Two-photon imaging of the trabecular meshwork
Two-photon imaging of the trabecular meshwork
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DOI：
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发表时间：
2010-05
期刊：
影响因子：
2.2
通讯作者：
D. Ammar;Tim C. Lei;E. Gibson;M. Kahook
中科院分区：
文献类型：
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作者：
D. Ammar;Tim C. Lei;E. Gibson;M. Kahook
目的采用非侵入性、非破坏性技术对天然未固定状态下的小梁网进行成像。方法采用双光子激发荧光（2PEF）和二次谐波产生（SHG）双光子显微镜（2PM）对人眼小梁网标本进行成像。沿组织轴（z轴）沿着分析多个图像以生成该区域的三维（3D）模型。结果通过固有荧光（2PEF）和SHG检测到大的胶原纤维（直径约10 µm）的网格。存在紧密重叠束的区域以及从TM表面可见的充满流体的区域。多个图像的3D分析显示，TM深处的开放区域穿透小管TM（JTM）并连接到Schlemm管（IWSC）的内壁。这些开放区域可以代表前房和施累姆氏管（SC）之间的低阻力流体通路。结论：人眼流出道系统的2PM成像仅从固有光学特性记录胶原结构，而不添加外源性荧光标记。2PM成功成像到TM中，无需固定、包埋或组织学处理。使用先进的光学技术进行深度渗透，发现可能代表IWSC中孔隙的区域，这些区域已通过多项电子显微镜研究记录。我们的工作表明，2PM成像有潜力作为一个新的衡量标准，用于评估人类眼睛的水流出区，是值得进一步探索。
Purpose To image the trabecular meshwork (TM) in its native unfixed state using a non-invasive, non-destructive technique. Methods Two-photon microscopy (2PM), including two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG), was used to image flat-mounted trabecular meshwork samples from human cadaver eyes. Multiple images were analyzed along the tissue axis (z-axis) to generate a three-dimensional (3D) model of the region. Results A lattice of large collagen fibers (~10 µm in diameter) were detected by inherent fluorescence (2PEF) and SHG. There are regions of both tightly overlapping bundles as well as fluid-filled regions visible from the surface of the TM. 3D analysis of multiple images reveals that the open regions deep in the TM penetrate the juxtacanalicular TM (JTM) and connect to the inner wall of Schlemm’s canal (IWSC). These open regions may represent low-resistance fluid pathways between the anterior chamber and Schlemm’s canal (SC). Conclusions 2PM imaging of the outflow system of the human eye documented collagenous structures solely from inherent optical properties, without addition of an exogenous fluorescent label. 2PM successfully imaged into the TM without the need for fixation, embedding, or histological processing. Deep penetration using advanced optical techniques revealed regions likely representing pores in the IWSC that have been documented by multiple electron microscope studies. Our work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.