[Keratinocyte density of the cornea in vivo. Automated measurement with a modified confocal microscopy MICROPHTHAL]. / Keratozytendichte der In-vivo-Kornea. Automatische Messung mit einem modifizierten konfokalen Mikroskop MICROPHTHAL.

BACKGROUND: The MICROPHTHAL is a confocal slit light scanning microscope for a non-invasive in-vivo examination of corneal structures of human eyes. With this instrument even thin layers of corneal tissue can be imaged in good quality. Otherwise, blurring of single frames and deviations from the z-axis in video-sequences caused by high speed movements of the eye would normally prevent a measurement the density of keratocytes in the cornea. The goal of the investigation was optical pachymetry, the automatical measurement of the keratocytes density and a 3D-dimensional reconstruction of the central cornea in-vivo under constant imaging conditions. MATERIALS AND METHODS: We developed a low-vacuum suction cup system for stabilizing the eye in front of the microscope objective during the z-scan through the cornea. A stepmotor shifting system for the objective locates inside the suction cup with a central hole was installed underneath them icroscope. Control of this system via computer facilitated shifting the focal plane along the z-axis. The layer images were recorded using a S-VHS-tape and saved on the PC. The digital analysis was performed using a special software to automatically and off-line evaluate the density of keratocytes in combination with the 3D-reconstruction. The software also corrected the background illumination and small axial jitter. After this procedure the keratocytes density and the 3D-reconstruction in 70 images of the z-scan were calculated. We examined 47 corneas of 25 healthy probands. The range of age was 25-56 years. Independent control evaluation of the video sequences were taken manually on an INDIGO HIGH IMPACT workstation. RESULTS: By assign all keratocytes to the corneal measurement volume we found a averaged density of 15,730 cells/mm3 in the central cornea. The averaged thickness of the cornea was 0.556 mm. The control valuation of identical video-sequences on the workstation accomplished the same result of 16,000 keratocytes/mm3, also similar the result of the automatically measurement with the modified software. CONCLUSIONS: This modification of the microscope is a promising in-vivo tool for optical pachymetry and quantitative examination of corneal microstructures. The stabilization effect of the low-vacuum suction cup system in the front of the microscope for computer-controlled valuation of the density profile of keratocytes and the 3D-reconstruction of a central corneal volume element has produced encouraging results. Characterization of pathophysiological changes in the distribution of keratocytes after excimer laser ablation for phototherapeutic or photorefractive keratectomy, for example, can be estimated without pain for the patients.

[In vivo imaging of corneal innervation in the human using confocal microscopy]. / In-vivo-Darstellung der Hornhautinnervation des Menschen mit Hilfe der konfokalen Mikroskopie.

To date, descriptions of the structure of corneal innervation have only been possible on the basis of histological techniques. Confocal microscopy represents a new method for the structural examination of the cornea in vivo. Through our examinations we first defined the control group and then proceeded to record the reinnervation of donor tissue after perforating keratoplasty. We used the confocal slit-scanning video-microscope Microphthal to examine 40 corneas from 20 normal volunteers, 15 donor corneas and 5 eyes after enucleation for ocular tumors. These results were compared to our findings from postoperative checks on 14 patients after perforating keratoplasty (from 1 month to 2 years). With this system we were able to see nerves in the middle and in the superficial stroma. The course of these nerves can be followed, as well as their branching in the subepithelial plexus. Nerve fibers from superficial stromal nerves penetrate Bowman's membrane and create the basal epithelial plexus in the region of the basal epithelium. Seven months after perforating keratoplasty the first stromal nerves could be seen in the central corneal area. The first central reinnervation in the region of Bowman's membrane as well as in the basal epithelium was not detected until 15 months after operation. With confocal microscopy we have the potential to study the morphology of corneal innervation in vivo and in fresh donor tissue. For the first time it is possible to perform non-invasive morphological studies of reinnervation of the human cornea after surgical treatment.

