Wide-field three-photon excitation in biological samples
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...
| Main Authors: | , , , , , , , , |
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| Other Authors: | |
| Format: | Article |
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Springer Nature
2018
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| Online Access: | http://hdl.handle.net/1721.1/113016 https://orcid.org/0000-0002-8261-2371 https://orcid.org/0000-0002-5738-0126 https://orcid.org/0000-0003-2220-4365 https://orcid.org/0000-0002-0419-3351 https://orcid.org/0000-0003-4698-6488 |
| Summary: | 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. |
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