Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.
