Response properties of the genetically encoded optical H2O2 sensor HyPer

Weller, Jonathan; Kizina, Kathrin M.; Can, Karolina; Bao, Guobin; Müller, Michael

doi:10.1016/j.freeradbiomed.2014.07.045

Cited by 40 publications

(28 citation statements)

References 63 publications

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“…However, the following evidence supports direct apoplastic ROS perception: (1) ROS are highly reactive and unstable molecules and among other ROS molecules H 2 O 2 is the most stable form (Giorgio et al, 2007), with estimated half-life from 1 ms (most sources) to seconds. As inferred from the measurements utilizing the genetically encoded HyPer probe, the half-life of H 2 O 2 in Arabidopsis guard cells, including the effect of influx from the apoplast and degradation in the symplasm, is in the range of seconds (Costa et al, 2010), while in animal cells H 2 O 2 appears more stable (Weller et al, 2014). The half-life of O 2 c2 in biological systems is several orders of magnitude shorter (Giorgio et al, 2007), thus, the transport of ROS other than H 2 O 2 seems unlikely.…”

Section: Ros Sensing In Guard Cell Signalingmentioning

confidence: 99%

Reactive Oxygen Species in the Regulation of Stomatal Movements

Sierla

Waszczak²,

Vahisalu³

et al. 2016

Plant Physiol.

185

137

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Section: Ros Sensing In Guard Cell Signalingmentioning

confidence: 99%

Reactive Oxygen Species in the Regulation of Stomatal Movements

Sierla

Waszczak²,

Vahisalu³

et al. 2016

Plant Physiol.

185

137

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“…Most sensors that are used by excitation ratio measurement do not have a substantial change in fluorescence lifetime [e.g., (13,25)], as the fluorescence intensity change is predominantly a change between different absorbance states that produce the same, or similar excited states. Most sensors designed to exploit Förster resonance energy transfer (FRET) between two different FPs do show a change in fluorescence lifetime of the ''donor'' FP, as FRET provides an additional nonradiative pathway out of the excited state and thus speeds its decay [e.g., (10,14,15)].…”

Section: Innovationmentioning

confidence: 99%

Cytosolic NADH-NAD⁺Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

Mongeon

Venkatachalam

Yellen

2016

Antioxidants & Redox Signaling

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Aim: Cytosolic NADH-NAD + redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD + redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. Results: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD + ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Innovation: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Conclusion: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD + ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553-563.

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“…Dissociated hippocampal cell cultures were prepared from 2-to 4-day-old mice of either sex (55). They were incubated at 37°C in a humidified 5% CO 2 -containing atmosphere, and the medium was refreshed every 2-3 days.…”

Section: Preparationmentioning

confidence: 99%

“…During FLIM, fluorescence decay rates were monitored by a 76-MHz time-correlated single-photon counting detector (FLIM X16; LaVision BioTec) and underwent iterative reconvolution with the IRF, extracted from the background signal of cell cultures or the second harmonic generation emission of urea crystals (5,55). Exponential fitting was performed using a nonlinear Levenberg-Marquardt leastsquare fitting algorithm (4,43,55,57).…”

Section: Optical Recordingsmentioning

confidence: 99%

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Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

Wagener

Kolbrink

Dietrich

et al. 2016

Antioxidants & Redox Signaling

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Aims: Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. Results: Our redox indicator mice widely express Thy1-driven roGFP1 (reduction-oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. Innovation and Conclusion: Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell-and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding of cellular redox signals in their full complexity. Antioxid. Redox Signal. 25, 41-58.

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Response properties of the genetically encoded optical H2O2 sensor HyPer

Cited by 40 publications

References 63 publications

Reactive Oxygen Species in the Regulation of Stomatal Movements

Reactive Oxygen Species in the Regulation of Stomatal Movements

Cytosolic NADH-NAD⁺Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

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Response properties of the genetically encoded optical H2O2 sensor HyPer

Cited by 40 publications

References 63 publications

Reactive Oxygen Species in the Regulation of Stomatal Movements

Reactive Oxygen Species in the Regulation of Stomatal Movements

Cytosolic NADH-NAD+Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

Contact Info

Product

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About

Cytosolic NADH-NAD⁺Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging