Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

eLife

Dimitre G Ouzounov¹  Xusan Yang²  Fei Xia³  Chris Xu³  Minsu Kim⁴  Melissa R Warden⁵  Chunyan Wu⁵  Tianyu Wang⁵  Wenchao Gu⁵ 

[1] College of Veterinary Medicine, Cornell University, Ithaca, United States;College of Human Ecology, Cornell University, Ithaca, United States;Department of Neurobiology and Behavior, Cornell University, Ithaca, United States;Meining School of Biomedical Engineering, Cornell University, Ithaca, United States;School of Applied and Engineering Physics, Cornell University, Ithaca, United States;

关键词: calcium;    three-photon;    3-photon;    two-photon;    2-photon;    neural imaging;   

DOI  :  10.7554/eLife.53205

来源: DOAJ

【 摘 要 】

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 perturbation to the sample. 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. By considering the combined effects of in-focus signal attenuation and out-of-focus background generation, we quantified the cross-over depth beyond which three-photon microscopy outpeforms two-photon microscopy in recording fidelity. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. Increased immunoreactivity was observed with 150 mW excitation power at 1 and 1.2 mm imaging depths. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

【 授权许可】

Unknown