High-quality factor oscillators are often used in measurements of verysmall force since they exhibit an enhanced sensitivity in the narrow frequencyband around resonance. Forces containing frequencies outside this frequencyband are often not detectable and the total force acting on the oscillatorremains unknown. In this thesis we present methods to eciently use theavailable bandwidth around resonance to reconstruct the force from partialspectral information.We apply the methods to dynamic atomic force microscopy (AFM) wherea tip at the end of a small micro-cantilever oscillates close to a sample surface.By reconstructing the force between the tip and the surface we can deducedierent properties of the surface. In contrast, in conventional AFM only oneof the many frequency components of the time-dependent tip-surface forceallowing for only qualitative conclusions about the tip-surface force.To increase the number of measurable frequency components we developed Intermodulation AFM (ImAFM). ImAFM utilizes frequency mixing ofa multifrequency drive scheme which generates many frequencies in the response to the nonlinear character of the tip-surface interaction. ImAFM,amplitude-modulated AFM and frequency-modulated AFM can be considered as special cases of narrow-band AFM, where the tip motion can bedescribed by a rapidly oscillating part and a slowly-varying envelope function. Using the concept of force quadratures, each rapid oscillation cycle canbe analyzed individually and ImAFM measurements can be interpreted as arapid measurement of the dependence of the force quadratures on the oscillation amplitude or frequency. To explore the limits of the force quadraturesdescription we introduce the force disk which is a complete description of thetip-surface force in narrow-band AFM at xed static probe height.We present a polynomial force reconstruction method for multifrequencyAFM data. The polynomial force reconstruction is a linear approximativeforce reconstruction method which is based on nding the parameters of amodel force which best approximates the tip-surface force. Another classof reconstruction methods are integral techniques which aim to invert theintegral relation between the tip-surface force and the measured spectraldata. We present an integral method, amplitude-dependence force spectroscopy (ADFS), which reconstructs the conservative tip-surface force fromthe amplitude-dependence of the force quadratures. Together with ImAFMwe use ADFS to combine high-resolution AFM imaging at high speeds withhighly accurate force measurements in each point of an image. For the measurement of dissipative forces we discuss how methods from tomography canbe used to reconstruct forces that are a function of both tip position andvelocity.The methods developed in this thesis are not limited to dynamic AFM andwe describe them in the general context of a harmonic oscillator subject to anexternal force. We hope that theses methods contribute to the transformationof AFM from a qualitative imaging modality into quantitative microscopy andwe hope that they nd application in other measurements which exploit theenhanced sensitivity of a high-quality factor oscillator. / <p>QC 20130527</p>

Owing to atomic force microscopy’s (AFM) high-resolution in both imaging and force spectroscopy, it is very successful in probing not only structures, but also nanomechanics of biological samples in solution. In this thesis, the nanomechanical properties of lipid bilayers of biological relevance and proteins of the barnacle adhesive were examined using AFM indentation, AFM-based force mapping, and single-molecule pulling experiments. Through high-resolution AFM-based force mapping, the self-organized structures exhibited in phase-segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine / egg sphingomyelin / cholesterol (DEC) in the absence and presence of ceramide (DEC-Ceramide) were directly correlated with their breakthrough forces, elastic moduli, adhesion, and bilayer thickness. Results were presented as two-dimensional visual maps. The highly stable ceramide-enriched domains in DEC-Ceramide bilayers and the effect of different levels of cholesterol as well as of diblock copolymers, on the nanomechanical stability of the model systems studied were further examined. For the proteins of the barnacle adhesive, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and chemical staining with amyloid-selective dyes, in addition to AFM imaging, indentation, and pulling experiments were performed to study the structure and nanomechanics of the polymerized barnacle glue. Nanoscale structures exhibiting rod-shaped, globular, and irregularly shaped morphologies were observed in the bulk barnacle cement by AFM. SEM coupled with energy dispersive x-ray (EDX) makes evident the organic nature of the rod-shaped nanoscale structures while FTIR spectroscopy on the bulk cement gave signatures of β-sheet and random coil conformations. Indentation data yielded higher elastic moduli for the rod-shaped structures as compared to the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo Red and Thioflavin-T) revealed that about 5% of the bulk cement are amyloids.

