Deep tissue multi-photon imaging using adaptive optics with direct focus sensing and shaping.

Area of Science:

• Biomedical Optics
• Neuroimaging
• Microscopy

Background:

• High-resolution optical imaging in living tissues is limited by light scattering and aberrations.
• Penetrating deep into tissues for microscopy requires advanced techniques to overcome these challenges.

Purpose of the Study:

• To develop a novel adaptive optics microscope for deep tissue imaging.
• To enable high-resolution, in vivo visualization of neuronal structures with minimal invasiveness.

Main Methods:

• An adaptive optics three-photon microscope utilizing analog lock-in phase detection for focus sensing and shaping (ALPHA-FSS) was developed.
• A conjugate adaptive optics configuration with remote focusing was employed.
• In vivo imaging, functional calcium imaging, and laser-mediated microsurgery were performed.

Main Results:

• ALPHA-FSS effectively measured and compensated for optical aberrations and scattering.
• Subcellular resolution was achieved at depths up to 750 µm in the mouse cortex through the intact skull.
• High-resolution imaging extended to 1.1 mm depth in the deep cortex and hippocampus of the intact brain.

Conclusions:

• The ALPHA-FSS microscope provides a near-non-invasive method for high-resolution in vivo imaging deep within the intact brain.
• This technology advances neuroimaging capabilities, allowing detailed study of neuronal structures and functions at unprecedented depths.