In vivo confocal microscopy of the bulbar conjunctiva

Background:  The aim of this work is to develop a more complete qualitative and quantitative understanding of the in vivo histology of the human bulbar conjunctiva.
Methods:  Laser scanning confocal microscopy (LSCM) was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 healthy human volunteer subjects.
Results:  The superficial epithelial layer of the bulbar conjunctiva is seen as a mass of small cell nuclei. Cell borders are sometimes visible. The light grey borders of basal epithelial cells are clearly visible, but nuclei can not be seen. The conjunctival stroma is comprised of a dense meshwork of white fibres, through which traverse blood vessels containing cellular elements. Orifices at the epithelial surface may represent goblet cells that have opened and expelled their contents. Goblet cells are also observed in the deeper epithelial layers, as well as conjunctival microcysts and mature forms of Langerhans cells. The bulbar conjunctiva has a mean thickness of 32.9 ± 1.1 µm, and a superficial and basal epithelial cell density of 2212 ± 782 and 2368 ± 741 cells/mm², respectively. Overall goblet and mature Langerhans cell densities are 111 ± 58 and 23 ± 25 cells/mm², respectively.
Conclusions:  LSCM is a powerful technique for studying the human bulbar conjunctiva in vivo and quantifying key aspects of cell morphology. The observations presented here may serve as a useful marker against which changes in conjunctival morphology due to disease, surgery, drug therapy or contact lens wear can be assessed.     

正常人球结膜激光共焦显微镜下的形态学分析

背景激光共焦显做镜可从细胞水平对正常和病变组织进行活体观察,在眼表系统中的应用越来越广泛,但国内对正常球结膜的活体激光共焦显微镜检测结果报道较少。目的利用活体激光共焦显微镜观察分析正常球结膜的形态。方法应用德国海德堡激光共焦显微镜（HRT3）对健康志愿者15例21眼的上方、鼻下方、鼻侧、颞侧球结膜进行扫描,分辨率为1μm,激光波长为670nm,观察视野为400μm×400Ixm。分析球结膜细胞的形态,对球结膜上皮细胞进行计数,并计算杯状细胞和树突状细胞（DE）的密度。结果球结膜表层上皮细胞胞核小,边界模糊。基底上皮细胞边界清晰但胞核不可见。类似杯状细胞的细胞呈卵圆形,胞体较大,细胞内充满高反光均一明亮颗粒,成群或散在分布,上皮表面的孔洞结构可能是已经分泌排出内容物的杯状细胞。DE呈高反光颗粒,伴树枝状突起,分布于球结膜上皮各层。球结膜基质由密集的白色网状纤维构成,其问可见含有细胞成分的血管穿过。球结膜表层上皮细胞密度和基底上皮细胞密度分别为（2556±692）mm^2。和（2985±376）mm^2,杯状细胞和DC的密度分别为（77±39）mm^2。和（26±35）mm^2。经统计学分析各方位上皮细胞密度差异无统计学意义（P=0．204,P=0．130）,各方位杯状细胞和DC的差异有统计学意义（P=0．001,P=0．000）。结论激光共焦显微镜是活体研究人类球结膜形态的有用工具,有助于眼表疾病的诊断和治疗。     

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     

In vivo confocal microscopy of meibomian glands and palpebral conjunctiva in vernal keratoconjunctivitis

Purpose:

To investigate the correlations between conjunctival inflammatory status and meibomian gland (MG) morphology in vernal keratoconjunctivitis (VKC) patients by using in vivo confocal microscopy (CM).

Materials and Methods:

Nineteen VKC patients (7 limbal, 7 tarsal, and 5 mixed forms) and 16 normal volunteers (controls) were enrolled. All subjects underwent CM scanning to obtain the images of upper palpebral conjunctiva and MGs. Inflammatory cell (IC) density in palpebral conjunctival epithelial and stromal layers, Langerhans cell (LC) density at lid margins and the stroma adjacent to the MG, and MG acinar unit density (MGAUD) were recorded. The longest and shortest diameters of MG acinar were measured. The Kruskal-Wallis test was used to compare the parameter differences whereas the Spearman''s rank correlation analysis was applied to determine their correlations.

Results:

Among all groups, no significant statistical differences were found in epithelial and stromal IC densities, mean values of MG acinar unit densities, or longest and shortest diameters. Both LC parameters in the tarsal-mixed groups were significantly higher than those in the limbal and control groups. All LC densities of VKC patients showed a positive correlation with MGAUD and shortest diameter.

Conclusions:

In VKC patients, the conjunctival inflammatory status could be associated with the MG status. In vivo CM is a noninvasive, efficient tool in the assessment of MG status and ocular surface.     

