Classically, mitochondria have been studied by biochemical, genetic and electron microscopic approaches. In the last two decades, it became evident that mitochondria are highly dynamic organelles that are frequently dividing and fusing, changing size and shape and traveling long distances throughout the life of a cell. The study of the complex structural changes of mitochondria in vivo became possible with the advent of fluorescent labeling techniques in combination with live cell imaging microscopy. This review aims to provide an overview on novel fluorescent markers that are used in combination with mitochondrial fusion assays and various live cell microscopy techniques to study mitochondrial dynamics. In particular, approaches to study the movement of mitochondrial proteins and novel imaging techniques (FRET imaging-, 4Pi- and STED-microscopy) that provide high spatial resolution are considered.