Norbert Koop

Influence of temperature and time on thermally induced forces in corneal collagen and the effect on laser thermokeratoplasty

PURPOSE To investigate thermomechanical aspects of corneal collagen denaturation as a function of temperature and time and the effect of the induced forces on refractive changes with laser thermokeratoplasty (LTK). SETTING Medical Laser Center Lübeck, Lübeck, Germany. METHODS In a material-test setup, porcine corneal strips were denatured in paraffin oil at various constant temperatures for 10 and 500 seconds, and the temporal course of the contractive forces was studied under isometric conditions. Typical LTK lesions were performed in porcine eyes in vitro with a continuous-wave infrared laser diode at a wavelength of 1.87 microm for 10 and 60 seconds. The laser power was chosen to achieve comparable denatured volumes at both irradiation times. The refractive changes were measured and analyzed by histologic evaluations and temperature calculations. RESULTS The time course of the induced forces was characterized by a maximal force, which increased almost linearly with temperature, and a residual lower force. After 500 seconds of heating, the highest force was achieved with a temperature of 75 degrees C. With a limited heating period of only 10 seconds, the forces steadily increased with temperature over the entire observation period. Laser thermokeratoplasty produced less refractive change after 10 seconds of irradiation than after 60 seconds, although the laser power was 25% higher in the short heating period. Polarization light microscopy of LTK lesions revealed different stages of thermal damage. CONCLUSION The course of the contractive forces during and after heating is a complicated function of the spatial time/temperature profile. Laser thermokeratoplasty lesions produced with 2 irradiation times showed different stages of denaturation and induced refractive change.

Optische Kohärenztomographie der Kornea und des vorderen Augenabschnitts

Ziel: Die Methode der optischen Kohärenztomographie (OCT) wurde bezüglich ihrer Eignung zur Vermessung des vorderen Augenabschnitts, der Kornea und thermischer Gewebeschädigungen untersucht. Beispielhaft wurden hierzu im Rahmen der Laserthermokeratoplastik (LTK) die Veränderungen der Hornhautdicke durch die Bestrahlung sowie die induzierten thermischen Läsionen vermessen.Material und Methoden: Mit dem von uns entwickelten experimentellen Scanning-OCT wurden mit einer optischen Auflösung von ca. 20 µm axial und 25 µm lateral x-z-Schnitte des vorderen Augenabschnitts vorgenommen. Als Modellauge dienten frisch enukleierte, tonisierte Schweineaugen. Thermische Läsionen wurden mit einer kontinuierlich emittierenden Laserdiode (λ = 1,86 µm) und verschiedenen Bestrahlungsparametern appliziert. Vor und nach Koagulation wurden die Hornhaut von Limbus zu Limbus in einem zentralen OCT-Schnittbild dargestellt sowie die einzelnen Koagulationsherde vermessen.Ergebnisse: Globale und lokale Hornhautdickenänderungen sowie der Abstand Hornhaut-Linse lassen sich mit hoher Genauigkeit bestimmen. Thermische Läsionen sind in ihren Ausdehnungen klar darstellbar und zeigen gute Übereinstimmungen mit histologischen Schnittbildern, sind jedoch aufgrund der optischen Auflösung an ihren Rändern nicht so scharf abgegrenzt, wie von histologischen Präparaten bekannt.Schlußfolgerung: Mit der Methode der OCT lassen sich in vitro und, mit reduzierter Auflösung, auch in vivo quantitative Vermessungen des vorderen Augenabschnitts vornehmen. Aufgrund der qualitativ guten Übereinstimmung des Ausmaßes thermischer Schädigungen der Hornhaut mit der histologischen Morphometrie läßt sich dieses Meßverfahren auch klinisch nach LTK zur Kontrolle der Ausdehnung und Denaturierungstiefen von Koagulationen sowie zur Untersuchung des individuellen Heilungsverlaufs verwenden.Target: The method of optical coherence tomography (OCT) was investigated regarding its suitability and limits for measuring the cornea and the anterior segment of the eye. Furthermore, the stromal expansion of thermally induced lesions in the cornea directly after irradiation was determined within the scope of the laser thermokeratoplasty (LTK).Material and methods: With the experimental scanning OCT system, x-z sections of the anterior eye segment were made with an optical resolution of about 20 µm axially and 25 µm laterally. Freshly enucleated, tonicized porcine eyes were used as model eyes. Thermal lesions were applied with a continuously emitting laser diode (λ = 1.86 µm) and various radiation parameters. Before and after coagulation, the cornea was viewed from limbus to limbus in a central OCT scan and the individual coagulation source was measured.Results: Global and local changes of the thickness of the cornea as well as the distance between cornea and lens were measured with high precision. Thermal lesions in their expansion can be clearly presented and matching well with the histologically stained sections, but are not as exactly defined at the edges due to the limited optical resolution, as known from histological preparations.Conclusion: With the OCT method quantitative measuring of the anterior eye segment can be performed in vitro and with reduced resolutions also in vivo. Due to the qualitatively good correspondence regarding the dimensions of thermal damage of the cornea with histologically obtained morphometric results, this method can be used for supervision of coagulation directly after LTK as well as for examination of the individual healing process.

