An instrument, adding the capability to measure 3D volumetric chemical composition, has been constructed by me as a member of the Sánchez Nano Laboratory. The laboratory's in situ atomic force microscope (AFM) and secondary ion mass spectrometry systems (SIMS) are functional and integrated as one instrument. The SIMS utilizes a Ga focused ion beam (FIB) combined with a quadrupole mass analyzer. The AFM is comprised of a 6-axis stage, three coarse axes and three fine. The coarse stage is used for placing the AFM tip anywhere inside a (13x13x5 mm3) (xyz) volume. Thus the tip can be moved in and out of the FIB processing region with ease. The planned range for the Z-axis piezo was 60 µm, but was reduced after it was damaged from arc events. The repaired Z-axis piezo is now operated at a smaller nominal range of 18 µm (16.7 µm after pre-loading), still quite respectable for an AFM. The noise floor of the AFM is approximately 0.4 nm Rq. The voxel size for the combined instrument is targeted at 50 nm or larger. Thus 0.4 nm of xyz uncertainty is acceptable. The instrument has been used for analyzing samples using FIB beam currents of 250 pA and 5.75 nA. Coarse tip approaches can take a long time so an abbreviated technique is employed. Because of the relatively long thro of the Z piezo, the tip can be disengaged by deactivating the servo PID. Once disengaged, it can be moved laterally out of the way of the FIB-SIMS using the coarse stage. This instrument has been used to acquire volumetric data on AlTiC using AFM tip diameters of 18.9 nm and 30.6 nm. Acquisition times are very long, requiring multiple days to acquire a 50-image stack. New features to be added include auto stigmation, auto beam shift, more software automation, etc. Longer term upgrades to include a new lower voltage Z-piezo with strain-gauge feedback and a new design to extend the life for the coarse XY nano-positioners. This AFM-SIMS instrument, as constructed, has proven to be a great proof of concept vehicle. In the future it will be used to analyze micro fossils and it will also be used as a part of an intensive teaching curriculum.

Therapeutics targeting the PI3K (phosphatidylinositol 3-kinase) and the Ras/MAPK (mitogen-activated protein kinases) pathways have potential as non-toxic treatments for triple-negative breast cancer due to their frequent over-activation in several forms of cancer. Interestingly, the PI3K and Ras/MAPK pathways have been shown to incite cancer dormancy behavior individually and tumorigenic behavior in unison when induced in healthy breast epithelial cells (MCF-10A) in vivo. Tumorigenesis and metastasis are heavily reliant on the specific mechanical and adhesive properties of cells, including decreased stiffness, increased mechanosensitivity, and decreased adhesion. However, the describe cellular behaviors are poorly understood for dormant cancer phenotypes. Understanding the mechanical and adhesive behaviors of MCF-10A cells as a function of PI3K and/or Ras/MAPK pathway over-activation further explores the cross-talk enabling unique dormant and tumorigenic characteristics. Cellular viscoelasticity and adhesion were measured for MCF-10A cells with PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression to activate the PI3K and Ras/MAPK pathways respectively with atomic force microscopy. PTEN knockout alone has no observable influence on cell adhesion but resulted in softer cells with less organized cytoskeleton. Activated KRAS overexpression increased cell stiffness and cell adhesion regardless of PTEN expression level. Moreover, the overexpression of activated KRAS enhanced the sensitivity of cells to the substrate stiffness. The findings suggest that the cancer-associated pathways PI3K and Ras/MAPK regulate cell adhesion and mechanics to promote tumor formation and metastasis. More importantly, the results that signify mutations of different molecular pathways associated with cancer dormancy regulate cell mechanics differently suggests that cell stiffness is a biomarker that detects and differentiates different types of dormant cancers.

The aim of this work is to better understand the factors and mechanisms leading to the supramolecular organization of collagen layers adsorbed on polymers. Native collagen adsorption on polystyrene (PS) and plasma-oxidized polystyrene (PSox) substrates revealed that the adsorbed layer consists in two parts: a dense and thin sheet (~ 10 nm) in which fibrils are formed, as revealed by atomic force microscopy, and an overlying thick layer (~ 200 nm) which contains protruding molecules, as revealed by quartz crystal microbalance with dissipation monitoring. The protruding molecules are in low density but modify noticeably the local viscosity. Faster and enhanced fibril formation takes place on hydrophobic compared to hydrophilic substrate. As a result of drastic thermal denaturation, the ability of collagen to assemble into fibrils is lost and the number of protruding molecules responsible for higher viscosity is reduced. Radiochemical measurements showed that collagen molecules are more easily displaced when adsorbed on a hydrophobic substrate compared to a hydrophilic substrate. This may explain why fibril formation occurs more readily on the more hydrophobic substrate, but is in contrast with higher surface affinity. The possible explanation of this paradox by the quick formation of a dense layer of collagen molecules having a smaller number of contact points with a very hydrophobic surface could not be demonstrated by a comparison of adsorption procedures. Comparing different collagen sources revealed various modes of aggregation with different characteristics regarding size and order (large fibers in solution, smaller fibrils to featureless underneath layer in the adsorbed phase). Moreover, collagen aggregation in the solution is a process competing with adsorption: more aggregated solutions behave like less concentrated solutions regarding the adsorbed amount and fibril formation in the adsorbed phase. It must be emphasized that interpretation of the QCM-D data, which is based on fitting physical quantities according to a model, has to be performed very carefully, and requires the examination of the sensitivity of the fitted data to the fitting parameters.

