Purpose: To qualify and quantify structural alterations in keratoconic corneas ex vivo by the use of second-harmonic generation (SHG) microscopy and two-dimensional fast Fourier transform (2D-FFT) analysis.
Methods: Two keratoconic and three normal human corneal specimens were imaged with a multiphoton microscope. Forward and backward second-harmonic generation images (FSHG and BSHG) of corneal stroma were obtained at high resolution and at different depths. The SHG images were further analyzed with a 2D-FFT algorithm to quantify the texture and orientation changes of collagen fibers.
Results: The results showed that the abnormality of collagen architecture was found through the whole layer of stroma. 2D-FFT analysis of SHG cornea images provided fiber orientation direction and an aspect ratio (AR) as a quantitative measure of fiber direction determination. It was found that for keratoconic cornea the average AR values are statistically greater than those of the normal cornea: 1.66 ± 0.42 (case I keratoconic cornea), 1.72 ± 0.44 (case II keratoconic cornea), and 1.34 ± 0.16 (average of three normal human corneas). Furthermore, the fiber directions determined by 2D-FFT analysis of BSHG and FSHG images were strongly correlated for large AR values (AR > 2).
Conclusions: The high correlation between FSHG and BSHG fiber direction for large AR values shows that BSHG imaging can provide qualitative and quantitative information of the extent of structural changes in a keratoconic cornea. The ability to use BSHG for diagnosing and monitoring stroma abnormalities, even when cornea transparency is retained, demonstrates the clinical potential of this method: