Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior.

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.

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.

[In vivo diagnosis of epithelial changes of the oropharynx using confocal microscopy]. / In-vivo-Diagnostik von Epithelveränderungen des Oropharynx mittels konfokaler Mikroskopie.

BACKGROUND: Confocal laser scanning microscopy enables the visualization of the anterior regions of the oropharynx mucosa. The specific aim of this investigation was to evaluate whether this in vivo tool supplies essential information for the surgeon prior to operation or not. PATIENTS AND METHODS: The laser scanning microscope HRT II and Rostock Cornea Module were used in this in vivo study. To obtain comparable images, the specifications of this tool used for all investigations were maintained (63 x water immersion objective lens). The investigations were performed on 9 patients with tongue cancer with primary tumor site and stage I (AJCC) and on 12 patients with head and neck cancer who underwent radiochemotherapy (RCT). Data from 21 patients were compared to those of healthy subjects. RESULTS: The following parameters can be detected using LSM: nuclear density, nuclear size, nucleus/cytoplasm relation, number of nuclei, regularity of cell layers, morphology of cells of a cell layer, and occurrence of cellular junctions. In regard to these parameters, dysplastic and cancerous lesions reveal significant differences compared to healthy tissue of the oropharynx. After RCT several epithelial changes were found, such as keratosis, mild dysplasia, increased vascularization and more cell edema and necrosis. CONCLUSIONS: Consistent differences exist between LSM findings of healthy subjects and those of patients with dysplastic, keratotic and cancerous lesions. Both strong hyperplasia and leukoplakia prevent a visualization of the basement cell layer in the oropharynx. The combination of LSM technology and endoscopy and following further investigations are needed for evaluation of LSM technology in the field of otorhinolaryngology.

[Confocal microscopic evaluation of epithelia of the larynx]. / Konfokalmikroskopische Untersuchungen des Kehlkopfepithels.

BACKGROUND: To evaluate mucosal changes of the true vocal cord, e. g. leukoplakia, microlaryngoscopy is needed to obtain tissue for histopathologic investigation. But there is a discrepancy between the amount of representative tissue necessary for pathology and the postoperative voice function. Thus, a diagnostic procedure that enables the surgeon to differentiate inflammation from dysplasia and carcinoma in situ intra-operatively is urgently needed. We are now reporting on our results using laser scanning microscopy to visualize larynx epithelia. METHODS: The laser scanning microscope used in this ex vivo study is a combination of the Heidelberg Retina Tomograph HRTII and the Rostock Cornea Module. A 63 x water immersion objective lens was used for imaging. PATIENTS: Seven larynx preparations of patients (age 54 to 62 years) with a T4 larynx carcinoma who underwent laryngectomy between 2003 and 2006 were examined. RESULTS: Areas of epithelia of the true vocal cord of macroscopically normal appearance supplied images of healthy, but also inflamed laryngeal epithelia. The corresponding histopathologic sections prepared horizontally confirmed the regularity and homogeneity of the mucosa. The different layers of the lamina propria can be separated. In contrast to healthy and inflamed laryngeal mucosa, dysplasia, precancerous lesions and carcinoma-atypical cells were visible. In the latter entity a loss of cellular junctions characterized by lack of visualization of the cell membrane was apparent. CONCLUSIONS: LSM supplies information of healthy, inflamed, but also pre-malignant and malignant mucosa changes of the larynx. The pilot study shows that both hyperkeratosis and extensive hyperplasia confine the visualization of the basement cell layer. Research projects have been initiated with the objective to develop a rigid endoscope with integrated LSM technology.

[Three-dimensional confocal laser scanning microscopy of the corneal nerve structure]. / 3D-konfokale Laser-Scanning-Mikroskopie der kornealen epithelialen Nervenstruktur.

PURPOSE: The aim of this study was the evaluation of a technology for in vivo visualization of distribution and morphology of corneal nerves by means of 3D confocal laser scanning microscopy (3D-CLSM). METHOD: The anterior corneas of four human volunteers were examined by an in-house developed confocal laser scanning microscope based on a commercially available instrument (Heidelberg Retina Tomograph II, Heidelberg Engineering GmbH, Germany). Raw stacks were converted using ImageJ (NIH, USA) for 3D-reconstruction using AMIRA 3.1 (TGS Inc, USA). RESULTS: The spatial arrangement of epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. After 3D-reconstruction of volunteers' corneas, volume rendering and selective oblique sections have been done to demonstrate the nerves in the central human cornea. 3D-imaging shows thick nerve bundles rising out of the deeper stroma. The nerves further divide, resulting in fibers that are arranged parallel to Bowman's layer and are partly interconnected. Branches rising up to the superficial cell layer cannot be visualized. Wound healing following refractive surgery can be evaluated. CONCLUSIONS: 3D-CLSM allows in vivo visualization and analysis of the spatial arrangement of the epithelium, nerves and keratocytes of the human cornea. The developed method provides a basis for further studies on the alterations of the cellular arrangement and epithelial innervation in corneal diseases. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