[Detection of morphological corneal changes caused by chloroquine therapy using confocal in vivo microscopy]. / Erfassung morphologischer Hornhautveränderungen infolge Chloroquintherapie mit Hilfe der konfokalen in-vivo-Mikroskopie.

BACKGROUND: Chloroquine keratopathy is known as a disease with epithelial deposits in the cornea. The appearance of corneal changes does not seem to be related to dose and duration of treatment. Chloroquine accumulates in lysosomes. The purpose of this study was to investigate microscopic changes of corneal morphology in patients treated with chloroquine in vivo. PATIENTS AND METHODS: Using the confocal slit scanning microscope, we examined 16 patients during chloroquine treatment and 4 patients after treatment. The patients' age was between 33 and 75 years. They were treated with chloroquine over a minimum period of 2 months up to a maximum period of 10 years. RESULTS: In 12 of 20 patients, corneal changes were visible. An atypical inverse reflectivity of basal cells in comparison to healthy volunteers was found. There were also deposits in the wing cell layer. We saw a higher density in the anterior stroma before the appearance of epithelial opacities, as well as in cases of keratopathy on slit-lamp examination and also after the cessation of chloroquine treatment. In one patient with long-term therapy, there were atypically shaped and branched nerves in the anterior stroma. CONCLUSIONS: Using confocal microscopy, it was in some cases possible to detect changes in the corneal morphology before these changes could be detected by slit-lamp examination. As a result of this study we found that chloroquine keratopathy is not limited to the epithelium but can affect the anterior stroma and possibly neural structures.

Fluorescence microscopy and three-dimensional imaging of the porcine corneal keratocyte network.

BACKGROUND: Little is known about the spatial arrangement and the corresponding morphometric data describing the living keratocyte network. For determination of alterations in corneal diseases it is crucial to know the morphology of the keratocyte network in the healthy state. Porcine cornea was used as a model tissue because it allows the study of species differences. METHODS: Corneas from freshly enucleated pig eyes were stained with calcein AM and ethidium homodimer and examined by confocal laser scanning microscopy. High-resolution fluorescence images were used for three-dimensional reconstructions from which cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished and visualized in their spatial arrangement. Significant differences with respect to both shape and fluorescence intensity distribution of the cell bodies were found. Cell volume density was 7.7% in the anterior stroma, 13.7% in the central stroma and 11.8% in the posterior stroma. CONCLUSION: The technique described allowed good visualization of the spatial arrangement of the keratocyte network. Combined with morphometric methods, the analysis of the state of the cornea yields a quantitative description. The method is expected to be useful for the determination of morphological alterations in corneal disease or following surgical treatment.

Corneal reinnervation following penetrating keratoplasty--correlation of esthesiometry and confocal microscopy.

Thus far assessment of corneal reinnervation after penetrating keratoplasty has been possible only by esthesiometry techniques. Since the introduction of confocal microscopy, we have been capable of performing structural corneal in vivo examinations. The purpose of our study was to correlate esthesiometry results with confocal microscopy findings. We used a Cochet-Bonnet esthesiometer and a Microphthal confocal microscope to investigate corneal grafts in vivo. A total of 46 eyes were examined preoperatively and for up to 3 years after penetrating keratoplasty. At 8 weeks after keratoplasty the first stromal nerves were detected in the periphery of the graft. The first nerves in the middle and superficial stroma of the graft center were observed at 7 months after surgery. Reinnervation of the central basal epithelium was found at 2 years after keratoplasty. The highest level of sensitivity was detected in young patients with reinnervation of the basal epithelium. Confocal microscopy enables us to correlate morphologically and functionally corneal reinnervation after surgery. The present study shows that corneal reinnervation is influenced by the amount of time elapsing after surgery, the patient's age, and the preoperative diagnosis. In none of our patients was normal nerve morphology or sensitivity observed during the follow-up period. Comparison of the morphology of nerves seen in eyes after nonsurgical trauma with that observed in corneal grafts indicates that surgically induced scar formation may limit nerve regeneration in grafts.

