Lee Po-Yi, Schilpp Hannah, Naylor Nathan, Watkins Simon C, Yang Bin, Sigal Ian A
Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA.
Department of Ophthalmology, School of Medicine, University of Pittsburgh, Pittsburgh, PA.
bioRxiv. 2023 Feb 27:2023.01.29.526111. doi: 10.1101/2023.01.29.526111.
Collagen architecture determines the biomechanical environment in the eye, and thus characterizing collagen fiber organization and biomechanics is essential to fully understand eye physiology and pathology. We recently introduced instant polarized light microscopy (IPOL) that encodes optically information about fiber orientation and retardance through a color snapshot. Although IPOL allows imaging collagen at the full acquisition speed of the camera, with excellent spatial and angular resolutions, a limitation is that the orientation-encoding color is cyclic every 90 degrees (π/2 radians). In consequence, two orthogonal fibers have the same color and therefore the same orientation when quantified by color-angle mapping. In this study, we demonstrate IPOLπ, a new variation of IPOL, in which the orientation-encoding color is cyclic every 180 degrees (π radians). Herein we present the fundamentals of IPOLπ, including a framework based on a Mueller-matrix formalism to characterize how fiber orientation and retardance determine the color. The improved quantitative capability of IPOLπ enables further study of essential biomechanical properties of collagen in ocular tissues, such as fiber anisotropy and crimp. We present a series of experimental calibrations and quantitative procedures to visualize and quantify ocular collagen orientation and microstructure in the optic nerve head, a region in the back of the eye. There are four important strengths of IPOLπ compared to IPOL. First, IPOLπ can distinguish the orientations of orthogonal collagen fibers via colors, whereas IPOL cannot. Second, IPOLπ requires a lower exposure time than IPOL, thus allowing faster imaging speed. Third, IPOLπ allows visualizing non-birefringent tissues and backgrounds from tissue absorption, whereas both appear dark in IPOL images. Fourth, IPOLπ is cheaper and less sensitive to imperfectly collimated light than IPOL. Altogether, the high spatial, angular, and temporal resolutions of IPOLπ enable a deeper insight into ocular biomechanics and eye physiology and pathology.
胶原蛋白结构决定了眼睛中的生物力学环境，因此表征胶原蛋白纤维组织和生物力学对于全面理解眼睛的生理和病理至关重要。我们最近引入了即时偏振光显微镜（IPOL），它通过彩色快照对有关纤维取向和相位延迟的光学信息进行编码。尽管IPOL能够以相机的全采集速度对胶原蛋白进行成像，具有出色的空间和角度分辨率，但一个局限性在于取向编码颜色每90度（π/2弧度）循环一次。因此，两条正交纤维具有相同的颜色，从而在通过颜色-角度映射进行量化时具有相同的取向。在本研究中，我们展示了IPOLπ，这是IPOL的一种新变体，其中取向编码颜色每180度（π弧度）循环一次。在此，我们介绍IPOLπ的基本原理，包括基于穆勒矩阵形式的框架，以表征纤维取向和相位延迟如何决定颜色。IPOLπ改进的定量能力能够进一步研究眼组织中胶原蛋白的基本生物力学特性，如纤维各向异性和卷曲。我们提出了一系列实验校准和定量程序，以可视化和量化视神经乳头（眼睛后部的一个区域）中的眼胶原蛋白取向和微观结构。与IPOL相比，IPOLπ有四个重要优势。第一，IPOLπ能够通过颜色区分正交胶原蛋白纤维的取向，而IPOL则不能。第二，IPOLπ比IPOL需要更低的曝光时间，从而允许更快的成像速度。第三，IPOLπ能够从组织吸收中可视化非双折射组织和背景，而在IPOL图像中两者均显示为暗的。第四，IPOLπ比IPOL更便宜，并且对光线准直不完善的敏感度更低。总之，IPOLπ的高空间、角度和时间分辨率能够更深入地洞察眼生物力学以及眼睛的生理和病理。