[In vivo imaging of the conjunctival epithelium using confocal laser scanning microscopy]. / In-vivo-Darstellung des Bindehautepithels. Eine vergleichende morphologische Untersuchung mittels konfokaler Laser-Scanning-Mikroskopie und Impressionszytologie.

BACKGROUND: In various ocular diseases, cytomorphological findings of the ocular surface are an essential component of clinical diagnostics. When evaluating the conjunctival epithelium, minimally invasive acquisition of biomaterial is necessary for lab and technical processing and in vitro histological examination. To examine corneal structures in vivo, confocal laser scanning microscopy is a successful standard method. Our aim was to employ in vivo confocal laser scanning microscopy also for examining the conjunctival epithelium. MATERIAL AND METHOD: Results were analyzed and compared with cytomorphological findings of impression cytology. Accordingly, the basic features of conjunctival in vivo examination using RLSM were described and defined. In vivo images were analyzed and compared with impression cytological slide preparations (n=110) of 23 healthy test persons. Examination was standardized. Finally, the confocal laser scan images were compared to the impression cytological patterns. RESULTS: Due to the distribution of reflectors (pixel brightness), diagnostic analysis of important morphological structures (cell nucleus, cytoplasm, nucleus/plasma relation) of the conjunctiva is possible. Secretory cells of the epithelium (goblet cells) can be easily recognized by their size. Highly reflective pixels depict cell walls or wide intercellular spaces with high contrast. CONCLUSIONS: The in vivo investigation of important anatomical and morphological structures of the conjunctival epithelium is possible using RLSM. The distribution pattern of goblet cell pixel brightness may correlate with various secretion contents or suggest distinct, recognizable, functional conditions (hypo- or hypersecretion).

Evaluation of biometric methods for measuring the anterior chamber depth in the non-contact mode.

PURPOSE: High-resolution biometry of the anterior ocular segment is now becoming more and more important against a background of refractive surgery and the evaluation of potentially accommodative lens replacement materials. The aim of this study was a systematic investigation of the currently available non-contact methods for measuring the anterior chamber depth (ACD). METHODS: The ACDs of 50 phakic eyes of 27 patients aged between 19 and 59 years were measured with the IOL-Master (Zeiss), the AC-Master (Zeiss), the Pentacam (Oculus) and slit-lamp pachymetry by Jaeger (Haag-Streit). RESULTS: The median anterior chamber depth in the investigated eyes was 3.63 mm for the IOL-Master (minimum 2.88 mm, maximum 4.22 mm), 3.802 mm for the AC-Master (2.816 mm-4.373 mm), 3.915 mm for the Pentacam (minimum 2.994 mm, maximum 4.614 mm) and 3.75 mm for Jaeger (2.887 mm-4.29 mm). With a probability of error of alpha=0.05 there were no significant differences concerning the ACD between the methods of Jaeger and AC-Master, Jaeger and IOL-Master, or Pentacam and AC-Master (Wilcoxon and Wilcox). The intra-individual variability was +/-5.4 microm for AC-Master, +/-12.7 microm for Pentacam, +/-24.5 microm for IOL-Master and +/-41.2 microm for Jaeger. The maximum method-dependent difference in ACD determination was 285 microm. CONCLUSIONS: All the methods allow non-contact biometry, but the results might differ due to measuring principles inherent to the system, experience of the examiner and compliance of the patient. Partial coherence interferometry with the AC-Master offers the advantage of measurement exactly along the optical axis with the highest reproducibility and patient compliance.

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 eta = 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.

[Epidemic keratoconjunctivitis. In vivo images of corneal structures with the confocal Rostocker laser scanning microscope (RLSM)]. / Keratoconjunctivitis epidemica. In-vivo-Darstellung kornealer Strukturen mit dem konfokalen Rostocker Laser-Scanning-Mikroskop (RLSM).

