Atomic force microscopy (AFM) has become an important medical and biological tool for the noninvasive imaging of cells and biomaterials in medical, biological, and biophysical research. The major advantages of AFM over conventional optical and electron microscopes for bio-imaging include the facts that no special coating is required and that imaging can be done in all environments-air, vacuum, or aqueous conditions. In addition, it can also precisely determine pico-nano Newton force interactions between the probe tip and the sample surface from force-distance curve measurements.It is widely known that mitochondrial swelling is one of the most important indicators of the opening of the mitochondrial permeability transition (MPT) pore. As mitochondrial swelling is an ultrastructural change, quantitative analysis of this change requires high-resolution microscopic methods such as AFM. Here, we describe the use of AFM-based shape analysis for the characterization of nanostructural changes in heart mitochondria resulting from myocardial ischemia-reperfusion injury.