffolds, reduction of MeRho-Az to the parent amine produces a 1200-fold fluorescenceturn on (F/F0) over 60min (440-fold without any background correction) (Figure 2a). This represents one of the strongest fluorescentresponses from H2S detection recorded to date. While thereaction of some probes with H2S may reach completion morequickly, the magnitude of response with MeRho-Az afteronly 5 min is significant. Furthermore, the fluorescence turn-on characteristicsof MeRho-Az are faster and stronger than a recently reportednitro-reduction rhodol platform,64 whichis consistent with previous findings from our group in which azidereduction on a naphthalimide scaffold proceeds faster and has a strongerturn-on than the corresponding nitro-functionalized analogue.65 After determining that MeRho-Az effectively reports on H2S, the sensitivity and detectionlimit of the probe was examined. A linear, concentration-dependentfluorescence relationship was observed between MeRho-Az fluorescence and increasing H2S concentrations (Figure 2b, Table S1). The detectionlimit was calculated to be the concentration at which the fluorescenceequals that of [blank + 3σ] according to a linear regressionfit of the data and determined to be 86 ± 7 nM. Supporting thevalidity of this detection limit, the MeRho-Az probecan differentiate between 1.0 and 0.10 μM H2S witha p value <0.01. Finally, to test the photostabilityof MeRho-Az, we prepared three common azide-based H2S detection probes HSN2, DNS-N3, and C7-Az,31,40,65 which are based on naphthalimide,dansyl, and coumarin fluorophores, respectively, and compared thephotoactivation of each azide under identical conditions in the absenceof H2S. As expected, the rhodol system in MeRho-Az exhibits significantly less photoactivation than the other azide-basedsystems (Figure 3c). Taken together, thesedata demonstrate the reactivity of MeRho-Az with H2S and highlights its sensitivity and potential for use inbiological applications.

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.


Related in: MedlinePlus