From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

Atomic force microscopy of biomimetic systems

This thesis was driven by the ambition to create suitable model systems that mimic complex processes in nature, like intramolecular transitions, such as unfolding and refolding of proteins, or intermolecular interactions between different cell compo-nents. Novel biophysical approaches were adopted by employing atomic force mi-croscopy (AFM) as the main measurement technique due to its broad diversity. Thus, high-resolution imaging, adhesion measurements, and single-molecule force distance experiments were performed on the verge of the instrumental capabilities. As first objective, the interaction between plasma membrane and cytoskeleton, me-diated by the linker protein ezrin, was pursued. Therefore, the adsorption process and the lateral organization of ezrin on PIP2 containing solid-supported membranes were characterized and quantified as a fundament for the establishment of a biomimetic model system. As second component of the model system, actin filaments were coated on functionalized colloidal probes attached on cantilevers, serving as sensor elements. The zealous endeavor of creating this complex biomimetic system was rewarded by successful investigation of the activation process of ezrin. As a result, it can be stated that ezrin is activated by solely binding to PIP2 without any further stimulating agents. Additional cofactors may stabilize and prolong the active conformation but are not essentially required for triggering ezrin’s transformation into an active conformation. In the second project, single-molecule force distance experiments were performed on bis-loop tetra-urea calix[4]arene-catenanes with different loading rates (increase in force per second). These macromolecules were specifically designed to investigate the rupture and rejoining mechanism of hydrogen bonds under external load. The entangled loops of capsule-like molecules locked the unbound state of intramolecular hydrogen bonds mechanically, rendering a rebinding observable on the experimental time scale. In conjunction with Molecular Dynamics simulations, a three-well potential of the bond rupture process was established and all kinetically relevant parameters of the experiments were determined by means of Monte Carlo simulations and stochastic modeling. In summary, it can be stated that atomic force microscopy is an invaluable tool to scrutinize relevant processes in nature, such as investigating activation mechanisms in proteins, as shown by analysis of the interaction between F-actin and ezrin, as well as exploring fundamental properties of single hydrogen bonds that are of paramount interest for the complete understanding of complex supramolecular structures.

Tailoring molecule nanostructures on insulating surfaces investigated by non-contact atomic force microscopy

Self-assembled molecular structures were investigated on insulating substrate surfaces using non-contact atomic force microscopy. Both, substrate preparation and molecule deposition, took place under ultra-high vacuum conditions. First, C60 molecules were investigated on the TiO2 (110) surface. This surface exhibits parallel running troughs at the nanometer scale, which strongly steer the assembly of the molecules. This is in contrast to the second investigated surface. The CaF2 (111) surface is atomically flat and the molecular assemblyrnwas observed to be far less affected by the surface. Basically different island structures were observed to what is typically know. Based on extensive experimental studies and theoretical considerations, a comprehensive picture of the processes responsible for the island formation of C60 molecules on this insulating surfaces was developed. The key process for the emergence of the observed novel island structures was made out to be the dewetting of molecules from the substrate. This new knowledge allows to further understand andrnexploit self-assembly techniques in structure fabrication on insulating substrate surfaces. To alter island formation and island structure, C60 molecules were codeposited with second molecule species (PTCDI and SubPc) on the CaF2 (111) surface. Depending on the order of deposition, quiet different structures were observed to arise. Thus, these are the first steps towards more complex functional arrangements consisting of two molecule species on insulating surfaces.