Owing to atomic force microscopy’s (AFM) high-resolution in both imaging and force spectroscopy, it is very successful in probing not only structures, but also nanomechanics of biological samples in solution. In this thesis, the nanomechanical properties of lipid bilayers of biological relevance and proteins of the barnacle adhesive were examined using AFM indentation, AFM-based force mapping, and single-molecule pulling experiments. Through high-resolution AFM-based force mapping, the self-organized structures exhibited in phase-segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine / egg sphingomyelin / cholesterol (DEC) in the absence and presence of ceramide (DEC-Ceramide) were directly correlated with their breakthrough forces, elastic moduli, adhesion, and bilayer thickness. Results were presented as two-dimensional visual maps. The highly stable ceramide-enriched domains in DEC-Ceramide bilayers and the effect of different levels of cholesterol as well as of diblock copolymers, on the nanomechanical stability of the model systems studied were further examined. For the proteins of the barnacle adhesive, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and chemical staining with amyloid-selective dyes, in addition to AFM imaging, indentation, and pulling experiments were performed to study the structure and nanomechanics of the polymerized barnacle glue. Nanoscale structures exhibiting rod-shaped, globular, and irregularly shaped morphologies were observed in the bulk barnacle cement by AFM. SEM coupled with energy dispersive x-ray (EDX) makes evident the organic nature of the rod-shaped nanoscale structures while FTIR spectroscopy on the bulk cement gave signatures of β-sheet and random coil conformations. Indentation data yielded higher elastic moduli for the rod-shaped structures as compared to the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo Red and Thioflavin-T) revealed that about 5% of the bulk cement are amyloids.

