Combined atomic force microscopy-scanning electrochemical microscopy (AFM-SECM) is capable of providing simultaneous topographical and electrochemical imaging at sample surfaces. Integration of amperometric biosensors at tip-integrated electrodes recessed from the apex of the AFM tip further enhances the versatility of such bifunctional probes. Of particular interest to this work was the detection of adenosine triphosphate (ATP) at a cellular level, since ATP is involved in many biologically relevant processes. There are challenges concerning the integration of biosensors into bifunctional AFM-SECM probes. This thesis focuses on addressing and advancing several of these limitations. Thin insulation layers are important for AFM-SECM based applications to enhance AFM and SECM performance. Plasma-polymerized fluorocarbon membranes are introduced as novel thin film insulation materials for AFM-SECM probes. Insulation layers with a thickness of < 300 nm were found to exhibit excellent insulating properties and satisfying temporal stability for successful application in AFM-SECM experiments. Furthermore new approaches for increasing the electrode area in conventionally focused ion beam (FIB) fabricated AFM-SECM probes were implemented, since enhancement of the current response in conjunction with biosensing experiments is required. Ion beam induced deposition (IBID) was used to generate platinum carbon (PtC) deposits at AFM-SECM probes, thereby successfully increasing the tip-integrated electrode area. PtC composites were thoroughly characterized in terms of their physical and electrochemical properties. Since a high carbon fraction in the PtC composite was inhibiting the charge transfer kinetics at the electrode surface for certain analytes, several pre-treatment strategies were investigated including annealing, UV/ozone treatment, and FIB milling. FIB milling proved to be the most promising procedure improving charge transfer properties at the electrode along with fabrication compatibility at AFM-SECM probes. The last part of this thesis aimed at providing fundamental studies on AFM-SECM application at live epithelial cell monolayers. AFM was used in different imaging modes to characterize the topography of epithelial cells. ATP detection at epithelial cells was achieved with amperometric biosensors combined with non-invasive SECM. Biosensors were further miniaturized at batch-fabricated AFM-SECM probes enabling laterally-resolved detection of ATP at epithelial cells. Additionally, PtC composite materials were evaluated for applicability as transducer platforms for enzymatic biosensors.

M.Sc. / Donor-acceptor (D-A) systems consist of a donor covalently or non-covalently linked to an acceptor. The simplest D–A system consists of a donor linked to an acceptor and is called a dyad system. Photoactive dyad systems are molecular devices designed to perform through the separation of charge separation states and the conversion of solar energy to chemical energy in analogy to photosynthesis.1 These dyad systems consist of a donor which is usually a chromophore and an acceptor. The design of these systems is guided to mimic photo-induced electron transfer (PET) and charge separation (CS), which are fundamental processes of photosynthesis. In nature, photosynthetic units are often built from dyads consisting of pigments like chlorophyll (donors), non–covalently linked to quinones, (acceptors). The donor harvests light energy and transfers the energy to the nearby pigment molecules until it eventually reaches a special region of the chlorophyll macrocycle called the reaction centre where this light energy is then converted to electrochemical energy. Photoactive dyad systems act as artificial photosynthetic models as they reproduce photo–induced electron transfer and charge separation of natural photosynthesis. In this project, dyad systems were made by covalently linking zinc pheophorbide, a modified chlorophyll derivative to double–walled carbon nanotubes (DWCNTs). Zinc pheophorbide acts as the donor and DWCNTs as the acceptors. Chlorophyll was modified by cutting the phytol chain and inserting zinc as the central metal to yield zinc pheophorbide. This derivative is stable against irradiation, has a good range of acceptor wavelength and is also a good light harvester. DWCNTs are one dimensional nanowires with two concentric tubes. They readily accept electrons because they have an extended π electron system. These electrons are then transported efficiently under ballistic conditions. DWCNTs were synthesised by catalytic chemical vapour deposition (CCVD) of methane over Mg0.99Co0.075Mo0.025O catalyst. In dyad system 1, amidated zinc pheophorbide molecules were covalently attached to oxidised DWCNTs in the presence of N–ethyl–N’–(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) \ and N–hydroxysuccinnimide (NHS) as a catalysts. Dyad system 2 was synthesized by attaching zinc pheophorbide molecules to amidated DWCNTs using the same catalysts.

