Reactive oxygen species (ROS) are considered to play a key damaging role in brain during the development of ischemic stroke. To clarify how different ROS contribute to ischemic pathogenesis, innovative approaches for real-time detection of redox parameters are necessary. Using highly sensitive genetically encoded biosensor HyPer7 and a fiber-optic neurointerface technology, we demonstrated that the level of hydrogen peroxide (HO) slowly increases in neurons and astrocytes of the ischemic area of the rat brain after middle cerebral artery occlusion during next 40 h; notably, in astrocytes the level is somewhat higher. Raman microspectroscopy in awake mice also revealed redox differences between mitochondria of neurons and astrocytes during acute ischemia caused by photothrombosis. Astrocytes demonstrated the overloading of the electron transport chain (ETC) with electrons after 1 h of ischemia, whereas neurons do not demonstrate changes in the amount of reduced electron carries. The combination of novel approaches allows to detail redox events with spatiotemporal resolution. We demonstrated redox difference between neurons and astrocytes in damaged brain areas . An elevated loading of astrocytic ETC with electrons during the acute ischemia phase provides basis for the increased generation of superoxide anion radical (O) with its following conversion to other reactive species. However, we observed increased HO concentrations in astrocytes and neurons at later pathogenesis stages. During this period, ETC did not demonstrate an elevated loading with electrons, and therefore, increased HO generation may be a phenomenon of secondary redox events. 00, 000-000.