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.     

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.     

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.     

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。     

In vivo laser and white-light confocal microscopic findings of human conjunctiva.

BACKGROUND AND OBJECTIVE: To report in vivo laser and white-light confocal microscopic findings of human conjunctiva to investigate the potential application of these confocal microscopes as diagnostic devices for normal and pathologic conjunctiva. PATIENTS AND METHODS: Two healthy volunteers, 28- and 35-year-old men, participated in this study. The temporal bulbar conjunctiva, which was approximately 5 mm away from the limbus, was examined by in vivo laser and white-light confocal microscopic analysis. RESULTS: Using laser confocal microscopy, at least two different cell types could be distinguished in both subjects: conjunctival superficial epithelial cells and conjunctival basal epithelial cells. In contrast, conjunctival epithelial cells could not be visualized using white-light confocal microscopy. CONCLUSIONS: These results indicate that laser confocal microscopy, but not white-light confocal microscopy, can be used to visualize in vivo human conjunctiva. Further investigations in a large number of normal subjects and in patients with conjunctival pathologies are required to fully evaluate the usefulness of this device in daily clinical ophthalmology.     

In vivo confocal microscopy of normal conjunctiva and conjunctivitis

PURPOSE: To analyze the appearance of normal conjunctiva and conjunctival inflammation by in vivo confocal microscopy. METHODS: Conjunctiva of 15 normal patients and 21 patients with conjunctivitis including bacterial, papillary, follicular, granulomatous, and cicatrizing disease were analyzed by the Heidelberg retina tomograph (HRTII)/Rostock cornea modul (RCM). RESULTS: Scans of normal bulbar and tarsal conjunctiva corresponded well to the established anatomy except for a prominent, thickened epithelial basement membrane observed by in vivo microscopy. Presumed goblet cells were visible throughout the conjunctival epithelium. Adenoid structures and hair follicles were discernible in the tarsal conjunctiva in vivo. Conjunctival perfusion could be observed directly. Acute and chronic inflammatory cells, conjunctival papillary, and follicular reactions, as well as conjunctival cicatrization, could be discriminated. In a patient with conjunctival granuloma, in vivo confocal microscopy disclosed suture material inside the lesion. CONCLUSION: Confocal microscopy using near-infrared laser light is a useful new tool in the analysis of conjunctival tissue in vivo. It is a valuable aid in the differential diagnosis of conjunctival inflammation and thus may guide therapeutical decisions.     

In vivo confocal laser scanning microscopy of the cornea in dry eye

Background We carried out an investigation into the morphological and quantitative corneal properties in dry eye with various underlying pathologies.Methods Ten patients with aqueous tear deficiency, 8 with dysthyroid ophthalmopathy, 8 with chronic lagophthalmos and 10 normal participants were examined. Confocal microscope images were taken at the centre and at the lower and upper periphery of the cornea. Quantitative and morphological assessments of the epithelium, of the sub-basal nerves, of the stroma and the endothelium were made. The epithelial and corneal thicknesses were measured.Results The mean superficial and intermediate epithelial cell densities in the central cornea in the patient groups were significantly lower than in normal participants (p<0.01). The peripheral epithelial thickness was smaller (p<0.01); it was smallest in the lagophthalmos group. The cornea was thinner in the patient groups (p<0.01). For sub-basal nerves, the density had decreased (p<0.05), and in lagophthalmos the number of beadlike formations had increased (p<0.001); in some patients we found irregular branching patterns.Conclusions Dry eye patients showed significant alterations in the cornea, presumably due to increased desquamation of the superficial cell layer. This was most pronounced at the lower periphery of the cornea in patients with exposure keratopathy.The authors had no financial relationship with the organisation that supported the research.The authors had full control of all primary data and agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review the data if requested.The results were partly presented at DOG in 2005.     

In vivo laser confocal microscopy findings of cryopreserved and fresh human amniotic membrane

BACKGROUND AND OBJECTIVE: Recently, the cornea-specific in vivo laser confocal microscope has become available. The objective of the current study was to obtain laser confocal images of fresh and cryopreserved human amniotic membrane. MATERIALS AND METHODS: Fresh and cryopreserved amniotic membranes from different donors were examined using the in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph II Rostock Cornea Module [HRT II-RCM]; Heidelberg Engineering GmbH, Dossenheim, Germany). RESULTS: In all samples of both fresh and cryopreserved amniotic membranes, the HRT II-RCM resolved four distinct layers: the epithelium, a basement membrane, a cell-rich stromal layer (fibroblast layer), and a spongy stromal layer. In a posterior aspect of the basement membrane layer (facing the fibroblast layer), microfolds were more prominent in fresh amniotic membranes than in cryopreserved amniotic membranes. For the cell-rich stromal layer, mesenchymal cells were observed more distinctively in fresh amniotic membranes than in cryopreserved amniotic membranes. CONCLUSION: These baseline laser confocal images can be used for future investigation of in vivo remodeling after amniotic membrane transplantation.     

