The photosynthetic bacterial reaction center in native and artificial envirnoments: effects on light-induced electron transfer

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex.

Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.

Design of optical networks for advanced applications

Nowadays, computing is migrating from traditional high performance and distributed computing to pervasive and utility computing based on heterogeneous networks and clients. The current trend suggests that future IT services will rely on distributed resources and on fast communication of heterogeneous contents. The success of this new range of services is directly linked to the effectiveness of the infrastructure in delivering them. The communication infrastructure will be the aggregation of different technologies even though the current trend suggests the emergence of single IP based transport service. Optical networking is a key technology to answer the increasing requests for dynamic bandwidth allocation and configure multiple topologies over the same physical layer infrastructure, optical networks today are still “far” from accessible from directly configure and offer network services and need to be enriched with more “user oriented” functionalities. However, current Control Plane architectures only facilitate efficient end-to-end connectivity provisioning and certainly cannot meet future network service requirements, e.g. the coordinated control of resources. The overall objective of this work is to provide the network with the improved usability and accessibility of the services provided by the Optical Network. More precisely, the definition of a service-oriented architecture is the enable technology to allow user applications to gain benefit of advanced services over an underlying dynamic optical layer. The definition of a service oriented networking architecture based on advanced optical network technologies facilitates users and applications access to abstracted levels of information regarding offered advanced network services. This thesis faces the problem to define a Service Oriented Architecture and its relevant building blocks, protocols and languages. In particular, this work has been focused on the use of the SIP protocol as a inter-layers signalling protocol which defines the Session Plane in conjunction with the Network Resource Description language. On the other hand, an advantage optical network must accommodate high data bandwidth with different granularities. Currently, two main technologies are emerging promoting the development of the future optical transport network, Optical Burst and Packet Switching. Both technologies respectively promise to provide all optical burst or packet switching instead of the current circuit switching. However, the electronic domain is still present in the scheduler forwarding and routing decision. Because of the high optics transmission frequency the burst or packet scheduler faces a difficult challenge, consequentially, high performance and time focused design of both memory and forwarding logic is need. This open issue has been faced in this thesis proposing an high efficiently implementation of burst and packet scheduler. The main novelty of the proposed implementation is that the scheduling problem has turned into simple calculation of a min/max function and the function complexity is almost independent of on the traffic conditions.

Atmospheric radiative transfer in multiple scattering conditions. Application to NWP models.

High spectral resolution radiative transfer (RT) codes are essential tools in the study of the radiative energy transfer in the Earth atmosphere and a support for the development of parameterizations for fast RT codes used in climate and weather prediction models. Cirrus clouds cover permanently 30% of the Earth's surface, representing an important contribution to the Earth-atmosphere radiation balance. The work has been focussed on the development of the RT model LBLMS. The model, widely tested in the infra-red spectral range, has been extended to the short wave spectrum and it has been used in comparison with airborne and satellite measurements to study the optical properties of cirrus clouds. A new database of single scattering properties has been developed for mid latitude cirrus clouds. Ice clouds are treated as a mixture of ice crystals with various habits. The optical properties of the mixture are tested in comparison to radiometric measurements in selected case studies. Finally, a parameterization of the mixture for application to weather prediction and global circulation models has been developed. The bulk optical properties of ice crystals are parameterized as functions of the effective dimension of measured particle size distributions that are representative of mid latitude cirrus clouds. Tests with the Limited Area Weather Prediction model COSMO have shown the impact of the new parameterization with respect to cirrus cloud optical properties based on ice spheres.

