title: Multiphoton microscopy of ex vivo corneas after collagen cross-linking.
abstract: OBJECTIVE
  To investigate changes in the morphology of the corneal stroma after collagen cross-linking (CXL) treatment in bovine and porcine eyes using a nonlinear microscope providing both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) corneal images.
  
  
  METHODS
  Freshly enucleated eyes were imaged using a tomographic nonlinear imaging method that was highly suitable to track temporal changes in corneal structures. CXL (riboflavin instillation plus UV irradiation) was applied on the enucleated eyes using similar protocols as in the clinic. A set of eyes without treatment were measured to be used as control.
  
  
  RESULTS
  In control corneas, SHG images showed a regular distribution of lamellae across the stroma that appeared stable for at least 6 hours postmortem. CXL changed the collagen distribution pattern showing some abnormal structures. TPEF revealed a large reduction in corneal thickness in CXL corneas immediately after treatment. The changes in the distribution of collagen bundles appeared also in corneas treated with riboflavin only, but not followed by UV irradiation. SHG tomography also revealed a partial recovery of the corneal thickness with time.
  
  
  CONCLUSIONS
  Nonlinear microscopy (in both tomographic and regular XY imaging configurations) was used to study spatial and temporal changes in the cornea during and after CXL on intact ocular globes. SHG imaging showed changes in the morphology of anterior corneal stroma after CXL. Regular collagen patterns turned into random distributed structures with thicker bundles at some localized areas. This might be a consequence of the corneal thickness decrease as a result of riboflavin-dextran instillation.
authors.affiliation: Laboratorio de Óptica, Centro de Investigación en Óptica y Nanofísica, Universidad de Murcia, Campus de Espinardo (Ed. 34), Murcia, Spain. bueno@um.es
authors.forename: Juan M
authors.initials: JM
authors.lastname: Bueno
authors.forename: Emilio J
authors.initials: EJ
authors.lastname: Gualda
authors.forename: Anastasia
authors.initials: A
authors.lastname: Giakoumaki
authors.forename: Pablo
authors.initials: P
authors.lastname: Pérez-Merino
authors.forename: Susana
authors.initials: S
authors.lastname: Marcos
authors.forename: Pablo
authors.initials: P
authors.lastname: Artal
mesh_terms: D000818:Animals; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology; D012256:Riboflavin / Q000494:pharmacology; D013552:Swine; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D003094:Collagen; D012256:Riboflavin