PURPOSE: Epidemic keratoconjunctivitis (EKC) is a highly contagious infection caused by adenovirus types 8, 19, and 37. The formation of subepithelial nummular infiltrates may lead to permanent visual disturbance. These infiltrates consist histopathologically of an accumulation of lymphocytes, histiocytes, and fibroblasts. PATIENT AND METHOD: The reasons for the persistence of these nummular infiltrates are not fully understood. We examined a 28-year-old female patient with typical signs of EKC infection clinically and additionally with the RLSM. RESULTS: Nummuli were identified by the RLSM as areas with an accumulation of dendritic cells (supposed to be Langerhans cells) as well as so far unidentified punctiform structures located in the basal cell layer and in the region of the subepithelial nerve plexus. CONCLUSIONS: The RLSM allows us to investigate corneal microstructures in vivo with precise depth localization related to epidemic keratoconjunctivitis. Further investigations may disclose the role of dendritic cells in the pathogenesis of the nummular formation as well as their contribution to the variable persistence of partially threatening vision opacities.

[Three-dimensional ultrasound biomicroscopic examinations for haptic differentiation of potentially accommodative intraocular lenses]. / 3D-ultraschallbiomikroskopische Untersuchungen zur Differenzierung der Haptiken von potenziell akkommodationsfähigen Intraokularlinsen.

PURPOSE: The principal ability of potentially accommodative IOLs is based on an axial shift of the IOL optics induced by the ciliary body action in interaction with a reversible change in haptic angulation. The aim of this study was to investigate the accommodative performance of this new IOL generation. METHOD: The authors have designed a test device to study IOL performance experimentally. These results were extrapolated to the in vivo situation in patients with accommodative implants. RESULTS: The 3D high-resolution presentation of the anterior segment of the eye allows a separation of the IOL haptic elements. In vitro and in the selected in vivo cases a change in haptic angulation <10 degrees in combination with a maximal IOL shift of 0.5 mm was found. CONCLUSIONS: The simulation model used in combination with 3D ultrasound biomicroscopy provides information about the potential of accommodative IOL designs. Conclusions corresponding to changes in haptic angulation during accommodation can be drawn and applied to the in vivo situation.

[Confocal laser-scanning microscopy to analyse the epithelium of the tongue]. / Konfokale Laser-Scanning-Mikroskopie zur Beurteilung des Zungenepithels.

BACKGROUND: Superficial changes of the epithelium of the tongue are diagnosed by biopsy. The aim of this in-vivo study was to show the normal epithelium, the subepithelial structures and the subepithelial vessels of the tongue using confocal microscopy. METHODS: A digital confocal Laser-Scanning Microscope (LSM), a combination of the Heidelberg Retina Tomograph HRT II and the Rostock Cornea Module (Heidelberg Engineering GmbH) was used. This digital microscope makes computer-based external hydraulic z-scan possible and the automatic collection of patient-based video sequences and the 3D reconstruction of the structures of the fungiform papilla. 800-fold magnifications are possible using a water immersion lens (Zeiss, 63-fold). A parabolic plexiglass disc with holes and a video camera were used to stabilize the tongue and to control the contact between the tongue surface and the contact element of the lens. PATIENTS: Confocal microscopy was performed on 17 healthy test volunteers aged 21 to 56. RESULTS: First findings with confocal LSM as a contact procedure show that a precise identification of the fungiform and filiform papillae and 3D reconstruction of the pores is possible. Furthermore, different cell layers of the tongue epithelium, subcellular structures of the epithelium and the subepithelial vessels to depth of 50 microm can be visualized by the erythrocytic flow and a max. 800-fold magnification. CONCLUSIONS: The confocal LSM is a nondestructive contact procedure and allows the in-vivo analysis of the tongue epithelium.

[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.

[Effect of intraocular lens design on posterior capsule opacification: an in-vitro model]. / In-vitro-Kapselsackmodell zur Testung des Einflusses des Linsendesigns auf die Entstehung einer posterioren Kapselfibrose--eine In-vitro-Studie.