Atomic-scale characterization of diamond surfaces and fullerene self-assembly

In this thesis, elemental research towards the implantation of a diamond-based molecular quantum computer is presented. The approach followed requires linear alignment of endohedral fullerenes on the diamond C(100) surface in the vicinity of subsurface NV-centers. From this, four fundamental experimental challenges arise: 1) The well-controlled deposition of endohedral fullerenes on a diamond surface. 2) The creation of NV-centers in diamond close to the surface. 3) Preparation and characterization of atomically-flat diamondsurfaces. 4) Assembly of linear chains of endohedral fullerenes. First steps to overcome all these challenges were taken in the framework of this thesis. Therefore, a so-called “pulse injection” technique was implemented and tested in a UHV chamber that was custom-designed for this and further tasks. Pulse injection in principle allows for the deposition of molecules from solution onto a substrate and can therefore be used to deposit molecular species that are not stable to sublimation under UHV conditions, such as the endohedral fullerenes needed for a quantum register. Regarding the targeted creation of NV-centers, FIB experiments were carried out in cooperation with the group of Prof. Schmidt-Kaler (AG Quantum, Physics Department, Johannes Gutenberg-Universität Mainz). As an entry into this challenging task, argon cations were implanted into (111) surface-oriented CaF2 crystals. The resulting implantation spots on the surface were imaged and characterized using AFM. In this context, general relations between the impact of the ions on the surface and their valency or kinetic energy, respectively, could be established. The main part of this thesis, however, is constituted by NCAFM studies on both, bare and hydrogen-terminated diamond C(100) surfaces. In cooperation with the group of Prof. Dujardin (Molecular Nanoscience Group, ISMO, Université de Paris XI), clean and atomically-flat diamond surfaces were prepared by exposure of the substrate to a microwave hydrogen plasma. Subsequently, both surface modifications were imaged in high resolution with NC-AFM. In the process, both hydrogen atoms in the unit cell of the hydrogenated surface were resolved individually, which was not achieved in previous STM studies of this surface. The NC-AFM images also reveal, for the first time, atomic-resolution contrast on the clean, insulating diamond surface and provide real-space experimental evidence for a (2×1) surface reconstruction. With regard to the quantum computing concept, high-resolution NC-AFM imaging was also used to study the adsorption and self-assembly potential of two different kinds of fullerenes (C60 and C60F48) on aforementioned diamond surfaces. In case of the hydrogenated surface, particular attention was paid to the influence of charge transfer doping on the fullerene-substrate interaction and the morphology emerging from self-assembly. Finally, self-assembled C60 islands on the hydrogen-terminated diamond surface were subject to active manipulation by an NC-AFM tip. Two different kinds of tip-induced island growth modes have been induced and were presented. In conclusion, the results obtained provide fundamental informations mandatory for the realization of a molecular quantum computer. In the process it was shown that NC-AFM is, under proper circumstances, a very capable tool for imaging diamond surfaces with highest resolution, surpassing even what has been achieved with STM up to now. Particular attention was paid to the influence of transfer doping on the morphology of fullerenes on the hydrogenated diamond surface, revealing new possibilities for tailoring the self-assembly of molecules that have a high electron affinity.

Revealing atomic-scale details in the force fields above carbonate surfaces

Diese Arbeit stellt eine ausführliche Studie fundamentaler Eigenschaften der Kalzit CaCO3(10.4) und verwandter Mineraloberflächen dar, welche nicht nur durch die Verwendung von Nichtkontakt Rasterkraftmikroskopie, sondern hauptsächlich durch die Messung von Kraftfeldern ermöglicht wurde. Die absolute Oberflächenorientierung sowie der hierfür zugrundeliegende Prozess auf atomarer Skala konnten erfolgreich für die Kalzit (10.4) Oberfläche identifiziert werden.rnDie Adsorption chiraler Moleküle auf Kalzit ist relevant im Bereich der Biomineralisation, was ein Verständnis der Oberflächensymmetrie unumgänglich macht. Die Messung des Oberflächenkraftfeldes auf atomarer Ebene ist hierfür ein zentraler Aspekt. Eine solche Kraftkarte beleuchtet nicht nur die für die Biomineralisation wichtige Wechselwirkung der Oberfläche mit Molekülen, sondern enthält auch die Möglichkeit, Prozesse auf atomarer Skala und damit Oberflächeneigenschaften zu identifizieren.rnDie Einführung eines höchst flexiblen Messprotokolls gewährleistet die zuverlässige und kommerziell nicht erhältliche Messung des Oberflächenkraftfeldes. Die Konversion der rohen ∆f Daten in die vertikale Kraft Fz ist jedoch kein trivialer Vorgang, insbesondere wenn Glätten der Daten in Frage kommt. Diese Arbeit beschreibt detailreich, wie Fz korrekt für die experimentellen Bedingungen dieser Arbeit berechnet werden können. Weiterhin ist beschrieben, wie Lateralkräfte Fy und Dissipation Γ erhalten wurden, um das volle Potential dieser Messmethode auszureizen.rnUm Prozesse auf atomarer Skala auf Oberflächen zu verstehen sind die kurzreichweitigen, chemischen Kräfte Fz,SR von größter Wichtigkeit. Langreichweitige Beiträge müssen hierzu an Fz angefittet und davon abgezogen werden. Dies ist jedoch eine fehleranfällige Aufgabe, die in dieser Arbeit dadurch gemeistert werden konnte, dass drei unabhängige Kriterien gefunden wurden, die den Beginn zcut von Fz,SR bestimmen, was für diese Aufgabe von zentraler Bedeutung ist. Eine ausführliche Fehleranalyse zeigt, dass als Kriterium die Abweichung der lateralen Kräfte voneinander vertrauenswürdige Fz,SR liefert. Dies ist das erste Mal, dass in einer Studie ein Kriterium für die Bestimmung von zcut gegeben werden konnte, vervollständigt mit einer detailreichen Fehleranalyse.rnMit der Kenntniss von Fz,SR und Fy war es möglich, eine der fundamentalen Eigenschaften der CaCO3(10.4) Oberfläche zu identifizieren: die absolute Oberflächenorientierung. Eine starke Verkippung der abgebildeten Objekte