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.

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.

<p><font face="Times-Roman"> <p align="left">The growth of the Internet has led to many interesting developments for both educational and commercial purposes. In this project an attempt was made to use the Internet for a research purpose to facilitate the determination of the thermal stability of diffusion barriers. Another purpose of this thesis is to investigate the teaching and training use of the Internet through the development of online interactive tools and activities as well as materials. The training aspects are mentioned as it is hoped that this thesis can serve as a form of documentation of the use of the Internet, while the central part was the determination of thermal stability of TiN, TaN and TiW diffusion <font face="Times-Roman">barriers on Ag.</font></p> </font></p>

Microscopic imaging in the biomedical sciences allows for detailed study of the structure and function of normal and abnormal (i.e., diseased) states of cells and tissues. The expression patterns of proteins and/or physiological parameters within these specimens can be related to disease progression and prognosis, and are often heterogeneously spread throughout the entire specimen. With conventional microscopy, a large number of individual image ‘tiles’ must be captured and subsequently combined into a mosaic of the entire specimen. This has the potential to introduce artefacts at the image seams, as well as introducing non-uniform illumination of the entire specimen. A further limitation often encountered in biomedical fluorescence microscopy is the high background due to the autofluorescence (AF) of endogenous compounds within cells and tissues. Often, AF can prevent the detection and/or accurate quantification in fluorescently- labelled tissues and, in general, can reduce the reliability of results obtained from such specimens. AF spectra are relatively broad and so can be present across a large number of image spectral channels. The intensity of AF also increases as the excitation wavelength is decreased, causing increasing amounts of autofluorescence when exciting in the blue and near-UV range of the spectrum (400 - 500 nm). This thesis reports the development of hyperspectral, fluorescence and brightfield imaging of entire, paraffin-embedded, formalin-fixed (PEFF) tissue slides using a prototype confocal scanner with a large field of view (FOV). This technology addresses the challenges of imaging large tissue sections through the use of a telecentric f-theta laser scan lens thus allowing an entire microscope slide (22x70 mm) to be imaged in a single scan at resolution equivalent to a 10x microscope objective. The development and optimization of brightfield and single-channel fluorescence imaging modes are discussed in the first half of this thesis, while the second half and appendices concentrate on the spectral properties of the system and removal of AF from PEFF tissue sections. The hyperspectral imaging mode designed for this system allows the fluorescence emission spectrum of each image pixel to be sampled at 6.7 nm/channel over a spectral range of 500-700 nm. This results in the ability to separate distinct fluorescence signatures from each other, and enables quantification even in situations where the AF completely masks the signal from the applied labels.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein &alpha; (C/EBP&alpha;) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBP&alpha;. Additionally, the original clone found to interact with C/EBP&alpha; consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBP&alpha; mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBP&alpha;, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBP&alpha;-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBP&alpha;-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBP&alpha;. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBP&alpha; was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBP&alpha;, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBP&alpha;.

Microscopic imaging in the biomedical sciences allows for detailed study of the structure and function of normal and abnormal (i.e., diseased) states of cells and tissues. The expression patterns of proteins and/or physiological parameters within these specimens can be related to disease progression and prognosis, and are often heterogeneously spread throughout the entire specimen. With conventional microscopy, a large number of individual image ‘tiles’ must be captured and subsequently combined into a mosaic of the entire specimen. This has the potential to introduce artefacts at the image seams, as well as introducing non-uniform illumination of the entire specimen. A further limitation often encountered in biomedical fluorescence microscopy is the high background due to the autofluorescence (AF) of endogenous compounds within cells and tissues. Often, AF can prevent the detection and/or accurate quantification in fluorescently- labelled tissues and, in general, can reduce the reliability of results obtained from such specimens. AF spectra are relatively broad and so can be present across a large number of image spectral channels. The intensity of AF also increases as the excitation wavelength is decreased, causing increasing amounts of autofluorescence when exciting in the blue and near-UV range of the spectrum (400 - 500 nm). This thesis reports the development of hyperspectral, fluorescence and brightfield imaging of entire, paraffin-embedded, formalin-fixed (PEFF) tissue slides using a prototype confocal scanner with a large field of view (FOV). This technology addresses the challenges of imaging large tissue sections through the use of a telecentric f-theta laser scan lens thus allowing an entire microscope slide (22x70 mm) to be imaged in a single scan at resolution equivalent to a 10x microscope objective. The development and optimization of brightfield and single-channel fluorescence imaging modes are discussed in the first half of this thesis, while the second half and appendices concentrate on the spectral properties of the system and removal of AF from PEFF tissue sections. The hyperspectral imaging mode designed for this system allows the fluorescence emission spectrum of each image pixel to be sampled at 6.7 nm/channel over a spectral range of 500-700 nm. This results in the ability to separate distinct fluorescence signatures from each other, and enables quantification even in situations where the AF completely masks the signal from the applied labels.

Nanolithography is a rapidly evolving field that requires new combinations of techniques to improve patterning and to assist in fabricating electromechanical devices. An increasing number of applications require surfaces with defined regions of different chemical functionality. In our previous project optimum conditions for lithographic patterning were determined and potential blockers were identified to reduce force on the tip. This work is focused on identifying good chemical modifications that will allow better control of basic patterning and to investigate the minimum force of patterning required while using each chemical system. The primary aim is to gain better control of basic pattern techniques in order to create more intricate patterns such as interdigitated arrays, which can subsequently be used in sensors. An atomic force microscope (AFM) is used to pattern the prepared colloid-coated glass slides. Several compounds were used in the investigation, including sodium sulphate, potassium sulphate, magnesium sulphate, sodium fluoride, sodium chloride, sodium bromide, and sodium iodide, potassium chloride, potassium bromide, potassium iodide, potassium dihydrogen phosphate, and potassium hydrogen phosphate. In Summary, the following were found as a result of this work:  The groups of sulphates were determined to require minimum patterning forces as indicated. Sodium sulphate took a force of 49 n Potassium sulphate took a force of 45 nN Magnesium sulphate took a force of 744.4 nN  The group of sodium and potassium halides were determined the minimum patterning forces as indicated. Sodium fluoride took a force of 8.42 nN Sodium chloride and potassium chloride took a force of 20.19 and 61.9nN Sodium bromide and potassium bromide took a force of 601.4 nN and 37.2 nN, respectively Sodium iodide and potassium iodide took a force of 953.7 nN and 47.2 nN, respectively  The phosphates were determined to require the minimum patterning forces as indicated. Potassium hydrogen phosphate took a force of 25nN Potassium dihydrogen phosphate took a force of 43 nN

A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation. / May 2007

It is my hope that this thesis may serve as a guide for future students wishing to build a microscope from scratch. The design and construction of a scanning, confocal fluorescence microscope equipped with shaped microwave excitation is detailed. The use of the microscope is demonstrated by coherently manipulating single Nitrogen-Vacancy centres in diamond. Further the instrument is used to investigate a dual Halbach array magnet system.