Park Choul Yong, Marando Catherine M, Liao Jason A, Lee Jimmy K, Kwon Jiwon, Chuck Roy S
Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, Gyunggido, South Korea.
Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, United States.
Invest Ophthalmol Vis Sci. 2016 Oct 1;57(13):5602-5610. doi: 10.1167/iovs.16-19706.
To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera.
The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF.
The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were <10 μm in width, in a loose, disorganized arrangement. In deeper image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 μm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus.
Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.
研究人角膜缘结膜、Tenon囊和巩膜中胶原蛋白和弹性蛋白的结构及分布。
使用倒置双光子激发荧光显微镜对人供体角膜植片的角膜缘结膜、Tenon囊和巩膜进行成像。无需固定过程。将激光（钛宝石激光器）调谐至850nm进行双光子激发。分别通过425/30nm发射滤光片和525/45nm发射滤光片收集二次谐波产生（SHG）的背散射信号和自发荧光（AF）。采集多个连续且重叠的（z轴堆叠）图像。胶原蛋白信号通过SHG收集，而弹性蛋白信号通过AF收集。
胶原束的大小和密度因深度而异：从结膜到巩膜逐渐增加。在浅表图像平面中，胶原束宽度<10μm，排列松散、无序。在较深的图像平面（巩膜上层和巩膜浅层），胶原束更粗（宽度接近100μm）且紧密排列。相比之下，弹性纤维更细且稀疏。弹性纤维在表层的取向与胶原纤维无关；但在巩膜深层，弹性纤维穿过胶原束间间隙。在角膜缘，胶原蛋白和弹性蛋白纤维都相对紧密，且垂直于角膜缘环分布。
双光子激发荧光显微镜使我们能够更详细地了解人角膜缘结膜、Tenon囊和巩膜的胶原蛋白和弹性蛋白结构。
