Rudolf Guthoff

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 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 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.

Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

Purpose: Evaluation of a new method to visualize distribution and morphology of human corneal nerves (Aδ- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. Methods: Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 μm thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope “Odyssey XL,” step size between 0.5 and 1 μm, and optical sections were digitally 3D-reconstructed. Results: Immediate staining of explanted corneas by the Life/Dead-Kit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, Aδ- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. Aδ-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, Aδ-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. Conclusions: Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

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.

The Keratocyte Network of Human Cornea: A Three-dimensional Study Using Confocal Laser Scanning Fluorescence Microscopy

Purpose. Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. Methods. Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of ≤270 &mgr;m in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. Results. Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 ± 5.05%) and lowest in the central stroma (9.31 ± 1.09%). In the anterior stroma, volume density was found to be 10.19 ± 4.37%. Conclusion. Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.

Improved penetration of aminoglycosides and fluoroquinolones into the aqueous humour of patients by means of Acuvue contact lenses

AbstractObjectives: In order to improve the penetration of topically applied drugs in ophthalmology, the suitability of hydrophilic contact lenses (Acuvue, Vistacon, power −1.0 D) as a drug delivery system for antibiotics was tested. A prospective study was undertaken to determine the transcorneal penetration of five topically applied aminoglycosides and fluoroquinolones into the aqueous humour of patients. Methods: Two hundred and sixty-five patients undergoing cataract extraction received 0.3% gentamicin, kanamycin, tobramycin, ciprofloxacin or ofloxacin solution by two different modes of administration: either as eye drops (nine drops every 15 min, starting 2 h prior to surgery) or by means of a drug delivery system (Acuvue contact lenses soaked for 1 h in eye drop solution without preservatives, 1–5 h prior to surgery). At the beginning of cataract extraction, 50–100 μl aqueous fluid was aspirated from the anterior chamber and immediately stored at −80 °C. Antibiotic concentrations were measured using fluorescence polarisation immuno-assays (aminoglycosides) or high-performance liquid chromatography (fluoroquinolones). Results: After soaking for 1 h in 0.3% eye drop solutions, Acuvue contact lenses released about 190–250 μg aminoglycoside and ofloxacin and 1000 μg ciprofloxacin. These amounts are considerably lower or in the same order of magnitude than obtained with application of eye drops (1350 μg).From the aminoglycosides tested, only gentamicin and tobramycin, but not kanamycin, were able to penetrate into the aqueous humour of patients. After the wearing of antibiotic-soaked lenses, mean aqueous humour concentrations were higher than after the use of eye drops. This difference reached significance in tobramycin (1.09 (1.30) μg · ml−1 vs 0.49 (0.79) μg · ml−1), ciprofloxacin (1.23 (0.60) μg · ml−1 vs 0.38 (0.33) μg · ml−1) and ofloxacin (5.55 (2.53) μg · ml−1 vs 0.56 (0.37) μg · ml−1).The percentage of patients with aqueous humour concentration above the MIC90 of Staphylococcus epidermidis, the most common cause of postoperative endophthalmitis, was 92% and 100% after wearing ciprofloxacin- or ofloxacin-soaked lenses, respectively. Conclusion: Gentamicin and tobramycin penetrated into the aqueous humour of patients, whereas kanamycin was not able to overcome the corneal barrier. Acuvue contact lenses soaked in 0.3% eye drop solutions can release sufficient amounts of gentamicin, ciprofloxacin and ofloxacin to produce bacteriostatic concentrations in the humor aquosus. Acuvue contact lenses can be recommended as a drug delivery system for fluoroquinolones.

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.