Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution...

Area of Science:

• Biophysics
• Optical Microscopy
• Biomedical Imaging

Background:

• Light scattering in thick specimens degrades 3D super-resolution imaging quality.
• Increased background noise and reduced optical sectioning are key challenges.
• Existing methods often require hardware modifications or are limited in application.

Purpose of the Study:

• To present a simple, hardware-independent digital method to mitigate light scattering issues in 3D super-resolution microscopy.
• To enhance image contrast, reduce background noise, and improve optical sectioning in thick biological samples.
• To demonstrate the effectiveness of the method in various biological specimens using a two-photon instant structured illumination microscope.

Main Methods:

• Utilized three laterally-structured, phase-shifted illumination patterns for excitation.
• Employed post-processing algorithms to digitally remove scattered and out-of-focus light.
• Integrated the method into a two-photon instant structured illumination microscope without hardware alterations.

Main Results:

• Successfully mitigated light scattering, significantly improving image quality in thick specimens.
• Demonstrated enhanced contrast and optical sectioning compared to standard imaging.
• Validated the approach in diverse biological samples, including pollen, endothelial cells, and cell spheroids.

Conclusions:

• The described digital processing method offers a simple yet effective solution for improving 3D super-resolution imaging in scattering biological tissues.
• This technique enhances image fidelity without requiring instrument modifications, making it broadly applicable.
• The method shows promise for advancing deep-tissue imaging and analysis in various biological research areas.