Quantitative Analysis of Collagen Lamellae in the Normal and Keratoconic Human Cornea by Second Harmonic Generation Imaging Microscopy

PURPOSE. To characterize the structural properties of collagen lamellae in the normal and keratoconic human corneal stroma, we measured their width and angle relative to Bowman's layer (BL). METHODS. Thirteen normal and four keratoconic corneas were examined. Collagen lamellae in tissue blocks from the central cornea were visualized by second harmonic generation imaging microscopy. Images obtained in 1-mu m steps from BL to Descemet's membrane (DM) were subjected to three- dimensional reconstruction. The reconstructed data sets were divided into 10 layers of equal depth ( L1-L10) for analysis. The width of lamellae adherent to BL (L0) was also determined. RESULTS. For the normal cornea, the width (mean 6 SD) of collagen lamellae was 6.5 +/- 1.7 mu m at L0, decreased to 4.3 +/- 1.3 mu m at L1, and then increased gradually with progression toward DM to 122.2 +/- 34.5 mu m at L10, whereas the angle of lamellae was 20.9 degrees +/- 5.4 degrees at L1 and decreased initially to 10.6 degrees +/- 3.2 degrees at L2 before declining gradually to 2.7 degrees +/- 2.2 degrees at L10. The width and angle of collagen lamellae in the keratoconic cornea were significantly larger and smaller, respectively, relative to those in the normal cornea. CONCLUSIONS. In the normal human cornea, collagen lamellae adjacent to BL are narrow and form a steep angle with BL, whereas they increase in width and their angle relative to BL flattens with progression toward DM. These properties of collagen lamellae are altered in keratoconus and are likely related to abnormalities of corneal shape.