Simulative Investigation on the Electronic, Vibrational and Optical Properties of the Ge2Sb2Te5 Chalcogenide

Chalcogenides are chemical compounds with at least one of the following three chemical elements: Sulfur (S), Selenium (Sn), and Tellurium (Te). As opposed to other materials, chalcogenide atomic arrangement can quickly and reversibly inter-change between crystalline, amorphous and liquid phases. Therefore they are also called phase change materials. As a results, chalcogenide thermal, optical, structural, electronic, electrical properties change pronouncedly and significantly with the phase they are in, leading to a host of different applications in different areas. The noticeable optical reflectivity difference between crystalline and amorphous phases has allowed optical storage devices to be made. Their very high thermal conductivity and heat fusion provided remarkable benefits in the frame of thermal energy storage for heating and cooling in residential and commercial buildings. The outstanding resistivity difference between crystalline and amorphous phases led to a significant improvement of solid state storage devices from the power consumption to the re-writability to say nothing of the shrinkability. This work focuses on a better understanding from a simulative stand point of the electronic, vibrational and optical properties for the crystalline phases (hexagonal and faced-centered cubic). The electronic properties are calculated implementing the density functional theory combined with pseudo-potentials, plane waves and the local density approximation. The phonon properties are computed using the density functional perturbation theory. The phonon dispersion and spectrum are calculated using the density functional perturbation theory. As it relates to the optical constants, the real part dielectric function is calculated through the Drude-Lorentz expression. The imaginary part results from the real part through the Kramers-Kronig transformation. The refractive index, the extinctive and absorption coefficients are analytically calculated from the dielectric function. The transmission and reflection coefficients are calculated using the Fresnel equations. All calculated optical constants compare well the experimental ones.

Electrochemistry of carbon based engineered structures

The main aims of my PhD research work have been the investigation of the redox, photophysical and electronic properties of carbon nanotubes (CNT) and their possible uses as functional substrates for the (electro)catalytic production of oxygen and as molecular connectors for Quantum-dot Molecular Automata. While for CNT many and diverse applications in electronics, in sensors and biosensors field, as a structural reinforcing in composite materials have long been proposed, the study of their properties as individual species has been for long a challenging task. CNT are in fact virtually insoluble in any solvent and, for years, most of the studies has been carried out on bulk samples (bundles). In Chapter 2 an appropriate description of carbon nanotubes is reported, about their production methods and the functionalization strategies for their solubilization. In Chapter 3 an extensive voltammetric and vis-NIR spectroelectrochemical investigation of true solutions of unfunctionalized individual single wall CNT (SWNT) is reported that permitted to determine for the first time the standard electrochemical potentials of reduction and oxidation as a function of the tube diameter of a large number of semiconducting SWNTs. We also established the Fermi energy and the exciton binding energy for individual tubes in solution and, from the linear correlation found between the potentials and the optical transition energies, one to calculate the redox potentials of SWNTs that are insufficiently abundant or absent in the samples. In Chapter 4 we report on very efficient and stable nano-structured, oxygen-evolving anodes (OEA) that were obtained by the assembly of an oxygen evolving polyoxometalate cluster, (a totally inorganic ruthenium catalyst) with a conducting bed of multiwalled carbon nanotubes (MWCNT). Here, MWCNT were effectively used as carrier of the polyoxometallate for the electrocatalytic production of oxygen and turned out to greatly increase both the efficiency and stability of the device avoiding the release of the catalysts. Our bioinspired electrode addresses the major challenge of artificial photosynthesis, i.e. efficient water oxidation, taking us closer to when we might power the planet with carbon-free fuels. In Chapter 5 a study on surface-active chiral bis-ferrocenes conveniently designed in order to act as prototypical units for molecular computing devices is reported. Preliminary electrochemical studies in liquid environment demonstrated the capability of such molecules to enter three indistinguishable oxidation states. Side chains introduction allowed to organize them in the form of self-assembled monolayers (SAM) onto a surface and to study the molecular and redox properties on solid substrates. Electrochemical studies on SAMs of these molecules confirmed their attitude to undergo fast (Nernstian) electron transfer processes generating, in the positive potential region, either the full oxidized Fc+-Fc+ or the partly oxidized Fc+-Fc species. Finally, in Chapter 6 we report on a preliminary electrochemical study of graphene solutions prepared according to an original procedure recently described in the literature. Graphene is the newly-born of carbon nanomaterials and is certainly bound to be among the most promising materials for the next nanoelectronic generation.

