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Response properties of the genetically encoded optical H2O2 sensor HyPer
Jonathan Weller1, Kathrin M Kizina1, Karolina Can1
1Institut 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.
The genetically engineered HyPer sensor monitors cellular redox conditions but shows pH sensitivity and adaptation to oxidation. Careful interpretation and pH co-monitoring are essential for accurate results in neuroscience research.
Area of Science:
- Cellular biology
- Neuroscience
- Biochemistry
Background:
- Reactive oxygen species (ROS) are crucial in cellular signaling and neuropathologies.
- Accurate monitoring of (sub)cellular redox states is of significant interest.
- Genetically encoded sensors offer advantages for dynamic redox measurements.
Purpose of the Study:
- To evaluate the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures.
- To assess HyPer's response to hydrogen peroxide (H2O2) and other oxidants.
- To determine HyPer's suitability for dynamic redox monitoring in complex biological systems.
Main Methods:
- Lipofection of mouse hippocampal cell cultures with HyPer.
- Treatment with H2O2 and assessment of fluorescence ratio changes.
- Control experiments using SypHer pH sensor.
- Anoxia/reoxygenation and Cl(-) replacement experiments.
- Mitochondria-targeted HyPer evaluation.
- Ratiometric two-photon excitation and fluorescence-lifetime imaging microscopy (FLIM).
Main Results:
- HyPer exhibited dose-dependent and reversible H2O2 responses in neurons and glia.
- Repeated H2O2 exposure led to declining responses and apparent recovery during oxidant presence.
- HyPer showed sensitivity to pH changes, other oxidants, and endogenous superoxide production.
- FLIM revealed H2O2-induced decreases in HyPer fluorescence lifetimes.
- HyPer demonstrated limitations including pH sensitivity and insensitivity to reducing agents.
Conclusions:
- HyPer enables dynamic redox recordings, surpassing synthetic dyes for certain applications.
- Two-photon excitation compatibility allows studies in more complex preparations.
- Quantitative analysis may be achievable with FLIM, independent of excitation wavelength switching.
- HyPer's pH sensitivity and response characteristics necessitate careful interpretation and co-monitoring with pH sensors like SypHer for reliable data.
