Tomographic reconstruction of mitochondria

Abstract

During oxidative phosphorylation, free energy of substrate oxidation is used to generate electrochemical gradients across the mitochondrial inner membrane. Full understanding of this process requires knowing the pathways for internal diffusion of ions and metabolites inside the organelle. This, in turn, requires detailed information about the organization of the membranes that compartmentalize the mitochondrion and the distribution of transport proteins (proton pumps, ion channels, metabolite carriers) on these membranes.We have undertaken the study of the compartmentation of rat-liver mitochondria (conventionally fixed and plastic-embedded) using electron microscopic tomography. Reconstructions of isolated mitochondria have been computed using projection images collected on the Albany high-voltage electron microscope from sections (0.5-1-μm thick) tilted around one axis over +/− 70° at 2° increments. A problem encountered with this approach is the directional loss of resolution due to the “missing wedge” of information in Fourier space in the direction parallel to the tilt axis. To overcome this problem, we have developed an alignment algorithm that allows projections collected over two or more tilting directions to be used in the same modified back-projection calculation.