We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

Reactive oxygen species (ROS) is a generic term that defines a wide variety of oxidant molecules with vastly different properties and biological functions that range from signalling to causing pathological damage. Consequently, the molecular description of oxidants needs to be chemically precise for rational translation of research on their biological effects into therapeutic benefit in redox medicine. This Expert Recommendation article pinpoints key issues associated with identifying and explaining the physiological roles of oxidants, focusing on H2O2 and O2 .-. The generic term ROS should not be used as if it were describing a specific molecular agent, and greater precision in measurement of H2O2, O2 .and other oxidants, along with more specific identification of their respective signalling targets are needed. Future work should also focus more on inter-organellar communication in oxidant signalling and on the interactions of redox-sensitive signalling targets within complex biological systems, including whole organisms with their lifetime environmental exposures. To that effect, we recommend that new tools be used which enable site-specific and real-time detection and quantification of individual oxidants in cells and model organisms. We also stress that physiological O2 levels should be maintained in cell culture to better mimic in vivo redox reactions associated with specific cell types.Use of more precise definitions and analytical tools will help harmonize research among the many disciplines towards the common goal of understanding redox biology and medicine.

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.