Abstract
The spatial organization of stromal collagen of ex-vivo corneas has been quantified in adaptive-optics second harmonic generation (SHG) images by means of an optimized Fourier transform (FT) based analysis. At a particular depth location, adjacent lamellae often present similar orientations and run parallel to the corneal surface. However this pattern might be combined with interweaved collagen bundles leading to crosshatched structures with different orientations. The procedure here reported provides us with both principal and crosshatched angles. This is also able to automatically distinguish a random distribution from a cross-shaped one, since it uses the ratio of the axes lengths of the best-fitted ellipse of the FT data as an auxiliary parameter. The technique has successfully been applied to SHG images of healthy corneas (both stroma and Bowman’s layer) of different species and to corneas undergoing cross-linking treatment.
Highlights
Collagen is the main structural element of the corneal stroma
The regular Fourier transform (FT) analysis based on computing the aspect ratio (AR) of the best fitted ellipse provides a value of 0.15 for Fig. 3(a) and a dominant direction of the lamellae of ~30° (90° apart from the set of points of the FT image of Fig. 3(d))
It can be observed that the corresponding FT image (Fig. 3(e)) shows two preferential directions at 111° and ~33° due to the crosshatched appearance of collagen fibers
Summary
Collagen (type I) is the main structural element of the corneal stroma. It presents a natural non-centrosymmetric organization with a triple helix fibril structure which generates a strong second harmonic generation (SHG) signal [1]. These fibrils assemble to form collagen bundles or lamellae [2]. SHG microscopy enables imaging of corneal collagen structures without staining procedures [1,3,4,5,6]. Corneal lamellae are often organized and their orientations are often analyzed in a qualitative manner. Quantification facilitates extraction of sensitive changes in collagen fiber organization due to pathology [4,7], surgery [8,9,10,11] or damage [12,13], and, may help in early diagnosis and follow-up processes
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