Laser Scanning In Vivo Confocal Microscopy Research Articles

Does Systemic Hematological Therapy Influence the Course of Paraproteinemic Keratopathy?

The purpose of this article is to evaluate the course of paraproteinemic keratopathy (PPK) in patients undergoing systemic therapy for the underlying hematological disease. Baseline and follow-up examinations included hematological work-up, best-corrected visual acuity, slit-lamp biomicroscopy, and in vivo confocal laser scanning microscopy (IVCM). We included 22 patients with bilateral PPK (aged 68 ± 10.4 years, 11 males). Ten patients with multiple myeloma (MM) underwent on-label systemic therapy. During follow-up, we observed a regression of corneal opacities in three patients under slit-lamp examination and under IVCM, while PPK remained unchanged in seven patients. In three patients with monoclonal gammopathy of ocular significance (MGOS), systemic therapy was initiated off-label to reduce the serum paraprotein load before penetrating keratoplasty (PKP). These patients showed no signs of PPK recurrence for up to 24 months after PKP. In one patient without systemic therapy, a recurrence in corneal grafts occurred within 12 months of PKP. In eight patients without systemic therapy, PPK remained stable. In conclusion, systemic therapy for MM patients reduced corneal opacity in 30% of treated patients. Furthermore, systemic therapy performed before PKP in patients without conventional systemic therapy indication (MGOS) likely postpones PPK recurrence in the corneal graft.

The sub‐basal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal microscopy

Purpose: Congenital aniridia is a rare disease, with a global prevalence of 1 in 40 000 to 1 in 100 000. During life up to 70% of aniridia sufferers develop aniridia‐associated keratopathy (AAK) with limbal stem cell insufficiency, impaired corneal epithelial cell differentiation, abnormal cell adhesion and wound healing. AAK is characterized by centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. We examined the sub‐basal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy (IVCM).Methods: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 47 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph‐III. At the sub‐basal nerve plexus, corneal nerve fibre density (CNFD), corneal nerve fibre length (CNFL), corneal total branch density (CTBD) and corneal nerve fibre width (CNFW) were analysed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually.Results: The CNFD (2.02 ± 4.08 vs. 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs. 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs. 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects, than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects, than in controls (0.03 ± 0.004 vs. 0.02 ± 0.003 mm/mm2) (p < 0.003). Keratocyte density was significantly lower in all stromal layers of congenital aniridia subjects, than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in congenital aniridia.Conclusions: CNFD, CNFL and CTBD decrease and CNFW increase well refer to the congenital aniridia associated neuropathy. The decreased keratocyte density contradicts the inflammatory hypothesis of congenital aniridia. Corneal stromal changes in different stages of AAK have to be further analysed in detail0.

Identification of presumed corneal neuromas and microneuromas using laser-scanning in vivo confocal microscopy: a systematic review

Background/aimsThis systematic review critically evaluated peer-reviewed publications describing morphological features consistent with, or using terms related to, a ‘neuroma’ or ‘microneuroma’ in the human cornea using laser-scanning in vivo confocal...

Factors Influencing the Diagnostic Accuracy of Laser-Scanning In Vivo Confocal Microscopy for Acanthamoeba Keratitis.

To determine the factors that influence the sensitivity and specificity of laser-scanning in vivo confocal microscopy (IVCM) for diagnosing Acanthamoeba keratitis (AK). This retrospective, controlled study included 28 eyes of 27 patients with AK and 34 eyes of 34 patients with bacterial keratitis (as the control group). All patients had undergone corneal imaging with a laser-scanning IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module). The IVCM images were independently evaluated by 2 experienced and 2 inexperienced masked observers. Sensitivity and specificity of IVCM for diagnosing AK and the effects of various clinical and imaging parameters on the sensitivity were then investigated. Overall, IVCM had average sensitivity and specificity of 69.7% ± 2.5% and 97.1% ± 4.2% for experienced observers and 59.0% ± 7.6% and 92.7% ± 10.4% for inexperienced observers, respectively. However, the sensitivity did not show any significant association with the duration of disease, size of ulcer, depth of involvement, culture results, or cyst morphology. Although interobserver agreement was good (κ = 0.60, P < 0.001) for the experienced observers, it was only at a moderate level (κ = 0.48, P < 0.001) for the inexperienced observers. IVCM has a moderate sensitivity and a high specificity for diagnosis of AK. Although clinical parameters do not affect this diagnostic accuracy, a higher sensitivity is seen when images are interpreted by experienced observers.