Untersuchungen zur Visuomotorik des Menschen

Motorische Bewegungen werden über die visuelle Rückmeldung auf ihre Genauigkeit kontrolliert und ggf. korrigiert. Über einen technischen Eingriff, wie beispielsweise einer Prismenbrille, kann man eine Differenz zwischen optisch wahrgenommener und haptisch erlebter Umwelt erzeugen, um die Fähigkeiten des visuomotorischen Systems zu testen. In dieser Arbeit wurde eine computergestützte Methode entwickelt, eine solche visuomotorische Differenz zu simulieren. Die Versuchspersonen führen eine ballistische Bewegung mit Arm und Hand aus in der Absicht, ein vorgegebenes Ziel zu treffen. Die Trefferpunkte werden durch einen Computer mit Hilfe eines Digitalisierungstablettes aufgenommen. Die visuelle Umwelt, welche den Versuchspersonen präsentiert wird, ist auf einem Monitor dargestellt. Das Monitorabbild – ein Kreuz auf weißem Hintergrund – betrachten die Testpersonen über einen Spiegel. Dieser ist in einem entsprechenden Winkel zwischen Monitor und Digitalisierungstablett angebracht, so dass das Zielbild auf dem Digitalisierungstablett projiziert wird. Die Testpersonen nehmen das Zielkreuz auf dem Digitalisierungstablett liegend wahr. Führt die Versuchsperson eine Zielbewegung aus, können die aufgenommenen Koordinaten als Punkte auf dem Monitor dargestellt werden und die Testperson erhält über diese Punktanzeige ein visuelles Feedback ihrer Bewegung. Der Arbeitsbereich des Digitalisierungstabletts kann über den Computer eingerichtet und so motorische Verschiebungen simuliert werden. Die verschiedenartigen Möglichkeiten dieses Aufbaus wurden zum Teil in Vorversuchen getestet um Fragestellungen, Methodik und technische Einrichtungen aufeinander abzustimmen. Den Hauptversuchen galt besonderes Interesse an der zeitlichen Verzögerung des visuellen Feedbacks sowie dem intermanuellen Transfer. Hierbei ergaben sich folgende Ergebnisse: ● Die Versuchspersonen adaptieren an eine räumlich verschobene Umwelt. Der Adaptationsverlauf lässt sich mit einer Exponentialfunktion mathematisch berechnen und darstellen. ● Dieser Verlauf ist unabhängig von der Art des visuellen Feedbacks. Die Beobachtung der Handbewegung während der Adaptation zeigt die gleiche Zielabfolge wie eine einfache Punktprojektion, die den Trefferort der Bewegung darstellt. ● Der exponentielle Verlauf der Adaptationsbewegung ist unabhängig von den getesteten zeitlichen Verzögerungen des visuellen Feedbacks. ● Die Ergebnisse des Folgeeffektes zeigen, dass bei zunehmender zeitlicher Verzögerung des visuellen Feedbacks während der Adaptationsphase, die Größe des Folgeeffektwertes geringer wird, d.h. die anhaltende Anpassungsleistung an eine visuomotorische Differenz sinkt. ● Die Folgeeffekte weisen individuelle Eigenheiten auf. Die Testpersonen adaptieren verschieden stark an eine simulierte Verschiebung. Ein Vergleich mit den visuomotorischen Herausforderungen im Vorleben der Versuchspersonen ließ vermuten, dass das visuomotorische System des Menschen trainierbar ist und sich - je nach Trainingszustand – unterschiedlich an wahrgenommene Differenzen anpasst. ● Der intermanuelle Transfer konnte unter verschiedenen Bedingungen nachgewiesen werden. ● Ein deutlich stärkerer Folgeeffekt kann beobachtet werden, wenn die wahrgenommene visuomotorische Differenz zwischen Ziel und Trefferpunkt in eine Gehirnhälfte projiziert wird und der Folgeeffekt mit der Hand erfolgt, welche von dieser Hirnhemisphäre gesteuert wird. Der intermanuelle Transfer wird demnach begünstigt, wenn die visuelle Projektion der Fehlerbeobachtung in die Gehirnhälfte erfolgt, die während der Adaptationsphase motorisch passiv ist.

