Imaging intracellular H2O2 with the genetically encoded PerFRET and OxyFRET probes

Hydrogen peroxide (H2O2) is an important signaling intermediate with various regulatory and effector functions. Despite its significance, the subcellular organization of H2O2 signals is poorly understood. Introducing novel techniques for the intracellular detection of H2O2 would be essential for a more complete understanding of its role in cellular signaling. We previously reported the development of two novel fluorescence resonance energy transfer (FRET)-based protein sensors that showed opposite emission ratio changes upon reaction with H2O2. In this chapter, we detail the methods for using OxyFRET and PerFRET for the assessment of changes in subcellular H2O2 levels. © Springer Science+Business Media, LLC, part of Springer Nature 2019.