Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo.
OBJECTIVE
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.
Tan
Hsin-Yuan
HY
Department of Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Linko, Taiwan.
Sun
Yen
Y
Lo
Wen
W
Lin
Sung-Jan
SJ
Hsiao
Ching-Hsi
CH
Chen
Yeong-Fong
YF
Huang
Samuel Chao-Ming
SC
Lin
Wei-Chou
WC
Jee
Shiou-Hwa
SH
Yu
Hsin-Su
HS
Dong
Chen-Yuan
CY
D003315:Cornea / Q000473:pathology*; D019781:Corneal Topography; D006801:Humans; D021621:Imaging, Three-Dimensional; D007640:Keratoconus / Q000473:pathology* / Q000601:surgery; D015948:Keratoplasty, Penetrating; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*