En face, single-plane SHG images from the great white shark cornea are shown
Figure 2. Each SHG image (
Figs. 2A–C) represents a 225 × 225-μm section of the anterior corneal stroma shown at a resolution of 0.44 μm/pixel taken at a stromal depth of 114 μm (
Fig. 2A), 123 μm (
Fig. 2B), and 129 μm (
Fig. 2C) from the corneal epithelium. Through-focus SHG images showed alternating sheets of highly organized, parallel collagen fibers running in parallel and oriented nearly perpendicular or orthogonal to each other (
Figs. 2A,
2C). At the interface between collagen sheets, the orthogonal orientation of two adjacent sheets could be imaged (
Fig. 2B) and an FTT generated showing the predominant directions of the collagen fibers in the two planes (
Fig. 2D). The angular offset in fiber directionality between neighboring sheets measured by FTT analysis detected an average offset of 82.6° ± 5.5° over the 3-D image data set for the great white shark. This pattern of nearly orthogonally arranged sheets of collagen fibers also was detected in other cartilaginous fish (sharpnose shark, not shown), boney fish (
Fig. 3A1, salmon), and amphibians (
Fig. 3B1, bullfrog). In reptiles (
Fig. 3C1, alligator) and birds (
Fig. 3D1, falcon), the collagen sheets appeared to be broken down into ribbons, but maintained an orthogonal orientation. Measurement of the angular offset (
Figs. 3A2–D2) using FTT measurements also showed an average offset ranging from 77.7° to 88.2° for these different clades. As shown previously,
7 SHG images taken from representative mammals (
Figs. 3E1, 3E2, rabbit) showed lamellae to be organized into collagen ribbons having a random angle offset between lamellae in the same plane that was distinctly different from that of nonmammalian vertebrate corneas.