活体共焦显微镜下观察正常人眼球结膜组织结构

目的 探讨应用活体激光共焦显微镜观察正常人球结膜的组织结构.方法 横断而研究.2008年2月至7月选择50名无眼部外伤、感染及配戴接触镜史,且裂隙灯显微镜检查无异常的正常人的50只眼作为研究对象.使用激光共焦显微镜对其上方、下方、鼻侧及颞侧球结膜进行检查,各层图像均被记录,分析球结膜各层形态,并对上皮细胞、杯状细胞及树突状细胞密度进行计数.运用单因素方差分析对各层上皮细胞密度和各方位杯状细胞密度进行统计学分析,运用最小显著性差异分析组间差异.结果 球结膜上皮浅表层细胞体积较大,排列松散,胞核呈低反光,细胞平均密度为(1643±206)个/mm2.中间层细胞呈卵圆形,体积较小,排列紧密,胞核呈点状高反光,细胞平均密度为(4693±228)个/mm2.基底层细胞呈多边形,排列规整,有清晰而高亮的细胞边界,细胞平均密度为(4420±230)个/mm2.经统计学分析3种上皮细胞密度存在显著件差异(F=1160.312,P=0.000).类似杯状细胞的细胞呈圆形,胞体较大,胞内充满透亮颗粒,成团或散在分布,细胞的平均密度为(432±72)个/mm2.树突状细胞呈高反光颗粒,伴树枝状突起,分散于结膜各层,细胞的平均密度为(22±25)个/mm2.在结膜上皮与固有层之间,存在一层致密的高反光的基底膜.球结膜固有层由高度血管化的疏松结缔组织组成,可见大量小规则的条状纤维或大片的网状纤维,弥散分布小圆周高亮的游走细胞,还可清晰观察到血管中血液的流动.结论 活体激光共焦显微镜是研究球结膜组织结构的一种有效上具,为眼表疾病的临床诊断提供了快速而无创的检查手段.     

In vivo confocal microscopy in blepharitis

BACKGROUND: Dysfunction of the meibomian glands with inflammation and obstruction has been suggested to be an important factor in the pathogenesis of chronic blepharitis. Few objective tests are, however, available to examine the meibomian glands directly. PATIENTS AND METHODS: Nineteen patients with anterior blepharitis, meibomitis, meibomian gland dysfunction or severe keratoconjunctivitis sicca associated with blepharitis as well as 10 patients with normal lid margins were examined with the HRTII/RCM in vivo confocal microscope. Scans of the tear film, the tarsal conjunctiva, the hair follicles and the meibomian glands were analysed by a masked observer. RESULTS: Patients with normal lid margins exhibited a minimal round cell infiltrate in the tarsal conjunctival epithelium and largely normal ducts of the meibomian glands lined with a multilayered epithelium as well as normal gland acini. In patients with anterior blepharitis, blepharitis associated with autoimmune peripheral ulcerative keratitis and blepharitis in the context of severe dry eye, confocal microscopy disclosed normal meibomian glands. In 12 patients with blepharitis/meibomitis or meibomian gland dysfunction, profound pathology was visible with dilatation and obstruction of the meibomian gland ducts. In 15 of 19 patients with blepharitis/meibomitis, but not in meibomian gland dysfunction, an intense inflammation was observed in the tarsal conjunctival epithelium and stroma. In one patient, demodex folliculorum was evident in vivo. In patients with normal lid margins as well as in patients with blepharitis, hair follicles appeared within normal limits. CONCLUSIONS: In vivo confocal microscopy allowed the examination of the tear film, the tarsal conjunctiva, the lid margin including the lash follicles and the meibomian glands. In patients with meibomian gland disease pathological changes could be visualised and documented objectively. The presence of an inflammatory infiltrate permitted us to differentiate between meibomitis and meibomian gland dysfunction. Changes of the lash follicles do not seem to play an important role in blepharitis. Thus, in vivo confocal microscopy represents an objective technique in the classification and follow-up of patients with blepharitis.     

