Faculty of Engineering and Health, University of Applied Science and Arts, 37085, Goettingen, Germany.
Department of Diagnostic and Interventional Radiology, University Medical Center Göttingen, Goettingen, 37075, Germany.
Sci Rep. 2024 Nov 16;14(1):28330. doi: 10.1038/s41598-024-78516-x.
Stiffness-related eye diseases such as keratoconus require comprehensive visualization of the complex morphological matrix changes. The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze unlabeled corneal tissue samples, qualitatively visualizing changes in corneal stiffness. Isolated porcine corneal tissue samples were treated with either NaCl or 0.1% glutaraldehyde (GTA) prior to clearing with benzyl alcohol/benzyl benzoate (BABB) and subsequently scanned with LSFM. After analysis of the LSFM data sets, the samples were embedded in paraffin to validate the results by conventional planar microscopy. In the unlabeled corneal tissue samples the 2D/3D morphology of the entire tissue volume was identified by specific autofluorescence signals. An enhancement of collagen crosslinking was induced by applying GTA to the corneal tissue. Subsequent LSFM scans showed specific morphological changes due to altered autofluorescence signals of the corneal stroma, which were confirmed by conventional histology. Therefore, LSFM analysis of corneal tissue samples allowed label-free 3D autofluorescence assessment of the corneal morphology in its anatomical context. It provides the technical basis for the examination of the pathologically altered cornea and facilitates ophthalmologic examinations of corneal diseases based on the altered tissue stiffness.
与僵硬相关的眼部疾病，如圆锥角膜，需要全面观察复杂的形态基质变化。本研究旨在使用三维（3D）光片荧光显微镜（LSFM）分析未经标记的角膜组织样本，定性地观察角膜硬度的变化。在使用苯甲醇/苯甲酸苄酯（BABB）进行透明化之前，先用 NaCl 或 0.1%戊二醛（GTA）处理分离的猪眼角膜组织样本，然后用 LSFM 进行扫描。对 LSFM 数据集进行分析后，将样本包埋在石蜡中，通过常规平面显微镜验证结果。在未经标记的角膜组织样本中，通过特定的自发荧光信号识别整个组织体积的 2D/3D 形态。将 GTA 应用于角膜组织可诱导胶原交联增强。随后的 LSFM 扫描显示由于角膜基质的自发荧光信号改变而导致的特定形态变化，这通过常规组织学得到证实。因此，LSFM 分析角膜组织样本允许在解剖学背景下对角膜形态进行无标记的 3D 自发荧光评估。它为病理性改变的角膜检查提供了技术基础，并促进了基于组织硬度改变的角膜疾病的眼科检查。