Laser scanning in vivo confocal microscopy (IVCM) for evaluating human corneal sub-basal nerve plexus parameters: protocol for a systematic review

IntroductionLaser scanning in vivo confocal microscopy (IVCM) enables non-invasive, high-resolution imaging of the cornea. In recent years, there has been a vast increase in researchers using laser scanning IVCM to...

Sensitivity and Specificity of Laser-Scanning In Vivo Confocal Microscopy for Filamentous Fungal Keratitis: Role of Observer Experience

To determine sensitivity and specificity of laser-scanning invivo confocal microscopy (LS-IVCM) for detection of filamentous fungi in patients with microbial keratitis and to evaluate the effect of observer's imaging experience on these parameters. Retrospective reliability study. This study included 21 patients with filamentous fungal keratitis and 24 patients with bacterial keratitis (as controls). The etiology of infection was confirmed based on the response to specific therapy regardless of culture results. All patients had undergone full-thickness corneal imaging by a LS-IVCM (Heidelberg Retina Tomograph 3 with Rostock Cornea Module; Heidelberg Engineering, Heidelberg, Germany). The images were evaluated for the presence of fungal filaments by 2 experienced observers and 2 inexperienced observers. All observers were masked to the clinical and microbiologic data. The mean number of images obtained per eye was 917 ± 353. The average sensitivity of LS-IVCM for detecting fungal filaments was 71.4% ± 0% for the experienced observers and 42.9% ± 6.7% for the inexperienced observers. The average specificity was 89.6% ± 3.0% and 87.5% ± 17.7% for these 2 groups of observers, respectively. Although there was a good agreement between the 2 experienced observers (κ= 0.77), the inexperienced observers showed only a moderate interobserver agreement (κ= 0.51). The LS-IVCM sensitivity was higher in patients with fungal infections who had positive culture or longer duration of the disease. Although LS-IVCM has a high specificity for diagnosing filamentous fungal keratitis, its sensitivity is moderate and highly dependent on the level of theobserver's experience and training with this imagingmodality.

Amniotic membrane transplantation - analysis of structural characteristics in amniotic membrane transplant and corneal ulcers

AIM: To analyze the structural characteristics in the cornea and the amniotic membrane (AM) with the help of laser scanning in-vivo confocal microscopy (IVCM) and to evaluate the morphometric changes in the cornea and the integration pattern of amniotic membrane into the host tissue using anterior-segment optical coherence tomography (AS-OCT) in patients with persistent corneal defect treated with amniotic membrane transplantation (AMT). MATERIALS AND METHODS: Nine eyes of six consecutive patients (mean age 44.9 ± 8.7 years) with corneal defects and stromal thinning unresponsive to topical treatment were enrolled in this study. Transplantation of cryopreserved amniotic membrane was performed. The time of healing of the corneal defect, morphometric analysis of the cornea and transplanted amniotic membrane using AS-OCT and the structural characteristics in the amniotic membrane grafts and corneal ulcers assessed by in vivo confocal microscopy were evaluated. RESULTS: Successful results after amniotic membrane transplantation (AMT) were observed in 8 of the 9 eyes (91.2%). On the 2nd day after transplantation, IVCM showed that under the amniotic membrane a new epithelium with large, flat, immature cells of the superficial corneal epithelium with an average density of 598.4 ± 66.38cells/mm2 was observed. The basal cells also showed immaturity and their average density was 1804 ± 93.32cells/mm2. In the corneal stroma edema and activated corneal cells were visualized. The average corneal epithelium thickness increased to 24.60 ± 2.07μm, the average density of epithelial cells increased to 657.6 ± 78.41cells/mm2, while the mean basal epithelium cells density was 2541 ± 540.13 cells/mm2. AS-OCT showed that the preoperative corneal thickness at the ulcer was 418.91 ± 96.56 μm. On the 2nd day after the surgery, the amniotic membrane (AM) thickness was measured to be 268 ±105 μm. On the 8th day it was two times smaller and was measured to be 123 ±39 μm. On the 25th day post-surgery the corneal thickness was measured at 494.03 ± 67.35 μm. In two of the cases integration of AM was found. CONCLUSIONS: AMT leads to recovery of corneal defects unresponsive to conservative treatment. The amniotic membrane graft is effective in promoting re-epithelization, nonetheless, it can also integrate into the host corneal tissue which results in an increase in corneal thickness, stabilization of the epithelium and reduction of the subjective signs. The integration of amniotic membrane into the damaged cornea proves that AMT is an effective and irreplaceable procedure for difficult-to-treat anterior ocular surface diseases.