Nonviral In Situ Green Fluorescent Protein Labeling and Culture of Primary, Adult Human Hair Follicle Epithelial Progenitor Cells

In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin‐resistance cassette and transfection of microdissected, organ‐cultured adult human scalp hair follicles generates specific K15 promoter–driven GFP expression in their stem cell–rich bulge region. K15‐GFP+ cells can be visualized in situ by GFP fluorescence and 2‐photon laser scanning microscopy. Vital K15‐GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15‐GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter–driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues. STEM CELLS 2009;27:2793–2803

Experimental induction and three-dimensional two-photon imaging of conjunctiva-associated lymphoid tissue.

PURPOSE Conjunctiva-associated lymphoid tissue (CALT) is assumed to be a key location for the generation of adaptive immune mechanisms of the ocular surface, but functional studies of CALT are still lacking. The purpose of this study was to establish an animal model that enables functional analysis of immune mechanisms going on within CALT. In addition, the use of two-photon microscopy, a new optical method, was evaluated for examining complex immunologic interactions of CALT by volume (three-dimensional [3-D]) and time-dependence (four-dimensional [4-D]) in vivo. METHODS The conjunctiva of female BALB/c mice was repeatedly challenged with topical Chlamydia trachomatis serovar C or a solution of ovalbumin and cholera toxin B. Two-photon microscopy was conducted on explanted, unfixed, and unstained eyes with adjacent nictitating membranes. RESULTS After three to five stimulations, CALT was detected exclusively in the nictitating membrane of 73% (C. trachomatis) or 70% (ovalbumin/ cholera toxin) of the animals. CALT mainly consisted of CD45R/B220+ B cells and CD4+ and CD8+ T cells. Electron microscopy showed intraepithelial lymphocytes and follicles consisting of lymphocytes, dendritic cells, and macrophages. Two-photon microscopy based on tissue autofluorescence allowed all components of CALT to be detected three dimensionally. High-resolution images were generated in tissue depths of 65 microm below the mucosal surface. CONCLUSIONS This study introduces a novel mouse model for functional investigations of CALT. Topical stimulation with C. trachomatis or ovalbumin/cholera toxin B reliably leads to CALT generation at the nictitating membrane. The use of two-photon microscopy enables groundbreaking 3-D and, in the future, intravital 4-D investigations of immunologic processes initiated in CALT.

Non-Viral in Situ GFP-Labelling and Culture of Primary, Adult Human Hair Follicle Epithelial Progenitor Cells.

In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin‐resistance cassette and transfection of microdissected, organ‐cultured adult human scalp hair follicles generates specific K15 promoter–driven GFP expression in their stem cell–rich bulge region. K15‐GFP+ cells can be visualized in situ by GFP fluorescence and 2‐photon laser scanning microscopy. Vital K15‐GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15‐GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter–driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues. STEM CELLS 2009;27:2793–2803

Comparison of Cornea Module and DermaInspect for noninvasive imaging of ocular surface pathologies.