Organic heterostructure approach for multifunctional light-emitting field-effect transistors

The possibility of combining different functionalities in a single device is of great relevance for further development of organic electronics in integrated components and circuitry. Organic light-emitting transistors (OLETs) have been demonstrated to be able to combine in a single device the electrical switching functionality of a field-effect transistor and the capability of light generation. A novel strategy in OLET realization is the tri-layer vertical hetero-junction. This configuration is similar to the bi-layer except for the presence of a new middle layer between the two transport layers. This “recombination” layer presents high emission quantum efficiency and OLED-like (Organic Light-Emitting Diode) vertical bulk mobility value. The key idea of the vertical tri-layer hetero-junction approach in realizing OLETs is that each layer has to be optimized according to its specific function (charge transport, energy transfer, radiative exciton recombination). Clearly, matching the overall device characteristics with the functional properties of the single materials composing the active region of the OFET, is a great challenge that requires a deep investigation of the morphological, optical and electrical features of the system. As in the case of the bi-layer based OLETs, it is clear that the interfaces between the dielectric and the bottom transport layer and between the recombination and the top transport layer are crucial for guaranteeing good ambipolar field-effect electrical characteristics. Moreover interfaces between the bottom transport and the recombination layer and between the recombination and the top transport layer should provide the favourable conditions for the charge percolation to happen in the recombination layer and form excitons. Organic light emitting transistor based on the tri-layer approach with external quantum efficiency out-performing the OLED state of the art has been recently demonstrated [Capelli et al., Nat. Mater. 9 (2010) 496-503] widening the scientific and technological interest in this field of research.

Optical Design for Automatic Identification and Portable Systems

This thesis proposes design methods and test tools, for optical systems, which may be used in an industrial environment, where not only precision and reliability but also ease of use is important. The approach to the problem has been conceived to be as general as possible, although in the present work, the design of a portable device for automatic identification applications has been studied, because this doctorate has been funded by Datalogic Scanning Group s.r.l., a world-class producer of barcode readers. The main functional components of the complete device are: electro-optical imaging, illumination and pattern generator systems. For what concerns the electro-optical imaging system, a characterization tool and an analysis one has been developed to check if the desired performance of the system has been achieved. Moreover, two design tools for optimizing the imaging system have been implemented. The first optimizes just the core of the system, the optical part, improving its performance ignoring all other contributions and generating a good starting point for the optimization of the whole complex system. The second tool optimizes the system taking into account its behavior with a model as near as possible to reality including optics, electronics and detection. For what concerns the illumination and the pattern generator systems, two tools have been implemented. The first allows the design of free-form lenses described by an arbitrary analytical function exited by an incoherent source and is able to provide custom illumination conditions for all kind of applications. The second tool consists of a new method to design Diffractive Optical Elements excited by a coherent source for large pattern angles using the Iterative Fourier Transform Algorithm. Validation of the design tools has been obtained, whenever possible, comparing the performance of the designed systems with those of fabricated prototypes. In other cases simulations have been used.

