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.

Visual scanning in sports actions: comparison between soccer goalkeepers and judo fighters

Visual search and oculomotor behaviour are believed to be very relevant for athlete performance, especially for sports requiring refined visuo-motor coordination skills. Modern coaches believe that a correct visuo-motor strategy may be part of advanced training programs. In this thesis two experiments are reported in which gaze behaviour of expert and novice athletes were investigated while they were doing a real sport specific task. The experiments concern two different sports: judo and soccer. In each experiment, number of fixations, fixation locations and mean fixation duration (ms) were considered. An observational analysis was done at the end of the paper to see perceptual differences between near and far space. Purpose: The aim of the judo study was to delineate differences in gaze behaviour characteristics between a population of athletes and one of non athletes. Aspects specifically investigated were: search rate, search order and viewing time across different conditions in a real-world task. The second study was aimed at identifying gaze behaviour in varsity soccer goalkeepers while facing a penalty kick executed with instep and inside foot. Then an attempt has been done to compare the gaze strategies of expert judoka and soccer goalkeepers in order to delineate possible differences related to the different conditions of reacting to events occurring in near (peripersonal) or far (extrapersonal) space. Judo Methods: A sample of 9 judoka (black belt) and 11 near judoka (white belt) were studied. Eye movements were recorded at 500Hz using a video based eye tracker (EyeLink II). Each subject participated in 40 sessions for about 40 minutes. Gaze behaviour was considered as average number of locations fixated per trial, the average number of fixations per trial, and mean fixation duration. Soccer Methods: Seven (n = 7) intermediate level male volunteered for the experiment. The kickers and goalkeepers, had at least varsity level soccer experience. The vision-in-action (VIA) system (Vickers 1996; Vickers 2007) was used to collect the coupled gaze and motor behaviours of the goalkeepers. This system integrated input from a mobile eye tracking system (Applied Sciences Laboratories) with an external video of the goalkeeper’s saving actions. The goalkeepers took 30 penalty kicks on a synthetic pitch in accordance with FIFA (2008) laws. Judo Results: Results indicate that experts group differed significantly from near expert for fixations duration, and number of fixations per trial. The expert judokas used a less exhaustive search strategy involving fewer fixations of longer duration than their novice counterparts and focused on central regions of the body. The results showed that in defence and attack situation expert group did a greater number of transitions with respect to their novice counterpart. Soccer Results: We found significant main effect for the number of locations fixated across outcome (goal/save) but not for foot contact (instep/inside). Participants spent more time fixating the areas in instep than inside kick and in goal than in save situation. Mean and standard error in search strategy as a result of foot contact and outcome indicate that the most gaze behaviour start and finish on ball interest areas. Conclusions: Expert goalkeepers tend to spend more time in inside-save than instep-save penalty, differences that was opposite in scored penalty kick. Judo results show that differences in visual behaviour related to the level of expertise appear mainly when the test presentation is continuous, last for a relatively long period of time and present a high level of uncertainty with regard to the chronology and the nature of events. Expert judoist performers “anchor” the fovea on central regions of the scene (lapel and face) while using peripheral vision to monitor opponents’ limb movements. The differences between judo and soccer gaze strategies are discussed on the light of physiological and neuropsychological differences between near and far space perception.

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.

Nanoscopic studies of conjugated polymer blends by (electric) scanning probe microscopy