Cette thèse porte sur la définition d'un schéma de recalage automatique en microscopie corrélative 2D et 3D, en particulier pour des images de microscopie optique et électronique (CLEM). Au cours des dernières années, la CLEM est devenue un outil d'investigation important et puissant dans le domaine de la bio-imagerie. En utilisant la CLEM, des informations complémentaires peuvent être collectées à partir d'un échantillon biologique. La superposition des différentes images microscopiques est généralement réalisée à l'aide de techniques impliquant une assistance manuelle à plusieurs étapes, ce qui est exigeant et prend beaucoup de temps pour les biologistes. Pour faciliter et diffuser le procédé de CLEM, notre travail de thèse est axé sur la création de méthodes de recalage automatique qui soient fiables, faciles à utiliser et qui ne nécessitent pas d'ajustement de paramètres ou de connaissances complexes. Le recalage CLEM doit faire face à de nombreux problèmes dus aux différences entre les images de microscopie électronique et optique et leur mode d'acquisition, tant en termes de résolution du pixel, de taille des images, de contenu, de champ de vision et d'apparence. Nous avons conçu des méthodes basées sur l'intensité des images pour aligner les images CLEM en 2D et 3D. Elles comprennent plusieurs étapes : représentation commune des images LM et EM à l'aide de la transformation LoG, pré-alignement exploitant des mesures de similarité à partir d'histogrammes avec une recherche exhaustive, et un recalage fin basé sur l'information mutuelle. De plus, nous avons défini une méthode de sélection robuste de modèles de mouvement, et un méthode de détection multi-échelle de spots, que nous avons exploitées dans le recalage CLEM 2D. Notre schéma de recalage automatisé pour la CLEM a été testé avec succès sur plusieurs ensembles de données CLEM réelles 2D et 3D. Les résultats ont été validés par des biologistes, offrant une excellente perspective sur l'utilité de nos développements. / This thesis is concerned with the definition of an automated registration framework for 2D and 3D correlative microscopy images, in particular for correlative light and electron microscopy (CLEM) images. In recent years, CLEM has become an important and powerful tool in the bioimaging field. By using CLEM, complementary information can be collected from a biological sample. An overlay of the different microscopy images is commonly achieved using techniques involving manual assistance at several steps, which is demanding and time consuming for biologists. To facilitate and disseminate the CLEM process for biologists, the thesis work is focused on creating automatic registration methods that are reliable, easy to use and do not require parameter tuning or complex knowledge. CLEM registration has to deal with many issues due to the differences between electron microscopy and light microscopy images and their acquisition, both in terms of pixel resolution, image size, content, field of view and appearance. We have designed intensity-based methods to align CLEM images in 2D and 3D. They involved a common representation of the LM and EM images using the LoG transform, a pre-alignment step exploiting histogram-based similarities within an exhaustive search, and a fine mutual information-based registration. In addition, we have defined a robust motion model selection method, and a multiscale spot detection method which were exploited in the 2D CLEM registration. Our automated CLEM registration framework was successfully tested on several real 2D and 3D CLEM datasets and the results were validated by biologists, offering an excellent perspective in the usefulness of our methods.

No description available.

