title: Fast Fourier transform-based analysis of second-harmonic generation image in keratoconic cornea.
abstract: OBJECTIVE
  To qualify and quantify structural alterations in keratoconic corneas ex vivo by the use of second-harmonic generation (SHG) microscopy and two-dimensional fast Fourier transform (2D-FFT) analysis.
  
  
  METHODS
  Two keratoconic and three normal human corneal specimens were imaged with a multiphoton microscope. Forward and backward second-harmonic generation images (FSHG and BSHG) of corneal stroma were obtained at high resolution and at different depths. The SHG images were further analyzed with a 2D-FFT algorithm to quantify the texture and orientation changes of collagen fibers.
  
  
  RESULTS
  The results showed that the abnormality of collagen architecture was found through the whole layer of stroma. 2D-FFT analysis of SHG cornea images provided fiber orientation direction and an aspect ratio (AR) as a quantitative measure of fiber direction determination. It was found that for keratoconic cornea the average AR values are statistically greater than those of the normal cornea: 1.66 ± 0.42 (case I keratoconic cornea), 1.72 ± 0.44 (case II keratoconic cornea), and 1.34 ± 0.16 (average of three normal human corneas). Furthermore, the fiber directions determined by 2D-FFT analysis of BSHG and FSHG images were strongly correlated for large AR values (AR > 2).
  
  
  CONCLUSIONS
  The high correlation between FSHG and BSHG fiber direction for large AR values shows that BSHG imaging can provide qualitative and quantitative information of the extent of structural changes in a keratoconic cornea. The ability to use BSHG for diagnosing and monitoring stroma abnormalities, even when cornea transparency is retained, demonstrates the clinical potential of this method:
authors.affiliation: Department of Engineering Science, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Wen
authors.initials: W
authors.lastname: Lo
authors.forename: Wei-Liang
authors.initials: WL
authors.lastname: Chen
authors.forename: Chiu-Mei
authors.initials: CM
authors.lastname: Hsueh
authors.forename: Ara A
authors.initials: AA
authors.lastname: Ghazaryan
authors.forename: Shean-Jen
authors.initials: SJ
authors.lastname: Chen
authors.forename: David Hui-Kang
authors.initials: DH
authors.lastname: Ma
authors.forename: Chen-Yuan
authors.initials: CY
authors.lastname: Dong
authors.forename: Hsin-Yuan
authors.initials: HY
authors.lastname: Tan
mesh_terms: D000465:Algorithms; D003094:Collagen / Q000648:ultrastructure*; D003319:Corneal Stroma / Q000473:pathology*; D005583:Fourier Analysis; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007640:Keratoconus / Q000473:pathology*; D018613:Microscopy, Confocal / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D003094:Collagen