共焦显微镜与OrbscanⅡ角膜地形图系统在角膜厚度测量中的差异

最近出现的Confoecan 2．0共焦显微镜是一种新型、无创伤性角膜影像学检查仪器，能在活体上对角膜从四维(三维空间和时间)水平进行扫描成像，并能通过Z-Scan系统测量角膜各层组织厚度，这为角膜厚度的测量提供了新的检测手段。为评价共焦显微镜在角膜厚度测量方面的价值，本研究利用该技术测量了62例85眼正常和病变角膜中央厚度，并与Orbscan Ⅱ裂隙扫描角膜地形图系统检测结果进行比较，现将结果报告如下。     

兔角膜中央厚度与角膜上皮厚度的相关性

目的:探讨兔角膜上皮厚度在A型超声下与共焦下测量结果的相关性,及表面麻醉药对角膜上皮的作用。方法:将20只兔随机分为两组,分析左右眼之间的统计学差别;再将左眼做为参照组,右眼滴用表面麻醉药,分析表面麻醉药对角膜上皮的影响;最后将参照组用同样方法处理后,将40眼的角膜上皮在A超下与共焦下的测量值进行统计学分析。结果:兔眼别与角膜中央厚度的测量没有显著差别;倍诺喜表面麻醉液会使角膜上皮厚度增加;A型超声下与生物共聚焦显微镜下测量的角膜上皮厚度呈正相关。结论:倍诺喜可使角膜上皮松解;A超下测量角膜上皮越厚,其共焦下测量角膜上皮也越厚。     

中国人正常角膜的共焦显微镜检查

摘要目的探讨正常人共焦显微镜下角膜各层细胞的活体细胞形态学特征及密度.方法对28例(年龄36.6士14.5岁,范围19～68岁)41眼(男14眼,女27眼)正常人中央部角膜进行活体共焦显微镜检查,描述角膜各层结构的图象特点,并分析角膜各层细胞的密度与年龄的相关性.结果上皮表层细胞排列疏松,边界清楚,核反光较强;基底层细胞排列紧密,胞质和核反光弱,偶尔可见点状细胞核,细胞平均密度为5724.41士562.5个/mm2.Bowman's膜为无一定形状的膜状物,仅可分辨的结构是上皮下的念珠状神经纤维.基质层中可见相对较暗的背景下明亮的角膜细胞核.前基质内有放射状分布的神经纤维.前基质层角膜细胞平均密度为1099.10士164.90个/mm     

正常中国汉族儿童角膜的共焦显微镜研究

目的采用角膜激光共焦显微镜对正常中国汉族儿童活体角膜各层组织结构进行观察,分析正常中国汉族儿童角膜各层细胞的活体细胞形态学特征及密度。方法 49例6～13岁正常中国汉族儿童中央部角膜进行活体共焦显微镜检查,研究角膜各层结构的图象特点,并分析角膜各层细胞的密度,与成年人进行对比。结果中国汉族儿童正常角膜上皮表层细胞排列疏松,边界清楚,胞体较大,核反光较强,又称翼状细胞;基底层细胞排列紧密,呈规则的蜂巢状,胞质反光弱,正常情况下未见细胞核,细胞平均密度为5947.58±769.3个/mm2。前弹力膜为无细胞结构的膜状物,可见串珠状的神经纤维走行。基质层中可见在相对较暗的背景下明亮的角膜基质细胞核。角膜基质内有神经纤维分布。前基质层角膜细胞平均密度为1621.12±123.26个/mm2,后基质层角膜细胞平均密度为984.71±113.17个/mm2,前后基质层细胞密度有显著性差别（P〈0.05）。后弹力层中无细胞结构,为均一无结构组织。内皮细胞层表现为规则的六边形结构,细胞结构清晰,细胞平均密度为3682.38±251.87个/mm2。左右眼及男女之间角膜各层细胞密度的差异无统计学意义（P=0.341～0.611和P=0.194～0.855）。各层角膜细胞的面积和密度与性别和眼别无差异。中国汉族儿童角膜各层细胞的密度较正常成人高。结论角膜激光共焦显微镜能够在实时、活体和三维空间从细胞水平清晰观察中国汉族儿童活体角膜各层细胞的形态结构,可以对角膜各层结构进行定性和定量分析,在儿童角膜病的研究和临床诊断方面是十分有用的工具。     

