Lo Wen, Teng Shu-Wen, Tan Hsin-Yuan, Kim Ki Hean, Chen Hsiao-Ching, Lee Hsuan-Shu, Chen Yang-Fan, So Peter T C, Dong Chen-Yuan
Department of Physics, National Taiwan University, Taipei 106, and Chang-Gung Memorial Hospital, Linko, Taiwan, Republic of China.
Microsc Res Tech. 2006 Dec;69(12):973-5. doi: 10.1002/jemt.20373.
The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.
这项工作的目的是证明多光子显微镜是一种无需切片和染色就能研究完整角膜结构的首选技术。在微米分辨率下，多光子成像可以提供上皮细胞、角膜胶原纤维束和角膜细胞的大尺度形态特征和详细结构。通过一台自制的多光子显微镜，获得了从绿色荧光蛋白（GFP）小鼠摘除的完整眼睛的大面积多光子横截面图像。飞秒钛宝石激光器输出的760 nm激光产生了宽带多光子荧光（435 - 700 nm）和二次谐波产生（SHG，360 - 400 nm）信号。使用水浸物镜（Fluor，40X，NA 0.8；尼康）来便于对弯曲的眼表面进行成像。整个角膜的多光子图像提供了上皮细胞、角膜细胞和整体胶原纤维方向的形态学信息。具体而言，我们的平面、大面积多光子图像揭示了基质胶原的同心图案，这表明整个基质中胶原呈层状排列。此外，绿色荧光蛋白（GFP）标记增强了细胞区域的荧光对比度，并有助于观察静止的角膜细胞。我们的结果表明，GFP标记的小鼠角膜的多光子成像既具有形态学意义又能显示结构细节。二次谐波产生成像揭示了胶原纤维方向，而多光子荧光成像则显示了角膜中细胞的形态和分布。我们的结果支持多光子显微镜是用于角膜进一步体内研究和诊断的合适技术。
