Still displaying characteristics of their bacterial origin, such as autonomous division, motility, and their own genome, mitochondria remain an elusive component of modern eukaryotes. They produce most of the cell's energy in the form of adenosine triphosphate (ATP), thus dysfunction on the mitochondrial level can lead to severe health issues. From the biological point of view their involvement in every major activity of the cell, well past energy production, makes them a fascinating, yet complex target to explore. Many advances were made in recent years to understand the molecular players and the intricate interactions mitochondria undergo with other cellular structures. Several of these findings were enabled by developments in microscope technology, such as improved labelling, gentler imaging modalities, and higher throughput imaging and analysis. Unsurprisingly, open questions remain, particularly surrounding single mitochondria and their biological variations. Mitochondrial fission is considered a key process for both the proliferation and degradation of mitochondria. How these diametrically opposed outcomes are mediated by the same process has remained an open question in the field. Using live-cell multi-color instant structured illumination microscopy (iSIM) to resolve the details of mitochondrial fission at high temporal resolution we identified two distinct types of mitochondrial division. Midzone divisions contribute to mitochondrial proliferation, while peripheral divisions give rise to smaller mitochondria targeted for degradation through mitophagy. With mitochondria distributed throughout the cell, maintaining homeostasis occurs via series of mitochondrial dynamics. Fissions and fusions lead to rapid, local diffusion of content, while long-range microtubule transport disperses content more directly over larger distances. It remained unknown how cells balance directed transport with local fusion and diffusion to accomplish efficient mitochondrial content exchange in the cell. Thus far, a major obstacle in acquiring reproducible measurements of mitochondrial transport is the inherent variation of cellular morphologies. We addressed this challenge by standardizing cell shapes on protein micropatterns and following subgroups of photoconvertibly labelled mitochondria, enabling reproducible and quantitative measurements of mitochondrial transport. Our findings revealed an interconnected, proliferative population of mitochondria near the nucleus, engaged in continuous exchange and releasing short mitochondria for long-distance content transfer. In contrast, peripheral mitochondria exhibited less directed trafficking towards the perinuclear space, with most content exchange occurring through fission and fusion events. Altogether, this thesis sheds light om some fascinating ways in which cells maintain their mitochondrial population. Using advanced microscopy and image analysis we underscore the potential of these tools to unravel further intriguing puzzles of mitochondrial biology.