title: Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye.
abstract: OBJECTIVE
  The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes.
  
  
  METHODS
  The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye.
  
  
  RESULTS
  Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes.
  
  
  CONCLUSIONS
  Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.
authors.affiliation: Microscopic Biophysics Laboratory, Department of Physics, National Taiwan University, Teipei.
authors.forename: Shu-Wen
authors.initials: SW
authors.lastname: Teng
authors.forename: Hsin-Yuan
authors.initials: HY
authors.lastname: Tan
authors.forename: Ju-Li
authors.initials: JL
authors.lastname: Peng
authors.forename: Huei-Hsing
authors.initials: HH
authors.lastname: Lin
authors.forename: Ki Hean
authors.initials: KH
authors.lastname: Kim
authors.forename: Wen
authors.initials: W
authors.lastname: Lo
authors.forename: Yen
authors.initials: Y
authors.lastname: Sun
authors.forename: Wei-Chou
authors.initials: WC
authors.lastname: Lin
authors.forename: Sung-Jan
authors.initials: SJ
authors.lastname: Lin
authors.forename: Shiou-Hwa
authors.initials: SH
authors.lastname: Jee
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
authors.forename: Chen-Yuan
authors.initials: CY
authors.lastname: Dong
mesh_terms: D000818:Animals; D003094:Collagen / Q000648:ultrastructure*; D003228:Conjunctiva / Q000737:chemistry / Q000648:ultrastructure*; D003315:Cornea / Q000737:chemistry / Q000648:ultrastructure*; D007089:Image Enhancement / Q000379:methods; D016850:Limbus Corneae / Q000737:chemistry / Q000648:ultrastructure; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D012590:Sclera / Q000737:chemistry / Q000648:ultrastructure*; D013552:Swine
chemical_list: D003094:Collagen