Overestimation of Corneal Endothelial Cell Density in Smaller Frame Sizes in In Vivo Confocal Microscopy

To evaluate the effect of frame size on the calculated corneal endothelial cell density (CECD) in images of laser scanning in vivo confocal microscopy (IVCM). Forty-nine corneal endothelial images acquired by laser scanning IVCM (Heidelberg Retina Tomograph 3 with Rostock Corneal Module) with different endothelial cell densities were analyzed. In each image (160,000 μm), the CECD was calculated using the fixed-frame method by counting cells in the following frame sizes: 80,000 μm, 40,000 μm, 20,000 μm, 10,000 μm, 5000 μm, and 2500 μm. The calculated CECD was then compared with that of the variable-frame method as the reference value. There was no significant difference in the calculated CECD between the variable-frame method (2004 ± 832 cells/mm), and the fixed-frame method using a 40,000-μm frame (2023 ± 810 cells/mm). On the other hand, the calculated CECD showed significant overestimations in frame sizes of 20,000 μm (2066 ± 820 cells/mm), 10,000 μm (2156 ± 785 cells/mm), 5000 μm (2352 ± 783 cells/mm), and 2500 μm (2715 ± 754 cells/mm), with P < 0.001 in all. This resulted in overestimations of 4.8 ± 9.8%, 11.9 ± 16.2%, 24.9 ± 23.1%, and 49.1 ± 38.8% for these frame sizes, respectively. Images with lower CECD demonstrated higher overestimations of cell density in smaller frame sizes. In laser scanning IVCM images, there is significant overestimation of CECD if the cells are counted in frames smaller than 25% of the image. Similar frame sizes should be used when monitoring CECD over time.

In vivo corneal confocal microscopic analysis in patients with keratoconus.

To quantify corneal ultrastructure using laser scanning in vivo confocal microscopy (IVCM) in patients with keratoconus and control subjects. Unscarred corneas of 78 keratoconic subjects without a history of contact lens use and 36 age-matched control subjects were evaluated with slit-lamp examination (SLE), corneal topography and laser scanning IVCM. One eye was randomly chosen for analysis. Anterior and posterior stromal keratocyte, endothelial cell and basal epithelial cell densities and sub-basal nerve structure were evaluated. IVCM qualitatively demonstrated enlarged basal epithelial cells, structural changes in sub-basal and stromal nerve fibers, abnormal stromal keratocytes and keratocyte nuclei, and pleomorphism and enlargement of endothelial cells. Compared with control subjects, significant reductions in basal epithelial cell density (5817±306 cells/mm(2) vs 4802±508 cells/mm(2), P<0.001), anterior stromal keratocyte density (800±111 cells/mm(2) vs 555±115 cells/mm(2), P<0.001), posterior stromal keratocyte density (333±34 cells/mm(2) vs 270±47 cells/mm(2), P<0.001), endothelial cell density (2875±223 cells/mm(2) vs 2686±265 cells/mm(2), P<0.001), sub-basal nerve fiber density (31.2±8.4 nerves/mm(2) vs 18.1±9.2 nerves/mm(2), P<0.001), sub-basal nerve fiber length (21.4±3.4 mm/mm(2) vs 16.1±5.1 mm/mm(2), P<0.001), and sub-basal nerve branch density (median 50.0 (first quartile 31.2 - third quartile 68.7) nerve branches/mm(2) vs median 25.0 (first quartile 6.2 - third quartile 45.3) nerve branches/mm(2), P<0.001) were observed in patients with keratoconus. Significant microstructural abnormalities were identified in all corneal layers in the eyes of subjects with keratoconus using IVCM. This non-invasive in vivo technique provides an important means to define and follow progress of microstructural changes in patients with keratoconus.

