IOVS Molecular Pharmacology
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(Investigative Ophthalmology and Visual Science. 2006;47:5251-5259.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0386
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Multiphoton Fluorescence and Second Harmonic Generation Imaging of the Structural Alterations in Keratoconus Ex Vivo

Hsin-Yuan Tan,1,2 Yen Sun,3 Wen Lo,3 Sung-Jan Lin,4 Ching-Hsi Hsiao,1 Yeong-Fong Chen,1 Samuel Chao-Ming Huang,1 Wei-Chou Lin,5 Shiou-Hwa Jee,4 Hsin-Su Yu,4 and Chen-Yuan Dong3

1From the Department of Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Linko, Taiwan; the 2Institute of Biomedical Engineering, College of Medicine and Engineering, and the 3Department of Physics, National Taiwan University, Taipei, Taiwan; the 4Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; and the 5Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan.

PURPOSE. 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.




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