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Ultra-deep through-skull mouse brain imaging via the combination of skull optical clearing and three-photon microscopy

By Mubin He, Dongyu Li, Zheng Zheng, Hequn Zhang, Tianxiang Wu, Weihang Geng, Zhengwu Hu, Zhe Feng, Shiyi Peng, Liang Zhu, Wang Xi, Dan Zhu, Jun Qian

Posted 21 Dec 2021
bioRxiv DOI: 10.1101/2021.12.20.473469

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and thee-photon fluorescence microscopy (3PM). The Visible-NIR-II compatible Skull Optical Clearing Agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1 mm and neuronal imaging depth of >700 m through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.

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