Reduced Corneal Endothelial Cell Density in Patients With Dry Eye Disease

To evaluate corneal endothelial cell density (ECD) in patients with dry eye disease (DED) compared to an age-matched control group. Cross-sectional, controlled study. This study included 90 eyes of 45 patients with moderate to severe DED (aged 53.7 ± 9.8 years) and 30 eyes of 15 normal controls (aged 50.7 ± 9.8 years). All subjects had a complete ophthalmic evaluation including symptom assessment using the Ocular Surface Disease Index (OSDI) and corneal fluorescein staining. In addition, laser scanning invivo confocal microscopy was performed to measure the density of the following parameters in the central cornea: endothelial cells, subbasal nerves, and subbasal immune dendritic cells. Corneal ECD was significantly lower in the DED group (2595.8 ± 356.1 cells/mm(2)) than in the control group (2812.7 ± 395.2 cells/mm(2), P= .046). The DED group showed significantly lower corneal subbasal nerve density (17.1 ± 6.9mm/mm(2)) compared to the control group (24.7 ± 4.4mm/mm(2), P < .001). Dendritic cell density was significantly higher in the DED group than in the controls (111.7 ± 137.3 vs 32.0 ± 24.4 cells/mm(2), respectively, P= .002). There were statistically significant correlations between corneal ECD and dry eye severity parameters including the OSDI score (rs=-0.26, P= .03), and corneal fluorescein staining (rs=-0.28, P= .008). There is a significant reduction in corneal ECD in DED that correlates with clinical severity of the disease.

In Vivo Confocal Microscopy Analyses of Corneal Microstructural Changes in a Prospective Study of Collagen Cross-linking in Keratoconus

To use in vivo confocal microscopy (IVCM) to quantitatively analyze microstructural changes over time, after corneal collagen cross-linking for keratoconus. Prospective cohort study. A total of 38 eyes of 38 patients undergoing collagen cross-linking for keratoconus. Prospective, clinical cohort study of corneal collagen cross-linking in progressive keratoconus. Laser scanning IVCM performed preoperatively and at 1, 3, 6, and 12 months postoperatively. Density of corneal sub-basal nerves, anterior and posterior keratocytes, and corneal endothelium. Compared with baseline values, the mean sub-basal nerve density decreased significantly at 1, 3, and 6 months postoperatively (P < 0.01); however, this returned to preoperative values at 12 months (P = 0.57). One month postoperatively, there was complete absence of keratocyte nuclei in 86% of corneas. Anterior stromal edema with hyper-reflective cytoplasm and extracellular lacunae in a honeycomb-like appearance was observed and persisted at 3 months postoperatively. Scattered, presumed fragmented keratocyte nuclei, were observed at 1 and 3 months, but by 6 months, keratocyte repopulation of the anterior stroma was apparent. Quantitative analysis confirmed a significant decrease in the mean anterior keratocyte density 1, 3, and 6 months postoperatively (P ≤ 0.01) with return to baseline values at 12 months postoperatively (P = 0.57). The demarcation between treated and untreated corneal stroma appeared as a region where normal keratocytes transitioned into elongated, hyper-reflective, needle-like structures and then into large hyper-reflective stromal bands. There was no significant change in posterior keratocyte density or endothelial density at any postoperative time point. This prospective IVCM study revealed complete loss of the sub-basal nerve plexus and loss of anterior stromal keratocytes in the early postoperative period, with complete regeneration of the sub-basal nerve plexus and keratocyte repopulation by 12 months postoperatively. The posterior stroma and corneal endothelium were unaffected.