Conjugated polymers and conjugated polymer blends have attracted great interest due to their potential applications in biosensors and organic electronics. The sub-100 nm morphology of these materials is known to heavily influence their electromechanical properties and the performance of devices they are part of. Electromechanical properties include charge injection, transport, recombination, and trapping, the phase behavior and the mechanical robustness of polymers and blends. Electrical scanning probe microscopy techniques are ideal tools to measure simultaneously electric (conductivity and surface potential) and dielectric (dielectric constant) properties, surface morphology, and mechanical properties of thin films of conjugated polymers and their blends.rnIn this thesis, I first present a combined topography, Kelvin probe force microscopy (KPFM), and scanning conductive torsion mode microscopy (SCTMM) study on a gold/polystyrene model system. This system is a mimic for conjugated polymer blends where conductive domains (gold nanoparticles) are embedded in a non-conductive matrix (polystyrene film), like for polypyrrole:polystyrene sulfonate (PPy:PSS), and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). I controlled the nanoscale morphology of the model by varying the distribution of gold nanoparticles in the polystyrene films. I studied the influence of different morphologies on the surface potential measured by KPFM and on the conductivity measured by SCTMM. By the knowledge I gained from analyzing the data of the model system I was able to predict the nanostructure of a homemade PPy:PSS blend.rnThe morphologic, electric, and dielectric properties of water based conjugated polymer blends, e.g. PPy:PSS or PEDOT:PSS, are known to be influenced by their water content. These properties also influence the macroscopic performance when the polymer blends are employed in a device. In the second part I therefore present an in situ humidity-dependence study on PPy:PSS films spin-coated and drop-coated on hydrophobic highly ordered pyrolytic graphite substrates by KPFM. I additionally used a particular KPFM mode that detects the second harmonic electrostatic force. With this, I obtained images of dielectric constants of samples. Upon increasing relative humidity, the surface morphology and composition of the films changed. I also observed that relative humidity affected thermally unannealed and annealed PPy:PSS films differently. rnThe conductivity of a conjugated polymer may change once it is embedded in a non-conductive matrix, like for PPy embedded in PSS. To measure the conductivity of single conjugated polymer particles, in the third part, I present a direct method based on microscopic four-point probes. I started with metal core-shell and metal bulk particles as models, and measured their conductivities. The study could be extended to measure conductivity of single PPy particles (core-shell and bulk) with a diameter of a few micrometers.

A scanning electron microscope for ultracold quantum gases

This thesis presents a new imaging technique for ultracold quantum gases. Since the first observation of Bose-Einstein condensation, ultracold atoms have proven to be an interesting system to study fundamental quantum effects in many-body systems. Most of the experiments use optical imaging rnmethods to extract the information from the system and are therefore restricted to the fundamental limitation of this technique: the best achievable spatial resolution that can be achieved is comparable to the wavelength of the employed light field. Since the average atomic distance and the length scale of characteristic spatial structures in Bose-Einstein condensates such as vortices and solitons is between 100 nm and 500 nm, an imaging technique with an adequate spatial resolution is needed. This is achieved in this work by extending the method of scanning electron microscopy to ultracold quantum gases. A focused electron beam is scanned over the atom cloud and locally produces ions which are subsequently detected. The new imaging technique allows for the precise measurement of the density distribution of a trapped Bose-Einstein condensate. Furthermore, the spatial resolution is determined by imaging the atomic distribution in one-dimensional and two-dimensional optical lattices. Finally, the variety of the imaging method is demonstrated by the selective removal of single lattice site. rn

