Miniature three-photon microscopy maximized for scattered fluorescence collection.

Area of Science:

• Neuroscience
• Microscopy
• Biomedical Engineering

Background:

• Deep-tissue multiphoton microscopy is challenged by photon diffusion and scattering.
• Imaging neural activity in deep brain structures requires advanced techniques.

Purpose of the Study:

• To develop a miniature three-photon microscope (m3PM) for enhanced deep-tissue imaging.
• To overcome scattering limitations in biological samples.

Main Methods:

• Designed a miniature three-photon microscope (m3PM) prioritizing fluorescence collection efficiency.
• Utilized safe laser power for imaging deep brain regions.

Main Results:

• Successfully imaged calcium activity throughout the entire cortex and dorsal hippocampal CA1 up to 1.2 mm depth.
• Detected sensorimotor behavior-correlated neural activity in freely moving mice during behavioral tasks.
• Demonstrated m3PM's capability in highly scattering regimes.

Conclusions:

• The m3PM enables unprecedented deep-brain imaging in vivo.
• Facilitates the study of neural mechanisms in deep cortex and subcortical structures in behaving animals.