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Methods/New Tools, Novel Tools and Methods

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

Kevin Takasaki, Reza Abbasi-Asl and Jack Waters
eNeuro 6 January 2020, ENEURO.0255-19.2019; DOI: https://doi.org/10.1523/ENEURO.0255-19.2019
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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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 postulated that the 2-photon depth limit, beyond which results become inaccurate, is where in- and out-of-focus fluorescence are equal, which we term the balance depth. Calculations suggest that the balance depth should be at ∼600 µm in mouse cortex. Neither the 2-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth 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- and 3-photon results from superficial locations were almost identical. 2-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for 2-photon excitation. However, the 2-photon depth limit and balance depth were at 450 µm, shallower than predicted by calculations. Our results were from tissue with a largely homogenous distribution of fluorophores. The expected balance depth is deeper in tissue with fewer fluorophores outside the focal plane and our results therefore establish a superficial bound on the 2-photon depth limit in mouse visual cortex.

SIGNIFICANCE STATEMENT This study measures the maximum depth in the mouse brain to which it is possible to obtain quantitatively accurate results with 2-photon microscopy, a form of non-linear fluorescence microscopy.

Footnotes

  • The authors declare no competing financial interests.

  • Allen Institute for Brain Science

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Superficial bound of the depth limit of 2-photon imaging in mouse brain
Kevin Takasaki, Reza Abbasi-Asl, Jack Waters
eNeuro 6 January 2020, ENEURO.0255-19.2019; DOI: 10.1523/ENEURO.0255-19.2019

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Superficial bound of the depth limit of 2-photon imaging in mouse brain
Kevin Takasaki, Reza Abbasi-Asl, Jack Waters
eNeuro 6 January 2020, ENEURO.0255-19.2019; DOI: 10.1523/ENEURO.0255-19.2019
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