The cytoskeleton is a diverse, multi-protein framework that plays a fundamental role in many cellular activities including mitosis, cell division, intracellular transport, cell motility, muscle contraction, and the regulation of cell polarity and organization. Furthermore, cytoskeletal filaments have been implicated in the pathogenesis of a wide variety of diseases including cancer, blood disease, cardiovascular disease, inflammatory disease, neurodegenerative disease, and problems with skin, nail, cornea, hair, liver and colon. Increasing evidence suggests that the distribution and organization of the cytoskeleton in living cells are affected by mechanical stresses and the cytoskeleton determines cell stiffness. We developed a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the primary structural constituents of the cytoskeleton and extended a prior solution from the finite elasticity literature for use in a sub-class of atomic force microscopy (AFM) studies of cell mechanics. The model showed that the degree of substrate stretch and the geometry of the AFM tip dramatically affect the measured cell stiffness. Consistent with previous studies, the model showed that disruption of the actin filaments can reduce the stiffness substantially, whereas there can be little contribution to the overall cell stiffness by the microtubules or intermediate filaments. To investigate the effect of mechanical stretching on cytoskeletal remodeling and cell stiffness, we developed a simple cell-stretching device that can be combined with an AFM and confocal microscopy. Results demonstrate that cyclic stretching significantly and rapidly alters both cell stiffness and focal adhesion associated vinculin and paxillin, suggesting that focal adhesion remodeling plays a critical role in cell stiffness by recruiting and anchoring F-actin. Finally, we estimated cytoskeletal remodeling by synthesizing data on stretch-induced dynamic changes in cell stiffness and focal adhesion area using constrained mixture approach. Results suggest that the acute increase in stiffness in response to an increased cyclic stretch was probably due to an increased stretch of the original filaments whereas the subsequent decrease back towards normalcy was consistent with a replacement of the highly stretched original filaments with less stretched new filaments.

This work explores the effects of weak nonlinearity on harmonic oscillators.Two particular systems are studied experimentally: A superconductingresonator formed from a coplanar waveguide that oscillates at microwave frequencies,and the cantilever of an atomic force microscope (AFM) vibratingat ultrasonic frequencies. Both of these systems are described in the introduction,followed by a theory chapter giving a general theoretical framework for nonlinear oscillators. Basic properties of nonlinear oscillators, such asbifurcation and intermodulation, are explained using simple models. Experimental methods, including cryogenic and microwave measurement techniques,are described in some detail. The nonlinear superconducting resonator is studied for use as a parametric amplifier. A strong drive tone, called the pump, drives the oscillator nearthe point of bifurcation. A second, much weaker drive signal that is slightlydetuned from the pump, will cause energy to move from the pump to the signal, giving signal amplification. We have measured a signal gain greaterthan 22 dB in a bandwidth of 30 kHz, for a resonator pumped at 7.6 GHz.This type of amplifier is phase-sensitive, meaning that signals in phase withthe pump will be amplified, but signals in quadrature phase of the pump will be deamplified. Phase-sensitivity has important implications on the amplifier’snoise properties. With a parametric amplifier, a signal can be amplified without any additional noise being added by the amplifier, something that is fundamentally impossible for a standard amplifier. The vibrating AFM cantilever becomes a nonlinear oscillator when it is interacting with a surface. When driven with two frequencies, the amplitudeand phase of the cantilever’s response will develop mixing products, or intermodulation products, that are very sensitive to the exact form of the nonlinearity. Very small changes in the surface properties will be detectable when measuring the intermodulation products. Simultaneously measuring many intermodulation products, or acquiring an intermodulation spectrum,allows one to reconstruct the tip-surface interaction. Intermodulation AFM increases the sensitivity of the measurement or the contrast of the acquiredimages, and provides a means of rapidly measuring the nonlinear tip-surface interaction. The method promises to enhance the functionality of the AFM beyond simple topography measurement, towards quantitative analysis of the chemical or material properties of the surface. / <p>QC 20100812</p>

