Wide-field three-photon excitation in biological samples

摘要

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 700μm 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 threephoton optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin;action potentials could reliably be excited without causing photodamage.