For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly (dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (≥600 ps), high spatial resolution (1.4 µm), calibrated detection (0 to 300±8 µM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.