Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence.

Area of Science:

• Biomedical Optics
• Ophthalmology
• Microscopy

Background:

• Corneal imaging is crucial for diagnosing eye diseases.
• Current imaging techniques may require exogenous dyes or lack resolution.
• Non-invasive, high-resolution imaging methods are needed for detailed corneal structure analysis.

Purpose of the Study:

• To develop and apply a multiphoton microscopy technique for high-resolution ex vivo rabbit cornea imaging.
• To differentiate corneal cell types and extracellular matrix components using endogenous signals.
• To characterize collagen structure and organization within the corneal stroma.

Main Methods:

• Utilized a multiphoton microscope with second-harmonic generation (SHG) and two-photon excited fluorescence (TPF).
• Performed ex vivo imaging of rabbit cornea in a backscattering geometry without exogenous dyes.
• Employed spectral characterization and imaging, along with polarization-dependent SHG and 3D SHG tomography.

Main Results:

• Clearly visualized endogenous TPF from corneal cells and SHG from the extracellular matrix.
• Confirmed the structural origin of TPF and SHG signals through spectral analysis.
• Successfully separated keratocyte and epithelial cells from the collagen-rich stroma using spectral imaging.
• Revealed collagen fiber orientation via SHG polarization dependence.
• 3D SHG tomography demonstrated that collagen lamellae occupy approximately 88% of the stromal volume.

Conclusions:

• Multiphoton microscopy with SHG and TPF provides high-resolution, label-free imaging of corneal microstructure.
• This technique enables differentiation of corneal cell types and detailed analysis of collagen organization.
• The findings offer a valuable tool for studying corneal structure and potential disease-related alterations.