Standardized Baseline Human Corneal Subbasal Nerve Density for Clinical Investigations With Laser-Scanning in Vivo Confocal Microscopy

We established a baseline value for central corneal subbasal nerve density in a large, healthy cohort. A total of 106 healthy volunteers (207 eyes) underwent full ophthalmic examination, including laser-scanning in vivo confocal microscopy (IVCM) of the central cornea. Images of the corneal subbasal nerve plexus were acquired and analyzed based on defined criteria. Nerve tracing was performed by two human observers and by a fully automated method. Subbasal nerve density was stratified by eye, observer, tracing method, calculation method, and age group. Association of nerve density with age was examined by linear regression and population distribution was examined by nonlinear regression. We analyzed 892 distinct, high quality images of the subbasal nerve plexus (mean, 4.3 images/eye) from 207 eyes. An overall mean central subbasal nerve density of 19 mm/mm(2) was found in 106 subjects aged 15 to 88 years, independent of eye, sex, or nerve tracing method, while the SD was a consistent 4 to 5 mm/mm(2). Subbasal nerve density followed a normal Gaussian distribution, and correlated negatively with age, with a mean decline of 0.25% to 0.30% per year, independent of eye, observer, or nerve tracing method. Moreover, the use of automated tracing techniques and randomized sampling may improve the speed and reproducibility of subbasal nerve density assessment for clinical applications. A baseline human corneal subbasal nerve density has been determined by laser-scanning IVCM using rigorous methods. The methods and results could aid in the future assessment of corneal nerves in various patient populations.

In Vivo Confocal Microscopic Findings in Posterior Polymorphous Corneal Dystrophy

To describe the corneal findings in posterior polymorphous corneal dystrophy (PPCD) as imaged with laser scanning in vivo confocal microscopy (IVCM). IVCM images of 7 subjects with PPCD who had typical slit-lamp biomicroscopic findings of endothelial vesicular, band, and/or placoid lesions were evaluated. Five women and 2 men aged 7 to 64 years were included in this study. Laser scanning IVCM (Heidelberg Retina Tomograph II, Rostock Cornea Module) revealed hyporeflective, round, vesicular lesions with diameters ranging between 20 and 200 µm in 3 subjects, combined vesicular and curvilinear hyperreflective band-like lesions in 3 subjects, and combined vesicular and placoid hyperreflective lesions in 1 subject at the level of Descemet membrane (DM), endothelial cell layer, and posterior stroma adjacent to DM. One subject had coassociated epithelial basement membrane dystrophy. Additional findings included posterior stromal keratocytes with elongated spindle-like nucleus, giant and nucleated endothelial cells, endothelial deposits, and guttae-like dark spots. The mean endothelial cell density was 1485.7 ± 486.3 cells per square millimeter (range, 990-2365 cells/mm). The mean central corneal thickness was 585.3 ± 37.17 μm (range, 534-643 μm). Laser scanning IVCM is able to highlight the characteristic microstructural alterations at the level of endothelium and DM in the setting of PPCD and may have diagnostic utility in equivocal cases with borderline biomicroscopic findings. The possible association of PPCD with epithelial basement membrane dystrophy warrants further investigation.

