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.

[pt] Nesta tese, apresentamos um procedimento utilizado para a calibração do fotodetector e dos cantileveres utilizados em nosso AFM para a medida de força lateral. Desenvolvemos um código em Matlab para o controle do microscópio que permitiu a realização do estudo da influência da força normal na fricção. Também foi desenvolvido um segundo código em Matlab para a medida automatizada da adesão. Apresentamos e discutimos a influência da energia livre superficial na fricção e adesão de várias superfícies. Neste trabalho um estudo da influência da condensação capilar na forca lateral foi estudado para superfícies hidrofílicas, e hidrofóbicas. Encontramos que as nano asperezas podem realizar contatos singulares descritos pelo modelo de Hertz ou múltiplos contatos de acordo com o modelo de Greenwood. O tipo de contato entre as nano asperezas pode ser controlado através da hidrofobicidade e da umidade relativa no ambiente de medida. É verificado que os meniscos formados entre ponta e superfície influenciam a força lateral, através do aumento da força normal e também através da energia gasta pela ponta para arrastar ou deformar o capilar durante seu deslocamento sobre a superfície. O efeito da cinética de condensação capilar da água sobre a fricção foi também estudado. É mostrado que a molhabilidade é determinante para a definição dos mecanismos da dissipação de energia entre as nanoasperezas. Apresentamos também a influência da hidrofobicidade superficial no coeficiente de atrito. A correlação observada entre o ângulo de contato e o coeficiente de atrito reforça a importância da cinética da condensação capilar nos processos de fricção que ocorre na escala de nanômetros. / [en] In this work, the procedures developed to the calibration of the AFM photodetector and cantilevers for lateral force measurements in our AFM is presented. A Matlab code that controls the microscope allows the study of the influence of the normal force on the lateral one. A second Matlab code was developed in order to study the adhesion forces in an automated way. We present and discuss the influence of the surface free energy on the friction and adhesion forces. In this work, the lateral forces were measured at hydrophilic and hydrophobic surfaces. It was observed that the nano asperities may form single asperity contacts described by the Hertz model as well as multi-asperity type of contacts described by the Greenwood model. The nanoasperity contact may be controlled by the wettability and ambient relative humidity. It is seen that the capillar formed between the tip and the surface influences the tip-surface normal force and the friction forces due to the dissipation of energy caused by the drag or brake of the capillar meniscous. The effect of capillary condensation kinetics was studied as well. It is shown that the surface wettability is determinant to the energy dissipation mechanism in nanoscale. The influence of the surface wettability on the friction coefficient is presented. The observed correlation between the friction coefficient and contact angle enhances the influence of the surface wettability and its kinetics in the friction forces at nanoscale.

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.

Surface characteristics such as topography and critical dimensions serve as important indicators of product quality and manufacturing process performance especially at the micrometer and the nanometer scales. This paper first reviews different technologies used for obtaining high precision 3-D images of surfaces, along with some selected applications. Atomic force microscopy (AFM) is one of such methods. These images are commonly distorted by convolution effects, which become more prominent when the sample surface contains high aspect ratio features. In addition, data artifacts can result from poor dynamic response of the instrument used. In order to achieve reliable data at high throughput, dynamic interactions between the instrument's components need to be well understood and controlled, and novel image deconvolution schemes need to be developed. Our work aims at mitigating these distortions and achieving reliable data to recover metrology soundness. A summary of our findings will be presented. / Singapore-MIT Alliance (SMA)

In this paper, the problem of dual actuation in the atomic force microscope (AFM) is analyzed. The use of two actuators to balance the trade-off between bandwidth, range, and precision has been recently extended to nano-positioning systems. Despite existing demands, this concept undergoes fundamental limitations towards its extension to AFMs. This is attributed to the non-conventional requirement imposed on the control signal response, as it used to create the image of the characterized surface. / Singapore-MIT Alliance (SMA)

The chemokine, Monocyte Chemoattractant Protein (MCP-1), enhances integrin mediated monocyte adhesion to the vascular endothelium during inflammation. In this study, we demonstrate that MCP-1 promotes rapid sub-second adhesion of THP-1 cells to Vascular Cell Adhesion Molecule-1 (VCAM-1), but not to Intercellular Cell Adhesion Molecule-1 (ICAM-1). MCP-1 activates membrane tethered Very Late Antigen 4 (VLA-4, α4β1), but not necessarily cytoskeleton anchored VLA-4. Activated tethered VLA-4 bonds tremendously increased the period of time monocytes remain bound from hundreds of milliseconds to several seconds and also increased the distance over which immunologic surveillance occurs from several microns up to 20 microns along the endothelium. Lastly at the single molecule level, MCP-1 stimulated tethered VLA-4 bonds exhibit increased resistance to pulling force. In conclusion MCP-1 increased tethered VLA-4 bond resistance to force providing a mechanism for monocyte recruitment to the endothelium.

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.