The work presented here has a multidisciplinary character, having as a common factor the characterization of self-assembled nanostructures through force spectroscopy. Exploring AFM as a tool for characterizing self-assembly and interaction forces in soft matter nanostructures, three different Bio and nonbiological systems where investigated, all of them share the common characteristic of being soft matter molecular structures at the nanoscale. The studied systems in question are: a) Polyelectrolyte – lipid nanocomposites. Single polyelectrolyte adsorption-desorption from supported lipid bilayers, b) Polyelectrolyte brushes and c) Virus-Like particles (VLPs). The scientific interest and industrial applications for each of these different nanostructures is broad, and their potential uses in the near future ranges from smart nanocontainers for drug and gene delivery, surface platforms for molecular recognition to the development of new nanodevices with ultrasensitive external stimuli responsiveness. These nano-structures are constructed following assembly of smaller subunits and belong to representative examples of soft matter in modern nanotechnology. The stability, behavior, properties and long term durability of these self-organized structures depends strongly on the environmental conditions to which they are exposed since their building mechanism is a balance between attractive noncovalent interactions and momentum transmitted collisions due Brownian motion of the solvent molecules. For example a set of long chain molecules firmly attached to one end to a surface will alter their conformation as the space between them is reduced or the environmental conditions are modified (i.e. ionic strength, pH or temperature). For a highly packed condition, this fuzzy surface known as a polyelectrolyte brush will then behave as a responsive material with tunable responsiveness. Thus the objective in the present case was to investigate the change in morphology and the mechanical response of a polyelectrolyte brush to external forces by application of AFM nanoindentations under different ionic strength conditions. The degree of penetration of the AFM tip through the brush will provide insights into the forces exerted by the brush against the tip. Compressions on the brush should aid to characterize its changes in compressibility for different salt concentrations. For the second chosen system, the interaction between two assembled interfaces was investigated at the single molecular level. A multilayered film formed by the consecutive assembly of oppositely charged polyelectrolytes and subsequently coated with a lipid membrane represents a fascinating soft composite material resembling more than a few structural components emerging in living organisms. The fluid bilayer, thus provide a biocompatible interface where additional functionalities can further be integrated (fusion peptides for instance). The smooth polymer cushion confers not only structural flexibility but also adaptability of the chosen substrate properties to be coated. This type of interface could be useful in the development of novel molecular biosensors with single molecule recognition capacities or in the fabrication of assays against pathogenic agents. The aim of this project was to study the molecular binding mechanism between the last polyelectrolyte layer and the lipid head group of the lower lipid leaflet. Understanding this adsorption mechanism between both interfaces, should likewise contribute to improve the fabrication of lipid coated polymeric nano/micro capsules with targeting properties. For example this could be critical in the field of nonviral gene therapy, where the improvement in the design of condensates of nucleic acids and other polymers with lipids (lipoplexes) are of main interest for its posterior use as delivery vectors. Finally, viral capsids were investigated. These naturally occurring assembled nanocontainers within living organisms stand for a remarkable example of nature’s morphological designs. These structures self-assemble from a small number of different proteins occurring in identical copies. The capsid as a self-assembled structure carries multiple functions: compaction of the genome, protection against external chemical threats, target recognition, structural support and finally facilitating the release of the genome into the host cell. It is highly interesting how these different functions are organized within the capsid which consists, for example, in the case of the norovirus of 180 identical copies of one single protein. Therefore, the mechanical stability and elastic properties of virus-like particles of Rubella and Norovirus were investigated by external application of loading forces with an AFM tip. The measurements were performed under conditions relevant for the virus infection mechanism. The applied compressions on these protein shells at pH values mimicking the virus life cycle will aid to learn about possible internal transitions among proteins which may be important for switching between the various functions of the capsid. The choice of two unrelated viral systems with different entry pathways into the cell and with different morphological architectures is expected to reveal crucial information about the stability and mechanical resistance to deformation of these empty membrane-coated and bare viral capsids. This last might provide clues on the stage of particle disassembly and cargo release during the final step of the infection process.

Single molecule force spectroscopy is an important technique to determine the interaction forces between biomolecules. Atomic force microscopy (AFM) is one of the tools used for this purpose. So far, AFMs usually use cantilevers as the force sensors and piezoelectrics as the actuators which may have some drawbacks in terms of speed and noise. In this research, a micromachined membrane actuator was used in two important types of experiments, namely the single molecule pulling and force-clamp based force spectroscopy. These two methods permit a more direct way of probing the forces of biomolecules, giving a detailed insight into binding potentials, and allowing the detection of discrete unbinding forces. To improve the quality of the experiments there is a need for high force resolution, high time resolution and increase in the throughput. This research focuses on using the combination of AFM and membrane based probe structures that have electrostatic actuation capability. The membrane actuators are characterized for range, dynamics, and noise to illustrate their adequacy for these experiments and to show that the complexity they introduce does not affect the noise level in the system. The control system described in this thesis utilizes the novel membrane actuator structures and integrates it into the current AFM setup. This is a very useful tool which can be implemented on any AFM without changing its mechanical architecture. To perform an experiment, all that is needed is to place the membrane actuator on the AFM stage, under the imagining head, and run the control system, which was implemented using LabVIEW. The system allows the user to maintain a precise and continuous control of the force. This was demonstrated by performing a life time experiment using biomolecules. Moreover, by slightly modifying the control scheme, the system allows us to linearize the membrane motion, which is inherently non-linear. The feasibility of using this control system for a variety of loading rate experiments are also demonstrated.

