title: Response properties of the genetically encoded optical H2O2 sensor HyPer.
abstract: Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H2O2 reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H2O2 treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H2O2 was still present. Although HyPer should be H2O2 specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Cl(-) exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H2O2-albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H2O2 increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H2O2 decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.
authors.affiliation: Institut für Neuro- und Sinnesphysiologie, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Universitätsmedizin, D-37073 Göttingen, Germany.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Weller
authors.affiliation: Institut für Neuro- und Sinnesphysiologie, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Universitätsmedizin, D-37073 Göttingen, Germany.
authors.forename: Kathrin M
authors.initials: KM
authors.lastname: Kizina
authors.affiliation: Institut für Neuro- und Sinnesphysiologie, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Universitätsmedizin, D-37073 Göttingen, Germany.
authors.forename: Karolina
authors.initials: K
authors.lastname: Can
authors.affiliation: Institut für Neurophysiologie und Zelluläre Biophysik, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Universitätsmedizin, D-37073 Göttingen, Germany.
authors.forename: Guobin
authors.initials: G
authors.lastname: Bao
authors.affiliation: Institut für Neuro- und Sinnesphysiologie, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Universitätsmedizin, D-37073 Göttingen, Germany. Electronic address: mmuelle7@gwdg.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D005453:Fluorescence; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000032:analysis*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D006861:Hydrogen Peroxide
keywords: Fluorescence lifetime imaging; Free radicals; Optical redox probes; Oxidative stress; Reactive oxygen species; Redox signaling; Redox-imaging; Two-photon microscopy