Three-dimensional resolution enhancement of two-photon microscopy by combining point spread function engineering and...

Area of Science:

• Biomedical Imaging
• Optical Microscopy
• Biophysics

Background:

• Two-photon microscopy (TPM) offers deep tissue imaging but suffers from limited 3D spatial resolution due to diffraction.
• Axial resolution is a significant bottleneck in TPM, hindering the visualization of intricate biological structures.

Purpose of the Study:

• To develop and demonstrate a 3D resolution enhancement strategy for TPM.
• To improve the axial and lateral resolution beyond conventional TPM capabilities.

Main Methods:

• Point spread function (PSF) engineering by modulating the objective pupil to enhance axial high-frequency components.
• Acquisition of complementary volumetric datasets using conventional Gaussian and modulated illumination.
• Joint reconstruction of datasets using a Hessian-regularized deconvolution framework.

Main Results:

• Achieved 1.56× lateral and 1.67× axial resolution improvements over conventional TPM using 200-nm fluorescent beads.
• Demonstrated an additional 1.26× axial improvement with PSF engineering and deconvolution compared to deconvolution with Gaussian-PSF alone.
• Successfully visualized fine 3D structures in HeLa cells and restored resolution enhancement in brain slices with aberration correction.

Conclusions:

• PSF engineering combined with multi-image deconvolution is an effective strategy for enhancing 3D resolution in TPM.
• The developed method improves contrast and visualization of fine structures in biological tissues.
• Aberration correction is crucial for maintaining the performance of this resolution enhancement technique.