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。     

应用活体共焦显微镜观察翼状胬肉组织结构

目的 应用活体共焦显微镜(德国海德堡视网膜激光断层扫描系统Ⅱ代与Rostock角膜模 块组成)对人眼翼状胬肉进行活体组织成像分析.方法 系列病例分析.采取2010年3月门诊就诊的翼 状胬肉患者中选择11例14只眼,应用活体共焦显微镜对翼状胬肉头部、颈部和体部进行检查,记录各部分图像,分析其组织形态特点.结果 应用共焦显微镜观察到翼状胬肉头部两种上皮结构:一种是翼状胬肉上皮细胞,包括浅表上皮细胞和深层上皮细胞.浅表上皮细胞呈边界高反光的不规则多边形,浅表上皮细胞间见核/浆比显著失调的不规则细胞,深层上皮细胞间可见大量成熟的朗格汉斯细胞浸润;另一种是角膜上皮细胞,细胞间散在炎性细胞浸润.头部角膜浅基质层可见一些基质细胞和层间走行的纤维组织.翼状胬肉颈部和体部的上皮细胞结构与头部胬肉上皮结构相似,颈部血管较正常结膜血管丰富,在头部形成栏栅样结构,未跨越透明角膜,同时可以看到血管中流动的血细胞.体部基质层清晰可辨,由大量纤维组织构成,走行与基质平面基本平行并相互交错成致密的网状结构,可见新生血管横跨其间.结论 活体共焦显微镜是一种非侵入性的组织观察方法,提供了翼状胬肉的活体、实时、动态的图像资料,是研究翼状胬肉组织结构的一种有效工具.

Abstract：

Objective To study the histology of the pterygia in vivo by confocal microscopy. Methods Series of case study. Fourteen pterygium eyes in 11 patients were selected for using in vivo confocal mieroscopy. Results Two kinds of epithelial cells were observed in the transitional zone of cornea and pterygium. Superficial epithelium cells of the pterygium characterized as polygon cells with brightly refractive border. There were many mature Langerhans cells and big irregular cells with significantly decreased nuclear/cytoplasm ratio within the Epithelial. Irregularly brightly reflective cells presented in the corneal epithelia. Fibrous tissue from the pterygium and cornea stroma cells were found in the superficial stroma. The blood vessels became dilated in the neck and body of pterygium, terminated at the head as palisade-like structures with lots of blood cells. The stroma of the pterygium body was filled with a lot of fibers which formed a dense net with neovascular across. Conclusions IVCM is a powerful technique for studying the vivo histology of the pterygium.Noninvasive technique for living tissue has provided in vivo, real-time, dynamic image data. It is a useful tool for investigating the structures ofpterygium.     

In vivo confocal microscopy of pre-endothelial deposits

BACKGROUND: Deposits in various layers of the cornea might result from long-term medical therapy, photorefractive surgery, and longterm use of contact lenses or corneal dystrophies. METHODS: A 46-year-old woman was referred to our department with the suspected diagnosis of posterior polymorphous dystrophy. Slit-lamp biomicroscopy revealed bilateral small-sized deposits in the posterior part of the cornea. In vivo confocal microscopy was performed to evaluate these deposits in detail. RESULTS: In vivo confocal microscopy of the cornea identified hyperreflec-tive "dot-like" structures in the deep stromal layer and anterior to the endothelial cell layer. The morphology and number of keratocytes of the posterior stroma and of endothelial cells appeared normal. CONCLUSIONS: In vivo confocal microscopy is a very useful tool to analyze and visualize pre-endothelial deposits. Because there is no family history of corneal disease, the exact origin of the pre-endothelial deposits in our case remains unclear.     

In vivo confocal microscopy of fleck dystrophy

PURPOSE: To use in vivo confocal microscopy to evaluate corneas with fleck dystrophy. METHODS: Both eyes of three patients with corneal fleck dystrophy were examined with a scanning slit confocal microscope. Corneal epithelium, stroma, and endothelium were evaluated, as well as the basal epithelial and stromal nerves. RESULTS: The epithelium did not show any anomalies, but the basal nerves showed hyperreflective inclusions. Throughout the entire stroma, hyperreflective dots of various shapes were seen. These consisted mostly of spherical matter with a diameter of 3-5 microm and were sometimes enclosed in cyst-like structures. The majority of the stromal cells and stromal nerves appeared normal. The endothelial cell layer was unaffected. CONCLUSION: In vivo confocal microscopy demonstrates previously unreported inclusions in the basal nerves of fleck dystrophy corneas. In addition to this new finding, the study confirms earlier histopathologic reports, demonstrating accumulation of pathologic material in the stromal cells.     

In vivo confocal microscopy in hydroxychloroquine-induced keratopathy

Background Vortex keratopathy, arising as a side effect of several medications, is characterized by golden-brown deposits in the cornea. Methods A 41-year-old woman treated for sarcoidosis with hydroxychloroquine therapy and suffering from vortex keratopathy was examined by in vivo confocal microscopy. Scans of both corneas were performed. Results By slit lamp examination, the left but not the right eye showed a golden-brown deposit throughout the cornea. In vivo confocal microscopy revealed the presence of highly reflective, dot-like intracellular inclusions concentrated in the basal epithelial layer. They were also detected within the anterior and posterior stroma, but not within the endothelium. In regions of the anterior stroma, devoid of inclusions, hyperreflective ramified keratocytes were observed, forming an extended interconnecting network. Conclusion In addition to the granular deposits, in vivo confocal microscopy revealed hyperreflective, possibly phagocytic keratocytes.     

