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Ning-Jiun Jan, Jonathan Grimm, Kira Lathrop, Gadi Wollstein, Larry Kagemann, Joel Schuman, Hiroshi Ishikawa, Ian Sigal; Measuring Micron-Scale Collagen Fiber Orientation in the Lamina Cribrosa and Peripapillary Sclera. Invest. Ophthalmol. Vis. Sci. 2013;54(15):65.
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Collagen fiber orientation is a critical factor in determining the local biomechanical response to loading of the lamina cribrosa (LC) and peripapillary sclera (PPS). Our goal was to evaluate the repeatability of our novel technique for measuring micron-scale collagen fiber orientation in LC and PPS.
A sheep eye (<2 yo) was obtained from a local slaughterhouse and fixed overnight in formalin (10%) within 24 hours of death. Following fixation the LC and PPS were cryosectioned coronally into 30 µm sections. One section through the LC was selected and imaged using light microscopy (Olympus BX60, 12-bit grayscale 1600x1200 pixels) three times at high resolution (0.73 µm/pixel , 10x objective, NA 0.30) and three times at low resolution (1.48 µm/pixel, 4x objective, NA 0.13). Repeatability was measured by overlying stitched image sets and calculating the angle differences between fiber orientations in each image and the mean angle over all image sets of that resolution.
In both LC and PPS angle differences within resolutions were small, with 95% of the fiber orientations differing by less than 5°, independent of the resolution (Figure 1). Angle differences in the PPS were smaller than in the LC. Overlaid orientations obtained from the three high resolution image sets illustrate the small magnitude of the differences (Figure 2).
Our method was highly reproducible, with multiple measurements differing by only a few degrees. When the measurements are overlaid they are essentially indistinguishable. Micron-scale collagen fiber orientation data of the LC and PPS will enable better understanding of the effects of intraocular pressure on the optic nerve head.
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