title: Combined nonlinear and femtosecond confocal laser-scanning microscopy of rabbit corneas after photochemical cross-linking.
abstract: OBJECTIVE
  Photochemical cross-linking of corneal stromal collagen using riboflavin and ultraviolet irradiation is an evolving treatment for keratoconus. The purpose of the present study was to investigate the wound-healing process in rabbit corneas after cross-linking.
  
  
  METHODS
  Photochemical cross-linking was performed according to a standard protocol on the right eyes of eight male New Zealand White rabbits; the left eyes served as controls. Untreated controls and cross-linked rabbit corneas were imaged 3 days, 6 days, and 6 weeks after treatment using a customized setup for three-dimensional nonlinear microscopy and confocal laser-scanning microscopy of reflected femtosecond light (fs-CLSM).
  
  
  RESULTS
  The combination of fs-CLSM in reflective mode and two-photon-excited fluorescence permitted differentiation of the following zones in the lamina propria of treated corneas 3 and 6 days after cross-linking: (1) an anterior zone with postapoptotic keratocyte debris, visible only on fs-CLSM in reflective mode; (2) a posterior zone with activated keratocytes with strong autofluorescence; and (3) surviving or restored keratocytes with moderate autofluorescence beyond the intermediate zone. Repopulation with normal keratocytes was achieved by 6 weeks. Bi-directional, second-harmonic generation (SHG) imaging showed no global differences in the fiber orientation and lamellar structure of stromal collagen at any time point. A relatively strong additional two-photon excited fluorescence occurred in the treated corneas with a diffuse three-dimensional spatial distribution.
  
  
  CONCLUSIONS
  This combination of imaging modalities has the potential to become a new clinical instrument capable of visualizing corneal changes at the cellular and extracellular level.
authors.affiliation: Laser Zentrum Hannover eV, Hannover, Germany. a.krueger@lzh.de
authors.forename: Alexander
authors.initials: A
authors.lastname: Krüger
authors.forename: Marine
authors.initials: M
authors.lastname: Hovakimyan
authors.forename: Diego F
authors.initials: DF
authors.lastname: Ramírez Ojeda
authors.forename: Oliver
authors.initials: O
authors.lastname: Stachs
authors.forename: Andreas
authors.initials: A
authors.lastname: Wree
authors.forename: Rudolf F
authors.initials: RF
authors.lastname: Guthoff
authors.forename: Alexander
authors.initials: A
authors.lastname: Heisterkamp
mesh_terms: D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003315:Cornea / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D021621:Imaging, Three-Dimensional; D007640:Keratoconus / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D010778:Photochemotherapy; D011817:Rabbits; D014945:Wound Healing
chemical_list: D003094:Collagen