Analysis of corneal stroma organization with wavefront optimized nonlinear microscopy.

Area of Science:

• Ophthalmology
• Biomedical Engineering
• Microscopy

Background:

• The cornea's stromal collagen organization is crucial for its optical properties.
• Understanding interspecies variations in corneal structure is vital for translational research.

Purpose of the Study:

• To investigate and compare the stromal collagen organization in healthy ex vivo corneas of various species using second harmonic generation (SHG) microscopy.
• To characterize species-specific collagen distribution across different corneal depths.

Main Methods:

• Utilized a custom backscattered nonlinear microscope with a femtosecond laser and wavefront aberration control.
• Acquired second harmonic generation (SHG) signals from collagen in unfixed, unstained porcine, bovine, rabbit, rat, chicken, and human corneas.
• Performed optical sectioning across the entire corneal thickness to analyze collagen lamellae organization.

Main Results:

• SHG imaging revealed distinct microscopic collagen lamellae organization in the stroma of different species.
• Anterior stroma showed similar interwoven collagen patterns across all species.
• Posterior stroma exhibited species-specific collagen bundle arrangements, with longer, parallel-oriented bundles in some species and depth-dependent orientation changes in others.

Conclusions:

• A novel wavefront aberration-controlled microscope enabled detailed characterization of corneal collagen morphology across species.
• Findings provide a basis for comparing animal models to human corneas and understanding corneal diseases.