Mitochondria are critical for cellular adenosine triphosphate (ATP) production; however, recent studies suggest that these organelles fulfill a much broader range of tasks. For example, they are involved in the regulation of cytosolic Ca(2+) levels, intracellular pH and apoptosis, and are the major source of reactive oxygen species (ROS). Various reactive molecules that originate from mitochondria, such as ROS, are critical in pathological events, such as ischemia, as well as in physiological events such as long-term potentiation, neuronal-vascular coupling and neuronal-glial interactions. Due to their key roles in the regulation of several cellular functions, the dysfunction of mitochondria may be critical in various brain disorders. There has been increasing interest in the development of tools that modulate mitochondrial function, and the refinement of techniques that allow for real time monitoring of mitochondria, particularly within their intact cellular environment. Innovative imaging techniques are especially powerful since they allow for mitochondrial visualization at high resolution, tracking of mitochondrial structures and optical real time monitoring of parameters of mitochondrial function. The techniques discussed include classic imaging techniques, such as rhodamine-123, the highly advanced semi-conductor nanoparticles (quantum dots), and wide field microscopy as well as high-resolution multiphoton imaging. We have highlighted the use of these techniques to study mitochondrial function in brain tissue and have included studies from our laboratories in which these techniques have been successfully applied.