Three sensitive methodologies were used to measure and characterize hyperoxia-induced oxidative stress in CNS cells (in vitro). A commercially available AFM (Bioscope SZ; Veeco) was mated with an inverted fluorescence microscope (Nikon TE2000-E) for simultaneous acquisition of AFM and fluorescence data. The system is housed in a hyperbaric chamber (Riemers Systems Inc.), allowing for cellular measurements at normobaric and hyperbaric pressures (0-85 psig), including hyperbaric oxygen (HBO2). AFM was used to resolve changes in membrane ultrastructure in response to oxidative stimuli (e.g. hyperoxia, H2O2). Fluorescence microscopy was used to detect superoxide with Dihydroethidium (DHE). An amperometric biosensor (ISO-HPO-100; WPI) was used to measure nM levels of H2O2. Results from AFM scans of hyperoxia-treated glutaraldehyde-fixed (1%) U87 glioblastoma cells revealed nanoscopic membrane surface blebbing. Hyperoxia (95% O2 and 3.5 ATA O2) induced a dose-dependent increase in average membrane roughness (Ra), which correlated with elevated malondialdehyde (MDA) production. Similar results in Ra levels and MDA production were observed with exogenous H2O2 (0.2-2mM). Amperometric biosensors detected increased cellular H2O2 production during hyperoxia, which was elevated 156 ± 8% (95% O2) and 276 ± 52% (4 ATA O2) above baseline (20% O2). In conclusion, these novel techniques (used alone or in combination) have provided a highly sensitive and complimentary means to detect reactive oxygen species (ROS) and assess oxidative stress from hyperoxia. ONR grant N000140610105 (DPD), ONR-DURIP equipment grant N000140210643 (JBD).