Lei Tim C, Ammar David A, Masihzadeh Omid, Gibson Emily A, Kahook Malik Y
Department of Electrical Engineering, University of Colorado Denver, Denver, CO, USA.
Mol Vis. 2011;17:2628-33. Epub 2011 Oct 8.
To image the human trabecular meshwork (TM) using a non-invasive, non-destructive technique without the application of exogenous label.
Flat-mounted TM samples from a human cadaver eye were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). In TPAF, two optical photons are simultaneously absorbed and excite molecules in the sample that then emit a higher energy photon. The signal is predominately from collagen and elastin. The CARS technique uses two laser frequencies to specifically excite carbon-hydrogen bonds, allowing the visualization of lipid-rich cell membranes. Multiple images were taken along an axis perpendicular to the surface of the TM for subsequent analysis.
Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes. Simultaneous CARS imaging revealed elliptical structures of ~7×10 µm in diameter populating the meshwork which were consistent with TM cells. Irregularly shaped objects of ~4 µm diameter appeared in both the TPAF and CARS channels, and are consistent with melanin granules.
CARS techniques were successful in imaging live TM cells in freshly isolated human TM samples. Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols. CARS imaging of the TM represents a new avenue for exploring details of aqueous outflow and TM cell physiology.
使用一种非侵入性、非破坏性技术且不施加外源性标记来对人小梁网（TM）进行成像。
使用两种非线性光学技术对来自人尸体眼睛的平铺TM样本进行成像：相干反斯托克斯拉曼散射（CARS）和双光子自发荧光（TPAF）。在TPAF中，两个光学光子同时被吸收并激发样本中的分子，然后这些分子发射出更高能量的光子。该信号主要来自胶原蛋白和弹性蛋白。CARS技术使用两个激光频率来特异性激发碳氢键，从而实现富含脂质的细胞膜的可视化。沿着垂直于TM表面的轴拍摄多张图像以供后续分析。
对TM的多张TPAF图像分析揭示了各种大小的典型胶原重叠束。同时进行的CARS成像显示，直径约为7×10 µm的椭圆形结构遍布小梁网，与TM细胞一致。直径约4 µm的不规则形状物体出现在TPAF和CARS通道中，与黑色素颗粒一致。
CARS技术成功地对新鲜分离的人TM样本中的活TM细胞进行了成像。使用标准组织学技术也获得了类似的图像，然而本文所述方法的优点是在未处理、未固定的组织上进行，避免了由于注入固定剂和用酒精处理而可能出现的精细组织形态潜在扭曲。TM的CARS成像为探索房水流出和TM细胞生理学细节提供了一条新途径。
