title: Characterizing the morphologic changes in collagen crosslinked-treated corneas by Fourier transform-second harmonic generation imaging.
abstract: OBJECTIVE
  To evaluate the efficacy of using forward second harmonic generation (SHG) and 2-dimensional fast Fourier-transform (2D-FFT) analysis for the label-free characterization and quantification of morphologic changes in the corneal stroma after collagen crosslinking (CXL).
  
  
  METHODS
  Department of Physics, National Taiwan University, Taipei, Taiwan.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  En face forward SHG imaging and 2D-FFT analysis were performed on ex vivo porcine corneas at the depths of 100, 200, 400, and 800 μm. Morphologic changes in stromal collagen fiber in control, ultraviolet-A (UVA), riboflavin, and riboflavin-UVA treated porcine corneas were assessed. Hematoxylin-eosin staining and Sirius red staining were performed for comparison.
  
  
  RESULTS
  Corneas after CXL treatment tended to have collagen fibers that were wavy compared with the linear pattern in normal corneas. Quantitative 2D-FFT analysis of forward SHG images also showed an increase in the standard deviations of the distribution of stromal collagen fiber orientations, which is indicative of the changed pattern of crosslinked stromal collagen fibers.
  
  
  CONCLUSIONS
  Second harmonic generation imaging showed the morphologic changes in stromal collagen after CXL treatment. The linear collagen fibers in normal corneal stroma became wavy after treatment. With the introduction of 2D-FFT analysis, the morphologic changes can be quantified.
authors.affiliation: Institute of Biomedical Engineering, College of Medicine and Engineering, National Taiwan University, Taipei, Taiwan.
authors.forename: Hsin-Yuan
authors.initials: HY
authors.lastname: Tan
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Chang
authors.forename: Wen
authors.initials: W
authors.lastname: Lo
authors.forename: Chiu-Mei
authors.initials: CM
authors.lastname: Hsueh
authors.forename: Wei-Liang
authors.initials: WL
authors.lastname: Chen
authors.forename: Ara A
authors.initials: AA
authors.lastname: Ghazaryan
authors.forename: Po-Sheng
authors.initials: PS
authors.lastname: Hu
authors.forename: Tai-Horng
authors.initials: TH
authors.lastname: Young
authors.forename: Shean-Jen
authors.initials: SJ
authors.lastname: Chen
authors.forename: Chen-Yuan
authors.initials: CY
authors.lastname: Dong
mesh_terms: D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism / Q000473:pathology*; D003432:Cross-Linking Reagents / Q000627:therapeutic use*; D005583:Fourier Analysis; D008853:Microscopy; D017319:Photosensitizing Agents / Q000627:therapeutic use*; D012256:Riboflavin / Q000627:therapeutic use*; D013552:Swine; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D003094:Collagen; D012256:Riboflavin