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 a...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.展开更多
基金support from NIH-5-P41-EB015871-27,DP3-DK10102401,1-U01-NS090438-01,1-R01-EY017656-0,6A1,1-R01-HL121386-01A1the Biosym IRG of Singapore-MIT Alliance Research and Technology Center+6 种基金the Koch Institute for Integrative Cancer Research Bridge Initiativethe Hamamatsu Inc.,and the Samsung GRO programsupported by the Wellcome Trust 093831/Z/10/Zfunding from NIH 1R24MH106075,NIH 2R01DA029639,NIH 1R01MH103910,NIH 1R01GM104948,the MIT Media Lab,the New York Stem Cell Foundation-Robertson Award and NSF CBET 1053233an EMBO Longterm Fellowship to carry out this researchsupport from NIH 5U54 CA151884-04 and 9-P41-EB015871-26A1for NCI grants R35 CA197743 and P01 CA080124 to carry out this work.
文摘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.
