Andrey Zhivov

In vivo confocal microscopy, an inner vision of the cornea - a major review.

The demands of modern ophthalmology have evolved from descriptive findings from the slit lamp to in vivo assessment of cellular level changes. Nowadays, the latter can be provided by in vivo confocal microscopy. This article gives an overview of confocal principles using tandem scanning, scanning slit and laser scanning techniques used in ophthalmology. The main part of the paper describes the clinical applications emphasizing the anatomy of the normal and pathological cornea, and illustrates side‐effects of topical medication, contact lens wear, cross‐linking and refractive surgery. Finally, a summary about experimental applications, including animal studies, surface characterization and volume rendering as well as future developments, is given.

Early Detection of Nerve Fiber Loss by Corneal Confocal Microscopy and Skin Biopsy in Recently Diagnosed Type 2 Diabetes

We sought to determine whether early nerve damage may be detected by corneal confocal microscopy (CCM), skin biopsy, and neurophysiological tests in 86 recently diagnosed type 2 diabetic patients compared with 48 control subjects. CCM analysis using novel algorithms to reconstruct nerve fiber images was performed for all fibers and major nerve fibers (MNF) only. Intraepidermal nerve fiber density (IENFD) was assessed in skin specimens. Neurophysiological measures included nerve conduction studies (NCS), quantitative sensory testing (QST), and cardiovascular autonomic function tests (AFTs). Compared with control subjects, diabetic patients exhibited significantly reduced corneal nerve fiber length (CNFL-MNF), fiber density (CNFD-MNF), branch density (CNBD-MNF), connecting points (CNCP), IENFD, NCS, QST, and AFTs. CNFD-MNF and IENFD were reduced below the 2.5th percentile in 21% and 14% of the diabetic patients, respectively. However, the vast majority of patients with abnormal CNFD showed concomitantly normal IENFD and vice versa. In conclusion, CCM and skin biopsy both detect nerve fiber loss in recently diagnosed type 2 diabetes, but largely in different patients, suggesting a patchy manifestation pattern of small fiber neuropathy. Concomitant NCS impairment points to an early parallel involvement of small and large fibers, but the precise temporal sequence should be clarified in prospective studies.

In vivo confocal microscopic evaluation of langerhans cell density and distribution in the corneal epithelium of healthy volunteers and contact lens wearers.

Purpose: To examine and compare the density and distribution of Langerhans cells (LCs) in the corneal epithelium of healthy volunteers and contact lens wearers. Methods: A total of 225 eyes of 130 healthy volunteers (age, 17-81 years) without history of ocular inflammation, trauma, or surgery and 98 eyes of 55 contact lens wearers (age, 13-76 years) were examined in vivo with the combination of the Heidelberg Retina Tomograph II and in-house-invented Rostock Cornea Module. Results: In healthy volunteers, in vivo confocal microscopy revealed LCs in 31% of all volunteers, with 37 of these 43 volunteers presenting LCs both in the center and the periphery of the cornea with densities of 34 ± 3 and 98 ± 8 cells/mm2, respectively. In the group of contact lens wearers, 55% of all corneas presented with LCs, and 11 of these 33 corneas revealed LCs at central and peripheral locations. Although LC densities were markedly higher in both the central (78 ± 25 cells/mm2) and the peripheral cornea (210 ± 24 cells/mm2) of contact lens wearers, the gradient of LC density from peripheral to central cornea was found almost identical in both groups. In the central cornea, LC density decreased with duration of contact lens wear. LCs were located at the depth of 35 to 60 μm (ie, the level of lower intermediate cells, basal cells, and subepithelial nervous plexus). LCs presented as either large cells bearing long processes or smaller cells lacking cell dendrites, most supposedly indicating mature and immature phenotype, respectively. Conclusions: In vivo confocal microscopy enables evaluation of LC density and distribution in corneal epithelium. LCs were found present both in the center and the periphery of the cornea without difference in distribution between healthy volunteers and contact lens wearers. However, contact lens wearers revealed almost twofold higher LC densities in both locations, implying chronic mechanical irritation of the cornea in response to the contact lens as foreign body. Taken together, analysis of LC using in vivo confocal microscopy provides helpful information for a better understanding of contact lens-disturbed ocular homeostasis.

