Joachim Stave

Der modifizierte Heidelberg-Retina-Tomograph HRTErste Ergebnisse einer In-vivo-Darstellung von kornealen Strukturen

ZusammenfassungHintergrund. Zur konfokalen In-vivo-Abbildung von kornealen Strukturen werden gegenwärtig in der Regel Tandemschlitz-Scanning-Mikroskope mit Halogen- oder Hg-Lampen eingesetzt. Die systematisch ungleichmäßige Objektausleuchtung bei diesen Systemen gestattet eine automatische Bildauswertung nur unter erheblichem Korrekturaufwand. Unser Ziel war der Aufbau eines digital-konfokalen Laser-Rastermikroskops zur Darstellung der vorderen Augenabschnitte auf der Basis des Heidelberg-Retina-Tomographen HRT, gekoppelt mit einer speziellen Software zur automatischen Quantifizierung von Korneaparametern, z. B. der Keratozytendichte. Material und Methode. Wir entwickelten für den HRT einen Objektivvorsatz zur Fokussierung des Lasers auf die Kornea in Kombination mit einem computergesteuerten externen hydraulischen z-Scan-System. Durch eine programmierbare Adaptierungselektronik für den externen Schrittmotor des z-Antriebs unter Ausnutzung aller internen Steuer- und Bildspeicherfunktionen einschließlich der Patientenverwaltung sowie unter Beibehaltung der Originalfunktion des HRT entstand ein digital-konfokales Laser-Scanning-Mikroskop. Zur Bildauswertung und automatischen Keratozytenzählung wird eine spezielle 3D- bzw. CHEMOTAXIS®-Software eingesetzt. Ergebnisse. Erste Untersuchungen zeigen eine gleichmäßige Bildausleuchtung des Epithels, des Endothels sowie der Keratozytenkerne. Der hydraulische z-Scan gestattet eine präzise und ruckfreie Aufnahme von Bildserien in 0,7 s und damit eine reproduzierbare Bestimmung des Keratozytendichteprofils sowie eine dreidimensionale Darstellung aller Hornhautstrukturen.AbstractBackground. At present, confocal tandem scanning microscopes with halogen or mercury lamps are used to depict all corneal structures in vivo, e.g., before and after PRK or LASIK. Insufficient imaging quality and irregular corneal illumination are the main problems for automatic quantitative evaluation of the keratocyte density when applying this instrument. A high correction is required for correcting the background irregularities of pictures. Our aim was to find out whether it is possible to change the Heidelberg retina tomograph (HRT) into a high-resolution digital laser scanning microscope for the visualization of anterior segments of the eye, coupled with a special evaluation software. Material and method. We developed a lens adapter for the HRT that focusses the laser beam onto the cornea by combining with an external, computer-controlled hydraulic z-scan sledge. By using a programmable adaptation for the external stepmotor on the z-scan sledge in combination with all internal control functions and patient data, it is possible to create a digital confocal laser scanning microscope with retention of all the original HRT functions. For evaluation of the corneal images and automatic count of keratocytes, we used special 3D and Chemotaxis software. Results. First investigations show a regular illumination of all corneal structures as the epithelium, endothelium, and keratocytes. The hydraulic z-scan allowed a precise shift of the focus through the cornea to take series of images for the evaluation of the keratocyte profile and 3D reconstruction of all corneal structures.

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 investigations of the corneal epithelium with the confocal Rostock Laser Scanning Microscope (RLSM).

The confocal tandem scanning microscope was first used in 1985 by Lemp et al for in vitro and in 1990 by Cavanagh et al for in vivo investigation of human eyes. The aim of this study was to investigate the cells of the central and the peripheral portions of the corneal epithelium and to measure corneal epithelium thickness and the total thickness of the corneas of our volunteers with the new Rostock Laser Scanning Microscope. Material and Methods: A Heidelberg Retina Tomograph (HRT II) was used in combination with a water contact microscope lens (Zeiss, ×63, 0.95), the Rostock cornea module (RCM) developed at our institute for the in vivo examination of the cornea. In this study, 92 eyes of 68 subjects between the ages of 15 and 88 years were examined. Results: At the superficial cell layer, the average cell density in the central cornea was 840 ± 295 cells/mm2, and in the periphery it was 833 ± 223 cells/mm2. At the wing cell layer, the average cell density rises to 5070 ± 1150 cells/mm2 in the central and to 5582 ± 829 cells/mm2 in the peripheral cornea. At the basal cell layer, the cell density rises further to 8996 ± 1532 cells/mm2 in the central and 10,139 ± 1,479 cells/mm2 in the peripheral corneal epithelium. The average corneal thickness in the central region was found to be 545 ± 25 μm, and 652 ± 75 μm in the periphery. The average epithelium thickness was determined centrally to be 54 ± 7 μm, and peripherally 61 ± 5 μm. Conclusions: The Rostock Scanning Laser Microscope offers a standardized, reproducible, safe, and fast diagnostic procedure for the evaluation of the corneal epithelium. This technology allows better image quality compared with confocal-slit scanning microscopes and produces a precise depth measurement.

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.

