Anwendung der konfokalen Laser-Scanning-Mikroskopie zur Charakterisierung der Struktur von Zähnen und von oberflächenmodifiziertem Titan für Knochenimplantate

Zusammenfassung Mittels Fluoreszenzfarbstoffen können Strukturen sichtbar gemacht werden, die auf kon-ventionellem Weg nicht, oder nur schwer darzustellen sind. Besonders in Kombination mit der Konfokalen Laser Scanning Mikroskopie eröffnen sich neue Wege zum spezifischen Nachweis unterschiedlichster Komponenten biologischer Proben und gegebenenfalls deren dreidimensionale Widergabe.Die Visualisierung des Proteinanteils des Zahnhartgewebes kann mit Hilfe chemisch kopplungsfähiger Fluorochrome durchgeführt werden. Um zu zeigen, daß es sich bei dieser Markierung nicht um unspezifische Adsorption des Farbstoffes handelt, wurde zur Kontrolle die Proteinkomponente der Zahnproben durch enzymatischen Verdau beseitigt. Derartig behandelte Präparate wiesen eine sehr geringe Anfärbbarkeit auf.Weiterführend diente diese enzymatische Methode als Negativkontrolle zum Nachweis der Odontoblastenfortsätze im Dentin bzw. im Bereich der Schmelz-Dentin-Grenze. Hiermit konnte differenziert werden zwischen reinen Reflexionsbildern der Dentinkanäle und den Zellausläufern deren Membranen gezielt durch lipophile Fluoreszenzfarbstoffe markiert wurden.In einem weiteren Ansatz konnte gezeigt werden, daß reduzierte und daher nichtfluoreszente Fluoresceinabkömmlinge geeignet sind, die Penetration von Oxidationsmitteln (hier H2O2) in den Zahn nachzuweisen. Durch Oxidation dieser Verbindungen werden fluoreszierende Produkte generiert, die den Nachweis lieferten, daß die als Zahnbleichmittel eingesetzten Mittel rasch durch Schmelz und Dentin bis in die Pulpahöhle gelangen können.Die Abhängigkeit der Fluoreszenz bestimmter Fluorochrome von deren chemischer Um-gebung, im vorliegenden Fall dem pH-Wert, sollte eingesetzt werden, um den Säuregrad im Zahninneren fluoreszenzmikroskopisch darzustellen. Hierbei wurde versucht, ein ratio-metrisches Verfahren zu entwickeln, mit dem die pH-Bestimmung unter Verwendung eines pH-abhängigen und eines pH-unabhängigen Fluorochroms erfolgt. Diese Methode konnte nicht für diese spezielle Anwendung verifiziert werden, da Neutralisationseffekte der mineralischen Zahnsubstanz (Hydroxylapatit) die pH-Verteilung innerhalb der Probe beeinflußen. Fluoreszenztechniken wurden ebenfalls ergänzend eingesetzt zur Charakterisierung von kovalent modifizierten Implantatoberflächen. Die, durch Silanisierung von Titantestkörpern mit Triethoxyaminopropylsilan eingeführten freien Aminogruppen konnten qualitativ durch den Einsatz eines aminspezifischen Farbstoffes identifiziert werden. Diese Art der Funktionalisierung dient dem Zweck, Implantatoberflächen durch chemische Kopplung adhäsionsvermittelnder Proteine bzw. Peptide dem Einheilungsprozeß von Implantaten in den Knochen zugänglicher zu machen, indem knochenbildende Zellen zu verbessertem Anwachsverhalten stimuliert werden. Die Zellzahlbestimmung im Adhäsionstest wurde ebenfalls mittels Fluoreszenzfarbstoffen durchgeführt und lieferte Ergebnisse, die belegen, daß die durchgeführte Modifizierung einen günstigen Einfluß auf die Zelladhäsion besitzt.

High resolution study of local stress inside alumina - micro mechanical analysis using laser scanning confocal microscope

The aim of this work is to measure the stress inside a hard micro object under extreme compression. To measure the internal stress, we compressed ruby spheres (a-Al2O3: Cr3+, 150 µm diameter) between two sapphire plates. Ruby fluorescence spectrum shifts to longer wavelengths under compression and can be related to the internal stress by a conversion coefficient. A confocal laser scanning microscope was used to excite and collect fluorescence at desired local spots inside the ruby sphere with spatial resolution of about 1 µm3. Under static external loads, the stress distribution within the center plane of the ruby sphere was measured directly for the first time. The result agreed to Hertz’s law. The stress across the contact area showed a hemispherical profile. The measured contact radius was in accord with the calculation by Hertz’s equation. Stress-load curves showed spike-like decrease after entering non-elastic phase, indicating the formation and coalescence of microcracks, which led to relaxing of stress. In the vicinity of the contact area luminescence spectra with multiple peaks were observed. This indicated the presence of domains of different stress, which were mechanically decoupled. Repeated loading cycles were applied to study the fatigue of ruby at the contact region. Progressive fatigue was observed when the load exceeded 1 N. As long as the load did not exceed 2 N stress-load curves were still continuous and could be described by Hertz’s law with a reduced Young’s modulus. Once the load exceeded 2 N, periodical spike-like decreases of the stress could be observed, implying a “memory effect” under repeated loading cycles. Vibration loading with higher frequencies was applied by a piezo. Redistributions of intensity on the fluorescence spectra were observed and it was attributed to the repopulation of the micro domains of different elasticity. Two stages of under vibration loading were suggested. In the first stage continuous damage carried on until certain limit, by which the second stage, e.g. breakage, followed in a discontinuous manner.

