A wide-angle X-ray fibre diffraction method for quantifying collagen orientation across large tissue areas: application to the human eyeball coat

Abstract

A quantitative map of collagen fibril orientation across the human eyeball coat, including both the cornea and the sclera, has been obtained using a combination of synchrotron wide-angle X-ray scattering (WAXS) and three-dimensional point mapping. A macromolecular crystallography beamline, in a custommodified fibre diffraction setup, was used to record the 1.6 nm intermolecular equatorial reflection from fibrillar collagen at 0.5 mm spatial resolution across a flat-mounted human eyeball coat. Fibril orientation, derived as an average measure of the tissue thickness, was quantified by extraction of the azimuthal distribution of WAXS scatter intensity. Vector plots of preferential fibre orientation were remapped onto an idealized eyeball surface using a custombuilt numerical algorithm, to obtain a three-dimensional representation of the collagen fibril architecture.

Polarization microscopy for characterizing fiber orientation of ocular tissues

“…WAXS has recently been applied to the whole eye [50]. Like PLM, these methods are based on the molecular characteristics of collagen.…”

Collagen Structure and Mechanical Properties of the Human Sclera: Analysis for the Effects of Age

“…Winkler et al [8] measured collagen lamellar orientations within and around the optic nerve head (ONH) using second harmonic generation multiphoton microscopy. More recently, techniques have been developed to gain quantitative measures of bulk scleral fiber orientation and distribution, including small-angle light scattering (SALS) [9][10][11] and wide-angle X-ray scattering (WAXS) [12,13].…”

Peripapillary sclera architecture revisited: A tangential fiber model and its biomechanical implications

“…[5, 7, 13–17] Since collagen fibers stiffen when stretched, this ring would serve to restrict the opening of the canal that would be expected under elevated IOP. However, this current consensus is largely based on low-resolution, light scattering imaging techniques including small angle light scattering (SALS) and wide angle x-ray scattering (WAXS) [5, 7, 15, 16, 18, 19] or high resolution techniques such as second harmonic generation [17, 20] or electron microscopy [20–22] with excellent resolution but modest field of view. SALS and WAXS methods have been adopted in part due to their abilities to provide quantitative data that can be easily incorporated into mechanical models, however the resolution of these methods is on the order of 100–300 μm [5, 23], far below the resolution needed to observe individual collagen fibers or even bundles, on the order of 10–50 μm in width.…”