Mitochondria play an important role in apoptotic and necrotic cell death. Depending on the stimulus, a variety of mitochondrial dysfunctions can trigger onset of cell death. Assessment of mitochondrial dysfunction in living cells in situis important to understand the sequence of events producing cell injury and death. Laser scanning confocal microscopy, a technique that creates submicron thin optical slices through living cells and tissues, allows us to monitor several mitochondrial events simultaneously in intact single cells over time.

During oxidative stress, we monitored mitochondrial redox status, Ca2+ increase, reactive oxygen species (ROS), the mitochondrial permeability transition, and mitochondrial depolarization in cultured hepatocytes. When an oxidant chemical, t-BuOOH, was added to cultured hepatocytes, oxidation of mitochondrial pyridine nucleotides occurred as visualized directly by ultraviolet confocal microscopy of NAD(P)H autofluorescence. This event was followed by an increase of mitochondrial Ca2+ measured by the Ca2+-indicating fluorophore, Rhod-2.