Call number: LD2668 .T4 1985 Y67 / Master of Science

A study of the nucleation, growth and final microstructure of vacuum deposited nickel films has been performed using scanning tunneling microscopy (STM) as the primary research instrument. Typical nucleation conditions are reported for nickel films grown on partially shadowed highly-oriented pyrolytic graphite (HOPG), and techniques are developed for using the STM to catalog film islands instead of more conventional electron microscopes. Values for the activation energy of surface diffusion, critical nucleus size, changes in the saturation nucleation density with temperature, and spatial variations in the nucleation rate are included. Roughening and microstructure changes observed with STM are reported as functions of substrate temperature and deposition angle for nickel films grown on highly-oriented pyrolytic graphite and fused silica. Conventional film RMS roughness values are compared to microRMS values derived from STM data and STM images of film microstructure are compared with SEM and optical microscope photographs.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / In this dissertation, state-of-the-art fluorescence microscopy techniques are employed to probe unique nanoscale phenomena in poly(dimethylsiloxane) (PDMS) films and on single carbon nanofibers. In one study, the mobility and physical entrapment of single dye molecules in dry and solvent-loaded PDMS films is explored. Experiments are performed under dry nitrogen and at various levels of isopropyl alcohol (IPA) loading from the vapor phase, as monitored by a PDMS-coated quartz-crystal microbalance. Single molecules are shown to be predominantly immobile under dry conditions and mostly mobile under IPA-saturated conditions. FCS is used to measure the apparent diffusion coefficient, yielding a mean that is virtually independent of IPA loading and sample class. An increase in the population of mobile molecules under high IPA conditions is attributed to the filling of film micropores with solvent, rather than by incorporation of molecularly dispersed solvent into the PDMS. In a second study, the molecular mobility of both neutral and cationic molecules in cured PDMS films is studied as a function of oligomer extraction. Cross correlation and Bayesian burst analysis methods were used to quantify the populations of fixed and total molecules, respectively. The results show that the total concentration of dye increases with increased oligomer extraction, while the relative populations of fixed and mobile molecules decrease and increase, respectively. These results are relevant to the use of PDMS in microfluidics, nanofiltration and pervaporation membranes and solid phase microextraction fibers. In a final study, molecular beacons (MBs) were immobilized onto the ends of single, sol-gel encapsulated vertically-alligned carbon nanofibers (VACNFs) attached to a silicon electrode. MB fluorescence was monitored as a function of the potential applied to the VACNF in a three-electrode electrochemical cell. Application of positive potentials attracts the negatively charged backbone of the MB, causing hybridization of the stem and a reduction in beacon fluorescence. Negative potentials cause dehybridization of the stem, and an increase in MB fluorescence. This study presents the first measurement of potential-dependent dehybridization/rehybridization of MBs attached directly to the end of a single VACNF. These studies will help to characterize the mechanism by which future lab-on-a-chip devices will detect harmful bio-organisms.

Poster exhibited at GPSC Student Showcase, February 24th, 2016, University of Arizona. Recipient of the 2016 Katheryne B. Willock Library Research Award. / The nervous system is composed of two types of cells: neurons and glia. In neuronal circuits, neurons communicate through synapses and glia play a crucial modulatory role. To modulate chemical reuptake, glia send processes close to synapses and many glia directly appose or ensheathe a synapse. This structural motif is one of the elements often included in describing a vertebrate tripartite synapse, which includes a bidirectional functional neuron-glia relationship. The exact nature of this neuron-glia communication is not well understood. In the invertebrate fruit fly, we have also found that particular neurons and glia also have a bidirectional functional relationship. This allows us to ask new questions about glial morphology. Throughout multiple images, I identified particular neuronal synapses and surrounding glia. After creating a 3D reconstruction, I measured the distance between a particular neuronal synapse and its closest glial process. Interestingly, the neuronal synapses were not directly apposed or ensheathed by glia, and the distance to the closest glial process varied one-hundred-fold. With variable distance, functional communication is consistently present. These findings provide important insight into invertebrate neuron-glia communication, and offer new avenues to investigate the structural neuron-glia relationships that are required for reciprocal signaling between the two cell classes.

The cell membrane is a highly complex structure consisting of a large diversity of phospholipids and macromolecules. There exist a variety of diseases that compromise the integrity of this key component of the cell. This thesis considers the investigation of interactions between β-amyloid peptide (Aβ₄₂) and lipid bilayers. To facilitate understanding of this complex system, it is advantageous to employ a model sample; supported lipid bilayers (SLB) and giant multilamellar vesicles (MLVs) are used as proxy cell membranes. These nanostructures are widely used as models of cellular membranes in many areas of scientific research. Phospholipid molecules self-organise into bilayer structures containing phase-separated microdomains, which are believed to be important in many biological processes. This study aims to develop model systems and experimental tools to explore hypothetical mechanisms through which the β-amyloid interacts with the lipid membranes. A lack of mechanistic understanding is the major challenge to our efforts to elucidate not only the interactions of the Aβ42 with the lipid membranes, but also the behaviour of these systems towards the changes of the environmental conditions (pH, concentration, temperature). Our results suggest that there are various different methods, such as AFM, CARS microscopy and Raman spectroscopy as well as neutron scattering that are capable of fast imaging. Overall, all these techniques contributed in a complementary study of Aβ₄₂ aggregation states under extreme and physiological conditions as well as to image Aβ₄₂ interactions with lipid bilayers consisted of specific lipids.

No description available.