Hydrogen peroxide (H 2 O 2 ) is a key redox intermediate generated within cells. Existing probes for H 2 O 2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H 2 O 2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H 2 O 2 diffusion from the mitochondrial matrix, to find a functional output of H 2 O 2 gradients in polarized cells, and to prove the existence of H 2 O 2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for realtime H 2 O 2 imaging in various biological contexts.

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H 2 O 2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H 2 O 2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for singlewavelength fluorescent lifetime imaging of H 2 O 2 levels both in vitro and in vivo. R eactive oxygen species (ROS) are products of incomplete molecular oxygen reduction. Among ROS, the superoxide anion radical O 2 * − and hydrogen peroxide H 2 O 2 are the most investigated in biology because they are produced by a wide range of enzymes, specifically or as side-products, and have a number of well-known biological effects.1 For a long time, ROS were viewed mostly in a context of their nonspecific damaging action on DNA, lipids, and proteins.2 This point of view was strongly supported by the discovery of antioxidant enzymes decomposing ROS and by the fact that phagocytes produce ROS when killing pathogens.1,3 Later, the ROS toxicity dogma was challenged after specialized O 2 * − and H 2 O 2 producing enzymes were found to be expressed in most cell types.4,5 This fact gave a new impulse to ROS investigations but, now in the context of their regulatory function, as signaling molecules. It was shown that H 2 O 2 acted as a second messenger selectively oxidizing those cysteine residues in proteins that were ionized (deprotonated) at physiological pH values. 6,7 Initially, protein tyrosine phosphatases were shown to be reversibly inactivated by H 2 O 2 . 8,9 Since then, the list of known redox regulated proteins grew exponentially. During the last years, proteomics and computational approaches added a lot of new members to the list.

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca2+ uptake.