HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime...

Area of Science:

• Biomedical Engineering
• Cell Biology
• Biochemistry

Background:

• Reactive oxygen species (ROS) are crucial for cellular signaling and dynamic biological events.
• Accurate monitoring of hydrogen peroxide (H2O2), a key ROS, requires high-performance sensors.
• Existing sensors like HyPer and HyPer-2 have limitations in response time, speed, and dynamic range.

Purpose of the Study:

• To introduce HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2.
• To demonstrate HyPer-3's superior performance characteristics compared to previous HyPer variants.
• To validate HyPer-3's utility in advanced imaging applications for biological research.

Main Methods:

• Development of a genetically encoded fluorescent protein sensor, HyPer-3.
• Characterization of HyPer-3's response time, speed, and dynamic range.
• In vivo imaging of H2O2 gradients in zebrafish larvae.
• Single-wavelength fluorescent lifetime imaging (FLIM) of H2O2 levels in vitro and in vivo.

Main Results:

• HyPer-3 exhibits significantly improved response time and speed over HyPer and HyPer-2.
• HyPer-3 demonstrates an expanded dynamic range compared to the original HyPer indicator.
• Successful in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae was achieved.
• HyPer-3 enabled effective single-wavelength FLIM for H2O2 quantification.

Conclusions:

• HyPer-3 represents a significant advancement in genetically encoded H2O2 sensors.
• Its enhanced performance enables more precise monitoring of dynamic biological processes.
• HyPer-3 is a valuable tool for in vivo and in vitro studies of H2O2 signaling.