利用高分辨率非线性光学宏观技术评估角膜胶原组织。
Evaluating corneal collagen organization using high-resolution nonlinear optical macroscopy.
机构信息
Department of Ophthalmology, Gavin Herbert Eye Institute, University of California Irvine Medical Center, Orange, CA, USA.
出版信息
Eye Contact Lens. 2010 Sep;36(5):260-4. doi: 10.1097/ICL.0b013e3181ee8992.
PURPOSE
Recent developments in nonlinear optical (NLO) imaging using femtosecond lasers provides a noninvasive method for detecting collagen fibers by imaging second harmonic-generated (SHG) signals. However, this technique is limited by the small field of view necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the field of view to assess the entire collagen structure using high-resolution macroscopic (HRMac) imaging.
METHODS
Intact human eyes were fixed under pressure, and the whole cornea (13-mm diameter) was excised and embedded in low-melting point agar for vibratome sectioning (200-300 microm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser (Carl Zeiss Microimaging Inc., Thornwood, NY) to generate SHG images. For each vibratome section, an overlapping series of three-dimensional data sets (466 x 466 x 150 microm) were taken, covering the entire tissue (15 mm x 6 mm area) using a motorized, mechanical stage. The three-dimensional data sets were then concatenated to generate an NLO-based tomograph.
RESULTS
The HRMac of the cornea yielded large macroscopic (80 megapixels per plane), three-dimensional tomographs with high resolution (0.81 microm lateral, 2.0 microm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented, and extracted. Three-dimensional reconstructions suggested that the anterior cornea comprises highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's layer).
CONCLUSIONS
We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.
目的
飞秒激光的非线性光学（NLO）成像的最新发展提供了一种通过成像二次谐波产生（SHG）信号来检测胶原纤维的非侵入性方法。然而，这种技术受到产生 SHG 信号所需的小视场的限制。本报告的目的是回顾我们努力通过使用高分辨率宏观（HRMac）成像大大扩展视场以评估整个胶原结构的努力。
方法
在压力下固定完整的人眼，并切除整个角膜（直径 13 毫米）并嵌入低熔点琼脂中用于振动切片（200-300 微米）。然后使用蔡司 LSM 510 Meta 和变色龙飞秒激光器（卡尔蔡司微成像公司，Thornwood，NY）对切片进行光学扫描，以产生 SHG 图像。对于每个振动切片，使用电动机械台获取重叠的三维数据集（466 x 466 x 150 微米）系列，覆盖整个组织（15 毫米 x 6 毫米区域）。然后将三维数据集拼接在一起以生成基于 NLO 的断层扫描图。
结果
角膜的 HRMac 产生了大的宏观（每个平面 8000 万像素）、具有高分辨率（横向 0.81 微米，轴向 2.0 微米）的三维断层扫描图，其中可以追踪、分割和提取单个胶原纤维（基质层片）。三维重建表明，前角膜由高度交织的层片组成，这些层片插入前限界层（Bowman 层）。
结论
我们得出结论，基于 NLO 的 HRMac 断层扫描提供了一种强大的新工具，可用于评估角膜内胶原结构组织。
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