Confocal in vivo microscopy and confocal laser-scanning fluorescence microscopy in keratoconus.

The purpose of this study was the determination of morphological changes in the corneal epithelium and the keratocyte network in keratoconus. In all, 33 eyes of 19 patients were examined in vivo using the confocal slit-scanning microscope Microphthal. After penetrating keratoplasty, recipients' trephanates were stained with the Live/Dead kit and examined using the confocal laser-scanning fluorescence microscope Diaphot 300/Odyssey. The fluorescence images were reconstructed three-dimensionally. All findings were compared with data from healthy corneas. Morphological alterations were found only in the area of the corneal apex; obviously elongated superficial epithelial cells arranged in a whorl-like fashion were found. Near Bowman's membrane, highly reflective changes and fold-like structures were visible. The anterior stroma also showed an increased reflectivity. In the posterior stroma, typical findings were Vogt's striae and keratocytes with extremely long processes arranged nearly in parallel. In scarred stroma the keratocytes were spindle-shaped and arranged irregularly. The spatial organization of the living keratocyte network could be demonstrated through three-dimensional reconstructions.

Assessment of corneal alterations following laser in situ keratomileusis by confocal slit scanning microscopy.

This investigation was done to evaluate in vivo changes in corneal micromorphology occurring after laser in situ keratomileusis. By means of confocal microscopy, nine eyes of eight patients showing uncomplicated wound healing at 2-16 weeks after surgery and one eye of one patient at 30 weeks after initial surgery who had to undergo two retreatments because of a central island were examined. Video recording was used to register the data. The distribution of scattered light in the treated corneae was registered with the z-scan of the microscope. Slit-lamp findings obtained several weeks postoperatively showed only minor corneal alterations. Confocal microscopy revealed morphological changes as a result of the surgery, especially in the epithelium and in the anterior stroma. With the z-scan an additional peak determined by higher reflectivity in the anterior stroma was found. The retreated patient showed marked folds in the area of the interface and flap. Confocal in vivo microscopy is suitable for the noninvasive detection of micromorphological alterations following refractive corneal surgery and for observation of the postsurgical wound-healing phase, including nerve regeneration.

[Confocal in vivo microscopy after 15 mm sclerocorneoplasty à chaud in necrotizing keratitis]. / Konfokale In-vivo-Mikroskopie nach 15-mm-Sklerokorneoplastik à chaud bei nekrotisierender Keratitis.

BACKGROUND: Confocal microscopy allows an in-vivo visualization of corneal structures in frontal optical sections. By means of this method we show morphological changes in a corneal transplant after sclerocorneoplasty à chaud because of a necortizing keratitis. CASE REPORT AND METHOD: We report on a patient who had to undergo a 15 mm diameter sclerocorneoplasty à chaud because of a necrotizing keratitis of his right functionally last eye in October 1993. Five months after transplantation there was a circular vascularization of the scleral rim and a progressive crystalline deposition in the periphery of the cornea. The main part of the donor cornea remained clear with a vision of 0.6. After one year the patient had developed a mature complicated cataract and got a phacoemulsification with a tunnel in the transplanted cornea and an implantation of a posterior chamber lens. In May 1995 the visual acuity was 0.9. We examined the epithelium, stroma and endothelium of the graft by means of in-vivo confocal slit scanning microscopy. RESULTS: All layers of the cornea are demonstrated. There were crystalline needle-like structures in the peripheral stroma and round cystic bodies in the deep stroma. Nerves were visible one year after transplantation. The endothelial cells showed a mild polymorphism and still a density of 2200 cells/mm2. CONCLUSIONS: Confocal microscopy is a useful non invasive technique for routine examinations after keratoplasty. Microscopical changes in the tissue are also visible in case of absent postoperative complications. With growing knowledge the confocal microscopy will give more insight into processes of wound healing.