In Vivo Confocal Microscopy of the Ocular Surface

Over the past two decades, the applications of in vivo confocal microscopy to the investigation of ocular surface diseases in the living eye have been greatly extended. Confocal microscopy enables detailed investigation of tarsal and palpebral conjunctiva, central and peripheral cornea, tear film, and lids, and it allows evaluation of the ocular surface at the cellular level. High-quality imaging in both contact and noncontact modes has allowed new understanding of the functions of the ocular surface system, and in the coming years, such knowledge will become increasingly comprehensive and specific. Confocal microscopy may provide a link between well-established ex vivo histology and in vivo study of ocular pathology, not only in clinical science but also in clinical practice. The purpose of this review is to summarize the current knowledge about in vivo confocal microscopy of the ocular surface.

In vivo confocal microscopy of the ocular surface: from bench to bedside.

Abstract In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.

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

BackgroundWe carried out an investigation into the morphological and quantitative corneal properties in dry eye with various underlying pathologies.MethodsTen 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.ResultsThe 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.ConclusionsDry 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.

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

PurposeEvaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves.MethodThe 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.ResultsThe 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.Conclusions3D-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.

Imaging and Quantification of Subbasal Nerve Plexus in Healthy Volunteers and Diabetic Patients with or without Retinopathy

Background The alterations of subbasal nerve plexus (SBP) innervation and corneal sensation were estimated non-invasively and compared with the values in healthy volunteers. Additionally, this study addressed the relation of SBP changes to the retinal status, glycemic control and diabetes duration. Methodology/Principal Findings Eighteen eyes of diabetic patients with peripheral diabetic neuropathy aged 68.8±8.8 years and twenty eyes of healthy volunteers aged 66.3±13.3 yrs. were investigated with in vivo confocal laser-scanning microscopy (CLSM). An adapted algorithm for image analysis was used to quantify the morphological and topological properties of SBP. These properties were correlated to incidence of diabetic retinopathy (DR) and corneal sensation (Cochet-Bonnet esthesiometer). The developed algorithm allows a fully automated analysis of pre-segmented SBP structures. Altogether, 10 parameters were analysed, and all of them revealed significant differences between diabetic patients and healthy volunteers. The nerve fibre density, total fibre length and nerve branches were found to be significantly lower in patients with diabetes than those of control subjects (nerve fibre density 0.006±0.002 vs. 0.020±0.007 mm/mm2; total fibre length 6223±2419 vs. 19961±6553 µm; nerve branches 25.3±28.6 vs. 141.9±85.7 in healthy volunteers). Also the corneal sensation was significantly lower in diabetic group when compared to controls (43±11 vs. 59±18 mm). There was found no difference in SBP morphology or corneal sensation in the subgroups with (DR) or without (NDR) diabetic retinopathy. Conclusions/Significance SBP parameters were significantly reduced in diabetic patients, compared to control group. Interestingly, the SBP impairment could be shown even in the diabetic patients without DR. Although automatic adapted image analysis simplifies the evaluation of in vivo CLSM data, image acquisition and quantitative analysis should be optimised for the everyday clinical practice.

Image reconstruction of the subbasal nerve plexus with in vivo confocal microscopy.

PURPOSE To overcome the anterior corneal mosaic (ACM) phenomenon in in vivo confocal laser scanning microscopy (CLSM) and to reconstruct undistorted images of the subbasal nerve plexus (SNP), facilitating morphometric analysis in the presence of ACM ridges. METHODS CLSM was performed in five healthy volunteers. An original image processing algorithm based on phase correlation was used to analyze and reduce motion distortions in volume scan image sequences. Three-dimensional tracing of the SNP was performed to reconstruct images containing only the SNP layer, with nerve fibers clearly visible even in ACM areas. RESULTS Real-time mapping of the SNP revealed the presence of ridges with K-structures underneath them in all cases. The occurrence of K-structures correlated directly with development of ACM observed by slit lamp and resulted in massive deformation at the level of Bowmans membrane, seriously interfering with examination of SNP structures. The average elevation of ACM ridges was 20.6 μm (range, 8.7-34.0 μm). The novel method presented permitted reconstruction of the SNP layer in regions of ACM. CONCLUSIONS The described method allows the precise analysis and elimination of motion artifacts in CLSM volume scans, in conjunction with the capability to reconstruct SNP structures even in the presence of severe ACM. The robustness and automation of the described algorithms require ongoing development, but this will provide a sound basis for extended studies of corneal nerve regeneration or degeneration and for use in clinical practice.

Structural-functional correlations of corneal innervation after LASIK and penetrating keratoplasty.

