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Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain
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Three-photon imaging of mouse brain structure and function through the intact skull
Tianyu Wang1, Dimitre G Ouzounov2, Chunyan Wu2
1School of Applied and Engineering Physics, Cornell University, Ithaca, NY, USA. tw329@cornell.edu.
Nature Methods
|September 12, 2018
Summary
Three-photon microscopy offers superior optical sectioning for imaging through mouse skulls compared to two-photon methods. This technique enables deep brain imaging and long-term calcium activity monitoring in awake mice.
Area of Science:
- Neuroscience
- Optical Imaging
- Biomedical Engineering
Background:
- Optical imaging in the brain is hindered by skull-induced scattering and aberrations.
- Existing two-photon microscopy techniques face limitations in achieving sufficient depth and clarity through intact skulls.
Purpose of the Study:
- To evaluate the efficacy of three-photon excitation for overcoming skull-induced optical challenges in mouse brains.
- To demonstrate deep-tissue optical imaging and long-term neural activity monitoring in vivo.
Main Methods:
- Comparison of three-photon and two-photon excitation microscopy using identical systems and wavelengths.
- In vivo imaging of cerebral vasculature and GCaMP6s calcium signals in awake adult mice.
- Assessment of imaging depth, resolution, frame rate, and field of view.
Main Results:
- Three-photon excitation demonstrated significantly improved optical sectioning compared to two-photon excitation.
- Successful imaging of vasculature at depths exceeding 500 μm through the intact mouse skull.
- Weeks-long GCaMP6s calcium imaging in cortical layers 2/3 and 4 at 8.5 frames per second with a wide field of view.
Conclusions:
- Three-photon microscopy is a powerful tool for deep-brain optical imaging in vivo, overcoming limitations of skull penetration.
- This technique facilitates advanced neuroscientific research, including long-term monitoring of neural dynamics in awake, behaving animals.
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