In dieser Dissertation werden die Bindungseigenschaften von Rezeptor-Ligand-Komplexen mit Hilfe von Optischen Pinzetten untersucht. Aufgrund ihrer außerordentlichen Orts- (2nm) und Kraftauflösung (0,2pN) ist es möglich, diese spezifischen Interaktionen anhand einzelner Bindungsereignisse zu charakterisieren. Als Modellsysteme dienen die Wechselwirkungen zwischen den phosphorylierungsspezifischen, monoklonalen Antikörpern HPT-101 und HPT-104 und dem Morbus Alzheimer relevanten Tau-Peptid. Dieses pathogen veränderte Peptid wird krankheitsspezifisch an den Aminosäuren Threonin231 und Serin235 phosphoryliert, sodass die Detektion dieses Phosphorylierungsmusters mit Hilfe von monoklonalen Antikörpern eine mögliche Früherkennung der Alzheimer-Krankheit darstellt. Eine notwendige Voraussetzung dafür ist jedoch die exakte Kenntnis der Bindungsstellen des Liganden am Rezeptor. Ziel des ersten Teils dieser Arbeit ist es, das Epitop des monoklonalen Antikörpers HPT-101 zu bestimmen. Dazu werden mögliche bindungsrelevante Aminosäuren durch ein Alanin ausgetauscht (Alanin-Scan) und so insgesamt sieben neue Tau-Isoformen aus dem ursprünglichen doppelt-phosphorylierten Peptid Tau[pThr231/pSer235] hergestellt. Die jeweiligen Interaktionen zwischen den modifizierten Peptiden und dem Antikörper werden mit der dynamischen Kraftspektroskopie untersucht und mit Hilfe eines literaturbekannten Modells analysiert. Die sich daraus ergebenden Bindungsparameter (Lebensdauer der Bindung, charakteristische Bindungslänge, freie Aktivierungsenergie und Affinitätskonstante) werden zusammen mit den relativen Bindungshäufigkeiten erstmals genutzt, um Kriterien für essentielle, sekundäre und nicht-essentielle Aminosäuren im Tau-Peptid zu definieren. Bemerkenswerterweise existieren für insgesamt drei dieser Parameter (Bindungslebensdauer, Bindungslänge und Affinitätskonstante) scharfe Klassengrenzen, mit denen eine objektive Einteilung des Epitops von Antikörper HPT-101 möglich ist. Die erhaltenen Ergebnisse sind in überzeugender Weise im Einklang mit ELISA-Messungen zu diesem Antikörper-Peptid-Komplexen, sie liefern jedoch einen tieferen Einblick in die Natur einer spezifischen Bindung, da den kraftspektroskopischen Messungen auch die Bindungskinetik zugänglich ist. Das zweite Projekt der vorliegenden Dissertation etabliert eine Methodik, um die Datenvarianz in der Bestimmung der relativen Bindungshäufigkeit zu reduzieren. Anhand einer Kombination aus Fluoreszenz- und kraftspektroskopischen Messungen werden die Wechselwirkungen zwischen dem monoklonalen Antikörper HPT-104 und dem fluoreszenzmarkierten Peptid Tau[Fl-pThr231] untersucht. Es wird gezeigt, dass durch Vorsortieren der Peptid-beschichteten Kolloide, entsprechend ihrer Oberflächenbeladung, die Datenvarianz in den Bindungshäufigkeitsmessungen signifikant reduziert wird.

abstract: Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights. / Dissertation/Thesis / Doctoral Dissertation Physics 2016

No description available.

I develop an analytically tractable model of dynamic force spectroscopy by considering the forced escape of a Brownian particle out of a potential well, along a one-dimensional reaction pathway. I compute explicit expressions for pertinent experimental observables, such as average bond lifetimes and rupture force distributions. The results generalize conventional quasistatic theories to arbitrary forces and loading rates, thus covering the whole range of conditions found in experiments and all-atom simulations. The theory is extended to so-called catch-slip bonds that play an important role in biology, and to “hidden” degrees of freedom, which may bear significantly on the observed bond kinetics at high loading rates.

No description available.