不同光源共焦显微镜的活体正常角膜图像研究

目的 分别采用卤素灯光源共焦显微镜和激光光源共焦显微镜对中国正常成人活体角膜各层组织结构进行观察,对照研究其图像特征.方法 对35例(70只眼)中国成人(年龄18～55岁)中央部角膜分别采用卤素灯光源共焦显微镜和激光光源共焦显微镜进行检查,研究角膜各层结构的图像特点,并进行对比.结果 共焦显微镜检查的35例(70只眼)中,67只眼95.7％成功获得了角膜上皮翼状细胞层的图像,70只眼100％成功获得角膜上皮基底细胞层、前弹力层、前基质层、后基质层及角膜内皮细胞层的图像.结论 卤素灯光源共焦显微镜的放大倍数是1000倍,细胞放大倍数大,近似于初级电镜的放大倍数,但卤素灯光源的组织穿透力较弱,光源易衰减,所得的角膜各层细胞图像较激光角膜共焦显微镜的略微模糊,所得角膜各层图像总体颜色偏灰、结构偏模糊.激光角膜共焦显微镜的放大倍数是800倍,较卤素灯光源的共焦显微镜略小,但激光光源的组织穿透力强,所得角膜各层细胞图像较卤素灯光源的更清晰,而且在特定角度可获得非常类似角膜组织病理学切片的图像.     

圆锥角膜各期的共焦显微镜表现

目的评价共焦显微镜在圆锥角膜临床研究中的应用价值。方法应用共焦显微镜观察圆锥角膜患者32例（48眼）及正常对照组17例（28眼），分别比较早、中、晚期圆锥角膜与正常对照组的图像特点。结果早期圆锥角膜出现激活状态的角膜细胞、浅基质层的细小皱褶、深基质层的暗纹、部分内皮细胞异形性明显，中、晚期圆锥角膜出现角膜上皮细胞拉伸、细胞核皱缩；基质细胞排列紊乱、基质层暗纹；而对照组未发现上述表现。各期圆锥角膜的角膜基质层厚度、不同深度角膜基质细胞密度、内皮细胞密度与对照组之间差异有统计学意义（P〈0．05）。结论共焦显微镜对早期圆锥角膜的发现以及圆锥角膜病理发展的研究具有重要的临床价值。     

人体角膜内皮细胞的共焦显微镜研究

目的应用共焦显微镜观察活体角膜内皮细胞的形态学特征。方法应用共焦显微镜观察正常人（20例）、角膜炎（12例）、角膜移植术后（19例）、葡萄膜炎（5例）、大疱性角膜病变（3例）、角膜白斑（6例）、角膜营养不良（7例）、青光眼（10例）、圆锥角膜（17例）等患者的角膜内皮,分析所得活体角膜内皮图象,总结其形态学特征。结果活体共焦显微镜下角膜内皮的图象主要有8种：①完全正常角膜内皮。②水肿角膜内皮。③失代偿角膜内皮。④“激活”角膜内皮。⑤排斥角膜内皮。⑥有KP角膜内皮。⑦有“疣状物”的角膜内皮。⑧“双核”角膜内皮细胞。结论共焦显微镜能对活体角膜内皮进行实时、无创、准确以及可重复、可比较的观察。     

