title: Two-Photon Microscopy of the Mouse Peripheral Cornea Ex Vivo.
abstract: OBJECTIVE
  To investigate the 3-dimensional (3D) cell and extracellular matrix (ECM) structure of mouse peripheral corneas in normal and corneal neovascularization tissues using 2-photon microscopy (TPM) based on both intrinsic and extrinsic moxifloxacin contrasts.
  
  
  METHODS
  Peripheral corneas in freshly enucleated mouse eyes were imaged by TPM based on both intrinsic and extrinsic contrasts. Intrinsic autofluorescence and second harmonic generation were used to image cells and ECM collagen, respectively. Moxifloxacin ophthalmic solution was applied to image cells. The peripheral cornea, limbus, and sclera were imaged in 3D. In addition to normal mice, mouse models of suture-induced corneal neovascularization were imaged to visualize changes in the microstructure.
  
  
  RESULTS
  Complex 3D cell and ECM structures in the cornea, limbus, and sclera were visualized by TPM. TPM images based on intrinsic contrasts visualized both cell and ECM structures, and TPM images based on moxifloxacin visualized cell structures with enhanced contrast. On the limbus side of the mouse peripheral cornea, TPM images visualized the vasculature in the limbus, the trabecular meshwork/Schlemm canal, iris, and ciliary body. On the scleral side, TPM images visualized cell and ECM structures in the sclera and multiple cell layers below the sclera. TPM images of the peripheral cornea in the corneal neovascularization condition visualized the extension of vasculature from the limbus to the cornea.
  
  
  CONCLUSIONS
  TPM imaging based on both intrinsic and external moxifloxacin contrasts visualized detailed 3D cell and ECM microstructures in the mouse peripheral cornea. TPM based on moxifloxacin might be advantageous for studying cell structures by enhancing image contrast.
authors.affiliation: *Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Gyeoungbuk, Republic of Korea; †Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Songpa-gu, Seoul, Republic of Korea; and ‡Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeoungbuk, Republic of Korea.
authors.forename: Jun Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Ki Hean
authors.initials: KH
authors.lastname: Kim
authors.forename: Seunghun
authors.initials: S
authors.lastname: Lee
authors.forename: Hyerin
authors.initials: H
authors.lastname: Jeong
authors.forename: Myoung Joon
authors.initials: MJ
authors.lastname: Kim
mesh_terms: D000818:Animals; D003094:Collagen / Q000378:metabolism; D003287:Contrast Media / Q000737:chemistry; D003315:Cornea / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D016510:Corneal Neovascularization / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D004549:Elastin / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism / Q000473:pathology*; D005260:Female; D024841:Fluoroquinolones / Q000737:chemistry; D021621:Imaging, Three-Dimensional / Q000379:methods; D016850:Limbus Corneae / Q000166:cytology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D036641:Microscopy, Fluorescence, Multiphoton; D000077266:Moxifloxacin; D012590:Sclera / Q000166:cytology / Q000473:pathology
chemical_list: D003287:Contrast Media; D024841:Fluoroquinolones; D003094:Collagen; D004549:Elastin; D000077266:Moxifloxacin