Im Fokus dieser Arbeit stand der Aufbau einer auf DNA basierenden Nanostruktur. Der universelle Vier-Buchstaben-Code der DNA ermöglicht es, Bindungen auf molekularer Ebene zu adressieren. Die chemischen und physikalischen Eigenschaften der DNA prädestinieren dieses Makromolekül für den Einsatz und die Verwendung als Konstruktionselement zum Aufbau von Nanostrukturen. Das Ziel dieser Arbeit war das Aufspannen eines DNA-Stranges zwischen zwei Fixpunkten. Hierfür war es notwendig, eine Methode zu entwickeln, welche es ermöglicht, Funktionsmoleküle als Ankerelemente ortsaufgelöst auf eine Oberfläche zu deponieren. Das Deponieren dieser Moleküle sollte dabei im unteren Mikrometermaßstab erfolgen, um den Abmaßen der DNA und der angestrebten Nanostruktur gerecht zu werden. Das eigens für diese Aufgabe entwickelte Verfahren zum ortsaufgelösten Deponieren von Funktionsmolekülen nutzt das Bindungspaar Biotin-Neutravidin. Mit Hilfe eines Rasterkraftmikroskops (AFM) wurde eine zu einem „Stift“ umfunktionierte Rasterkraftmikroskopspitze so mit der zu deponierenden „Tinte“ beladen, dass das Absetzen von Neutravidin im unteren Mikrometermaßstab möglich war. Dieses Neutravidinmolekül übernahm die Funktion als Bindeglied zwischen der biotinylierten Glasoberfläche und dem eigentlichen Adressmolekül. Das somit generierte Neutravidin-Feld konnte dann mit einem biotinylierten Adressmolekül durch Inkubation funktionalisiert werden. Namensgebend für dieses Verfahren war die Möglichkeit, Neutravidin mehrmals zu deponieren und zu adressieren. Somit ließ sich sequenziell ein Mehrkomponenten-Feld aufbauen. Die Einschränkung, mit einem AFM nur eine Substanz deponieren zu können, wurde so umgangen. Ferner mußten Ankerelemente geschaffen werden, um die DNA an definierten Punkten immobilisieren zu können. Die Bearbeitung der DNA erfolgte mit molekularbiologischen Methoden und zielte darauf ab, einen DNA-Strang zu generieren, welcher an seinen beiden Enden komplementäre Adressequenzen enthält, um gezielt mit den oberflächenständigen Ankerelementen binden zu können. Entsprechend der Geometrie der mit dem AFM erzeugten Fixpunkte und den oligonukleotidvermittelten Adressen kommt es zur Ausbildung einer definierten DNA-Struktur. Mit Hilfe von fluoreszenzmikroskopischen Methoden wurde die aufgebaute DNA-Nanostruktur nachgewiesen. Der Nachweis der nanoskaligen Interaktion von DNA-bindenden Molekülen mit der generierten DNA-Struktur wurde durch die Bindung von PNA (peptide nucleic acid) an den DNA-Doppelstrang erbracht. Diese PNA-Bindung stellt ihrerseits ein funktionales Strukturelement im Nanometermaßstab dar und wird als Nanostrukturbaustein verstanden. / The main aim of this work was the development of a DNA-based nanostructure. The universal four-letter code of DNA allows addressing bonds at the molecular level. The chemical and physical property of DNA makes this macromolecule an ideal candidate as a construction element for nanostructures. The aim of this work was to span a DNA strand between two fixed points. For this purpose it was necessary to develop a method which makes it possible to deposit functional molecules as anchoring elements with highly spatial resolution on a surface. These molecules should be immobilized on the lower micrometer scale to meet the requirements of the desired nanostructure. The method that has been developed for this task, which enables to deposit functional molecules, uses the binding pair biotin-neutravidin. Using the tip of an atomic force microscope (AFM), which can be uses like a pen, it was possible to deposit neutravidin on the lower micrometer scale. This neutravidin molecule is the linking element between the biotinylated glass surface and the actual address molecule. The thus generated neutravidin field could then be functionalized with a biotinylated molecule by incubation. The method has been published as sequential spotting method because it enables a sequential functionalization of neutravidin after it has been deposited. It was so possible to build up a multi-component array. The limitation of being able to deposit only one single substance with an AFM has been circumvented. It also was necessary to create anchor elements in order to immobilize the DNA at defined positions. The processing of the DNA was carried out using molecular biological methods and aimed at generating a DNA strand, which at both ends has a complementary sequence for binding to the surface bound anchor elements. The defined structure is a result of the geometry of the fixed points, generated by the AFM. Using fluorescence microscopy, the constructed DNA nanostructure was detected. The proof of the interaction of DNA-binding molecules with the DNA structure was carried out by the binding of PNA (peptide nucleic acid), which is capable of binding to double stranded DNA. The PNA and its DNA-interaction is a functional building block in the nanometer scale and can be regarded as a promising nanostructure.

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>