Lee Jungbin, Kim Seonghan, Yoon Chang Ho, Kim Myoung Joon, Kim Ki Hean
Department of Mechanical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-gu, Pohang, Gyeongbuk 37673, South Korea.
These authors contributed equally.
Biomed Opt Express. 2020 Aug 6;11(9):4890-4900. doi: 10.1364/BOE.401896. eCollection 2020 Sep 1.
Goblet cells (GCs) in the conjunctiva are specialized epithelial cells producing mucins on the ocular surface. GCs play important roles in maintaining homeostasis of the ocular surface, and GC dysfunction is associated with various complications including dry eye diseases. Current GC examination methods, which are conjunctival impression cytology and confocal reflection microscopy, have limitations for routine examination. Fluorescence microscopy using moxifloxacin was recently introduced as a non-invasive and high-contrast imaging method, but further development is needed to be used for GC examination. Here we developed a non-invasive high-speed high-contrast GC imaging method, called moxifloxacin based axially swept wide-field fluorescence microscopy (MBAS-WFFM). This method acquired multiple fluorescence images with the axial sweeping of the focal plane to capture moxifloxacin labeled GCs on the tilted conjunctival surface in focus and generated all-in-focus images by combining the acquired images. The imaging field of view and imaging speed were increased to 1.6 mm × 1.6 mm and 30 fps. An image processing method was developed for the analysis of GC density. MBAS-WFFM was applied to alkali burn mouse models and detected GC damage and recovery via longitudinal imaging. MBAS-WFFM could assess the status of GCs rapidly and non-invasively. We anticipate MBAS-WFFM to be a starting point for non-invasive GC examination and the diagnosis of GC associated diseases. For example, MBAS-WFFM could be used to classify dry eye diseases into detail categories for effective treatment.
结膜中的杯状细胞（GCs）是在眼表产生黏蛋白的特化上皮细胞。杯状细胞在维持眼表稳态中发挥重要作用，而杯状细胞功能障碍与包括干眼疾病在内的各种并发症相关。当前的杯状细胞检测方法，即结膜印迹细胞学和共焦反射显微镜检查，在常规检查中存在局限性。最近引入的使用莫西沙星的荧光显微镜检查作为一种非侵入性且高对比度的成像方法，但要用于杯状细胞检测还需要进一步发展。在此，我们开发了一种非侵入性高速高对比度杯状细胞成像方法，称为基于莫西沙星的轴向扫描宽视野荧光显微镜检查（MBAS-WFFM）。该方法通过焦平面的轴向扫描获取多个荧光图像，以捕捉聚焦在倾斜结膜表面上的莫西沙星标记的杯状细胞，并通过组合所获取的图像生成全聚焦图像。成像视野和成像速度分别提高到1.6毫米×1.6毫米和30帧/秒。开发了一种图像处理方法用于分析杯状细胞密度。MBAS-WFFM应用于碱烧伤小鼠模型，并通过纵向成像检测杯状细胞损伤和恢复情况。MBAS-WFFM能够快速且非侵入性地评估杯状细胞的状态。我们期望MBAS-WFFM成为非侵入性杯状细胞检测及杯状细胞相关疾病诊断的起点。例如，MBAS-WFFM可用于将干眼疾病细分为不同类别以进行有效治疗。
