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Updated: Jul 18, 2026
A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva
Published on: May 17, 2020
Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo
Hsin-Yuan Tan1, Yen Sun, Wen Lo
1Department of Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Linko, 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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