【摘要】 目的：利用激光共焦显微镜观察正常人的中央角膜组织结构和细胞形态。方法：对54例（68眼）患者进行激光共焦显微镜连续扫描，使用激光共焦显微镜对其角膜中央区进行检查, 拍摄各层角膜图像,观察组织结构和细胞形态,对细胞密度进行计数并分析。结果：角膜组织学结构：上皮细胞较其它细胞的体积小，数量多，随深度增加细胞逐渐变小。Bowman膜较薄，无明显细胞结构，层间有神经走形。基质层的细胞核围成“网眼”状结构，随深度加深，“网眼”变大，排列变松。Descemet膜表现为一个与后基质层和内皮层交织的移行带。内皮细胞为六边形结构，排列规则。角膜细胞密度：在各年龄组间，上皮细胞表层与基底层之间的差异均有统计学意义（P<0.01），前基质层细胞密度与中、后基质层密度之间有统计学差异（P<0.05）。A组和D组的内皮细胞密度有统计学差异（P<0.05）。其余各年龄段的角膜各层细胞密度，随年龄的增加，数差异均无统计学意义。结论：激光共焦显微镜可以在实时、活体和三维空间从细胞水平对角膜各层结构进行无创的定性定量分析,较传统光学共焦显微镜能获得更清晰、更密集的角膜图像,对角膜疾病的基础研究与临床诊断具有很高的价值。
【关键词】 激光；共焦显微镜；角膜
Observation of normal human central corneas using laser confocal microscope
Yao Shi, Yan Wu
Department of Ophthalmology, Nanjing General Hospital, Nanjing Military Command of Chinese PLA, Nanjing 210002, Jiangsu Province, China
Abstract
AIM: To observe central corneal structure and cell morphology of normal human by laser confocal microscope.
METHODS: Fifty four cases (68 eyes) with laser scanning confocal microscope. To inspect central corneal zone by laser confocal microscopy. Photographing all levels of the corneal image and observing the organizational structure and cell morphology. Count cell density and analysis .
RESULTS: Histological structure of the cornea: the epithelial cells have little volumn and large amount than other cells. The basal cells were smaller than the surface ones. The Bowmans layer appeared as an amorphous membrane and could be distinguished by the dence nerve plexus.The keratocyte nuclei surrounded like many "meshes" in stroma layers. The "meshes" became larger and looser in the posterior stroma. Descemets membrance was a displaced zone between the posterior stroma and the endothelial layers. The mosaic of the regular hexagonal endothelial cells was seen. The cell densities: In each group, the statistical difference existed in the density of the surface epithelial cells and the basal ones (P<0.01). The difference in the keratocyte density of the anterior stroma from that of the middle and posterior stroma was statistically significant (P<0.05) . The endothelial cell densities in group A were significantly different from those of group D (P<0.05). No statistically significant differences were found in cell densities in any other corneal layers.
CONCLUSION: Laser confocal microscope can offer noninvasive observation and qualitative and quantitative analysis in realtime and 3D condition to all layers of cornea. The images have high definition and exact depth localization.
KEYWORDS: laser; confocal microscope; cornea
0引言
1955 年Minsky[1] 发明共焦显微镜,1990年，Cavanagh等[2]首次将光学共焦显微镜应用于临床，使对角膜活体组织结构和细胞形态进行显微镜下观察成为可能。在之后的20多年时间内，共焦显微镜因无创、动态的优点在临床和研究领域得到了广泛的应用[3],使医学界角膜的组织学结构有了更深的了解。但是，传统的光学共焦显微镜有其缺点[4]，使用常规光束所得到的图像清晰度不够，分辨率不高，无法对病变进行确切的深度定位，只能以临近组织作为参考等，使其在临床应用上受到限制。近两年来，激光共焦显微镜投入临床使用，使用激光作为光源，提高了分辨率，增加了扫描深度，能更精确的反映角膜组织学结构，并为角膜结构的三维重建提供了可能。本实验利用海德堡激光共焦显微镜对正常人群的角膜进行检查，以了解激光共焦显微镜下的角膜组织学表现。
1对象和方法
1.1对象 所有受检者均选自2006 03/11在南京军区南京总医院眼科就诊患者或常规体检者。选择54例正常人(共68眼，其中右眼40眼，左眼28眼),性别不限，年龄12～71岁,其中A组(<20岁)10例（16眼），B组(20～40岁)18例（22眼），C组(40～60岁)18例（20眼），D组(>60岁)8例（10眼）。所有受检者均为健康人，无全身慢性疾病或急性重症疾病。所有受检眼均无外伤、感染及配戴接触镜史,经裂隙灯显微镜检查角膜透明,检眼镜检查无异常,作为正常健康眼入选本研究。选用激光共焦显微镜检查，激光共焦显微镜(德国海德堡公司生产)由海德堡视网膜激光断层扫描系统Ⅱ代(HRT Ⅱ)和Rostock角膜模块组件(RCM)组成。后者主要包括有: Rostock 角膜显微适配器, CCD 摄像头,Rostock角膜模块操作软件(包括自动图像分析和细胞计数)等。其中激光波长为670nm。观察视野为380μm ×380μm, 放大倍数为800倍, 分辨率为1μm。
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