title: Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo.
abstract: OBJECTIVE
  To demonstrate the application of multiphoton fluorescence (MF) and second harmonic generation (SHG) microscopy for ex vivo characterization of the structural alterations of human corneas due to keratoconus.
  
  
  METHODS
  Buttons of keratoconic corneas derived from penetrating keratoplasty were sent for structural analysis with a custom-built multiphoton microscope. Fluorescence detected within the cytoplasm and the SHG signal obtained from collagen were used to demonstrate the morphologic changes in the corneal specimens.
  
  
  RESULTS
  The fluorescent epithelial cells around the apical area were elongated and were aligned parallel to the adjacent collagen fibers. Parallel and centripetal distribution patterns of stromal collagen bundles were demonstrated at different depths within the keratoconic corneas.
  
  
  CONCLUSIONS
  MF and SHG microscopy provides three-dimensional structural analysis of keratoconus ex vivo. It may provide important morphologic information for the investigation of the pathogenesis of keratoconus and may have potential in a clinical setting as an in vivo diagnostic and monitoring system for advancing keratoconus.
authors.affiliation: Department of Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Linko, Taiwan.
authors.forename: Hsin-Yuan
authors.initials: HY
authors.lastname: Tan
authors.forename: Yen
authors.initials: Y
authors.lastname: Sun
authors.forename: Wen
authors.initials: W
authors.lastname: Lo
authors.forename: Sung-Jan
authors.initials: SJ
authors.lastname: Lin
authors.forename: Ching-Hsi
authors.initials: CH
authors.lastname: Hsiao
authors.forename: Yeong-Fong
authors.initials: YF
authors.lastname: Chen
authors.forename: Samuel Chao-Ming
authors.initials: SC
authors.lastname: Huang
authors.forename: Wei-Chou
authors.initials: WC
authors.lastname: Lin
authors.forename: Shiou-Hwa
authors.initials: SH
authors.lastname: Jee
authors.forename: Hsin-Su
authors.initials: HS
authors.lastname: Yu
authors.forename: Chen-Yuan
authors.initials: CY
authors.lastname: Dong
mesh_terms: D003315:Cornea / Q000473:pathology*; D019781:Corneal Topography; D006801:Humans; D021621:Imaging, Three-Dimensional; D007640:Keratoconus / Q000473:pathology* / Q000601:surgery; D015948:Keratoplasty, Penetrating; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*