In vivo confocal microscopy in fungal keratitis

BACKGROUND: Fungal keratitis is a major blinding eye disease found throughout the world, particularly in developing countries. Given the recent increase in Fusarium keratitis infections in contact lens wearers owing to contact lens solutions, a warning was recently issued by the Food and Drug Administration, making it a public health concern in developed countries. OBJECTIVE: To show the advantages of in vivo confocal microscopy imaging using the Heidelberg Retina Tomograph II-Rostock Cornea Module (HRTII-RCM) in the early diagnosis of fungal keratitis. METHODS: HRTII-RCM confocal microscopy was performed on five patients presenting with fungal keratitis and on three donor corneas contaminated with Fusarium solani, Aspergillus fumigatus and Candida albicans. RESULTS: Direct microscopic evaluation of corneal smears and culture revealed the presence of F solani in four cases and C albicans in one case. HRTII-RCM examination of the infected patients and contaminated donor corneas revealed numerous high-contrast elements resembling Fusarium, Aspergillus hyphae or Candida pseudofilaments in the anterior stroma. CONCLUSION: HRTII-RCM in vivo confocal microscopy is a new, non-invasive and rapid technique for the early diagnosis of fungal keratitis, showing high-resolution images resembling fungal structures in the early phase of the disease.     

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.     

In vivo confocal microscopy after herpes keratitis

PURPOSE: To describe the confocal microscopic findings, with special reference to corneal subbasal nerves, after herpes simplex virus (HSV) keratitis. METHODS: In this study, 16 HSV eyes and 14 contralateral eyes of 16 patients, diagnosed with unilateral HSV keratitis 1-12 months earlier by the presence of dendritic corneal ulceration or microbiologic confirmation, were examined by in vivo confocal microscopy for evaluation of corneal morphology. RESULTS: Herpes simplex virus eyes: In 2 eyes the surface epithelial cells appeared large, and no abnormalities were observed in the basal epithelial cells. In 2 eyes subbasal nerve fiber bundles were completely absent, in 3 eyes there was a reduced number of long nerve fiber bundles, and in 11 eyes the subbasal nerve plexus appeared normal. In 10 corneas, highly reflective dendritic structures were found at the level of the basal epithelial cells. Frequently these structures were found in the vicinity of stromal fibrosis. Areas with increased abnormal extracellular matrix were found in 11 eyes. Stromal nerves were not visualized in all corneas, but appeared normal when observed. Contralateral eyes: No abnormalities were observed in the epithelium. All corneas presented with a normal subbasal nerve plexus, but in 2 eyes dendritic particles were observed. Three corneas presented with activated keratocytes and increased amounts of abnormal extracellular matrix. CONCLUSIONS: When visualized by confocal microscopy, the subbasal nerve plexus appears relatively unaffected in cases with resolved HSV keratitis. Unidentified dendritic structures, presumably Langerhans cells, are frequently seen at the level of the basal epithelium in corneas with a history of herpetic disease.     

活体共聚焦显微镜在角膜病临床研究中的新进展

活体共聚焦显微镜能在细胞水平实时、非侵入性、高清晰地检测角膜结构,它已广泛应用于角膜病的研究.本文对活体共聚焦显微镜在感染性角膜炎、圆锥角膜、角膜后沉着物、长期应用抗青光眼药物引起的角膜病变及糖尿病相关的角膜病变的临床研究新进展进行综述.     

In vivo confocal corneal microscopy after keratoplasty

BACKGROUND: Seven eyes with clear grafts after penetrating keratoplasty were examined with in vivo confocal corneal microscopy in 1999. Our aim was the confocal microscopic investigation of the subclinical changes in clear grafts after long-term follow-up.METHODS: The preoperative diagnoses were keratoconus (two), granular corneal dystrophy (two), pseudophakic bullous keratopathy due to ACL (two), and corneal ulcer (one). The epithelium, corneal nerves, keratocytes of the anterior and posterior stroma, and endothelium were evaluated with confocal microscopy.RESULTS: Mean density of basal epithelial cells was 3928+/-378 cells/mm(2) at 15 months and 3284+/-565 cells/mm(2) at 66 months postoperatively. At 15 months the keratocyte density was 750+/-113 cells/mm(2) in the anterior stroma and 601+/-98 cells/mm(2) in the posterior stroma, at 66 months 383+/-53 cells/mm(2) in the anterior stroma and 411+/-98 cells/mm(2) in the posterior stroma. Endothelial cell density decreased from 1719+/-576 cells/mm(2) (15 months) to 965+/-272 cells/mm(2) (66 months).CONCLUSIONS: In the follow-up period a significant decrease of keratocyte and endothelial cell density was detectable with confocal microscopy. The clinical importance of our findings must be clarified with further examinations on more patients.