2型糖尿病患者角膜基质细胞形态学特征的共焦显微镜观察

目的 探讨2型糖尿病患者在共焦显微镜下角膜基质细胞形态学特征。方法 应用Confoscan3．0共焦显微镜对120例（146眼）2型糖尿病患者和36例（36眼）同年龄对照组的中央角膜进行检查，依据双目间接眼底镜和荧光素眼底血管造影（FFA）检查的结果，将糖尿病患者分为3组：糖尿病无眼底改变（NDR）组、非增生型糖尿病视网膜病变（NPDR）组、增生型糖尿病视网膜病变（PDR）组，记录角膜基质细胞和基质神经的图像，并对其进行分析。结果 与对照组比较，糖尿病各组角膜的中基质细胞密度均较对照组降低，而前基质细胞的密度只在PDR组有明显的减少，后基质细胞在糖尿病视网膜病变组均明显降低。角膜基质中形态异常的神经纤维在糖尿病组中出现的概率也明显高于对照组，两组间有显著统计学意义（，=36．450，P=0．000）。结论 共焦显微镜是一种有效无创的角膜检查方法；2型糖尿病患者角膜基质细胞密度减少，基质细胞、基质神经均存在形态学的异常。     

激光共焦显微镜对正常人眼角膜缘和中央角膜的观察

目的应用激光共焦显微镜对正常人眼角膜缘和中央角膜的组织结构与细胞形态的观察和分析。方法选择15名正常人的28只眼接受常规裂隙灯显微镜和检眼镜检查后,作为正常健康眼入选本研究。使用激光共焦显微镜对其上、下方角膜缘和角膜中央区进行检查,各层角膜图像均被记录,观察组织结构和细胞形态,对细胞密度进行计数并分析。结果所获角膜缘和角膜中央各层平面图像(x,y轴)及纵向断层图像(z轴)均非常清晰,同时获取动态录像。上、下方角膜缘均呈现Vogt栅栏状结构,并动态观察到血细胞在血管内的流动。表层上皮细胞排列非常疏松,边界明亮,胞体发暗,上方和下方角膜缘表层上皮细胞平均密度分别为(812±297)个/mm2和(785±263)个/mm2,二者比较差异无统计学意义(P>0.05)。上皮下可见明亮的Langerhans细胞,形态不规则,呈树枝状,上方和下方角膜缘Langerhans细胞平均密度分别为(288±102)个/mm2和(254±127)/mm2,二者比较差异无统计学意义(P>0.05)。角膜中央表层上皮细胞排列疏松,边界发亮,胞体发暗,细胞平均密度为(1098±315)个/mm2,多于上方和下方角膜缘(P<0.05)。基底上皮细胞排列紧密。上皮下和前基质层可见反光强烈的神经纤维丛,旁边偶见明亮的Langerhans细胞,形态不规则,细胞密度难以计算。浅层的神经纤维细小、弯曲度大、多小分支,深层的神经纤维粗大、弯曲度小、少见分支。基质层暗背景下散在分布细长的基质细胞,边缘欠清,细胞核明亮呈纺锤形。内皮细胞为排列整齐的六边形细胞,胞体发亮,边界发暗。角膜中央全层、基质层、上皮层厚度分别为(543.0±62.9)、(462·0±69.5)、(59.9±11.2)μm。结论激光共焦显微镜不仅可以对角膜进行无创的、实时的、活体的检查,而且与传统的光学共焦显微镜相比,具有高清晰度、确切的深度定位、时间动态观察、纵向断层扫描等优势,更可提供理想的角膜缘图像,对角膜疾病尤其是角膜缘疾病的基础研究与临床诊断将更有价值。     

