Maeno Sayo, Lewis Philip N, Young Robert D, Oie Yoshinori, Nishida Kohji, Quantock Andrew J
Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Structural Biophysics Research Group, School of Optometry & Vision Sciences, Cardiff University, Cardiff, Wales, UK.
Sci Rep. 2024 Dec 30;14(1):31786. doi: 10.1038/s41598-024-82888-5.
Fuchs' endothelial corneal dystrophy (FECD) is a common sight-threatening condition characterised by pathological changes in the posterior cornea. Here we report observations by light, transmission and volume scanning electron microscopy on changes in the endothelium and matrix associated with the characteristic deformations of Descemet's membrane, termed guttae. Specimens were archived full-thickness human corneal tissue, removed during graft surgery, that had been fixed, stained and embedded by conventional processing methods for examination by transmission electron microscopy more than 40-years previously. Intact archived samples can be extremely valuable where, as with FECD, new cell-based methods of therapy now avoid excision of the full cornea thickness and any tissue excised is inferior for study. Volume electron microscopy, in particular serial block face scanning electron microscopy (SBF SEM), employing backscatter electron detection from resin-embedded specimens, has become an invaluable technique for 3D imaging of biological samples. However, archived specimens are normally considered unsuitable for imaging as conventional processing methods generate low backscatter electron yield. To overcome this for SBF SEM, we subjected epoxy resin-embedded specimens to de-plastination, then applied additional contrasting agents, uranyl acetate and lead acetate, prior to re-embedding. Selected regions of interest in the new resin blocks were examined in a scanning electron microscope equipped for SBF SEM and serial image datasets acquired. Enhanced contrast enabled 3D reconstruction of endothelium and guttae in Descemet's membrane over large tissue volumes.
富克斯角膜内皮营养不良（FECD）是一种常见的致盲性疾病，其特征是角膜后部发生病理变化。在此，我们报告通过光学显微镜、透射电子显微镜和体积扫描电子显微镜观察到的与Descemet膜特征性变形（称为角膜小滴）相关的内皮和基质变化。标本为移植手术中切除的全层人类角膜组织，采用传统处理方法固定、染色和包埋，40多年前用于透射电子显微镜检查。对于FECD等疾病，完整的存档样本可能极具价值，因为现在基于细胞的新治疗方法避免了全层角膜切除，任何切除的组织都不利于研究。体积电子显微镜，特别是连续块面扫描电子显微镜（SBF SEM），利用从树脂包埋标本中检测背散射电子，已成为生物样本三维成像的一项重要技术。然而，存档标本通常被认为不适合成像，因为传统处理方法产生的背散射电子产率较低。为了克服SBF SEM的这一问题，我们对环氧树脂包埋的标本进行去塑料处理，然后在重新包埋之前添加额外的造影剂醋酸铀酰和醋酸铅。在配备SBF SEM的扫描电子显微镜中检查新树脂块中选定的感兴趣区域，并获取系列图像数据集。增强的对比度使得能够在大组织体积上对Descemet膜中的内皮和角膜小滴进行三维重建。