Diagnostik konjunktivaler Neoplasien mittels konfokaler In-vivo-Mikroskopie

There is a wide spectrum of benign and malignant conjunctival neoplastic lesions that are often impossible to distinguish clinically by slit-lamp microscopy. The current study was undertaken to compare in-vivo confocal laser scanning microscopy (CLSM) and histology for the preoperative assessment of benign or malignant status. We present the clinical details of three patients. In two cases the neoplastic lesions were classified as benign (actinic keratosis). In-vivo CLSM revealed densely layered, sometimes hyperreflective conjunctival epithelial cells, together with multiple inflammatory cells and microcysts. Correlated findings on histology showed keratinisation with inflammatory infiltrates and intracellular oedema formation. In-vivo CLSM images in the third patient revealed interruptions of the layered epithelial structure with regular conjunctival epithelium co-existing with complexes of enlarged cells with polymorphic nuclei. Histology also showed an abrupt transition from regular squamous epithelium to hyperplastic, dysplastic squamous epithelium. In this case the neoplastic lesion was classified as carcinoma in situ. The in-vivo CLSM images correlated positively with histology findings. Although in-vivo CLSM offers the capability to perform non-invasive examinations over time, associated histological assessment (because of its more precise detail and additional staining techniques) remains indispensable for planning further action and determining the prognosis.

Influence of Benzalkonium Chloride on Langerhans Cells in Corneal Epithelium and Development of Dry Eye in Healthy Volunteers

Purpose: To investigate the influence of benzalkonium chloride (BAC) on corneal Langerhans cells (LCs) and on the development of dry eye.Methods: A randomized double-blind clinical trial was performed in 20 healthy volunteers. One eye of each subject was treated with a 0.01% BAC solution (and the fellow eye with a placebo solution) three times daily for 12 weeks. The distribution and density of LCs in the central and peripheral corneal epithelium were evaluated by in-vivo confocal laser-scanning microscopy (CLSM). The subjects were monitored for dry eye (subjective discomfort, slit-lamp biomicroscopy, tear film break-up time, Schirmer’s test).Results: In the BAC group, compared with placebo, a marked increase in LC density was found in the central cornea at Week 6 and in the central and peripheral cornea at Week 12. LC density then decreased again in both zones after the end of treatment, falling toward (or even below) baseline levels. Significant changes in the LC count relative to baseline were found at Week 12 in the central and peripheral cornea in the BAC group as well as in the peripheral cornea in the placebo group. LC density in the BAC group increased more rapidly in the central than in the peripheral cornea. During therapy the BAC group showed no signs of dry eye.Conclusion: 12-week application of a 0.01% BAC solution in healthy volunteers induces a significant increase in LCs in the central cornea at Week 12 without dry-eye changes. The return of LC counts toward (or even below) baseline levels just four weeks after the end of BAC administration demonstrates the rapid normalization of the inflammatory environment.

Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy

To determine whether in vivo confocal microscopy (IVCM) of the cornea can be used for the label-free detection and monitoring of lymph vessels in live corneas. Parallel corneal hemangiogenesis and lymphangiogenesis was induced by the placement of a single suture in one cornea of male Wistar rats. Fourteen days after suture placement and under general anesthesia, laser-scanning IVCM was performed in the vascularized region. Corneas were subsequently excised for flat-mount double immunofluorescence with a pan-endothelial marker (PECAM-1/CD31) and a lymphatic endothelial specific marker (LYVE-1). Using the suture area and prominent blood vessels as points of reference, the identical microscopic region was located in both fluorescent and archived in vivo images. Additionally, vessel diameter, lumen contrast, and cell diameter and velocity within vessels were quantified from in vivo images. Comparison of identical corneal regions in fluorescence and in vivo revealed prominent CD31(+)/LYVE-1(3+) lymph vessels that were visible in vivo. In vivo, corneal lymph vessels were located in the vascularized area in the same focal plane as blood vessels but had a darker lumen (P < 0.001) sparsely populated by highly reflective cells with diameters similar to those of leukocytes in blood vessels (P = 0.61). Cell velocity in lymph vessels was significantly reduced compared with blood particle velocity (P < 0.001). Morphologic characteristics enabled subsequent identification of corneal lymphatics in live, vascularized rat corneas before immunofluorescence labeling. IVCM enabled the nondestructive, label-free, in vivo detection of corneal lymphatics. IVCM provides the possibility of observing lymphatic activity in the same live corneas longitudinally and, as a clinical instrument, of monitoring corneal lymphatics in live human subjects.

