Abstract
Fluorescence intensity has historically been used to measure the concentration of NADH of in living cells and tissues. Fluorescence lifetime imaging has shown that changes in metabolism results in significant shifts in the lifetimes and concentration of bound and free forms of NADH. To compare the metabolic state as revealed by two-photon imaging of NADH fluorescence with other measures of cellular metabolism, we have developed a non-invasive phase-fluorimetric technique to quantify the oxygen consumption of adherent cells. This optical method can be used in conjunction with NADH imaging techniques to provide a more complete assessment of cellular energetics. To test this instrument. We have used mitochondrial uncouplers and inhibitors with varying substrate and oxygen concentrations to adjust the metabolic state. We learned that as the glucose concentration is increased, the cell's metabolic rate increases, as expected. We also varied the temperature at which our experiments we taking place, and found that cells at room temperature had significantly lower consumption rates than those at 37 degree C. We also tracked cell density, but found that this had a minimal effect on oxygen consumption rates.