Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain
- Cornell University, United States
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
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.
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.
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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Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain
eLife 9:e53205.
https://doi.org/10.7554/eLife.53205
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