Ammar David A, Lei Tim C, Masihzadeh Omid, Gibson Emily A, Kahook Malik Y
Department of Ophthalmology, University of Colorado Denver, Aurora, CO 80045, USA.
Mol Vis. 2011 Feb 24;17:583-90.
To image the native (unfixed) human trabecular meshwork (TM) through the overlying sclera using a non-invasive, non-destructive technique.
Two-photon microscopic (2PM) methods, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to image through the sclera of a human cadaver eye into the TM region. Multiple images were analyzed along the tissue axis (z-axis) to generate a three-dimensional (3D) model of the region. The tissue was subsequently fixed, paraffin embedded, and histological sections were photographed for comparison to the 2PM images.
3D analysis of multiple 2PM SHG images revealed an open region deep within the TM consistent with the location of Schlemm's canal (SC). Images of the scleral spur and surrounding tissues were also obtained. The SC, TM, scleral spur, and surrounding tissue images obtained with 2PM matched with histologically stained sections of the same tissue.
2PM imaging of the outflow system of the human eye documented collagenous structures solely from inherent optical properties. 2PM successfully imaged through the sclera into the SC/TM without the need for fixation, embedding, or histological processing. This 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.
使用非侵入性、非破坏性技术通过覆盖的巩膜对天然（未固定）的人小梁网（TM）进行成像。
采用双光子显微镜（2PM）方法，包括双光子自发荧光（2PAF）和二次谐波产生（SHG），通过人尸体眼睛的巩膜对TM区域进行成像。沿组织轴（z轴）分析多个图像，以生成该区域的三维（3D）模型。随后将组织固定、石蜡包埋，并拍摄组织学切片以与2PM图像进行比较。
对多个2PM SHG图像进行3D分析，发现TM深处有一个开放区域，与施莱姆管（SC）的位置一致。还获得了巩膜突及周围组织的图像。2PM获得的SC、TM、巩膜突和周围组织图像与同一组织的组织学染色切片相匹配。
人眼流出系统的2PM成像仅根据固有光学特性记录了胶原结构。2PM成功地通过巩膜对SC/TM进行了成像，无需固定、包埋或组织学处理。这项工作表明，2PM成像有潜力作为评估人眼房水流出区域的一种新方法，值得进一步探索。