Minimally invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without actual tissue probing. For this matter, confocal microscopy (Cornea Module) is in daily use in ophthalmic practice. Multiphoton microscopy is a new optical technique that enables high-resolution imaging and functional analysis of living tissues based on tissue autofluorescence. This study was set up to compare the potential of a multiphoton microscope (DermaInspect) to the Cornea Module. Ocular surface pathologies such as pterygia, papillomae, and nevi were investigated in vivo using the Cornea Module and imaged immediately after excision by DermaInspect. Two excitation wavelengths, fluorescence lifetime imaging and second-harmonic generation (SHG), were used to discriminate different tissue structures. Images were compared with the histopathological assessment of the samples. At wavelengths of 730 nm, multiphoton microscopy exclusively revealed cellular structures. Collagen fibrils were specifically demonstrated by second-harmonic generation. Measurements of fluorescent lifetimes enabled the highly specific detection of goblet cells, erythrocytes, and nevus-cell clusters. At the settings used, DermaInspect reaches higher resolutions than the Cornea Module and obtains additional structural information. The parallel detection of multiphoton excited autofluorescence and confocal imaging could expand the possibilities of minimally invasive investigation of the ocular surface toward functional analysis at higher resolutions.

Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions.

Imaging of cancer cells by multiphoton microscopy using gold nanoparticles and fluorescent dyes

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.

Continuous-wave diode laserthermokeratoplasty: First clinical experience in blind human eyes

PURPOSE To evaluate the safety and stability of laser thermokeratoplasty (LTK) with a continuous-wave diode laser in blind human eyes and to optimize parameters for a study in sighted eyes. SETTING Department of Ophthalmology, Medical University Lübeck, Germany. METHODS A continuous-wave diode laser was set to emit radiation with a wavelength of 1.854 microns (Group 1, n = 4) or 1.870 microns (Group 2, n = 4) and 100 to 150 mW power for 10 seconds. A focusing handpiece was coupled with an application mask and fixed by partial vacuum to the conjunctiva or cornea. The radiation was focused into the corneal stroma between 400 and 600 microns in Group 1 and set to 1000 microns in Group 2. Eight (Group 1, single ring) or 16 (Group 2, double ring) coagulations were applied. RESULTS The refractive change increased with higher laser power and smaller ring diameters. Two rings of coagulations provided higher and more stable refractive changes of up to 5.66 diopters (D) than a single ring. The refractive effect stabilized between 3 and 6 months postoperatively. At 1 year, mean refractive change was +0.99 D +/- 0.39 (SD) in Group 1 and +2.32 +/- 2.24 D in Group 2. Extensive endothelial damage occurred in Group 1 but was minimal in Group 2. CONCLUSIONS Diode LTK was used to treat hyperopia safely and effectively. Regression occurred mainly in the first 3 postoperative months. With a wavelength of 1.870 microns, corneal endothelial damage was limited.

Diode laser thermokeratoplasty: Application strategy and dosimetry

Purpose: To investigate suitable application parameters for efficient hyperopic correction by laser thermokeratoplasty (LTK) using mid‐infrared laser diodes. Setting: Medical Laser Center Lubeck, Lubeck, Germany. Method: A tunable continuous‐wave laser diode in the spectral range between 1.845 and 1.871 pm was used. Transmitted by waveguides, the laser energy was used to induce coagulations on freshly enucleated porcine eyes to increase corneal curvature. The coagulations were equidistantly applied by a fiber‐cornea contact and a noncontact focusing device that were adjusted on a ring concentric to the corneal apex. Different laser parameters and application geometries were evaluated. Refractive changes were measured by computer‐assisted corneal topography before and after treatment. Polarization light microscopy and temperature calculations were used to analyze the coagulations. Results: Because of the tunability of the laser diode, the influence of the corneal absorption coefficient (between 0.9 and 1.6 mm‐1) on the refractive change could be measured. A laser power between 125 and 200 mW was adequate to achieve refractive changes up to 10.0 diopters. In the preferable focusing device, the refractive change increased almost logarithmically with the irradiation time up to 15 seconds. The number of coagulations on a fixed application ring showed no significant influence on refractive change; however, it showed an almost linear decrease with increasing ring diameter from 5.0 to 10.0 mm. Histological analysis revealed 3 stages of thermal damage. Conclusion: Diode LTK provided defined and uniform coagulations when using a well‐adapted focusing device, resulting in sufficient refractive change. The results indicate that diode LTK is superior to pulsed holmium LTK.