BACKGROUND: The development of posterior capsule opacification (PCO) is one of the commonest complications of modern cataract surgery. The various designs of intraocular lenses (IOL) seem to exert a barrier effect on the proliferation and migration of lens epithelial cells and the following development of PCO. METHODS: We set up a cell culture model (advanced 3D capsular bag model) and investigated six differently designed IOL made of different materials as to their effect on cell proliferation. Proliferation and migration of the cells were analysed and documented over a period of 28 days. A cell viability test using the LIVE/DEAD kit (Molecular Probes) was carried out at the end of the investigation. RESULTS: In all tests, lens epithelial cells adhered to and migrated onto the capsular bag. During the culture period, lens epithelial cells migrated only to the optical rim of two of the implanted IOL. On the other four, lens epithelial cells migrated further and covered the whole optical area of the IOL. CONCLUSIONS: Certain lens designs seem to have a reducing effect on the development of PCO. Our advanced in vitro capsular bag model is a suitable cell culture model for the investigation of the reducing effect of various IOL on the development of PCO.

[A 3-D capsular bag model for describing biomechanical properties of neu intraocular lenses]. / Ein 3-D-Kapselsackmodell zur Beschreibung der biomechanischen Eigenschaften neuer Kunstlinsentypen.

BACKGROUND: This study quantified the geometric deformation of the capsular bag following implantation of various intraocular lenses (IOL) using a three-dimensional capsular bag model made of silicone caoutchouc. METHODS: After implantation of 13 different IOLs (polymethylmethacrylate, silicone acrygel) into the artificial bag, the induced capsular bag deformation was measured and analyzed. The posterior space between IOL and capsule was examined by ultrasonographic biomicroscopy. RESULTS: Polymethylmethacrylate IOLs with C-haptic design induced a greater deformation of the capsular bag than silicone lenses; however, both types showed a larger gap posterior to the IOL than acrygel lenses. Acrygel IOLs with different haptic design revealed only minimal deformation with close contact posteriorly. CONCLUSIONS: The presented three-dimensional model simulates the biomechanical and geometrical parameters of the vital capsular bag. Further investigations may determine a correlation of close posterior IOL contact and lens epithelial cell progression.

Validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study.

An immunoassay for detection of a specific genetically modified soybean (Roundup-Ready) was validated on dried soybean powder in an interlaboratory study. Different percentages of genetically modified soybeans in nonmodified soybean matrix were evaluated in a blind study. Thirty-eight laboratories from 13 countries participated. The immunoassay was evaluated for 2 endpoints: (1) To give a semiquantitative result, i.e., determination of a given sample above or below a given threshold, or (2) to compute a quantitative result, i.e., percentage of genetically modified soybeans in the sample. Semiquantitative results showed that a given sample which contained <2% genetically modified soybeans was identified as below 2% with a 99% confidence level. Quantitative use of the assay resulted in a repeatability (r) and reproducibility (R) that were computed to be RSDr = 7% and RSDR = 10%, respectively, for a sample containing 2% genetically modified soybeans. Application of this method depends on availability of appropriate reference materials for a specific food matrix. Only matrix-matched reference materials can be used for analysis of food or food fractions.

[Imaging of the microarchitecture and dynamics of the break-up phenomena of the preocular tear film with the aid of laser scanning microscopy]. / Darstellung der Mikroarchitektur und Dynamik der Aufreissphänomene des präkornealen Tränenfilms mit Hilfe der Laser-Rastermikroskopie.

BACKGROUND: Sufficient and regular wetting of the cornea with a normal preocular tear film is an essential requirement for the accurate visual functioning of the eye. Multiple factors may cause tear film instability. Measuring the break-up time of the preocular tear film is a necessary, clinically reliable means for evaluating tear film stability. We designed a new instrument to observe the preocular tear film at high magnification which can contribute to the in vivo imaging of the physiological and pathophysiological dynamics of the tear film and its break-up phenomena. MATERIALS AND METHODS: To image the tear film phenomena at sufficient magnification (x200) we used a confocal laser scanning microscope. Modification of an additional adaptor allows the tear film to be imaged at high magnification. This study evaluated the dynamics and microarchitecture of the break-up phenomena in the preocular tear film of normal patients and patients with "dry eye". RESULTS: Laser scanning microscopy allows high magnification imaging of the preocular tear film and evaluation of a number of break-up mechanisms. The dynamics and structure of the tear film was analyzed and documented. Based on our observations we formulate a new hypothesis on the break-up mechanism of the preocular tear film caused by alterations in the epithelial surface, for example, an elevation or tear film microparticle. By evaluating video sequences in the fluorescence and reflection modus of the laser scanning ophthalmoscope we also found early thinning of the aqueous layer simultaneously with an intact lipid layer covering the aqueous layer. CONCLUSION: Additional criteria result from using high-magnification microscopy, for example, laser scanning microscopy. This method reveals more microstructures of the corneal surface and the preocular tear film. The morphological representation of break-up phenomena leads to a better understanding of the underlying mechanisms and can be used for further investigations. Confocal laser scanning microscopy supplements the examination facilities and can be used in addition to slit lamp microscopy and to diagnose "dry eye".