Diffusion in heterogeneous systems studied by laser scanning confocal microscopy and fluorescence correlation spectroscopy

Understanding and controlling the mechanism of the diffusion of small molecules, macromolecules and nanoparticles in heterogeneous environments is of paramount fundamental and technological importance. The aim of the thesis is to show, how by studying the tracer diffusion in complex systems, one can obtain information about the tracer itself, and the system where the tracer is diffusing. rnIn the first part of my thesis I will introduce the Fluorescence Correlation Spectroscopy (FCS) which is a powerful tool to investigate the diffusion of fluorescent species in various environments. By using the main advantage of FCS namely the very small probing volume (<1µm3) I was able to track the kinetics of phase separation in polymer blends at late stages by looking on the molecular tracer diffusion in individual domains of the heterogeneous structure of the blend. The phase separation process at intermediate stages was monitored with laser scanning confocal microscopy (LSCM) in real time providing images of droplet coalescence and growth. rnIn a further project described in my thesis I will show that even when the length scale of the heterogeneities becomes smaller than the FCS probing volume one can still obtain important microscopic information by studying small tracer diffusion. To do so, I will introduce a system of star shaped polymer solutions and will demonstrate that the mobility of small molecular tracers on microscopic level is nearly not affected by the transition of the polymer system to a “glassy” macroscopic state. rnIn the last part of the thesis I will introduce and describe a new stimuli responsive system which I have developed, that combines two levels of nanoporosity. The system is based on poly-N-isopropylacrylamide (PNIPAM) and silica inverse opals (iOpals), and allows controlling the diffusion of tracer molecules. rn

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

Laser scanning of active semiconductor devices--videotape script /

"This activity was supported by the Defense Advanced Research Projects Agency and the National Bureau of Standards."

Mapping mosquito breeding habitats (water) under mangrove canopy: contribution from airborne thermal scanning

Proceedings of the 11th Australasian Remote Sensing and Photogrammetry Conference

3D Laser Scanning for Digital Preservation and Dissemination of Cultural Heritage Main scanned objects - Four Temples of Different Religions in Sofia

The project demonstrates the use of modern technologies for preservation and presentation of the cultural and historical heritage. The idea is a database of cultural and historical heritage sites to be created applying three dimensional laser scanning technology and a combination of geodetic and photogrammetric methods and shooting techniques. For the purposes of carrying out this project, we have focused on some heritage sites in the central part of Sofia. We decided to include these particular buildings because of the fact that there is hardly another city in the world where within a radius of 400 m are located four temples of different religions - Jewish, Muslim, Orthodox and Catholic. In the recent years, preservation of cultural heritage has been increasingly linked to objectives of sustainable development. Today, it has become clear that cultural heritage is also an economic resource that should be used for further economic development (through compulsory preservation of its authentic cultural values). There has been a more active public debate on the role of cultural heritage, regarding the following topics: improving the quality of life through development of cultural tourism, leading to an increase of the employment rate, constantly improving the business climate, etc. Cultural heritage preservation is becoming one of the priority objectives of the urban development policy. The focus has been shifted to new ways of preservation, mainly combinations of sophisticated technological solutions and their application for the purposes of preservation and dissemination of the cultural heritage.

Airborne laser quantification of Florida shoreline and beach volume change caused by hurricanes

This dissertation combines three separate studies that measure coastal change using airborne laser data. The initial study develops a method for measuring subaerial and subaqueous volume change incrementally alongshore, and compares those measurements to shoreline change in order to quantify their relationship in Palm Beach County, Florida. A poor correlation (R2 = 0.39) was found between shoreline and volume change before the hurricane season in the northern section of Palm Beach County because of beach nourishment and inlet dynamics. However, a relatively high R2 value of 0.78 in the southern section of Palm Beach County was found due to little disturbance from tidal inlets and coastal engineering projects. The shoreline and volume change caused by the 2004 hurricane season was poorly correlated with R 2 values of 0.02 and 0.42 for the north and south sections, respectively. The second study uses airborne laser data to investigate if there is a significant relationship between shoreline migration before and after Hurricane Ivan near Panama City, Florida. In addition, the relationship between shoreline change and subaerial volume was quantified and a new method for quantifying subaqueous sediment change was developed. No significant spatial relationship was found between shoreline migration before and after the hurricane. Utilization of a single coefficient to represent all relationships between shoreline and subaerial volume change was found to be problematic due to the spatial variability in the linear relationship. Differences in bathymetric data show only a small portion of sediment was transported beyond the active zone and most sediment remained within the active zone despite the occurrence of a hurricane. The third study uses airborne laser bathymetry to measure the offshore limit of change, and compares that location with calculated depth of closures and subaqueous geomorphology. There appears to be strong geologic control of the depth of closure in Broward and Miami-Dade Counties. North of Hillsboro Inlet, hydrodynamics control the geomorphology which in turn indicates the location of the depth of closure.