Knowledge of the ultrastructural arrangement within wood fibres is important for understanding the mechanical properties of the fibres themselves, as well as for understanding and controlling the ultrastructural changes that occur during pulp processing. The object of this work was to explore the use of atomic force microscopy (AFM) in studies of the cell wall ultrastructure and to see how this structure is affected in the kraft pulp fibre line. This is done in order to eventually improve fibre properties for use in paper and other applications, such as composites. On the ultrastructural level of native spruce fibres (tracheids), it was found that cellulose fibril aggregates exist as agglomerates of individual cellulose microfibrils (with a width of 4 nm). Using AFM in combination with image processing, the average side length (assuming a square cross-section) for a cellulose fibril aggregate was found to be 15–16 nm although with a broad distribution. A concentric lamella structure (following the fibre curvature) within the secondary cell wall layer of native spruce fibres was confirmed. These concentric lamellae were formed of aligned cellulose fibril aggregates with a width of about 15 nm, i.e. of the order of a single cellulose fibril aggregate. It was further found that the cellulose fibril aggregates had a uniform size distribution across the fibre wall in the transverse direction. During the chemical processing of wood chips into kraft pulp fibres, a 25 % increase in cellulose fibril aggregate dimension was found, but no such cellulose fibril aggregate enlargement occurred during the low temperature delignification of wood into holocellulose fibres. The high temperature in the pulping process, over 100 ºC, was the most important factor for the cellulose fibril aggregate enlargement. Neither refining nor drying of kraft or holocellulose pulp changed the cellulose fibril aggregate dimensions. During kraft pulping, when lignin is removed, pores are formed in the fibre cell wall. These pores were uniformly distributed throughout the transverse direction of the wood cell wall. The lamellae consisting of both pores and matrix material (“pore and matrix lamella”) became wider and their numeral decreased after chemical pulping. In holocellulose pulp, no such changes were seen. Refining of kraft pulp increased the width of the pore and matrix lamellae in the outer parts of the fibre wall, but this was not seen in holocellulose. Upon drying of holocellulose, a small decrease in the width of the pore and matrix lamellae was seen, reflecting a probable hornification of the pulp. Refining of holocellulose pulp led to pore closure probably due to the enhanced mobility within the fibre wall. Enzymatic treatment using hemicellulases on xylan and glucomannan revealed that, during the hydrolysis of one type of hemicellulose, some of the other type was also dissolved, indicating that the two hemicelluloses were to some extent linked to each other in the structure. The enzymatic treatment also decreased the pore volume throughout the fibre wall in the transverse direction, indicating enzymatic accessibility to the entire fibre wall. The results presented in this thesis show that several changes in the fibre cell wall ultrastructure occur in the kraft pulp fibre line, although the effects of these ultrastructural changes on the fibre properties are not completely understood. / QC 20101012

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>

Physical forces in the cellular microenvironment play an important role in governing cell function. Forces transmitted through the cell cause distinct deformation of the nucleus, and possibly play a role in force-mediated gene expression. The work presented in this thesis drew upon innovative strategies employing simultaneous atomic force and laser-scanning confocal microscopy, as well as parallel optical stretching experiments, to gain unique insights into the response of eukaryotic cell nuclei to external force. Non-destructive approaches confirmed the existence of a clear anisotropy in nuclear mechanical properties, and showed that the nucleus' mechanical response to extracellular forces is differentially governed by both nuclear and cytoskeletal prestress: nuclear prestress regulates shape and anisotropic deformation, whereas cytoskeletal prestress modulates the magnitude and degree of deformation. Importantly, the anisotropic mechanical response was conserved among diverse differentiated cell types from multiple species, suggesting that nuclear mechanical anisotropy plays an important role in cell function.

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.

This research focuses on the nanoscopic investigation of the three-dimensional surface morphology of the neural growth cones from the snail Helisoma trivolvis, and InN and InGaN semiconductor material systems using Atomic Force Microscopy (AFM). In the analysis of the growth cones, the results obtained from AFM experiments have been used to construct a 3D architecture model for filopodia. The filopodia from B5 and B19 neurons have exhibited different tapering mechanisms. The volumetric analysis has been used to estimate free Ca2+ concentration in the filopodium. The Phase Contrast Microscopy (PCM) images of the growth cones have been corrected to thickness provided by AFM in order to analyze the spatial refractive index variations in the growth cone. AFM experiments have been carried out on InN and InGaN epilayers. Ternary InGaN alloys are promising for device applications tunable from ultraviolet (Eg[GaN]=3.4 eV) to near-infrared (Eg [InN]=0.7 eV). The real-time optical characteristics and ex-situ material properties of InGaN epilayers have been analyzed and compared to the surface morphological properties in order to investigate the relation between the growth conditions and overall physical properties. The effects of composition, group V/III molar ratio and temperature on the InGaN material characteristics have been studied and the growth of high quality indium-rich InGaN epilayers are demonstrated.