Publisher Summary This chapter discusses the advantages and limitations of the electron tomography technique as applied to mitochondrial research and also outlines the important steps in a typical tomographic investigation, with special attention to optimization of specimen preparation. Some of the biological applications of transmission electron microscopy (TEM) are investigations into the internal organization of the mitochondrion. The two dimensionality of conventional TEM imaging is imposed by the physics of the interaction of electrons with the specimen. Most structural information in electron micrographs is provided by electrons scattered elastically (without energy loss to the specimen) only once. However, for incident electrons of a given energy, as specimen thickness increases, the fraction of singly elastically scattered electrons drops and the fractions of singly inelastically and multiply elastically and inelastically scattered electrons increase, reducing image contrast greatly. Moreover, tomography of thick, plastic section specimens has the potential to become a routine tool for the three-dimensional (3D) analysis of biological structure. Furthermore, advances in methodology are leading to the extension of this 3D imaging technique to frozen-hydrated specimens.