Imaging of Cellular Oxygen and Analysis of Metabolic Responses of Mammalian Cells

Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca²⁺ , H⁺ ), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite – cellular oxygen – is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O₂ in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O₂ concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given.