Mok Aaron T, Wang Tianyu, Zhao Shitong, Kolkman Kristine E, Wu Danni, Ouzounov Dimitre G, Seo Changwoo, Wu Chunyan, Fetcho Joseph R, Xu Chris
School of Applied Engineering Physics, Cornell University, NY, USA.
Meining School of Biomedical Engineering, Cornell University, NY, USA.
bioRxiv. 2024 Apr 13:2023.11.14.566970. doi: 10.1101/2023.11.14.566970.
In vivo imaging of large-scale neuron activity plays a pivotal role in unraveling the function of the brain's network. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to overcome constraints on the field of view in three-photon microscopy and to perform deep imaging that is inaccessible to two-photon microscopy. These innovations enable us to image neuronal activities in a ~3.5-mm diameter field-of-view at 4 Hz with single-cell resolution and in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into any multiphoton microscope for large-field-of-view and system-level neural circuit research.
大规模神经元活动的体内成像在揭示大脑网络功能方面起着关键作用。多光子显微镜作为一种用于深部组织成像的强大工具，在提高其速度、视野和成像深度方面一直受到持续关注。然而，为避免在散射生物组织中产生热损伤，随着成像深度增加，视野呈指数级减小。我们提出了一系列创新方法，以克服三光子显微镜在视野方面的限制，并实现双光子显微镜无法达到的深度成像。这些创新使我们能够以单细胞分辨率在约3.5毫米直径的视野内以4赫兹的频率对小鼠大脑最深皮层的神经元活动进行成像。我们进一步展示了同时进行的大视野双光子和三光子成像、小鼠大脑的皮层下成像以及成年斑马鱼的全脑成像。所展示的技术可集成到任何多光子显微镜中，用于大视野和系统级神经回路研究。
