[Modified Heidelberg Retinal Tomograph HRT. Initial results of in vivo presentation of corneal structures]. / Der modifizierte Heidelberg-Retina-Tomograph HRT. Erste Ergebnisse einer In-vivo-Darstellung von kornealen Strukturen.

BACKGROUND: 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.

[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.

Electrophoretic fingerprinting and multiparameter analysis of cells and particles.

A recently developed electrophoretic instrumentation based on a real-time image processing system has been applied to electrophoretic fingerprinting and multiparameter analysis of cells and other particles. The comparison between theoretical and experimental electrophoretic fingerprints, completed by the analysis of differences between measured fingerprints, offers a new methodology for better understanding and controlling of the processes at solid/liquid interfaces. Moreover, the multi-parameter analysis including electrophoretic mobility, size, density and shape of cells can complete the diagnosis and prognosis of diseases.

New methods for the investigation of blood-biomaterial interaction.

Quantitative microscopy with integrated image processing is a useful tool for investigation of the interaction of blood components with biomaterials. We have developed new automated measuring devices suitable for simultaneously characterizing biological cells (size, shape, localization, migration, electrophoresis), synthetic particles (electrophoretic fingerprinting), and dialysis membranes (morphology, electric charge). These techniques are useful for the investigation of cell adherence on biomaterials, localization of cells in membrane filters (Chemotaxis), characterization of the protein adsorption on model systems, detection of cytokines (produced after lymphocyte-biomaterial contact), and estimation of morphological properties and charge distribution in dialysis membranes.

Electrophoretic mobility profiles: new aspects of characterization and discrimination of biological surfaces.

An advanced method of measuring and analyzing electrophoretic mobility profiles (electrophoretic fingerprints) was developed and for the first time applied to the cell surface. Dissociation constants calculated from a suitable mathematical model describe dissociation and, in part, adsorption processes on the surface and can thus be used for the characterization of complex processes on biological surfaces and the evaluation of small differences between cell surfaces.

[Susceptibility of leaves and fruits of tomato varieties and of their hybrids to attack by Phytophthora infestans]. / Die Prüfung von Tomatensorten und ihren Hybriden auf Blatt- und Fruchtbefall mit Phytophthora infestans.

1. The investigations were started to establish a correlation between the incidence of Phytophthora infestans (Mont.) de Bary on leaves and on fruits of tomatoes, the mechanism of the inheritance of the resistance and the number of responsible genes. 2. A close correlation was found between the spontaneous incidence on leaves and fruits and the results of artificial inoculation by the leaf-disc-test. The resistant types especially showed a remarkably low percentage of attack in all three tests. Resistant plants were successfully selected by means of the leaf-disc-test; the leaves as well as the fruits of the progeny were largely free from the disease. 3. The field-resistance of the variety 'Atom' was demonstrated to be due to a high degree of relative resistance of the leaves against the races T0 and T1. 4. The results obtained from F1-hybrids of 'Atom' with various more or less susceptible varieties indicate the participation of incompletely dominant genes. Since in the F2 a certain number of plants with a high degree of field-resistance could be selected, it is suggested that the field-resistance against Phytophthora infestans is based on a few genes only. From our results we conclude that principally two genes in the variety 'Atom' determine the field-resistance against the fungus. A participation of other modifier-genes can be supposed. We propose the gene-symbols Phf and Phf-2 for the Phytophthora-field-resistance.