Quantitative analysis of light scattering in polarization-resolved nonlinear microscopy

Area of Science:

• Biophotonics
• Microscopy
• Optical Physics

Background:

• Polarization resolved nonlinear microscopy (PRNM) provides structural insights in biological tissues.
• Deep tissue imaging is limited by light scattering, which degrades image quality and polarization purity.
• Understanding scattering effects is crucial for advancing PRNM in complex biological environments.

Purpose of the Study:

• To develop a robust characterization methodology for PRNM in thick scattering tissues.
• To quantify the impact of light scattering on nonlinear optical processes and polarization states.
• To establish a framework for reliable PRNM in challenging biological specimens.

Main Methods:

• Utilized a four wave mixing (FWM) process, leveraging its intrinsic in-depth local coherent source and depolarization quantification capabilities.
• Investigated diverse experimental layouts (epi/forward detection) using phantoms with varying scattering properties.
• Systematically studied the influence of scattering center size, geometry, and sample thickness on nonlinear excitation and emission.

Main Results:

• Depolarization of nonlinearly generated photons strongly depends on scattering center size, detection geometry, and sample thickness.
• An un-analyzed detection strategy demonstrated high robustness to scattering effects, preserving polarization-dependence readout even with degraded imaging.
• The methodology was successfully illustrated in polarization-resolved imaging of myelin lipid organization in mouse spinal cords.

Conclusions:

• The developed FWM-based characterization methodology provides a framework for robust PRNM in thick scattering tissues.
• Un-analyzed detection is a key strategy to overcome scattering-induced polarization degradation in nonlinear microscopy.
• This work enables improved deep tissue structural analysis using PRNM, particularly for myelin imaging.