In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis

OBJECTIVE: This study included in vivo and ex vivo investigations of patients with early-stage Acanthamoeba keratitis by using new-generation laser confocal microscopy (Heidelberg Retina Tomograph 2 Rostock Cornea Module [HRT 2-RCM]). METHODS: Three patients (2 men and 1 woman; mean age, 22.0 years) with early-stage Acanthamoeba keratitis diagnosed by direct examination (Parker ink-potassium hydroxide stain), culture from corneal epithelial scrapings, or both methods were enrolled in this study. All patients were examined by slit-lamp biomicroscopy. The area of the affected cornea was examined by HRT 2-RCM. Selected images of in vivo corneal layers and ex vivo cultured microorganisms were evaluated qualitatively for shape and degree of light reflection of the corneal structural changes or Acanthamoeba cysts. In addition, cultured Acanthamoeba were examined ex vivo by HRT 2-RCM. RESULTS: In vivo laser confocal microscopy showed highly reflective round-shaped, high-contrast Acanthamoeba cysts (10-20 microm in diameter) in the corneal epithelium in all cases, leading to rapid confirmation of the clinical diagnosis. In all culture samples of Acanthamoeba, ex vivo laser confocal microscopy showed highly reflective round- or stellate-shaped high-contrast particles (10-20 microm in diameter). CONCLUSIONS: In vivo laser confocal microscopy enables rapid and noninvasive diagnosis of early-stage Acanthamoeba keratitis with high resolution. In addition, ex vivo laser confocal images of Acanthamoeba cysts may be helpful when similar structures are identified and have to be interpreted under in vivo conditions.     

In vivo tandem scanning confocal microscopy in acanthamoeba keratitis

The in vivo confocal microscopy technique provides us with a real-time, non-invasive way of examining the human cornea. The most important advantage of this type of microscopy is to reveal the etiologic agents in infectious keratitis such as Acanthamoeba keratitis. We present several representative cases of Acanthamoeba keratitis, which were diagnosed in their early stages using in vivo confocal microscopy and managed based on that diagnosis. In our Acanthamoeba keratitis cases, highly-reflective round or ovoid organisms with a diameter of about 10-25 um were visualized distinctly against relatively-dark normal parenchymal structures, such as epithelial cells or keratocyte nuclei. Double-walled structures of Acanthamoeba cysts were clearly demonstrated in some cases. We can confirm that in vivo tandem scanning confocal microscopy is a powerful diagnostic tool for identifying the infecting organisms in Acanthamoeba keratitis.     

In vivo three-dimensional confocal laser scanning microscopy of the epithelial nerve structure in the human cornea

Purpose Evaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves. Method The anterior cornea was examined to a depth of 100 μm in four human volunteers with a confocal laser scanning microscope (CLSM) using a Rostock Cornea Module (developed in house) attached to a Heidelberg Retina Tomograph II (Heidelberg Engineering, Germany). Optical sections were digitally reconstructed in 3D using AMIRA (TGS Inc., USA). The scanned volumes had a greatest size of 300×300×40 μm and voxel size of 0.78×0.78×0.95 μm. Results The spatial arrangement of the epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. The 3D-reconstruction of the volunteers’ corneas in combination with the oblique sections gave a picture of the nerves in the central human cornea. Thin nerves run in the subepithelial plexus aligned parallel to Bowman’s layer and are partially interconnected. The diameter of these fibres varied between 1.0 and 5 μm. Thick fibres rose out of the deeper stroma. The diameter of the main nerve trunks was 12±2 μm. Branches penetrating the anterior epithelial cell layer could not be visualized. Conclusions 3D-CLSM allows analysis of the spatial arrangement of the anterior corneal nerves and visualization of the epithelium and keratocytes in the living human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal disease. This may help to clarify alterations of nerve fibre patterns under various clinical and experimental conditions.     

The evaluation of the treatment response in obstructive meibomian gland disease by in vivo laser confocal microscopy

Purpose  To evaluate the status of periglandular inflammation, ocular surface and tear function alterations in patients with obstructive meibomian gland disease (OMGD) by in vivo confocal microscopy before and after anti-inflammatory treatment, and to compare the results with patients receiving only topical non-preserved artificial tears and sodium hyaluronate eye drops without anti-inflammatory agents. Methods  Thirty-two eyes of 16 OMGD patients receiving anti-inflammatory treatment (treatment group) and 22 eyes of 11 OMGD patients receiving only topical non-preserved artificial tears and sodium hyaluronate eye drops (control group) were recruited in this prospective study. All subjects underwent slit-lamp examinations, tear film break-up time (BUT) measurements, fluorescein and Rose-Bengal stainings, Schirmer test І without anesthesia, transillumination of the lids (meibography), and in vivo laser confocal microscopy of the lids (HRTII-RCM). Results  The mean BUT, fluorescein staining scores, and inflammatory cell densities observed by in vivo confocal microscopy improved significantly in the group receiving anti-inflammatory treatment (p < 0.05), whereas no significant alterations of these parameters were observed in the group not receiving anti-inflammatory agents (p > 0.05). Conclusions  In vivo confocal microscopy was able to effectively demonstrate the treatment responses in patients with OMGD. Inflammatory cell density calculation seems to be a promising new parameter of in vivo confocal microscopy in the evaluation of treatment responses.