Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats

Area of Science:

• Neuroscience
• Biomedical Engineering
• Optical Imaging

Background:

• Imaging neuronal activity in deep cortical layers of freely moving animals is challenging.
• Existing microscopy techniques often lack the penetration depth or stability for in vivo studies of natural behaviors.

Purpose of the Study:

• To design and validate a head-mounted three-photon microscope for deep cortical layer imaging.
• To enable stable, long-term imaging of neuronal activity in freely moving rats.

Main Methods:

• Development of a head-mounted three-photon microscope system.
• Utilized a 1,320 nm excitation wavelength with a hollow-core fiber for stable pulse delivery.
• Implemented dispersion compensation for deep tissue penetration.
• Recorded neuronal activity in freely moving rats performing various behaviors.

Main Results:

• Achieved imaging depth greater than 1.1 mm below the cortical surface.
• Demonstrated stable imaging of layer 5 neuronal activity for over 1 hour.
• Successfully imaged neuronal activity during a range of natural behaviors in freely moving rats.

Conclusions:

• The developed three-photon microscope enables unprecedented deep cortical imaging in freely moving subjects.
• This technology facilitates the study of neural circuits underlying complex behaviors.
• Advances in fiber optics and dispersion compensation are crucial for deep-tissue in vivo imaging.