Calibration of FluBO for quantitative determination of intracellular oxygen. (A) During the logarithmic growth of E. coli cells, oxygen limitation was temporarily relieved twice by increasing the shaking frequency. The fitting curve of the time derivation of cyan-to-yellow fluorescence ratio (red) was drawn as described in the Methods. The pictograms symbolize the velocity of change of FRET ratio in dependence on cellular oxygen values: ⇨, no change; ⇧, fast increase; ⇗, slow increase. (B) The same data have been used to generate the calibration curve. In the used medium the value of 100% DOT corresponds to an oxygen concentration of 0.24 mmol/l at 30°C and an atmospheric pressure of 100 kPa. All parallel fermentation experiments in the microwells were performed in triplicates. The figures show a representative set of data.