Cyclophotocoagulation: experimental investigations of dosage problems.

BACKGROUND: During cyclophotocoagulation, transsclerally applied laser light reduces the aqueous-producing structures of the eye. One problem using this therapy is patient-specific dosage of the applied laser energy. The aim of our investigations was to obtain information about intensity and distribution of tissue destruction in the coagulation area. This may provide a basis for further on-line control of cyclophotocoagulation by ultrasound-controlled engineering of the diode laser. METHODS: To visualize the process of cyclophotocoagulation, a multifunctional measurement set-up was developed. It allowed the visualization of structural changes in the coagulation area using a common light microscope and comparison in the first set-up to the results detected by high-resolution ultrasound, applied in different working modes (B-mode, M-mode and RF signal analyses). In a second set-up an infrared thermography system showing temperature distribution on the scleral surface at the contact point of the laser probe was used. RESULTS: High resolution working in B- and M-mode was unsuitable to visualize structural changes within the therapeutic width. By analyzing RF ultrasound date, structural changes within the therapeutic width could be detected. Surface temperatures measured by infrared thermography correlated with visible structural changes when long exposure times and low laser power were applied. CONCLUSIONS: In certain cases the visualization of coagulation effects was possible with the help of either high-resolution ultrasound or infrared thermography. Spectrum analysis of RF ultrasound signals seems to be a potential method for successful control of cyclophotocoagulation.

[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.

[Imaging the tear film and in vivo cornea. Initial results with a modified confocal laser scanning ophthalmoscope]. / Darstellung von Tränenfilm und In-Vivo Kornea. Erste Untersuchungsergebnisse mit einem modifizierten konfokalen Laser-Scanning-Ophthalmoskop.

BACKGROUND: As the slit-lamp technique is an restrictive method for the in-vivo examination and documentation of the multi-layer structure and the dynamics of the tear film and the cornea, e.g. during wound-healing processes, our aim was to find out whether it is possible to change the confocal laser scanning ophthalmoscope (CLSO; Zeiss) into a high-resolution confocal laser scanning microscope (CLSM) for the visualization of the anterior segments of the eye and the tear film by adapting a special objective system. In order to gain information concerning the function of such a cornea microscope we examined selected patients with erosion or pterygium before and after operation and subsequent phototherapeutic keratectomy (PTK), as well as some in-vitro corneal surfaces after photorefractive keratectomy (PRK). MATERIAL AND METHODS: We developed an objective adapter for the CLSO in order to focus the laser beam onto anterior segments of the eye to visualize the tear film and the layer structure of the cornea. By combining the long-distance objective or a contract objective with different zoom-optic lenses it was possible to alter the scan field size and so the magnification of the CLSM by a factor of up to 1000. RESULTS: The CLSM provides a new method for the in-vivo examination of the tear film and its dynamics as well as the thin layers of the cornea in real-time imaging technique with high contrast and resolution in non-contact or contact procedures. CONCLUSION: This system is a unique took for evaluating and monitoring the effect of excimer laser ablation after PTK and PRK on the corneal surface and the dynamics of the tear film. The application of this method causes no pain for the patient.

[Binocular photometer for measuring light perception in diseases of the visual pathway]. / Binokulares Photometer zur Messung der Lichtempfindung bei Erkrankungen der Sehbahn.

In patients with a reduction in visual function because of optic nerve neuritis, light sensitivity is tested to detect differences between the right and left eye in combination with the swinging flashlight test and visual evoked potential (VEP). A binocular Zeiss microscope was modified to use integrated photometer-controlled binocular stimulus fields and a central light system for fixation and variation of the contrast (background illumination). For exact binocular measurements of light sensitivity on both eyes, electronically controlled test fields (visual angle 5 degrees-20 degrees) guarantees only macular or overfoveolar stimulation. In addition, splitting the light for each stimulus field from only one halogen lamp guarantees wave-length-independent testing. Patients with various retinal diseases and neuritis nervi optici pathology were examined using pattern VEP, testing of the absolute lower threshold of light sensitivity, and binocular comparison over the range of luminous density. This binocular optical photometer system allowed minimal differences to be identified in the present visual function and after the eye had recovered from optic nerve neuritis.