Abstract

1300-nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and tissue heating. We calculated and experimentally verified the excitation pulse energy to achieve the minimum photon count required for the detection of calcium transients in GCaMP6s-expressing neurons for 920-nm two-photon and 1320-nm three-photon excitation, respectively. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. We observed increased immunoreactivity with 150 mW excitation power at 1.0- and 1.2-mm imaging depths. Based on the data, we explained how three-photon excitation achieves better calcium imaging fidelity than two-photon excitation in the deep brain and quantified the imaging depth where three-photon microscopy should be applied. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

Data availability

All the parameters for calculation and models have been summarized as tables in the texts. The source data for all the figures have been provided. All the simulation codes have been uploaded and available for downloads.

Article and author information

Author details

  1. Tianyu Wang

    School of Applied and Engineering Physics, Cornell University, Ithaca, United States
    For correspondence
    tw329@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6087-6376
  2. Chunyan Wu

    School of Applied and Engineering Physics, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Dimitre G Ouzounov

    School of Applied and Engineering Physics, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Wenchao Gu

    Department of Neurobiology and Behavior, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Fei Xia

    Meining School of Biomedical Engineering, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6591-8769
  6. Minsu Kim

    College of Human Ecology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Xusan Yang

    School of Applied and Engineering Physics, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Melissa R Warden

    Department of Neurobiology and Behavior, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2240-3997
  9. Chris Xu

    School of Applied and Engineering Physics, Cornell University, Ithaca, United States
    For correspondence
    cx10@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Science Foundation (DBI-1707312)

  • Chunyan Wu
  • Dimitre G Ouzounov
  • Fei Xia
  • Xusan Yang

National Institutes of Health (DP2MH109982)

  • Wenchao Gu

Intelligence Advanced Research Projects Activity (D16PC00003)

  • Tianyu Wang
  • Dimitre G Ouzounov

Cornell Neurotech Mong Fellowships

  • Tianyu Wang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ryohei Yasuda, Max Planck Florida Institute for Neuroscience, United States

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#2010-0031) of Cornell University. All surgery was performed under isoflurane anesthesia, and every effort was made to minimize suffering.

Version history

  1. Received: October 31, 2019
  2. Accepted: January 29, 2020
  3. Accepted Manuscript published: January 30, 2020 (version 1)
  4. Version of Record published: February 18, 2020 (version 2)

Copyright

© 2020, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Tianyu Wang
  2. Chunyan Wu
  3. Dimitre G Ouzounov
  4. Wenchao Gu
  5. Fei Xia
  6. Minsu Kim
  7. Xusan Yang
  8. Melissa R Warden
  9. Chris Xu
(2020)
Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain
eLife 9:e53205.
https://doi.org/10.7554/eLife.53205

Share this article

https://doi.org/10.7554/eLife.53205

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