Oliver Stachs

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 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 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.

Spatially resolved Brillouin spectroscopy to determine the rheological properties of the eye lens

Presbyopia is closely associated with the loss of accommodation, and hence with a decline in the viscoelastic properties of the human eye lens. In this article we describe a method for obtaining spatially resolved in vivo measurements of the rheological properties of the eye lens, based on the spectroscopic analysis of spontaneous Brillouin scattering using a virtually imaged phased array (VIPA). The multi-pass configuration enhances resolution to the extent that measurements are possible in elastic biological tissue characterized by intense scattering. We also present spatially resolved measurements obtained in extracted animal eyes and lenses. The results yield entirely new insights into the aging process of the eye lens.

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.

Comparative in Vivo Confocal Microscopical Study of the Cornea Anatomy of Different Laboratory Animals

Purpose: The aim of the present study was to analyze and compare in vivo morphology of healthy cornea of six different laboratory animals. Matherials and Methods: One Pomeranian Coarsewool sheep, 5 Beagle dogs, 1 Norwegian and 2 Domestic Short-haired cats, 20 New Zealand White rabbits, 6 Wistar rats, and 10 Balb/c mice were included. The examination was performed bilaterally, using Heidelberg Retina Tomograph equipped with Rostock Cornea Module. The morphology of living corneal layers was visualized and compared between species. The central corneal thickness, density of keratocytes, and endothelial cells were quantified. Results: The epithelial multilayer showed a similarity in morphology between animal types, displaying three clearly distinguishable layers: superficial, intermediate, and basal. Subbasal nerve fibers were displayed as hyperreflective structures underneath basal cells. The subbasal fibers were confirmed in all species, however, the density varied between species. A pronounced Bowman’s membrane was visualized in sheep. In all other species, however, a thin acellular layer with overlying nerve fibers could be seen between basal epithelial cells and anterior stroma. The keratocytes nuclei could be demonstrated in all species except for mice, where no nuclei but only reflective structures resembling keratocytes cell bodies were detected. Overall, the density of keratocytes nuclei was significantly higher in the anterior than in the posterior stroma. Besides endothelial cells density, the endothelial cells morphology was very similar among all species, except for sheep. The endothelial cells were displayed as polygonal structures with bright cytoplasm and dark borders. In sheep, the appearance of the endothelium was very poor because of a thick hyperreflective Descemet’s membrane. Conclusions: The present study will help researchers consider appropriate models for animal experiments, depending on focus of investigation. In vivo CLSM can be used for the characterization of the living cornea over time, thus, reducing the number of animal experiments.

Real-time mapping of the subepithelial nerve plexus by in vivo confocal laser scanning microscopy

Aim To produce two-dimensional reconstruction maps of the subepithelial nerve plexus (SEP) in living cornea by in vivo laser scanning confocal microscopy in real time. Methods In vivo confocal laser scanning microscopy (Heidelberg Retinal Tomograph II in conjunction with the Rostock Cornea Module) was performed on normal eyes (n=6) and eyes after laser-assisted in situ keratomileusis (LASIK) (n=4). Source data (frame rate 30 Hz) were used to create large-scale maps of the scanned area in Automatic Real Time composite mode. The algorithm aligns single live images onto the previously mapped composite image using landmark feature-based image processing. Results Real-time mapping of the SEP was performed on a large-scale area up to 3.2×3.2 mm (3072×3072 pixels) in healthy subjects and in post-LASIK patients. Two-dimensional structures of the SEP were imaged in all 10 eyes. Mapping quality as well as acquisition time were dependent on subject compliance and examiner experience. Conclusion The described method permits real-time in vivo mapping of the SEP, thus providing the necessary basis for statistically robust conclusions concerning morphometric plexus alterations.