Abstract
Publisher Summary The chapter presents a study related to fluorescent imaging of mitochondrial nitric oxide in living cells. Nitric oxide (NO) is an important modulator of mitochondrial respiration and membrane potential. The presence of a mitochondrial NO synthase (mtNOS) has been described in the inner membrane of isolated rat liver mitochondria. However, because NO is a highly diffusible, short-lived (0.5–5 sec), highly reactive radical, its determination poses considerable technical problems. In fact, until a while ago, the production of NO by mtNOS has only been indirectly characterized in broken mitochondria using techniques, such as spectroscopy or the citrulline assay. Two novel fluorometric detection systems have been developed allowing the demonstration of the presence of NO within mitochondria via microscopy. 4,5-Diaminofluorescein diacetate (DAF-2/DA) permits the direct detection of NO production, and the MitoTracker allows the detection of mitochondrial potential changes. Thus, whereas DAF-2/DA can be used as a semiquantitative method of evaluation of NO production in living cells, the combination of these two systems provides an ideal tool for the spatial and direct visualization of NO within mitochondria. A variety of products from Molecular Probes allow the detection of mitochondrial activity.
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