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Wide-field Three-Photon Excitation in Biological Samples

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Rowlands, CJ 
Park, D 
Bruns, OT 
Piatkevich, KD 
Fukumura, D 


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.



multiphoton microscopy, temporal focusing, optogenetics, three-photon, biophotonics

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Light: Science & Applications

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Nature Publishing Group
Wellcome Trust (093831/B/10/Z)
CJR and PTCS acknowledge support from NIH -5-P41-EB015871-27, DP3-DK101024 01, 1-U01-NS090438-01, 1-R01-EY017656 -0, 6A1, 1-R01-HL121386-01A1, the Biosym IRG of Singapore-MIT Alliance Research and Technology Center, the Koch Institute for Integrative Cancer Research Bridge Initiative, the Hamamatsu Inc., and the Samsung GRO program. CJR is grateful for a fellowship to carry out this research; the fellowship was supported by the Wellcome Trust 093831/Z/10/Z. ESB acknowledges funding from NIH 1R24MH106075, NIH 2R01DA029639, NIH 1R01MH103910, NIH 1R01GM104948, the MIT Media Lab, the New York Stem Cell Foundation-Robertson Award, and NSF CBET 1053233. OTB is grateful for an EMBO Longterm Fellowship to carry out this research. MGB and OTB acknowledge support from NIH 5U54 CA151884-04 and 9-P41-EB015871-26A1. DF and RKJ are grateful for NCI grants R35 CA197743 and P01 CA080124 to carry out this work.