Electrical scanning probe microscopy on organic optoelectronic structures

In the field of organic optoelectronics, the nanoscale structure of the materials has huge im-pact on the device performance. Here, scanning force microscopy (SFM) techniques become increasingly important. In addition to topographic information, various surface properties can be recorded on a nanometer length scale, such as electrical conductivity (conductive scanning force microscopy, C-SFM) and surface potential (Kelvin probe force microscopy, KPFM).rnrnIn the context of this work, the electrical SFM modes were applied to study the interplay be-tween morphology and electrical properties in hybrid optoelectronic structures, developed in the group of Prof. J. Gutmann (MPI-P Mainz). In particular, I investigated the working prin-ciple of a novel integrated electron blocking layer system. A structure of electrically conduct-ing pathways along crystalline TiO2 particles in an insulating matrix of a polymer derived ceramic was found and insulating defect structures could be identified. In order to get insights into the internal structure of a device I investigated a working hybrid solar cell by preparing a cross cut with focused ion beam polishing. With C-SFM, the functional layers could be identified and the charge transport properties of the novel active layer composite material could be studied. rnrnIn C-SFM, soft surfaces can be permanently damaged by (i) tip induced forces, (ii) high elec-tric fields and (iii) high current densities close to the SFM-tip. Thus, an alternative operation based on torsion mode topography imaging in combination with current mapping was intro-duced. In torsion mode, the SFM-tip vibrates laterally and in close proximity to the sample surface. Thus, an electrical contact between tip and sample can be established. In a series of reference experiments on standard surfaces, the working mechanism of scanning conductive torsion mode microscopy (SCTMM) was investigated. Moreover, I studied samples covered with free standing semiconducting polymer nano-pillars that were developed in the group of Dr. P. Theato (University Mainz). The application of SCTMM allowed non-destructive imag-ing of the flexible surface at high resolution while measuring the conductance on individual pillarsrnrnIn order to study light induced electrical effects on the level of single nanostructures, a new SFM setup was built. It is equipped with a laser sample illumination and placed in inert at-mosphere. With this photoelectric SFM, I investigated the light induced response in function-alized nanorods that were developed in the group of Prof. R. Zentel (University Mainz). A block-copolymer containing an anchor block and dye moiety and a semiconducting conju-gated polymer moiety was synthesized and covalently bound to ZnO nanorods. This system forms an electron donor/acceptor interface and can thus be seen as a model system of a solar cell on the nanoscale. With a KPFM study on the illuminated samples, the light induced charge separation between the nanorod and the polymeric corona could not only be visualized, but also quantified.rnrnThe results demonstrate that electrical scanning force microscopy can study fundamental processes in nanostructures and give invaluable feedback to the synthetic chemists for the optimization of functional nanomaterials.rn

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients

In-situ and real time scanning probe microscopy of organic ultra thin films

In recent decades, Organic Thin Film Transistors (OTFTs) have attracted lots of interest due to their low cost, large area and flexible properties which have brought them to be considered the building blocks of the future organic electronics. Experimentally, devices based on the same organic material deposited in different ways, i.e. by varying the deposition rate of the molecules, show different electrical performance. As predicted theoretically, this is due to the speed and rate by which charge carriers can be transported by hopping in organic thin films, transport that depends on the molecular arrangement of the molecules. This strongly suggests a correlation between the morphology of the organic semiconductor and the performance of the OTFT and hence motivated us to carry out an in-situ real time SPM study of organic semiconductor growth as an almost unprecedent experiment with the aim to fully describe the morphological evolution of the ultra-thin film and find the relevant morphological parameters affecting the OTFT electrical response. For the case of 6T on silicon oxide, we have shown that the growth mechanism is 2D+3D, with a roughening transition at the third layer and a rapid roughening. Relevant morphological parameters have been extracted by the AFM images. We also developed an original mathematical model to estimate theoretically and more accurately than before, the capacitance of an EFM tip in front of a metallic substrate. Finally, we obtained Ultra High Vacuum (UHV) AFM images of 6T at lying molecules layer both on silicon oxide and on top of 6T islands. Moreover, we performed ex-situ AFM imaging on a bilayer film composed of pentacene (a p-type semiconductor) and C60 (an n-type semiconductor).

Studies of degradation of organic solar cell materials using electrical scanning probe microscopy