Continuos Flow Single Cell Separation into Open Microwell Arrays

A novel design based on electric field-free open microwell arrays for the automated continuous-flow sorting of single or small clusters of cells is presented. The main feature of the proposed device is the parallel analysis of cell-cell and cell-particle interactions in each microwell of the array. High throughput sample recovery with a fast and separate transfer from the microsites to standard microtiter plates is also possible thanks to the flexible printed circuit board technology which permits to produce cost effective large area arrays featuring geometries compatible with laboratory equipment. The particle isolation is performed via negative dielectrophoretic forces which convey the particles’ into the microwells. Particles such as cells and beads flow in electrically active microchannels on whose substrate the electrodes are patterned. The introduction of particles within the microwells is automatically performed by generating the required feedback signal by a microscope-based optical counting and detection routine. In order to isolate a controlled number of particles we created two particular configurations of the electric field within the structure. The first one permits their isolation whereas the second one creates a net force which repels the particles from the microwell entrance. To increase the parallelism at which the cell-isolation function is implemented, a new technique based on coplanar electrodes to detect particle presence was implemented. A lock-in amplifying scheme was used to monitor the impedance of the channel perturbed by flowing particles in high-conductivity suspension mediums. The impedance measurement module was also combined with the dielectrophoretic focusing stage situated upstream of the measurement stage, to limit the measured signal amplitude dispersion due to the particles position variation within the microchannel. In conclusion, the designed system complies with the initial specifications making it suitable for cellomics and biotechnology applications.

Influence of ice crystal habit and cirrus spatial inhomogeneities on the retrieval of cirrus optical thickness and effective radius

Diese Dissertation untersucht den Einfluss von Eiskristallform und räumlicher Inhomogenität von Zirren auf das Retrieval von optischer Wolkendicke und effektivem Eispartikelradius. Zu diesem Zweck werden flugzeuggetragene spektrale Messungen solarer Strahlung sowie solare und langwellige Strahlungstransfersimulationen durchgeführt. Flugzeuggetragene spektrale aufwärtsgerichtete Radianzen (Strahldichten) sind mit dem SMART-Albedometer (Spectral Modular Airborne Radiation measurement sysTem) während des CIRCLE-2 (CIRrus CLoud Experiment-2) Feldexperiments im Mai 2007 gemessen worden. Basierend auf diesen Radianzdaten werden mittels eines Wolkenretrievalalgorithmus optische Wolkendicken und effektive Eispartikelradien anhand von eindimensionalen Strahlungstransferrechnungen bestimmt. Die Auswirkung der Annahme unterschiedlicher Eiskristallformen auf die retrievten Parameter wird durch Variation der Einfachstreueigenschaften der Eispartikel untersucht. Darüber hinaus wird mittels Strahlungstransferrechnungen auch der Einfluss der Eiskristallform auf den Strahlungsantrieb von Eiswolken ermittelt. Die Frage nach dem relativen Einfluss von räumlicher Wolkeninhomogenität und Eiskristallform wird anhand von dreidimensionalen und independent pixel approximation (IPA) Strahlungssimulationen untersucht. Die Analyse basiert auf einer Modelleiswolke, die aus Daten des NASA (National Aeronautics and Space Administration) TC4 (Tropical Composition, Cloud, and Climate Coupling) Feldexperiments im Sommer 2007 in Costa Rica erzeugt wurde. Lokal gesehen können beide Effekte - Eiskristallform und räumliche Eiswolkeninhomogenität - die gleiche Grössenordnung haben und zu einer Unter- bzw. Überschätzung der retrievten Parameter um 40 – 60% führen. Gemittelt über die ganze Wolke ist jedoch der Einfluss der Eiskristallform viel bedeutender als der von räumlichen Inhomogenitäten.

