Wide-field three-photon excitation in biological samples

• Main Authors: Fukumura, Dai (Author), Jain, Rakesh K (Author), Rowlands, Christopher (Contributor), Park, Demian (Contributor), Bruns, Oliver Thomas (Contributor), Piatkevich, Kiryl (Contributor), Bawendi, Moungi G (Contributor), Boyden, Edward (Contributor), So, Peter T. C. (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences (Contributor), Massachusetts Institute of Technology. Department of Chemistry (Contributor), Massachusetts Institute of Technology. Department of Mechanical Engineering (Contributor), Massachusetts Institute of Technology. Media Laboratory (Contributor), McGovern Institute for Brain Research at MIT (Contributor), Massachusetts Institute of Technology. Center for Neurobiological Engineering (Contributor)
• Format: Article
• Language: English
• Published: Springer Nature, 2018-01-08T17:03:06Z.
• Subjects: Article
• Online Access: Get fulltext

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.
National Institutes of Health (U.S.) (Grant NIH-5-P41-EB015871-27)
National Institutes of Health (U.S.) (Grant DP3-DK101024 01)
National Institutes of Health (U.S.) (Grant 1-U01-NS090438-01)
National Institutes of Health (U.S.) (1-R01-HL121386-01A1)
National Institutes of Health (U.S.) (Grant 1-R01-EY017656-06A1)
National Institutes of Health (U.S.) (Grant 1R24MH106075)
National Institutes of Health (U.S.) (Grant 2R01DA029639)
National Institutes of Health (U.S.) (Grant 1R01MH103910)
National Institutes of Health (U.S.) (Grant 1R01GM104948)
National Science Foundation (U.S.) (Grant CBET 1053233)
National Institutes of Health (U.S.) (Grant 5U54 CA151884-04)
National Institutes of Health (U.S.) (Grant 9-P41-EB015871-26A1)