Quantitative Real-Time Imaging of Glutathione with Subcellular Resolution.
ABSTRACT: AIMS:Quantitative imaging of glutathione (GSH) with high spatial and temporal resolution is essential for studying the roles of GSH in redox biology. To study the long-standing question of compartmentalization of GSH, especially its distribution between the nucleus and cytosol, an organelle-targeted quantitative probe is needed. RESULTS:We developed a reversible reaction-based ratiometric fluorescent probe-HaloRT-that can quantitatively measure GSH dynamics with subcellular resolution in real time. Using HaloRT, we quantitatively measured the GSH concentrations in the nucleus and cytosol of HeLa cells and primary hepatocytes under different treatment conditions and found no appreciable concentration gradients between these two organelles. Innovation and Conclusion: We developed the first reversible ratiometric GSH probe that can be universally targeted to any organelle of interest. Taking advantage of this new tool, we provided definitive evidence showing that GSH concentrations are not significantly different between the nucleus and cytosol, challenging the view of nuclear compartmentalization of GSH.
Project description:The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition. In previous work, we described the FRET-based redox sensor CY-RL7 with a relatively high midpoint redox potential of -143 mV, which is required for monitoring glutathione potentials in the comparatively high oxidative environment of the ER. Here, the efficacy of the CY-RL7 probe was ascertained in the cytosol and ER of live cells with fluorescence microscopy and flow cytometry. The sensor was found to be fully reduced at steady state in the cytosol and became fully oxidized in response to treatment with 1-chloro-2,4-dinitrobenzene, a depletor of reduced glutathione (GSH). In contrast, the probe was strongly oxidized (88%) upon expression in the ER of cultured cells. We also examined the responsiveness of the ER sensor to perturbations in cellular glutathione homeostasis. We observed that the reductive level of the FRET sensor was increased two-fold to about 28% in cells pretreated with N-acetylcysteine, a substrate for GSH synthesis. Finally, we evaluated the responsiveness of CY-RL7 and roGFP1-iL to various perturbations of cellular glutathione homeostasis to address the divergence in the specificity of these two probes. Together, the present data generated with genetically encoded green fluorescent protein (GFP)-based glutathione probes highlight the complexity of the ER redox environment and indicate that the ER glutathione pool may be more oxidized than is currently considered.
Project description:During a minimally invasive tumor resection procedure, it is still a challenge to rapidly and accurately trace tiny malignant tumors in real time. Fluorescent molecular imaging is considered an efficient method of localizing tumors during surgery due to its high sensitivity and biosafety. On the basis of the fact that γ-glutamyltranspeptidase (GGT) is overexpressed in ovarian cancer, we herein designed a highly sensitive ratiometric fluorescent GGT-responsive probe Py-GSH for rapid tumor detection. Methods: The GGT response probe (Py-GSH) was constructed by using GSH group as a response group and pyrionin B as a fluorescent reporter. Py-GSH was characterized for photophysical properties, response speed and selectivity of GGT and response mechanism. The anti-interference ability of ratiometric probe Py-GSH to probe concentration and excitation power was evaluated both in vitro and in tissue. The biocompatibility and toxicity of the ratiometric probe was examined using cytoxicity test. The GGT levels in different lines of cells were determined by ratiometric fluorescence imaging and cytometry analysis. Results: The obtained probe capable to rapidly monitored GGT activity in aqueous solution with 170-fold ratio change. By ratiometric fluorescence imaging, the probe Py-GSH was also successfully used to detect high GGT activity in solid tumor tissues and small peritoneal metastatic tumors (~1 mm in diameter) in a mouse model. In particular, this probe was further used to determine whether the tissue margin following clinical ovarian cancer surgery contained tumor. Conclusion: In combination of ratiometric fluorescence probes with imaging instrument, a point-of-care imaging method was developed and may be used for surgical navigation and rapid diagnosis of tumor tissue during clinical tumor resection.
Project description:Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.
Project description:Magnesium is one of the most abundant metals in cells and is essential for a wide range of cellular processes. Magnesium imbalance has been linked to a variety of diseases, but the scarcity of sensors suitable for detection of Mg2+ with subcellular resolution has hampered the study of compartmentalization and mobilization of this ion in the context of physiological and pathological processes. We report herein a family of fluorescent probes for targeted detection of free Mg2+ in specific intracellular organelles, and its application in the study of programmed cell death. The new sensors feature a triazole unit that plays both structural and electronic roles by serving as an attachment group for targeting moieties, and modulating a possible internal charge transfer process for ratiometric ion sensing. A probe decorated with an alkylphosphonium group was employed for the detection of mitochondrial Mg2+ in live HeLa cells, providing the first direct observation of an increase in free Mg2+ levels in this organelle in the early stages of Staurosporine-induced apoptosis.
Project description:Selective Plane Illumination Microscopy (SPIM) is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET). Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+) probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+) dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+) signal percolation, predicted in previous studies, has been directly visualized.
Project description:We report a mitochondria-specific glutathione (GSH) probe-designated as Mito-RealThiol (MitoRT)-that can monitor in vivo real-time mitochondrial glutathione dynamics, and apply this probe to follow mitochondrial GSH dynamic changes in living cells for the first time. MitoRT can be utilized in confocal microscopy, super-resolution fluorescence imaging, and flow cytometry systems. Using MitoRT, we demonstrate that cells have a high priority to maintain the GSH level in mitochondria compared to the cytosol not only under normal growing conditions but also upon oxidative stress.
Project description:Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe-ThiolQuant Green (TQ Green)-for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies.
Project description:The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a pKa of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.
Project description:Biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a key role in an extensive range of physiological processes and biological functions. Therefore, the selective and sensitive detection of intracellular thiols is important for revealing cellular function. In this study, ethyl 2-(4-(acryloyloxy)-3-formylphenyl)-4-methylthiazole-5-carboxylate (NL-AC) was designed and synthesized as a colorimetric and ratiometric fluorescent probe that can be utilized to rapidly, sensitively and selectively detect biothiols in physiological media. The fluorescence intensity of this probe using the three target biothiols at a concentration of 20 equiv. of the probe increased by approximately 6~10-fold in comparison to that without the biothiols in aqueous solution. The limits of detection (LOD) for Cys, Hcy and GSH were 0.156, 0.185, and 1.838??M, respectively. In addition, both 1H-NMR and MS analyses suggested the mechanism of fluorescence sensing to be excited-state intramolecular proton transfer (ESIPT). The novel colorimetric and ratiometric probe is structurally simple and offers detection within 20?min. Furthermore, this probe can be successfully applied in bioimaging. The results indicate high application potential in analytical chemistry and diagnostics.
Project description:Alkaline phosphatase (ALP) is an important diagnostic indicator of many human diseases. To quantitatively track ALP in biosystems, herein, for the first time, we report an efficient two-photon ratiometric fluorescent probe, termed probe 1 and based on classic naphthalene derivatives with a donor-?-acceptor (D-?-A) structure and deprotection of the phosphoric acid moiety by ALP. The presence of ALP causes the cleave of the phosphate group from naphthalene derivatives and the phosphate group changes the ability of the intramolecular charge transfer (ICT) and remarkably alters the probe's photophysical properties, thus an obvious ratiometric signal with an isoemissive point is observed. The fluorescence intensity ratio displayed a linear relationship against the concentration of ALP in the concentration range from 20 to 180 U/L with the limit of detection of 2.3 U/L. Additionally, the probe 1 is further used for fluorescence imaging of ALP in living cells under one-photon excitation (405 nm) or two-photon excitation (720 nm), which showed a high resolution imaging, thus demonstrating its practical application in biological systems.