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Superficial bound of the depth limit of 2-photon imaging in mouse brain

Kevin Takasaki, Reza Abbasi-Asl, View ORCID ProfileJack Waters
doi: https://doi.org/10.1101/618454
Kevin Takasaki
Allen Institute for Brain Science, 615 Westlake Ave N, Seattle WA 98109
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Reza Abbasi-Asl
Allen Institute for Brain Science, 615 Westlake Ave N, Seattle WA 98109
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Jack Waters
Allen Institute for Brain Science, 615 Westlake Ave N, Seattle WA 98109
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  • ORCID record for Jack Waters
  • For correspondence: jackw@alleninstitute.org
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ABSTRACT

2-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. 2-photon excitation generates fluorescence from the focal plane, but also from outside the focal plane, with out-of-focus fluorescence increasing as the focus is pushed deeper into tissue. It has been suggested that the 2-photon depth limit, beyond which results become inaccurate, is where in- and out-of-focus fluorescence are equal. We found the depth limit of 2-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous 2- and 3-photon excitation. 2-photon results were accurate only superficial to 450 μm, matching the depth at which in-and out-of-focus fluorescence were equal. The expected depth limit is deeper in tissue with fewer fluorophores outside the plane of interest. Our results, from tissue with a largely homogenous distribution of fluorophores, establish a superficial bound on the 2-photon depth limit in the mouse visual cortex.

Footnotes

  • e-mail: kevint{at}alleninstitute.org, jackw{at}alleninstitute.org

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 25, 2019.
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Superficial bound of the depth limit of 2-photon imaging in mouse brain
Kevin Takasaki, Reza Abbasi-Asl, Jack Waters
bioRxiv 618454; doi: https://doi.org/10.1101/618454
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Superficial bound of the depth limit of 2-photon imaging in mouse brain
Kevin Takasaki, Reza Abbasi-Asl, Jack Waters
bioRxiv 618454; doi: https://doi.org/10.1101/618454

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