1. Culture cells in growth medium until 70% confluence. 2. Wash cells with 10 ml PBS. 3. Detach cells with 2 ml pre-warmed trypsin at room temperature for 4 min, resuspend in 8 ml growth medium. 4. Determine cell count using a Casy cell counter or hemocytometer. 5. Seed 30,000 cells in 400 µl growth medium in a Matek glass-bottomed dish, incubate overnight in a humidified incubator (37°C, 5% CO2). 6. Mix 25 µl Opti-MEM medium with 0.5 µg HyPerER plasmid and 1.5 µl Fugene® HD solution, incubate for 15 min at room temperature, add to cells. 7. Add 2.6 ml growth medium 6 h after transfection, incubate cells at 37°C for 24-48 h. 8. Perform imaging on an Olympus Fluoview 3000 microscope with temperature and CO2 control. Excite samples sequentially with 405 nm and 488 nm lasers, record emission in a 500-600 nm window. 9. Remove culture medium, wash dish twice with 1 ml pre-warmed HEPES Imaging Buffer, add 1 ml HEPES Imaging Buffer, place dish on microscope stage. 10. Search for a field of view with several healthy-looking cells, optimize photomultiplier tube voltage. 11. Measure baseline oxidative status every 20 s for at least 5 min, add stimulating substances (e.g., thapsigargin) and record until the signal stabilizes. 12. Conclude measurement with 100 µM H2O2 as a positive control.