PT  - JOURNAL ARTICLE
AU  - Tianyu Wang
AU  - Chunyan Wu
AU  - Dimitre G. Ouzounov
AU  - Wenchao Gu
AU  - Fei Xia
AU  - Minsu Kim
AU  - Xusan Yang
AU  - Melissa R. Warden
AU  - Chris Xu
TI  - Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain
AID  - 10.1101/846535
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 846535
4099  - http://biorxiv.org/content/early/2019/11/18/846535.short
4100  - http://biorxiv.org/content/early/2019/11/18/846535.full
AB  - 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.