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Bin Yang, Ning-Jiun Jan, Po Lam, Kira L Lathrop, Ian A Sigal; Collagen architecture in the third dimension:3D polarized light microscopy (3DPLM) for mapping in-plane (IP) and out-of-plane (OOP) collagen fiber architecture. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4825. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The collagen fibers of the eye have a complex 3D organization critical to its mechanical functions, however, quantification of 3D fiber orientation of the eye in high-resolution over wide fields has remained a challenge. Here, we demonstrate the application to the posterior sclera of 3DPLM techniques that we have developed that allows determination of collagen fiber orientation in 3D, including IP and OOP components.
A goat eye (<2 yo) was obtained from a local slaughterhouse and fixed overnight in formalin (10%) within 24 hours of death. Following fixation, the eye was cryosectioned sagittally into 25µm sections. A section through the optic nerve head was selected and imaged using polarized light microscopy (Olympus IX70 with 4X, and 10X objectives) as previously described (Jan et al., BOE, 2015). Three sclera regions of interest (ROIs) shown in Fig. 1a were selected for analysis of IP and OOP orientation: adjacent to the sclera/dura interface, innermost and the mid-sclera 400µm from the canal.
The polarization enhanced, IP and OOP fiber orientation maps are shown in Fig. 1a-c, respectively. The OOP fiber orientation varies with location in respect to the canal. The mean OOP angles were 56.2±17.2°, 26.6±15° and 37.5±21.7°, in ROIs 1, 2 and 3, respectively. Higher magnification revealed fiber bundles and fascicles (Fig. 2a-b) with a clear distinction between interweaving fibers in and out of plane (Fig. 2c-d). Fig. 2e-f show 3D fiber orientation maps with shorter lines indicating larger OOP angles.
We demonstrated a novel 3DPLM imaging approach to quantify IP and OOP fiber orientation in ocular tissues. The technique allowed identification of regions with predominantly circumferential, radial or mixed-orientation. 3DPLM can be used to acquire high magnification images with a wide field of view, making it ideal for studying 3D ocular tissue microstructures.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Figure 1 (a) polarization enhanced image, (b) OOP fiber orientation map with 0 degree being in-plane and 90 degrees being out-of-plane and (c) in-plane fiber orientation map. Scale bar=300µm.
Figure 2 (a-b) polarization enhanced image and OOP orientation map of ROI with single white arrow, (c-d) polarization enhanced image and OOP orientation map of ROI with double white arrow, (e-f) vector map. Red arrows indicate fiber fascicles. Scale bar=50µm.
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