The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic
generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as
being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser
pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton
autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial
microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button
from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The
specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of
lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In
addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the
cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover,
increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area.
In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological
features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future
the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.