Quantum dot-dye hybrid systems for energy transfer applications

In this thesis, we focus on the preparation of energy transfer-based quantum dot (QD)-dye hybrid systems. Two kinds of QD-dye hybrid systems have been successfully synthesized: QD-silica-dye and QD-dye hybrid systems.rn rnIn the QD-silica-dye hybrid system, multishell CdSe/CdS/ZnS QDs were adsorbed onto monodisperse Stöber silica particles with an outer silica shell of thickness 2 - 24 nm containing organic dye molecules (Texas Red). The thickness of this dye layer has a strong effect on the total sensitized acceptor emission, which is explained by the increase in the number of dye molecules homogeneously distributed within the silica shell, in combination with an enhanced surface adsorption of QDs with increasing dye amount. Our conclusions were underlined by comparison of the experimental results with Monte-Carlo simulations, and by control experiments confirming attractive interactions between QDs and Texas Red freely dissolved in solution. rnrnNew QD-dye hybrid system consisting of multishell QDs and organic perylene dyes have been synthesized. We developed a versatile approach to assemble extraordinarily stable QD-dye hybrids, which uses dicarboxylate anchors to bind rylene dyes to QD. This system yields a good basis to study the energy transfer between QD and dye because of its simple and compact design: there is no third kind of molecule linking QD and dye; no spacer; and the affinity of the functional group to the QD surface is strong. The FRET signal was measured for these complexes as a function of both dye to QD ratio and center-to-center distance between QD and dye by controlling number of covered ZnS layers. Data showed that fluorescence resonance energy transfer (FRET) was the dominant mechanism of the energy transfer in our QD-dye hybrid system. FRET efficiency can be controlled by not only adjusting the number of dyes on the QD surface or the QD to dye distance, but also properly choosing different dye and QD components. Due to the strong stability, our QD-dye complexes can also be easily transferred into water. Our approach can apply to not only dye molecules but also other organic molecules. As an example, the QDs have been complexed with calixarene molecules and the QD-calixarene complexes also have potential for QD-based energy transfer study. rn

New biosensor applications of surface plasmon and hydrogel optical waveguide spectroscopy

Rapid and sensitive detection of chemical and biological analytes becomes increasingly important in areas such as medical diagnostics, food control and environmental monitoring. Optical biosensors based on surface plasmon resonance (SPR) and optical waveguide spectroscopy have been extensively pushed forward in these fields. In this study, we combine SPR, surface plasmon-enhanced fluorescence spectroscopy (SPFS) and optical waveguide spectroscopy with hydrogel thin film for highly sensitive detection of molecular analytes.rnrnA novel biosensor based on SPFS which was advanced through the excitation of long range surface plasmons (LRSPs) is reported in this study. LRSPs are special surface plasmon waves propagating along thin metal films with orders of magnitude higher electromagnetic field intensity and lower damping than conventional SPs. Therefore, their excitation on the sensor surface provides further increased fluorescence signal. An inhibition immunoassay based on LRSP-enhanced fluorescence spectroscopy (LRSP-FS) was developed for the detection of aflatoxin M1 (AFM1) in milk. The biosensor allowed for the detection of AFM1 in milk at concentrations as low as 0.6 pg mL-1, which is about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.rnrnIn addition, LRSPs probe the medium adjacent to the metallic surface with more extended evanescent field than regular SPs. Therefore, three-dimensional binding matrices with up to micrometer thickness have been proposed for the immobilization of biomolecular recognition elements with large surface density that allows to exploit the whole evanescent field of LRSP. A photocrosslinkable carboxymethyl dextran (PCDM) hydrogel thin film is used as a binding matrix, and it is applied for the detection of free prostate specific antigen (f-PSA) based on the LRSP-FS and sandwich immunoassay. We show that this approach allows for the detection of f-PSA at low femto-molar range, which is approximately four orders of magnitude lower than that for direct detection of f-PSA based on the monitoring of binding-induced refractive index changes.rnrnHowever, a three dimensional hydrogel binding matrix with micrometer thickness can also serve as an optical waveguide. Based on the measurement of binding-induced refractive index changes, a hydrogel optical waveguide spectroscopy (HOWS) is reported for a label-free biosensor. This biosensor is implemented by using a SPR optical setup in which a carboxylated poly(N-isoproprylacrylamide) (PNIPAAm) hydrogel film is attached on a metallic surface and modified by protein catcher molecules. Compared to regular SPR biosensor with thiol self-assembled monolayer (SAM), HOWS provides an order of magnitude improved resolution in the refractive index measurements and enlarged binding capacity owing to its low damping and large swelling ratio, respectively. A model immunoassay experiment revealed that HOWS allowed detection of IgG molecules with a 10 pM limit of detection (LOD) that was five-fold lower than that achieved for SPR with thiol SAM. For the high capacity hydrogel matrix, the affinity binding was mass transport limited.rnrnThe mass transport of target molecules to the sensor surface can play as critical a role as the chemical reaction itself. In order to overcome the diffusion-limited mass transfer, magnetic iron oxide nanoparticles were employed. The magnetic nanoparticles (MNPs) can serve both as labels providing enhancement of the refractive index changes, and “vehicles” for rapidly delivering the analytes from sample solution to an SPR sensor surface with a gradient magnetic field. A model sandwich assay for the detection of β human chorionic gonadotropin (βhCG) has been utilized on a gold sensor surface with metallic diffraction grating structure supporting the excitation of SPs. Various detection formats including a) direct detection, b) sandwich assay, c) MNPs immunoassay without and d) with applied magnetic field were compared. The results show that the highly-sensitive MNPs immunoassay improves the LOD on the detection of βhCG by a factor of 5 orders of magnitude with respect to the direct detection.rn

