Two-photon microscopy (2PM) provides three-dimensional (3D) and four-dimensional (4D) (x, y, z, t) imaging in living specimens or under experimental physiological conditions very close to live. In conjunction with fluorescent labels, 2PM provides a powerful means of investigating the relationships between structure and function at the microscopic level that are key to understanding biological systems. This technique is able to provide time-resolved, 3D images of dynamic systems with near-diffraction-limited resolution and highly specific structural contrast.