Depth and age-dependent distribution of keratocytes in healthy human corneas: A study using scanning-slit confocal microscopy in vivo

Purpose: To document keratocyte distribution and changes with age in the cellular network of the human cornea in vivo. Setting: Department of Ophthalmology, University of Rostock, Rostock, Germany. Methods: Forty‐nine eyes of 31 healthy subjects of various ages were examined with a modified Microphthal® scanning‐slit confocal microscope (SSCM) (Hund) to document keratocyte distribution in the intact living cornea. Optical sections made by confocal microscopy were recorded on videotape, and the keratocyte density was determined for the total volume of the cornea and for the stromal sublayers. Results: The highest cell density was in the anterior stroma of the cornea immediately posterior to Bowmans membrane (24 320 cells/mm3 ± 6740 [SD]), the lowest in the central area (11 610 ± 4290 cells/mm3), and an intermediate density in the posterior stroma immediately adjacent to Descemets membrane (18 850 ± 4610 cells/mm3). The differences were statistically significant (P < .005). The keratocyte density was significantly lower in the anterior and posterior regions in the group older than 50 years: Cell density at 4% depth was 20 960 ± 8200 cells/mm3 and at 96%, 15 520 ± 4290 cells/mm3 (P < .05). Conclusions: In healthy living corneas, the keratocyte density was high in the areas adjacent to Bowmans and Descemets membranes and was lower in patients older than 50 years than in those younger than 50 years. Further studies are needed to document the rate of change with age and to better understand the role and capacity of aging keratocytes in regenerative processes following corneal diseases or surgical procedures.

In-vivo Three-Dimensional Confocal Laser Scanning Microscopy of Corneal Surface and Epithelium

Background/aims: To evaluate in vivo three-dimensional (3-D) confocal laser scanning microscopy (CLSM) as a technique for visualising the corneal surface and epithelium. Methods: Ten human corneas (three from healthy volunteers, three with bullous keratopathy, three from patients following penetrating keratoplasty, and one with corneal erosion) were examined by 3-D CLSM. A novel polymethyl methacrylate (PMMA) contact cap was designed to minimise artefacts due to applanation pressure. Results: 3-D reconstruction and different visualisation techniques (volume rendering, cross-section, en face view, oblique section and surface reconstruction) were performed to demonstrate alterations to corneal surface and epithelium. Image quality (cell identification, motion blur, absence of compression artefacts, imaging of superficial structures and of subepithelial nerve plexus) was considerably superior to that obtained using a conventional contact cap with a planar surface. Conclusions: 3-D CLSM permits in vivo visualisation and analysis of the corneal surface and of spatial arrangement at the cellular level in epithelium in normal and pathological corneas. The novel design of the contact cap minimises artefacts due to applanation pressure and improves the image quality of epithelial structures. The method provides a basis for further in vivo studies of alterations to corneal surface structure and its cellular arrangement.

Potentially accommodating intraocular lenses--an in vitro and in vivo study using three-dimensional high-frequency ultrasound.

PURPOSE To investigate the accommodative performance of new intraocular lenses (IOL) using the advantages of three-dimensional ultrasound biomicroscopy. METHODS An in vitro simulation device was designed to study IOL performance using an artificial capsular bag and a stretching device. The haptic region of the Akkommodative 1CU (HumanOptics AG) and CrystaLens AT-45 (Eyeonics Inc) was visualized in vitro in three dimensions, using an in-house developed three-dimensional ultrasound biomicroscope. The in vitro results were used to describe the in vivo situation in four patients with accommodative implants. RESULTS The haptic position and angulation in consideration of the accommodation state was distinguished and analyzed. In the simulation model, a maximal angulation change of 4.5 degrees and 4.3 degrees and a maximal forward shift of 0.33 mm and 0.28 mm was observed for the AT-45 and 1CU, respectively. In vivo, a change in haptic angulation <100 and a maximal forward shift of 0.50 mm was observed for the 1CU. These changes correspond to a theoretical approximate value of 0.50 diopters. CONCLUSIONS The in vitro simulation device examined with three-dimensional ultrasound biomicroscopy provided information on the accommodative performance of these potentially accommodative IOL designs. Using three-dimensional ultrasound biomicroscopy, corresponding changes in haptic angulation during pharmacological-induced accommodation were observed.

In vivo observation of papillae of the human tongue using confocal laser scanning microscopy.

The aim of this investigation was to visualize the epithelial structures of the tongue using confocal laser scanning microscopy (LSM). The human tongue epithelium of 28 healthy subjects, aged 21–67 years, mean age 38 years, 14 women and 14 men, was examined in vivo by LSM. Using LSM, a combination of the Heidelberg Retina Tomograph HRT II and the Rostock Cornea Module, up to 800-fold magnifications were obtained. On the tongue surface both filiform and fungiform papillae and their taste pores were easily identified. The epithelium of the tongue with its subcellular structures could be observed up to a depth of 50 µm, cellular structures up to 150 µm and subepithelial vessels up to 300 µm. Additionally the papillary crests and blood flow were visible. Confocal LSM seems suitable for noninvasive in vivo examination of the tongue. The hydraulic z scan, the manual start setting and the measurement of the depth allow a clear classification of the observed structures.

Contribution to comprehension of image formation in confocal microscopy of cornea with Rostock cornea module

Aim: To investigate the influence of refractive index of aqueous humour on imaging of corneal endothelium in confocal microscopy. To clarify the phenomenon of dark endothelial and bright epithelial cell membranes in confocal images of corneas. Methods: Use of a novel digital confocal laser scanning microscope, a combination of the Heidelberg retina tomograph (HRT II) and the Rostock cornea module. Exchange of aqueous humour solution from domestic pigs against glycerol/water solutions (refractive indices η = 1.337−1.47). Transelectron microscopy of endothelial and epithelial cell morphology. Results: Under the terms of variable refractive indices no differences were observed for general imaging of endothelium. Bright cells were bordered by dark cell membranes in all experiments. Electron microscopy of endothelium and epithelium revealed differences in intracellular and cell membrane structure of both cell types. Conclusion: Source of specific confocal optical behaviour of endothelium does not come from interface conditions to aqueous humour, but may result from intracellular variations and ultrastructure of cell membranes.