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Wen Lo, Wei-Liang Chen, Chiu-Mei Hsueh, Ara A Ghazaryan, Shean-Jen Chen, David Hui-Kang Ma, Chen-Yuan Dong, Hsin-Yuan Tan; Fast Fourier Transform–Based Analysis of Second-Harmonic Generation Image in Keratoconic Cornea. Invest. Ophthalmol. Vis. Sci. 2012;53(7):3501-3507. doi: https://doi.org/10.1167/iovs.10-6697.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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).
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.
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