PURPOSE To report a case in which a tissue saving program in an aberrated eye was used. METHODS A new algorithm for the selection of an optimized set of Zernike terms in customized treatments for laser corneal refractive surgery was developed and clinically tested. Ablation was performed using the SCHWIND ESIRIS excimer laser. Pre- and postoperative corneal wave aberrations were analyzed using the Keratron Scout videokeratoscope (Optikon 2000). RESULTS Required ablation was reduced by approximately 15% compared to full customized correction. Refraction was corrected to subclinical levels, uncorrected distance visual acuity improved to 20/20, corrected distance visual acuity gained 2 lines, aberrations were reduced by approximately 40% compared to preoperative baseline levels, and the functional optical zone of the cornea was enlarged by approximately 40% compared to preoperative baseline levels. Trefoil, coma, spherical aberration, and the root-mean-square value of the higher order aberrations were reduced. CONCLUSIONS Eliminating all higher order aberrations may not optimize visual function in highly aberrated eyes. The new algorithm minimized tissue removal in refractive surgery but further clinical evaluations are required to confi rm preliminary results.PURPOSE To compare corneal subbasal nerve fiber distribution and corneal sensation in healthy humans with findings obtained in regenerated subbasal nerves after LASIK and penetrating keratoplasty (PK). METHODS In a comparative case series study, in vivo confocal laser-scanning microscopy was used to investigate subbasal nerve fiber bundles in healthy individuals and at various time points after surgery in patients who had undergone LASIK and corneal grafting. Corneal sensation was measured (Cochet-Bonnet esthesiometer). RESULTS In normal corneas investigated, image superimposition revealed the consistent appearance of curved nerve fibers showing a whorl-like pattern with clockwise orientation. Nerve fibers parallel to Bowmans layer originating peripherally traveled radially inwards to a point located at the lower nasal quadrant. This pattern was not seen in any of the patients after LASIK or PK. Regenerated nerve fibers were thinner, more curved, and showed abnormal branching in nearly all patients. Normal corneal neuro-anatomical architecture remained absent even months after total restoration of corneal sensation. After LASIK, normal sensation was regained independently of normal subbasal nerve anatomy. Corneal grafts have shown some recovery of subbasal nerve morphology, at least in the graft periphery, but not complete recovery of function. CONCLUSIONS It would appear that normal corneal sensation after LASIK or PK does not always depend on normal subbasal nerve anatomy but on the collateral organization of subbasal nerve fibers.PURPOSE To report a case in which a tissue saving program in an aberrated eye was used. METHODS A new algorithm for the selection of an optimized set of Zernike terms in customized treatments for laser corneal refractive surgery was developed and clinically tested. Ablation was performed using the SCHWIND ESIRIS excimer laser. Pre- and postoperative corneal wave aberrations were analyzed using the Keratron Scout videokeratoscope (Optikon 2000). RESULTS Required ablation was reduced by approximately 15% compared to full customized correction. Refraction was corrected to subclinical levels, uncorrected distance visual acuity improved to 20/20, corrected distance visual acuity gained 2 lines, aberrations were reduced by approximately 40% compared to preoperative baseline levels, and the functional optical zone of the cornea was enlarged by approximately 40% compared to preoperative baseline levels. Trefoil, coma, spherical aberration, and the root-mean-square value of the higher order aberrations were reduced. CONCLUSIONS Eliminating all higher order aberrations may not optimize visual function in highly aberrated eyes. The new algorithm minimized tissue removal in refractive surgery but further clinical evaluations are required to confi rm preliminary results.PURPOSE To report a case in which a tissue saving program in an aberrated eye was used. METHODS A new algorithm for the selection of an optimized set of Zernike terms in customized treatments for laser corneal refractive surgery was developed and clinically tested. Ablation was performed using the SCHWIND ESIRIS excimer laser. Pre- and postoperative corneal wave aberrations were analyzed using the Keratron Scout videokeratoscope (Optikon 2000). RESULTS Required ablation was reduced by approximately 15% compared to full customized correction. Refraction was corrected to subclinical levels, uncorrected distance visual acuity improved to 20/20, corrected distance visual acuity gained 2 lines, aberrations were reduced by approximately 40% compared to preoperative baseline levels, and the functional optical zone of the cornea was enlarged by approximately 40% compared to preoperative baseline levels. Trefoil, coma, spherical aberration, and the root-mean-square value of the higher order aberrations were reduced. CONCLUSIONS Eliminating all higher order aberrations may not optimize visual function in highly aberrated eyes. The new algorithm minimized tissue removal in refractive surgery but further clinical evaluations are required to confi rm preliminary results.