Airborne Laser Quantification of Florida Shoreline and Beach Volume Change Caused by Hurricanes

This dissertation combines three separate studies that measure coastal change using airborne laser data. The initial study develops a method for measuring subaerial and subaqueous volume change incrementally alongshore, and compares those measurements to shoreline change in order to quantify their relationship in Palm Beach County, Florida. A poor correlation (R2 = 0.39) was found between shoreline and volume change before the hurricane season in the northern section of Palm Beach County because of beach nourishment and inlet dynamics. However, a relatively high R2 value of 0.78 in the southern section of Palm Beach County was found due to little disturbance from tidal inlets and coastal engineering projects. The shoreline and volume change caused by the 2004 hurricane season was poorly correlated with R2 values of 0.02 and 0.42 for the north and south sections, respectively. The second study uses airborne laser data to investigate if there is a significant relationship between shoreline migration before and after Hurricane Ivan near Panama City, Florida. In addition, the relationship between shoreline change and subaerial volume was quantified and a new method for quantifying subaqueous sediment change was developed. No significant spatial relationship was found between shoreline migration before and after the hurricane. Utilization of a single coefficient to represent all relationships between shoreline and subaerial volume change was found to be problematic due to the spatial variability in the linear relationship. Differences in bathymetric data show only a small portion of sediment was transported beyond the active zone and most sediment remained within the active zone despite the occurrence of a hurricane. The third study uses airborne laser bathymetry to measure the offshore limit of change, and compares that location with calculated depth of closures and subaqueous geomorphology. There appears to be strong geologic control of the depth of closure in Broward and Miami-Dade Counties. North of Hillsboro Inlet, hydrodynamics control the geomorphology which in turn indicates the location of the depth of closure.

Pilot Study on Improving the Efficiency of Transportation Projects Using Laser Scanning, 2003

Laser scanning is a terrestrial laser-imaging system that creates highly accurate three-dimensional images of objects for use in standard computer-aided design software packages. This report describes results of a pilot study to investigate the use of laser scanning for transportation applications in Iowa. After an initial training period on the use of the scanner and Cyclone software, pilot tests were performed on the following projects: intersection and railroad bridge for training purposes; section of highway to determine elevation accuracy and pair of bridges to determine level of detail that can be captured; new concrete pavement to determine smoothness; bridge beams to determine camber for deck-loading calculations; stockpile to determine volume; and borrow pit to determine volume. Results show that it is possible to obtain 2-6 mm precision with the laser scanner as claimed by the manufacturer compared to approximately one-inch precision with aerial photogrammetry using a helicopter. A cost comparison between helicopter photogrammetry and laser scanning showed that laser scanning was approximately 30 percent higher in cost depending on assumptions. Laser scanning can become more competitive to helicopter photogrammetry by elevating the scanner on a boom truck and capturing both sides of a divided roadway at the same time. Two- and three-dimensional drawings were created in MicroStation for one of the scanned highway bridges. It was demonstrated that it is possible to create such drawings within the accuracy of this technology. It was discovered that a significant amount of time is necessary to convert point cloud images into drawings. As this technology matures, this task should become less time consuming. It appears that laser scanning technology does indeed have a place in the Iowa Department of Transportation design and construction toolbox. Based on results from this study, laser scanning can be used cost effectively for preliminary surveys to develop TIN meshes of roadway surfaces. It also appears that this technique can be used quite effectively to measure bridge beam camber in a safer and quicker fashion compared to conventional approaches. Volume calculations are also possible using laser scanning. It seems that measuring quantities of rock could be an area where this technology would be quite beneficial since accuracy is more important with this material compared to soil. Other applications for laser scanning could include developing as-built drawings of historical structures such as the bridges of Madison County. This technology could also be useful where safety is a concern such as accurately measuring the surface of a highway active with traffic or scanning the underside of a bridge damaged by a truck. It is recommended that the Iowa Department of Transportation initially rent the scanner when it is needed and purchase the software. With time, it may be cost justifiable to purchase the scanner as well. Laser scanning consultants can be hired as well but at a higher cost.