Live-cell FLIM of a genetically encoded reporter reveals oxygen equilibration in cultured cells

Oxygen (O 2 ) availability critically governs cellular physiology, however, standard cell culture systems often fail to recapitulate physiological oxygenation due to limited O 2 diffusion across the culture medium. Poorly defined O 2 equilibration dynamics in vitro can compromise reproducibility and contribute to experimental variability. Here, we employ live-cell fluorescence lifetime imaging microscopy (FLIM) using the genetically encoded O 2 reporter CMV-dUnOFLS – a PEST-destabilized UnaG-mOrange2 fusion protein – to monitor O 2 equilibration in real time. Frequency-domain FLIM (FD-FLIM) revealed rapid and reversible changes in UnaG fluorescence lifetime in Gli36 and CHO cells during cyclic variations of ambient O 2 , reflecting culture medium oxygenation dynamics. Unexpectedly, in three different types of cell culture vessel, we noted a critical surface-area-to-volume (SA/V) ratio threshold of approximately 0.3 mm −1 , below which O 2 equilibration appeared to be delayed. Consequently, in 96-well plates, media volumes ≥100 µl significantly decelerated O 2 equilibration, while it occurred faster in 8-well imaging slides and 3.5 cm glass bottom dishes. Our findings demonstrate multiple factors critically influencing pericellular oxygenation and emphasize the necessity of real-time O 2 monitoring in vitro . The CMV-dUnOFLS reporter combined with FD-FLIM provides a powerful tool for assessing and optimizing O 2 dynamics in live-cell culture systems.