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双光子激发荧光显微镜显示的人角膜缘结膜,Tenon's囊和巩膜的胶原和弹性蛋白结构细节

Park CY, Marando CM, Liao JA, et al.

期刊名称:Investigative ophthalmology & visual science

卷期:2016年第57卷第13期

关键词:
巩膜 结膜 角膜

摘要

目的:旨在探讨人角膜缘结膜,Tenon's囊和巩膜的胶原和弹性蛋白结构细节胶原蛋白和弹性蛋白的结构和分布。方法:人角膜缘结膜,Tenon囊和巩膜采用倒置双光子激发荧光显微镜进行检查。操作不需要固定。激光(Tisapphire)调谐在850nm处进行双光子激发。分别通过425 / 30nm525 / 45nm发射滤光片收集二次谐波(SHG)和自发荧光(AF)后向散射信号。收集多个,连续和重叠的(z-stack)图像。使用SHG收集胶原信号,使用AF弹性蛋白信号。结果:胶原蛋白的大小和密度根据深度而变化:从结膜到巩膜逐渐增加。在浅图像平面中,胶原束宽度<10μm,松散,无组织排列。在深图像平面(巩膜和浅表巩膜)中,胶原束较厚(宽度近100μm)并且致密。相比之下,弹性蛋白纤维更薄,更稀疏。弹性蛋白纤维的来源与表层胶原纤维无关;但在深层巩膜中,弹性蛋白纤维填充在胶原蛋白间隙。角膜缘的胶原蛋白和弹性蛋白纤维相对紧凑,垂直于角膜缘环带分布。结论: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胶囊和巩膜的胶原和弹性蛋白结构。

Purpose:To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera.Methods: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.Results: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.Conclusions: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.


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