Laboratorio de Óptica, Centro de Investigación en Óptica y Nanofísica, Universidad de Murcia, Campus de Espinardo, Murcia, Spain.
Cornea. 2011 Jun;30(6):692-701. doi: 10.1097/ICO.0b013e3182000f94.
To investigate the organization of stromal collagen in healthy ex vivo corneas of different species from second harmonic generation (SHG) microscopy images.
A custom backscattered nonlinear microscope has been used to study the corneal structure of different species: porcine, bovine, rabbit, rat, chicken, and humans. The instrument uses a femtosecond laser for illumination, a scanning unit, and a photon-counting detection device. It also includes a wavefront aberration control module. SHG signals produced by collagen within the cornea were acquired. A motorized stage allowed optical sectioning across the entire corneal thickness. Samples were neither fixed nor stained, and they were fully scanned.
SHG images revealed the microscopic organization of the lamellae of collagen fibers. Despite absorption, for all corneal depths, images could be analyzed. The anterior stroma was similar in all samples, showing interwoven short bands of collagen randomly distributed. The lamellae at the central and posterior stroma were densely packed and often presented longer bundles lying predominantly parallel to the corneal surface with characteristic spatial distributions for each species. In particular, collagen bundles in bovine and porcine corneas were interweaved. In the chick cornea, the stromal arrangement had an orientation changing regularly with depth. In human corneas, lamellae were longer and had similar orientation than their neighbors.
Using a unique wavefront aberration-controlled backscattered nonlinear microscope, changes in corneal morphology as a function of depth were characterized for different species (including humans). This allowed a direct comparison among species, which might help to establish the basis of collagen distribution in animal models or to understand diseased corneas.
从二次谐波（SHG）显微镜图像研究不同物种健康离体角膜的基质胶原组织。
使用定制的背向散射非线性显微镜研究不同物种的角膜结构：猪、牛、兔、大鼠、鸡和人。该仪器使用飞秒激光照明、扫描单元和光子计数检测装置。它还包括一个波前像差控制模块。采集来自角膜内胶原的 SHG 信号。一个机动化平台允许在整个角膜厚度上进行光学切片。样本既未固定也未染色，并进行了全面扫描。
SHG 图像揭示了胶原纤维层的微观组织。尽管存在吸收，但可以分析所有角膜深度的图像。所有样本的前基质相似，显示交织的短胶原带随机分布。中央和后基质的层板排列紧密，通常呈现出与角膜表面平行的较长束，每个物种都有特征性的空间分布。特别是牛和猪角膜中的胶原束交织在一起。在鸡角膜中，基质排列随着深度的变化而规律变化。在人角膜中，层板比其相邻层板更长且取向相似。
使用独特的波前像差控制背向散射非线性显微镜，对不同物种（包括人类）的角膜形态随深度的变化进行了特征描述。这允许在物种之间进行直接比较，这可能有助于在动物模型中建立胶原分布的基础，或理解病变角膜。
