Small-angle X-ray scattering of muscles provides direct measurement of sarcomeric protein organization. Sarcomeres are highly ordered protein complexes that are pseudocrystalline in nature. (a) Synchrotron light sources produce high-powered X-rays that pass through muscle fibers to create diffraction patterns from their interaction with the sarcomere structures. (b) A representative diffraction pattern from rat tibialis anterior, permeabilized fiber bundle at rest. The structural properties of both thick (myosin) and thin (actin) filaments show up as reflections and layer lines along the meridional plane (perpendicular to the equatorial plane) and change in identifiable ways when the muscle is activated or under myopathies. (c and d) Geometric lattice planes and repeating periodic structures on the thick and thin filaments underlie the meridional and equatorial diffraction features.