Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

Area of Science:

• Ophthalmology
• Biophysics
• Materials Science

Background:

• Corneal stromal collagen organization is crucial for maintaining ocular biomechanics and transparency.
• Current methods for assessing corneal structure are often invasive or lack detailed resolution.
• Noninvasive techniques are needed to evaluate collagen organization in vivo.

Purpose of the Study:

• To determine the feasibility of using femtosecond-pulse lasers to generate second-harmonic generated (SHG) signals for noninvasive assessment of corneal stromal collagen organization.
• To characterize the SHG signal patterns in different species and correlate them with corneal structure.

Main Methods:

• Two-photon confocal microscopy was employed with variable-wavelength femtosecond lasers to acquire SHG signals from mouse, rabbit, and human corneas.
• Forward and backward scattered SHG signals were detected and spectrally analyzed.
• Cytoskeletal and nuclear staining were performed to verify the spatial relationship between SHG signals and corneal cells.

Main Results:

• The highest SHG signal intensity was observed at an 800 nm excitation wavelength across all species.
• Forward scattered SHG signals revealed a distinct fibrillar, lamellar pattern, while backward scattered signals were more diffuse.
• Corneal stroma exhibited two lamellar organization patterns: interwoven in the anterior and orthogonally arranged in the posterior stroma.
• Human corneas uniquely displayed transverse, sutural lamellae inserting into Bowman's layer, suggesting an anchoring function.

Conclusions:

• Two-photon confocal microscopy generating SHG signals offers a powerful, noninvasive method for studying corneal collagen structure.
• The unique sutural lamellae in human corneas provide significant biomechanical support, distinguishing them from mouse and rabbit corneas.