In Vivo Confocal Microscopy expanding horizons in corneal imaging

Confocal microscopy is an emerging optical technique that allows the living human cornea to be imaged on a cellular level. As such, confocal microscopy enables morphologic and quantitative analysis of corneal resident cells in health and disease and provides an exciting bridge between in vivo diagnosis and ex vivo histological confirmation of pathologic processes

Histological Evaluation of Corneal Scar Formation in Pseudophakic Bullous Keratopathy

PURPOSE: To evaluate histological changes in the corneal stroma in pseudophakic bullous keratopathy. METHODS: Twenty-eight patients (28 eyes) with pseudophakic bullous keratopathy underwent therapeutic penetrating keratoplasty at Shandong Eye Institute between January 2006 and November 2011. The patients were divided into two groups according to the duration of bullous keratopathy (<1.0 year group or >1.0 year group), and three buttons from enucleated eyes with choroidal melanoma served as a control. In vivo confocal microscopy examination, hematoxylin-eosin, Masson's trichrome stain and Van Gieson staining were used for microscopic examination. The histological evaluation and scoring of the buttons for morphological changes, including the degree of stromal scars, neovascularization and inflammatory cells within the corneal buttons, were compared. To study the underlying mechanism, connective tissue growth factor (CTGF) and TGF-β immunohistochemistry were performed. RESULTS: Confocal microscopy examination and histological evaluation and scoring of the buttons showed that compared with the <1.0 year group, stromal scars, neovascularization and inflammatory cells were more severe in the >1.0 year group (P<0.05). There was an increase in CTGF- and TGF-β1-positive stromal cells in the >1.0 year group. CONCLUSIONS: During the progression of pseudophakic bullous keratopathy, stromal scars occurred more often in the patients that had a longer duration of disease. Cytokines such as CTGF and TGF-β1 may play a role in this pathological process and deserve further investigation

Fully automatic evaluation of the corneal endothelium from in vivo confocal microscopy

Background Manual and semi-automatic analyses of images, acquired in vivo by confocal microscopy, are often used to determine the quality of corneal endothelium in the human eye. These procedures are highly time consuming. Here, we present two fully automatic methods to analyze and quantify corneal endothelium imaged by in vivo white light slit-scanning confocal microscopy. Methods In the first approach, endothelial cell density is estimated with the help of spatial frequency analysis. We evaluate published methods, and propose a new, parameter-free method. In the second approach, based on the stochastic watershed, cells are automatically segmented and the result is used to estimate cell density, polymegathism (cell size variability) and pleomorphism (cell shape variation). We show how to determine optimal values for the three parameters of this algorithm, and compare its results to a semi-automatic delineation by a trained observer. Results The frequency analysis method proposed here is more precise than any published method. The segmentation method outperforms the fully automatic method in the NAVIS software (Nidek Technologies Srl, Padova, Italy), which significantly overestimates the number of cells for cell densities below approximately 1200 mm?2, as well as previously publishedmethods. Conclusions The methods presented here provide a significant improvement over the state of the art, and make in vivo, automated assessment of corneal endothelium more accessible. The segmentation method proposed paves the way to many possible new morphometric parameters, which can quickly and precisely be determined from the segmented image.ImPhys/Imaging PhysicsApplied Science

Wide-Range Calibration of Corneal Backscatter Analysis by In Vivo Confocal Microscopy

PURPOSE. To report intra- and interinstrument calibration methods for corneal backscatter analysis by in vivo confocal microscopy. METHODS. Applicability of two reference standards was evaluated for corneal backscatter calibration. Repeated measurements of four concentrations of AMCO Clear (GFS Chemicals, Inc., Powell, OH) suspension and three transparencies (26%, 49%, and 65%) of polymethylmethacrylate (PMMA) slabs were performed to assess image intensity acquisition in a wide backscatter range. Intra- and intersession repeatability and lot-to-lot variation were determined for both standards. The effect of light intensity (LI) variation on image intensity acquisition was evaluated by examination of PMMA slabs with nonreference (60% and 80%) and reference (72%) LIs. Both reference standards were implemented in the protocol. Intrainstrument calibration was verified by measuring three normal corneas with 60%, 72%, and 80% LIs. Interinstrument calibration was tested by measuring PMMA slabs on a second, similar confocal microscope. RESULTS. AMCO Clear was used to express image intensity in absolute scatter units (SU), whereas the 49% transparent PMMA slab showed best repeatability, without image saturation, to adjust for LI variation. Intrainstrument calibration for LI variation reduced mean differences from -38.3% to 1.7% (60% LI) and from 33.9% to -0.6% (80% LI). The mean difference between similar microscopes decreased from 18.4% to 1.2%, after calibration of the second microscope. CONCLUSIONS. Large interinstrument differences necessitate calibration of corneal backscatter measurements. With AMCO Clear suspension and PMMA slabs, standardization was achieved in a wide backscatter range corresponding to normal and opaque corneas. These methods can easily be applied in ophthalmic practice. © 2011 The Association for Research in Vision and Ophthalmology, Inc

Normative database for corneal backscatter analysis by in vivo confocal microscopy

Purpose. To ascertain the sex and age relatedness, diurnal variation, and repeatability of backscatter measurement in the normal human cornea. Methods. Seven corneal backscatter variants were measured by in vivo confocal microscopy (IVCM) in both normal eyes (n = 314) of 157 healthy subjects. These subjects were assigned to one or more of three groups. The sex and age relatedness of corneal backscatter were assessed in group 1 (n = 300), which comprised 75 men and 75 women evenly distributed over five age categories. To assess diurnal variation, eyes in group 2 (n = 40) were measured four times a day, at 3-hour intervals. The eyes in group 3 (n = 50) were examined four times a year to determine intersession rep

In vivo confocal microscopy of the corneal endothelium: comparison of three morphometry methods after corneal transplantation

PURPOSE: The purpose of this study was to assess the endothelium of corneal grafts by in vivoconfocal microscopy (IVCM), and to evaluate an automated endothelial software system in comparison with a manual cell count and planimetry. PATIENTS AND METHODS: Overall, 40 corneal grafts (20 deep anterior lamellar keratoplasties (DALKs) and 20 penetrating keratoplasties (PKs)) were assessed by scanning-slit IVCM. The endothelial cell density (ECD) was estimated with the automated and the manual cell count method of the instrument's Nidek Advanced Vision Information System (NAVIS) software. The results were compared with planimetry as the reference method, and the agreement was assessed. RESULTS: The mean (±SD) automated ECD was 2278±524 cells/mm(2) (range 1167–3192 cells/mm(2)), whereas the manual cell count method gave significantly lower ECDs with a mean of 1213±677 cells/mm(2) (range 218–2440 cells/mm(2); P<0.001). The manual cell counts were also significantly lower than those by planimetry, with a mean ECD of 1617±813 cells/mm(2) (range 336–2941, P<0.001). Bland–Altman analyses indicated that the limits of agreement (LoA) between the automated and the planimetry method were −671 and +1992 cells/mm(2), whereas they were −1000 and +202 cells/mm(2) when comparing the manual cell counts with planimetry. CONCLUSION: Following keratoplasty, the NAVIS automated method is likely to overestimate endothelial cell counts due to oversegmenting of the cell domains. Automated ECDs are substantially higher than those by the manual counting method or planimetry. The differences are considerably larger post-keratoplasty than for normal corneas, and the methods should not be used interchangeably