The application of multiphoton fluorescence and second-harmonic generation microscopy in the evaluation of corneal wound healing after refractive corneal procedure
Date Issued
2008
Date
2008
Author(s)
Wang, Tsung-Jen
Abstract
Corneal refractive procedures correct patients’ refractive errors by reshaping corneas and change corneal curvature. The corneal wound healing process after operation is the key point of the recovery of the visual acuity and the reestablishment of the corneal transparency. It is a dynamic process involving epithelial healing and stromal healing. Although conventional histological examination is useful in characterizing the process, the fixation procedure prevents the study of tissue dynamics during the repair process. In addition, the regression and scar formation after refractive corneal procedure are related to the reactivation of the myofibroblasts and the rearrangement of the collagen fibers. Traditionally, the observation of myoflibroblasts and the collagen fibers depends on immunohistochemical stain.ecently, multiphoton microscopy, based on non-linear optical process, provides the feasibility of label-free pathological observation of corneas. The multiphoton excited autofluorescence from cytoplasma provides morphology of epithelial cells and keratocytes while second harmonic generation signals reveal collagen fibrils alignment in corneal stroma. There are a number of advantages associated with multiphoton microscopy. First, non-linear excitation of fluorescent species needs high photon density and only occurs at the objective focal point. Therefore, confocal-like image quality can be achieved without pinhole in multiphoton imaging. In addition, the typical excitation source in multiphoton microscopy is near infrared which scatters less in biological tissues. Therefore, multiphoton imaging has deeper penetration in biological specimens or turbid specimens. Due to the deeper penetration depth and optical section ability, three-dimensional reconstruction of biological specimens can also be achieved. Moreover, cytoplasmic autofluorescence and second harmonic generation from collagen which are the most abundant molecules in extracecullar matrices provide label-free and section-free observation of biological tissues. Using multiphoton microscopy in pathological imaging can avoid artificial effects in traditional histological process.n this project, conductive keratoplasty (CK) and photorefractive keratectomy (PRK) were performed in the rabbit eyes to serve as the animal models for studying the epithelial and stromal healing after refractive surgery. Ex vivo observations of wound healing process with multiphoton microscopic examination and conventional histological examination were done in different stages after operation. In the CK experiment, the re-epithelialization is accomplished within 1 week after surgical procedure. However, compensatory epithelial hyperplasia is observed at wounding sites. Structural alternation of corneal stroma can be visualized without extra labeling by second harmonic generation (SHG) imaging. In SHG images, the absence of SHG at wounding site indicates current-induced collagen thermal damage has lasted for at least 8 weeks after CK procedure. The collagen contraction caused by thermal damage can be immediately observed right after the CK procedure. n the PRK part, the re-epithelialization and keratocyte activation are observed by multiphoton excited autofluorescence (MAF) without labeling. Collagen regeneration in corneal stroma can be characterized by SHG imaging. To verify the correlation between activated keratocytes observed in MAF images and myofibroblasts observed in traditional immuohistochemical (IHC) imaging, keratocytes density are manually calculated in MAF and IHC. Positive correlation (0.9, p<0.05) is observed by using Spearman’s rank correlation analysis. The good correlation of activated keratocytes in MAF and myofibroblast in IHC suggest that MAF can be used to trace keratocyte activity during corneal wound healing process. Our results also suggest that, mytomicin-C can effectively reduce the popularity of keratocytes and the regeneration rate of collagenous stroma. ur results show the proof of principle of using multiphoton microscopy, a novel and nonlinear optical process, in observing corneas after refractive surgery. Multiphoton microscopy provides a less invasive way in observing cellular activities and extracecullar matrices alternation during corneal wound healing process. Comparing to traditional histology methods, the minimal invasion of mulitphoton observation provides the feasibility of applying it in in vivo, clinical examination in the future.
Subjects
Refractive corneal procedure
Photorefractive keratectomy
Conductive keratoplasty
Multiphoton microscopy
Corneal wound healing
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