Columbia University Irving Medical Center 1 article published in JoVE Biology Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells Emily Jie-Ning Yang1, Istvan R. Boldogh1,2, Haojie Ji2, Liza Pon1,2, Theresa C. Swayne1,2 1Department of Pathology and Cell Biology, Columbia University Irving Medical Center, 2Confocal and Specialized Microscopy Shared Resource in the Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.