Analysis of Corneal Change Mechanisms Through Second Harmonic Generation Microscopy
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Analysis of Corneal Change Mechanisms Through Second Harmonic Generation Microscopy


Second harmonic generation (SHG) microscopy has been a widely used method to image collagen-based tissues due to its non-invasive nature and ability to determine collagen changes with appropriate analysis. This technique has recently been applied to analyze the collagen structure of fresh corneal tissue, overcoming the limitations and challenges that occur with traditional microscopy and imaging techniques requiring fixation and dyes. Orientation and organization of the corneal collagen fibers is critical for optimal transparency and optical functionality. Corneal chemical injuries (CCIs) may result in the cornea to become opaque and thus obscuring vision. Other conditions, such as myopia or astigmatism, are a consequence of corneal shape deviations. Current treatments for vision correction are invasive, costly, or fail to fully restore a patient’s vision. Most corneal reshaping methods are either passive using rigid/semi-rigid contact lenses to gradually alter corneal shape (orthokeratology) or rely upon laser technology to perform precision ablation of corneal tissue. An alternative approach relies upon the in-situ generation of localized pH gradient that alter stress-strain relationships within the cornea, leading to potentially sustained shape change. This novel, minimally invasive approach (“electrochemical therapy”- ECT) is currently being evaluated to clear corneal opacification following a chemical injury and corneal reshaping. ECT has been previously used to reshape cartilage and chemically alter the collagen structure of skin. The objective of this thesis is to study corneal collagen structure using SHG microscopy to examine chemical injuries and electrochemical reshaping. Image processing techniques were developed to gauge structural changes observed with SHG imaging. Fresh ex-vivo rabbit corneas were imaged using SHG. Native fresh corneal tissue was examined and compared with alkaline-injured corneas before and after ECT-clearing. ECT reshaped cornea was examined as well. The extracted images were then processed through ImageJ and a customized MATLAB code to determine the collagen fiber orientation and compared to identify potential statistical indicators of structural change. Collagen distribution patterns throughout the thickness of the cornea were determined in all samples. Knowledge of the collagen structure obtained through SHG combined with white light photography and microscopic data provides information of the changing corneal extracellular matrix. This analysis can be utilized for collagen studies for future projects.

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