Two-photon excitation (2PE) overcomes many challenges in fluorescence microscopy. Compared to confocal microscopy, 2PE microscopy improves depth penetration, owing to the longer excitation wavelength required and to the ability to collect scattered emission photons as a useful signal. It also minimizes photodamage because lower energy photons are used and because fluorescence is confined to the geometrical focus of the laser spot. 2PE is therefore ideal for high-resolution, deep-tissue, time-lapse imaging of dynamic processes in cell biology. Here, we provide examples of important applications of 2PE for in vivo imaging of neuronal structure and signals; we also describe how it can be combined with optogenetics or photolysis of caged molecules to simultaneously probe and control neuronal activity.