Polarized light microscopy for 3-dimensional mapping of collagen fiber architecture in ocular tissues

Abstract

Collagen fibers play a central role in normal eye mechanics and pathology. In ocular tissues, collagen fibers exhibit a complex 3-dimensional (3D) fiber orientation, with both in-plane (IP) and out-of-plane (OP) orientations. Imaging techniques traditionally applied to the study of ocular tissues only quantify IP fiber orientation, providing little information on OP fiber orientation. Accurate description of the complex 3D fiber microstructures of the eye requires quantifying full 3D fiber orientation. Herein, we present 3dPLM, a technique based on polarized light microscopy developed to quantify both IP and OP collagen fiber orientations of ocular tissues. The performance of 3dPLM was examined by simulation and experimental verification and validation. The experiments demonstrated an excellent agreement between extracted and true 3D fiber orientation. Both IP and OP fiber orientations can be extracted from the sclera and the cornea, providing previously unavailable quantitative 3D measures and insight into the tissue microarchitecture. Together, the results demonstrate that 3dPLM is a powerful imaging technique for the analysis of ocular tissues.

Keywords: collagen; eye; orientation; polarized light microscopy; sclera.

Figure 1

The anatomy of the eye with major components and orientation illustrated. The red, green and blue boxes indicate the locations of three regions selected for imaging.

Figure 2

(a) Birefringence computed using both exact and approximate forms. (b) Calculated mean and dispersion (standard deviation) of OP angles with the presence of retardance approximation error. The plot demonstrates that mean calculated OP angles agree very well with reference OP angles. The dispersion of the calculated values was slightly larger for small OP angles than for larger ones. The error bar represents standard deviation.

Figure 3

OP fiber orientation mapping on 7 chicken tendon sections cryo-sectioned at known orientations from longitudinal (0°) to transverse (90°). The inset shows photos of tendons as set at various orientations (top) as well as images colored by the local OP. The line plot shows a quantitative comparison. Calculated mean OP fiber orientation matched reference fiber orientations. The error bar represents the standard deviation.

Figure 4

Application of 3dPLM to posterior pole of a sheep eye. (a) Bright field image with red and orange arrowheads pointing to long IP fiber bundles and OP fiber bundle fascicles, respectively; (b) IP fiber orientation map showing both IP fiber morphology and orientation; (c) OP fiber orientation map highlighting fiber bundle fascicles; (d) OP fiber orientation of small ROI shown in (c); (e) 3D visualization of collagen fibers

Figure 5

IP and OP fiber orientation map of the anterior sclera. (a) bright field image of the sclera. The red box indicates the approximate location of the imaged region. (b) IP fiber orientation map of the sclera. The insert indicates two perpendicular IP fiber orientation. (c) OP fiber orientation map of the sclera. (d) 3D fiber reconstruction on a small ROI indicated with a black box in (c). The orientation of the black lines represents the projected IP fiber orientation whereas the length of the line represents how out-of-plane the fiber is (large OP angles are shown by short lines and small OP angles by long lines). (e) 3D visualization of collagen fibers of a ROI indicated with a white box in (d).

Figure 6

IP and OP fiber orientation map of the cornea. (a) bright field image of the cornea. The red box indicates the approximate location of the imaged region. (b) IP fiber orientation map of the cornea. (c) OP fiber orientation map of the cornea. (d) 3D fiber orientation on a small ROI indicated with a white box in (c). The direction of the line indicates the fiber orientation, and the length of the line indicates inclination. (e) The IP and OP fiber orientation along a line ROI. (f) electronic micrograph showing collagen fiber organization in cornea adapted from . (g) 3D visualization of collagen fibers of a ROI indicated with a white box in (d).