Mitochondria are the main suppliers of energy for cells and their bioenergetic function is regulated by mitochondrial dynamics: the constant changes in mitochondria size, shape, and cristae morphology to secure cell homeostasis. Although mitochondrial dysfunction is implicated in a wide range of diseases, our understanding of mitochondrial function remains limited by the complexity of inferring these spatial features from 2D electron microscopical (EM) images of intact tissue. Here, we present a semi-automatic method for segmentation and 3D reconstruction of mitochondria, cristae, and intracristal spaces based on 2D EM images of the murine hippocampus. We show that our method provides a more accurate characterization of mitochondrial ultrastructure in 3D than common 2D approaches and propose an operational index of mitochondria's internal organization. We speculate that this tool may help increase our understanding of mitochondrial dynamics in health and disease.