Wide-field three-photon excitation in biological samples

• 来源期刊: 光：科学与应用（英文版）
• 关键词: biophotonics multiphoton microscopy optogenetics temporal focusing three-photon
• 年，卷（期）: 2017,（2）
• 页码范围: 143-151
• 页数: 9页
• 语种: 英文
• DOI: 10.1038/lsa.2016.255

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