Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production

Abstract Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement the oxidative burst is central to anti-microbial actions of macrophages. However, these highly reactive, short-lived radical also stimulate cells undergo programmed death at high concentrations, as well causing detrimental effects such oxidation macromolecules more moderate levels. Imaging ROS challenging, with many researchers working on challenge over past 10–15 years without producing a definitive method. We report new fluorescence microscopy-based technique, Bullseye Analysis. This methodology based concepts provided by FRAP (Fluorescence Recovery after Photobleaching) technique refined evidence spatiotemporal production ROS, subsequent consequences, subcellular scale. To exemplify we have used ROS-reporter dye, CellROX, ROS-inducing photosensitizer, LightOx58, potent source compared UV irradiation alone. Further validation was carried out using differing co-stains, notably Mitotracker JC-1.