The microstructure of cornea verticillata in Fabry disease and amiodarone-induced keratopathy: a confocal laser-scanning microscopy study

The purpose of this study is to describe cornea verticillata in Fabry disease and in amiodarone-induced keratopathy and to compare the corneal microstructure of both types. Ten eyes from ten normal subjects, 28 eyes from 22 patients with Fabry disease confirmed by molecular genetic studies, and 16 eyes from 11 patients receiving amiodarone were examined by slit-lamp microscopy and in-vivo confocal laser-scanning microscopy (CLSM) with following three-dimensional reconstruction of the individual corneal layers. Five patients with Fabry disease were monitored during the course of enzyme replacement therapy (ERT). Evidence of cornea verticillata was found by slit-lamp microscopy both in patients with Fabry disease and in those with amiodarone-induced keratopathy; CLSM revealed the same pattern of hyper-reflective deposits in the basal cell layer of corneal epithelium in both sets of patients. Microdot changes in the anterior stroma were more prevalent in patients receiving amiodarone but do not presuppose the simultaneous presence of cornea verticillata. The bulbar conjunctiva was found to be normal in all patients. The tarsal conjunctival epithelium contained round hyper-reflective structures, which are also encountered physiologically, but these were more common in patients with Fabry disease. In one out of the five patients examined, monitoring of corneal changes over time during ERT disclosed a regressive tendency of the deposits in the epithelial basal cell layer documented by CLSM. The microstructural corneal changes typically seen in cornea verticillata in both Fabry disease and in amiodarone-induced keratopathy can be successfully visualized by confocal in-vivo microscopy at the level of the basal cell layer. By analogy, with the grading system for cornea verticillata based on slit-lamp microscopy, staging of these deposits in the basal cell layer can also be performed following in-vivo CLSM. The microdots in the anterior stroma as well as the changes observed in the tarsal conjunctiva should be regarded as having less diagnostic value because such changes may also occur in normal subjects. The utility of CLSM as a tool for monitoring ERT in Fabry disease over time needs to be confirmed in studies with larger sample sizes conducted over a longer period.

Confocal microscopy: when is it helpful to diagnose corneal and conjunctival disease?

In vivo confocal microscopy (IVCM) is able to demonstrate the characteristic corneal and conjunctival anatomy in vivo at the cellular level. Normal corneal innervation and cell distribution, as well as changes associated with age, contact lens wear and systemic disease such as diabetes, can be documented in vivo with this technique. IVCM is helpful to differentiate corneal dystrophies and inclusion keratopathies. Infectious agents, such as fungi and Acanthameba, may be visualized directly in the cornea in keratitis. Postsurgical applications include its use following refractive surgery, UV-crosslinking, keratoplasty and amniotic membrane transplantation to evaluate corneal wound healing. Conjunctivalization of the cornea in presumed limbal insufficiency can also be shown by IVCM. Laser scanning IVCM also allows the examination of conjunctival structures, as well as the lid margin. Thus, IVCM may help to establish a final diagnosis in atypical conjunctival inflammation and uncharacteristic conjunctival tumors. Furthermore, IVCM has proven useful in the follow-up of filtering blebs after trabeculectomy.

Amniotic membrane transplantation for the management of corneal epithelial defects: an in vivo confocal microscopic study

Purpose:To evaluate amniotic membrane (AM) tissue morphology and corneal epithelial healing in human eyes after amniotic membrane transplantation (AMT), using laser scanning in vivo confocal microscopy (IVCM).Patients and methods:Twenty eyes...

Epithelial Dendritic Cell Distribution in Normal and Inflamed Human Cornea: In Vivo Confocal Microscopy Study

To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). Comparative case-controlled, observational confocal microscopy study. A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.