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.

Arising from E.-Z. Shen et al. Nature 508, 128–132 (2014); doi:10.1038/nature1301210.1038/nature13012 Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial; several studies suggest that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al.¹ provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of three-day old Caenorhabditis elegans correlated inversely with lifespan. The interpretation of mitoflashes as ‘bursts of superoxide radicals’ assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide², but this interpretation has been criticized because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide^3,4,5,6,7. Here we show that purified cpYFP is completely unresponsive to superoxide, and that mitoflashes do not reflect superoxide generation or provide a link between mitochondrial free radical dynamics and lifespan. There is a Reply to this Brief Communication Arising by Cheng, H. et al. Nature 514, http://dx.doi.org/10.1038/nature13859 (2014).

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen−vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca²⁺ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

Cellular antioxidant systems control the levels of hydrogen peroxide (H ₂ O ₂ ) within cells. Multiple theoretical models exist that predict the diffusion properties of H ₂ O ₂ depending on the rate of H ₂ O ₂ generation and amount and reaction rates of antioxidant machinery components. Despite these theoretical predictions, it has remained unknown how antioxidant systems shape intracellular H ₂ O ₂ gradients. The relative role of thioredoxin (Trx) and glutathione systems in H ₂ O ₂ pattern formation and maintenance is another disputed question. Here, we visualized cellular antioxidant activity and H ₂ O ₂ gradients formation by exploiting chemogenetic approaches to generate compartmentalized intracellular H ₂ O ₂ and using the H ₂ O ₂ biosensor HyPer to analyze the resulting H ₂ O ₂ distribution in specific subcellular compartments. Using human HeLa cells as a model system, we propose that the Trx system, but not the glutathione system, regulates intracellular H ₂ O ₂ gradients. Antioxid. Redox Signal. 31, 664–670.