Intense research is being done in the field of organic photovoltaics in order to synthesize low band-gap organic molecules. These molecules are electron donors which feature in combination with acceptor molecules, typically fullerene derivarntives, forming an active blend. This active blend has phase separated bicontinuous morphology on a nanometer scale. The highest recorded power conversionrnefficiencies for such cells have been 10.6%. Organic semiconductors differ from inorganic ones due to the presence of tightly bonded excitons (electron-hole pairs)resulting from their low dielectric constant (εr ≈2-4). An additional driving force is required to separate such Frenkel excitons since their binding energy (0.3-1 eV) is too large to be dissociated by an electric field alone. This additional driving force arises from the energy difference between the lowest unoccupied molecular orbital (LUMO) of the donor and the acceptor materials. Moreover, the efficiency of the cells also depends on the difference between the highest occupied molecular orbital (HOMO) of the donor and LUMO of the acceptor. Therefore, a precise control and estimation of these energy levels are required. Furthermore any external influences that change the energy levels will cause a degradation of the power conversion efficiency of organic solar cell materials. In particular, the role of photo-induced degradation on the morphology and electrical performance is a major contribution to degradation and needs to be understood on a nanometer scale. Scanning Probe Microscopy (SPM) offers the resolution to image the nanometer scale bicontinuous morphology. In addition SPM can be operated to measure the local contact potential difference (CPD) of materials from which energy levels in the materials can be derived. Thus SPM is an unique method for the characterization of surface morphology, potential changes and conductivity changes under operating conditions. In the present work, I describe investigations of organic photovoltaic materials upon photo-oxidation which is one of the major causes of degradation of these solar cell materials. SPM, Nuclear Magnetic Resonance (NMR) and UV-Vis spectroscopy studies allowed me to identify the chemical reactions occurring inside the active layer upon photo-oxidation. From the measured data, it was possible to deduce the energy levels and explain the various shifts which gave a better understanding of the physics of the device. In addition, I was able to quantify the degradation by correlating the local changes in the CPD and conductivity to the device characteristics, i.e., open circuit voltage and short circuit current. Furthermore, time-resolved electrostatic force microscopy (tr-EFM) allowed us to probe dynamic processes like the charging rate of the individual donor and acceptor domains within the active blend. Upon photo-oxidation, it was observed, that the acceptor molecules got oxidized first preventing the donor polymer from degrading. Work functions of electrodes can be tailored by modifying the interface with monomolecular thin layers of molecules which are made by a chemical reaction in liquids. These modifications in the work function are particularly attractive for opto-electronic devices whose performance depends on the band alignment between the electrodes and the active material. In order to measure the shift in work function on a nanometer scale, I used KPFM in situ, which means in liquids, to follow changes in the work function of Au upon hexadecanethiol adsorption from decane. All the above investigations give us a better understanding of the photo-degradation processes of the active material at the nanoscale. Also, a method to compare various new materials used for organic solar cells for stability is proposed which eliminates the requirement to make fully functional devices saving time and additional engineering efforts.

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

IDCardOCR: Studio di tecniche di OCR per acquisire dati anagrafici mediante scanning di documenti di identità

Il progetto IDCardOCR si propone di investigare e realizzare le tecnologie per la messa in opera di un servizio avanzato di scanning di documenti di identità e acquisizione automatica dei dati anagrafici in formato strutturato tramite dispositivi mobili. In particolare si vuole realizzare una App Android in grado di: • Acquisire immagini di documenti di identità in diversi formati e rilevare tramite OCR i dati anagrafici. I dati dovranno poi essere salvati in formato strutturato. • Permettere la definizione di diversi template per l’acquisizione di documenti di tipo diverso (patenti, passaporti, IDCard straniere,… ) • Predisporre la possibilità di caricamento dei dati acquisiti su un server.

Analisi di attacchi basati su port scanning

Nella tesi vengono presentate le analisi, con le relative considerazioni, di attacchi basati su port scanning, in particolar modo ci si sofferma sull'utilizzo di risorse, come impiego di CPU, di banda, ecc., in relazione alle diverse tecniche adoperate. Gli attacchi sono stati rivolti attraverso l'applicativo Nmap contro una rete di honeypot, gestiti e configurati attraverso il software Honeyd. Inoltre vengono fornite tutta una serie di conoscenze necessarie al lettore, le quali risulteranno essenziali per poter affrontare tutti gli argomenti trattati, senza doversi necessariamente informare da altre fonti.