Bottle microresonators for applications in quantum optics and all-optical signal processing

This thesis reports on the experimental realization, characterization and application of a novel microresonator design. The so-called “bottle microresonator” sustains whispering-gallery modes in which light fields are confined near the surface of the micron-sized silica structure by continuous total internal reflection. While whispering-gallery mode resonators in general exhibit outstanding properties in terms of both temporal and spatial confinement of light fields, their monolithic design makes tuning of their resonance frequency difficult. This impedes their use, e.g., in cavity quantum electrodynamics (CQED) experiments, which investigate the interaction of single quantum mechanical emitters of predetermined resonance frequency with a cavity mode. In contrast, the highly prolate shape of the bottle microresonators gives rise to a customizable mode structure, enabling full tunability. The thesis is organized as follows: In chapter I, I give a brief overview of different types of optical microresonators. Important quantities, such as the quality factor Q and the mode volume V, which characterize the temporal and spatial confinement of the light field are introduced. In chapter II, a wave equation calculation of the modes of a bottle microresonator is presented. The intensity distribution of different bottle modes is derived and their mode volume is calculated. A brief description of light propagation in ultra-thin optical fibers, which are used to couple light into and out of bottle modes, is given as well. The chapter concludes with a presentation of the fabrication techniques of both structures. Chapter III presents experimental results on highly efficient, nearly lossless coupling of light into bottle modes as well as their spatial and spectral characterization. Ultra-high intrinsic quality factors exceeding 360 million as well as full tunability are demonstrated. In chapter IV, the bottle microresonator in add-drop configuration, i.e., with two ultra-thin fibers coupled to one bottle mode, is discussed. The highly efficient, nearly lossless coupling characteristics of each fiber combined with the resonator's high intrinsic quality factor, enable resonant power transfers between both fibers with efficiencies exceeding 90%. Moreover, the favorable ratio of absorption and the nonlinear refractive index of silica yields optical Kerr bistability at record low powers on the order of 50 µW. Combined with the add-drop configuration, this allows one to route optical signals between the outputs of both ultra-thin fibers, simply by varying the input power, thereby enabling applications in all-optical signal processing. Finally, in chapter V, I discuss the potential of the bottle microresonator for CQED experiments with single atoms. Its Q/V-ratio, which determines the ratio of the atom-cavity coupling rate to the dissipative rates of the subsystems, aligns with the values obtained for state-of-the-art CQED microresonators. In combination with its full tunability and the possibility of highly efficient light transfer to and from the bottle mode, this makes the bottle microresonator a unique tool for quantum optics applications.