角膜厚度的共焦显微镜测量分析

目的　研究共焦显微镜测量角膜厚度的准确性和稳定性。方法　非随机性对照临床试验 ,用裂隙扫描共焦显微镜和 A超测厚仪分别测量 2 0人的 2 7个正常角膜中央厚度。结果　中央角膜厚度测量平均值 (± SD)裂隙扫描共焦显微镜为 (5 2 9.6± 33.9)μm ,A超测厚仪为 (5 36 .7± 38.2 )μm,两者差别有显著意义 (P <0 .0 0 1,配对资料 t检验 ) ,两方法差值所在范围 (± SD)是 (7.0± 7.1)μm ;各眼测量值间标准差的平均值共焦显微镜为 17.8μm ,A超测厚仪为 1.7μm ,共焦显微镜的变异度较 A超测厚仪大。结论　裂隙扫描共焦显微镜可以测量角膜厚度 ,但其测量值与 A超测厚仪所测值有差别 ,且测量变异度较 A超测厚仪大 ,稳定性相对差。     

In vivo corneal confocal microscopy in keratoconus

PURPOSE: To evaluate the corneas of keratoconic subjects using in vivo confocal microscopy. METHODS: Slit scanning confocal microscopy was used to evaluate the central cornea of one eye of each of 29 keratoconic subjects (mean age 31 +/- 10 years; range 16-49 years). Quantitative aspects of corneal morphology were compared against data from control subjects. RESULTS: Compared with normal control corneas, epithelial wing cell nuclei were larger (p < 0.0001) and epithelial basal cell diameter was larger (p < 0.05) in the keratoconic cornea. Many of the keratoconic corneas investigated showed increased levels of stromal haze and reflectivity, which appeared to be related to the presence of apical scarring on slit lamp examination. A grading scale was devised to quantify the levels of haze. This scale was shown to provide a measure of the level of scarring present. The anterior keratocyte density (AKD) and posterior keratocyte density were 19% lower (p < 0.0001) and 10% lower (p = 0.004) than in controls, respectively. The reduction in AKD was significantly associated with three factors: a history of atopy, eye rubbing and the presence of corneal staining. The mean endothelial cell density in keratoconus was 6% greater than that of normal controls (p = 0.05). The level of endothelial polymegethism was shown not to be different between keratoconic subjects and matched controls (paired t-test: t = 1.82, p = 0.08). CONCLUSIONS: Confocal microscopy demonstrates significant quantitative alterations of corneal morphology in keratoconus.     

Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo

PURPOSE: To quantify keratocyte density according to stromal region and subject age and to measure the thickness of the normal human cornea and its layers in vivo. METHODS: Seventy normal corneas of 70 subjects were examined by confocal microscopy (contact lens wearers were excluded). Ages of subjects ranged from 12 to 80 years, with 10 subjects per decade. Images were recorded by continuously focusing the optical section through the full-thickness central cornea. Two independent human observers manually identified bright objects (keratocyte nuclei) against a dark background to quantify keratocyte density. This method was validated histologically in three human corneas. Thickness measurements were obtained by plotting mean reflected light intensity in images against corneal depth, and calculating distances between intensity peaks that corresponded to corneal layers. RESULTS: Full-thickness central keratocyte density was 20,522 +/- 2,981 cells/mm(3) (mean +/- SD, n = 69). The number of keratocytes in a full-thickness column of central stroma, which had a cross-sectional area of 1 mm(2), was 9624 +/- 1385 cells. Keratocyte density was highest in the anterior 10% of the stroma. Full-thickness keratocyte density was correlated with age (r = -0.62, P < 0.001), decreasing 0.45% per year. Central corneal thickness was 563.0 +/- 31.1 microm (mean +/- SD) and central epithelial thickness was 48.6 +/- 5.1 microm. CONCLUSIONS: This is the first study to quantify regional keratocyte density comprehensively in vivo across a broad age range of normal human subjects. The method was acceptable to both subject and observer, and may prove useful for quantifying keratocyte density in patients with corneal disorders or after corneal surgery.     

