Aberration correction considering curved sample surface shape for non-contact two-photon excitation microscopy with...

Area of Science:

• Biomedical Optics
• Microscopy Techniques
• Optical Engineering

Background:

• Deep tissue imaging with two-photon excitation microscopy (TPM) is often limited by aberrations.
• Curved sample surfaces and refractive index mismatches induce spherical aberrations, degrading image quality and resolution.
• Existing methods struggle to correct these aberrations effectively for deep observation.

Purpose of the Study:

• To develop a method for high-quality deep observation using TPM with a dry objective lens.
• To correct aberrations caused by curved sample surfaces and refractive index mismatches.
• To improve fluorescence intensity and resolution in deep imaging applications.

Main Methods:

• Excitation light wavefront modulation using a spatial light modulator.
• Implementation of a novel optical path length difference calculation for pre-distortion wavefront design.
• Application of the technique to a two-photon excitation microscopy system with a dry objective lens.

Main Results:

• Successfully corrected aberrations caused by curved sample surfaces and refractive index mismatches.
• Achieved high-quality deep imaging up to 2,000 μm in a cleared mouse brain.
• Demonstrated improved fluorescence intensity and resolution in deep tissue observations.

Conclusions:

• Wavefront modulation is an effective strategy for overcoming deep imaging limitations in TPM.
• The developed method enables aberration-free excitation light condensation at the sample interface.
• This technique significantly enhances the capability of dry objective-based TPM for deep biological sample visualization.