Direct electron transfer to cytochrome c oxidase investigated by electrochemistry and time-resolved surface-enhanced infrared absorption spectroscopy

Cytochrom c Oxidase (CcO), der Komplex IV der Atmungskette, ist eine der Häm-Kupfer enthaltenden Oxidasen und hat eine wichtige Funktion im Zellmetabolismus. Das Enzym enthält vier prosthetische Gruppen und befindet sich in der inneren Membran von Mitochondrien und in der Zellmembran einiger aerober Bakterien. Die CcO katalysiert den Elektronentransfer (ET) von Cytochrom c zu O2, wobei die eigentliche Reaktion am binuklearen Zentrum (CuB-Häm a3) erfolgt. Bei der Reduktion von O2 zu zwei H2O werden vier Protonen verbraucht. Zudem werden vier Protonen über die Membran transportiert, wodurch eine elektrochemische Potentialdifferenz dieser Ionen zwischen Matrix und Intermembranphase entsteht. Trotz ihrer Wichtigkeit sind Membranproteine wie die CcO noch wenig untersucht, weshalb auch der Mechanismus der Atmungskette noch nicht vollständig aufgeklärt ist. Das Ziel dieser Arbeit ist, einen Beitrag zum Verständnis der Funktion der CcO zu leisten. Hierzu wurde die CcO aus Rhodobacter sphaeroides über einen His-Anker, der am C-Terminus der Untereinheit II angebracht wurde, an eine funktionalisierte Metallelektrode in definierter Orientierung gebunden. Der erste Elektronenakzeptor, das CuA, liegt dabei am nächsten zur Metalloberfläche. Dann wurde eine Doppelschicht aus Lipiden insitu zwischen die gebundenen Proteine eingefügt, was zur sog. proteingebundenen Lipid-Doppelschicht Membran (ptBLM) führt. Dabei musste die optimale Oberflächenkonzentration der gebundenen Proteine herausgefunden werden. Elektrochemische Impedanzspektroskopie(EIS), Oberflächenplasmonenresonanzspektroskopie (SPR) und zyklische Voltammetrie (CV) wurden angewandt um die Aktivität der CcO als Funktion der Packungsdichte zu charakterisieren. Der Hauptteil der Arbeit betrifft die Untersuchung des direkten ET zur CcO unter anaeroben Bedingungen. Die Kombination aus zeitaufgelöster oberflächenverstärkter Infrarot-Absorptionsspektroskopie (tr-SEIRAS) und Elektrochemie hat sich dafür als besonders geeignet erwiesen. In einer ersten Studie wurde der ET mit Hilfe von fast scan CV untersucht, wobei CVs von nicht-aktivierter sowie aktivierter CcO mit verschiedenen Vorschubgeschwindigkeiten gemessen wurden. Die aktivierte Form wurde nach dem katalytischen Umsatz des Proteins in Anwesenheit von O2 erhalten. Ein vier-ET-modell wurde entwickelt um die CVs zu analysieren. Die Methode erlaubt zwischen dem Mechanismus des sequentiellen und des unabhängigen ET zu den vier Zentren CuA, Häm a, Häm a3 und CuB zu unterscheiden. Zudem lassen sich die Standardredoxpotentiale und die kinetischen Koeffizienten des ET bestimmen. In einer zweiten Studie wurde tr-SEIRAS im step scan Modus angewandt. Dafür wurden Rechteckpulse an die CcO angelegt und SEIRAS im ART-Modus verwendet um Spektren bei definierten Zeitscheiben aufzunehmen. Aus diesen Spektren wurden einzelne Banden isoliert, die Veränderungen von Vibrationsmoden der Aminosäuren und Peptidgruppen in Abhängigkeit des Redoxzustands der Zentren zeigen. Aufgrund von Zuordnungen aus der Literatur, die durch potentiometrische Titration der CcO ermittelt wurden, konnten die Banden versuchsweise den Redoxzentren zugeordnet werden. Die Bandenflächen gegen die Zeit aufgetragen geben dann die Redox-Kinetik der Zentren wieder und wurden wiederum mit dem vier-ET-Modell ausgewertet. Die Ergebnisse beider Studien erlauben die Schlussfolgerung, dass der ET zur CcO in einer ptBLM mit größter Wahrscheinlichkeit dem sequentiellen Mechanismus folgt, was dem natürlichen ET von Cytochrom c zur CcO entspricht.