New perspectives on keratoconus as revealed by corneal confocal microscopy

Confocal microscopy (CM) of keratoconus is reviewed. In the Manchester Keratoconus Study (MKS), slit scanning CM was used to evaluate 29 keratoconic patients and light microscopy (LM) was performed on two of the keratoconic corneas post‐keratoplasty. The findings of the MKS are compared with other CM studies. Consideration of the differences between studies of cell counts is confounded by the use of different experimental controls. A consensus exists among studies with respect to qualitative observations. The epithelium appears more abnormal with increasing severity of keratoconus. In severe disease, the superficial epithelial cells are elongated and spindle shaped, epithelial wing cell nuclei are larger and more irregularly spaced and basal epithelial cells are flattened. Bowman’s layer is disrupted and split in the region of the cone and intermixed with epithelial cells and stromal keratocytes. Stromal haze and hyper‐reflectivity observed with CM correspond with apical scarring seen with the slitlamp biomicroscope (SLB). Hyper‐reflective keratocyte nuclei are thought to indicate the presence of fibroblastic cells. Increased haze detected with CM is found with LM to be due to fibroblastic accumulation and irregular collagen fibres. Dark stromal bands observed with CM correlate with the appearance of Vogt’s striae with SLB. Desçemet’s membrane appears normal with both CM and LM. Some evidence of endothelial cell elongation is observed with CM. The application of CM to ophthalmic practice has facilitated a greater understanding of medical and surgical approaches that are used to treat keratoconus. This review offers new perspectives on keratoconus and provides a framework, against which tissue changes in this visually debilitating condition can be studied in a clinical context in vivo using CM.     

LASIK术后远期角膜基质细胞及内皮细胞的共焦显微镜观察

目的应用共焦显微镜在细胞水平观察术后远期角膜基质细胞及内皮细胞变化。方法选择我院门诊行LASIK手术患者68例68眼,根据术前球镜度数,分为高度近视眼组(36例36眼)和低、中度近视眼组(32例32眼),术后随访6～44月,同时选择门诊行屈光检查的近视患者61例122眼作为对照组,对所有患者行共焦显微镜检查,并对结果进行统计学分析,比较LASIK手术对角膜基质及内皮细胞的影响。结果LASIK术后,高度近视眼组角膜前、后基质角膜细胞密度均减少,内皮细胞密度下降,内皮细胞多形性百分比增加,异型性百分比下降,与对照组之间差异有统计学意义(t=0.4077,P=0.000)。LASIK术后界面下基质角膜细胞密度明显减少(562个·mm-2±86个·mm-2),“无细胞区”长期存在;含“无细胞区”的基质深度与激光能量、削切深度、手术时间、年龄均无相关性。结论术后远期,适量激光切削对低、中度近视眼的角膜深层组织影响较小,对高度近视眼影响较大,所以高度近视眼应慎行LASIK手术。     

In vivo confocal microscopy of pre-Descemet's membrane corneal dystrophy

Pre-Descemet's membrane corneal dystrophy is clinically characterized by the presence of numerous tiny pleomorphic opacities located in the deep stroma immediately anterior to Descemet's membrane. A 35-year-old man, clinically diagnosed with pre-Descemet's corneal dystrophy, was examined by in vivo slit scan confocal microscopy. The pleomorphic structures containing dense hyperreflective inclusions in the posterior stroma were revealed in vivo. To the best of the authors' knowledge, it is consistent with the result of the previous histological study, but different from other reports using in vivo confocal microscopy.     

Imaging posterior polymorphous corneal dystrophy by in vivo confocal microscopy

To identify features of posterior polymorphous dystrophy (PPMD) by in vivo confocal microscopy, the corneas of a female patient with PPMD were examined using slit‐lamp biomicroscopy and slit‐scanning in vivo confocal microscopy. Characteristic endothelial vesicular and band lesions were seen clinically and easily identified using in vivo confocal microscopy. However, endothelial pleomorphism, an increased density and reflectance of posterior stromal keratocytes, and prominence of corneal nerves were also delineated. In vivo confocal microscopy enhances clinicopathological diagnosis and follow up of corneal dystrophies with subtle clinical presentations, such as PPMD.     

