Intracellular imaging of metmyoglobin and oxygen using new dual purpose probe EYFP‐Myoglobin‐mCherry

Abstract The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2] sensor Myoglobin‐mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2] sensing available to a wider group of laboratories.