How to use brighter 3d free#
(18, 19) Alternatively, reaction-based fluorescent probes offer the potential for in vivo compatibility, high sensitivity, and high spatiotemporal resolution while maintaining selectivity for H 2S over other RSONs including free thiols, which are abundant in much higher concentrations than H 2S in cellular milieu. Similarly, the usefulness of other techniques including gas chromatography and sulfur-selective electrodes are limited by complex workups and/or insufficient sensitivity. (17) Although the mBB method offers a robust platform for H 2S quantification, this technique still requires sample destruction and additional HPLC analysis. (16) By contrast, H 2S quantification using monobromobimane (mBB) has better detection limits and enables separation of free, sulfane, and acid-labile sulfide pools.
These conditions also liberate sulfur from acid-labile sulfur pools and are thus not selective for H 2S. (15) This technique, however, requires sample homogenization and a harshly acidic workup that precludes real-time detection or live-animal imaging. (13, 14) Historically the most widely utilized assay for H 2S detection and quantification has been the methylene blue assay. In recent years chemists have answered the call for improved tools for H 2S detection by developing small-molecule fluorescent probes and similar methods to investigate biological H 2S. (9-12) The intricacy of physiological H 2S reactivity requires that researchers utilize advanced chemical and technological tools for H 2S detection and imaging in order to gain a more detailed understanding of the interconnectivity of these networks. For example, oxidative S-sulfhydration (or persulfidation) of protein cysteine residues is proposed to constitute a significant sulfide storage mechanism, which modifies protein function and the signaling activity of H 2S. (8) Once generated, H 2S undergoes complex catabolism through its interactions with cellular oxidants, protein transition-metal centers, and reactive sulfur, oxygen, and nitrogen species (RSONs), all of which are sensitive to internal and external redox stimuli. (1, 3-7) Biosynthesized by three main enzymes, cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), H 2S is generated enzymatically in the heart, brain, liver, and kidneys however, localized H 2S concentrations in the body are tissue-dependent, suggesting differential activation and action in various H 2S-producing pathways. Since 1996, when Abe and Kimura first suggested that H 2S acts as a neuromodulator in hippocampal long-term potentiation, (2) H 2S has been recognized as an essential gasotransmitter that regulates many important physiological functions in the cardiovascular, nervous, endocrine, immune, and gastrointestinal systems. (1) No longer viewed as merely a toxic geological and environmental pollutant, H 2S is now at the center of a rich and expanding field focused on investigating its biological and physiological significance. The perception of hydrogen sulfide (H 2S) in the scientific community has shifted dramatically in the 21st century. 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.
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 H 2S in the intestinal tract of live zebrafish. To demonstrate the efficacy of this probe for H 2S detection, we demonstrate the ability of MeRho-Az to detect differences in H 2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H 2S synthesis. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. 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 H 2S, is selective for H 2S over other biological analytes, and has a detection limit of 86 nM. As our understanding of the complexity of physiological H 2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Hydrogen sulfide (H 2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research.
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