Decreased keratocyte density and central corneal thickness in primary open-angle glaucoma patients undergoing treatment with topical prostaglandin analogues

Purpose:

To evaluate whether prostaglandin (PG) analogue use is associated with alterations in keratocyte density and central corneal thickness (CCT) in subjects with primary open-angle glaucoma (POAG).

Materials and Methods:

Thirty-five POAG patients treated with PG analogues for >2 years and 35 control subjects without glaucoma were included in this cross-sectional study. All subjects were underwent CCT measurements using ultrasound pachymetry. Keratocyte densities of each stromal layer were determined by in vivo confocal microscopy. Student''s t-test and Chi-square test were used for statistical evaluations. Correlations between keratocyte densities and CCT were analyzed using Pearson''s correlation analysis.

Results:

Keratocyte densities in each stromal layer were significantly lower in glaucoma patients receiving PG analogues as compared to those of controls (P < 0.001). The mean CCT was also lower in glaucoma patients (515.2 ± 18.8 μ) than control subjects (549.6 ± 21.1 μ, P < 0.001). A positive correlation between keratocyte densities in each stromal layer and CCT was observed in POAG patients.

Conclusions:

Long-term administration of topical PG analogues may adversely influence keratocyte densities and CCT. Further prospective studies are required clarify the relationship between PG analogues and their effects on the cornea.     

Clinical corneal confocal microscopy

Confocal microscopy allows non-invasive in vivo imaging of the ocular surface. Its unique physical properties enable microscopic examination of all layers of the cornea and have been used to investigate numerous corneal diseases: epithelial changes, numerous stromal degenerative or dystrophic diseases, endothelial pathologies, corneal deposits, infections, and traumatic lesions. It offers a new approach to study the physiological reactions of the cornea to different stimuli and the pathophysiologic events leading to corneal dysfunction in certain diseases. Confocal microscopy proves to be a powerful diagnostic tool and is especially of value in certain corneal diseases by allowing straightforward and non-invasive recognition of the pathologic conditions.     

Fluorescein enhanced confocal microscopy in vivo for the evaluation of corneal epithelium

BACKGROUND: In vivo confocal microscopy is being increasingly used to evaluate corneal disorders. This study aimed to evaluate whether topical fluorescein application prior to in vivo confocal microscopy had any effect on the imaging characteristics of the corneal epithelium. METHODS: Confocal microscopy in vivo with Confoscan 3.0 (Vigonza, Italy) was performed on 22 corneas of 22 study patients before and 1 min after application of unpreserved 2% fluorescein solution on the cornea. Ten patients with normal corneal findings served as control. The quality of superficial epithelial (SE) visualization, the SE density, the image intensity and the presence of hyperreflective SE cells were evaluated. RESULTS: Eleven patients had normal corneal epithelium (group 1), six had keratoconus (group 2) and five had overt epitheliopathy (group 3). The visibility of SE layers of all subjects appeared to be enhanced in the post-fluorescein images. There was a significant difference between the mean pre-fluorescein (857.2 + 319.4 cells/mm(2)) and post-fluorescein (1378.0 +/- 292.1 cells/mm(2)) SE densities of all study subjects (paired t-test, P < 0.001). Hyperreflective SE cells were more common in the post-fluorescein images of groups 2 (83.3%), and group 3 (80%) compared with group 1 patients (36.4%). These differences were not significant between the two consecutive corneal images of control patients who received unpreserved artificial tears instead of fluorescein. CONCLUSIONS: Topical fluorescein application appears to enhance the visualization of the corneal epithelium. This technique may enable better evaluation of the corneal epithelium quantitatively and qualitatively in both normal subjects and patients with epithelial involvement.     

In vivo confocal microscopy of human cornea covered with human amniotic membrane

Purpose  Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Methods  Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22–24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Results  Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. Conclusions  We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.