This thesis is intended as an introductory lab manual on the experimental methods using fluorescence microscopy in modern radiobiology research. It is written for those who are unfamiliar with biology research. It first covers the proper use of laboratory equipment and growth of cell cultures in the lab. Subsequent chapters provide overviews of relevant modern experimental techniques for the quantification of radiation induced DNA damage in cells, and detailed protocols for performing these procedures. Techniques covered include immunostaining with fluorescent antibodies, the comet assay, and plasmid DNA transfections. Results of some straightforward experiments using these techniques are presented.

Polymer brushes provide the responsive smart surfaces which can be used for fabrication of various devices. In this thesis work, adhesion, friction, and wear of polystyrene (PS) - poly(2-vinyl pyridine) (P2VP) and polystyrene - poly(acrylic acid) (PAA) binary brushes and corresponding monobrushes were investigated in dried state under controlled environment. Spin-coated films were also investigated for comparison. The aim was to explore possibilities to control/tune adhesion, friction, and wear between inorganic or polymeric surfaces by use of polymer brushes. Atomic force microscopy (AFM) with sharp silicon nitride tip and colloidal probes was employed to investigate the nanoscale adhesion and friction forces between different inorganic and polymeric surfaces. Adhesion and friction on the polymer brushes were comparable to that on the spin-coated films. Adhesion and friction force values were correlated, and were in accordance with the wettability of the brush surfaces for most of the samples. Switching in the adhesion and friction forces was observed for the PS+P2VP and PS+PAA binary brushes on treatment with selective solvents. Maximum switching in adhesion force and friction coefficient was by a factor of 2.7 and 5.4, respectively. Furthermore, switching of friction for mixed brush surface was observed during macroscale friction measurements using nanoindenter. Friction coefficients at macroscale were higher than those at the nanoscale. Moreover, adhesion and friction forces between the surfaces were significantly influenced by the humidity, grafting density of polymer brushes, chemical composition of top of the binary brush surface, and tip scan velocity. Nanowear studies were carried out with AFM using sharp silicon nitride tip while macrowear studies were carried out using nanoindenter. Nanowear on the surfaces was affected by molecular entanglements, adhesion and friction forces as well as shape and status of the tip. It was observed that the typical wear mode for PS brushes (treated with toluene) was ripple formation. In case of P2VP brushes (treated with ethanol) and PAA brushes (treated with pH 10 water), wear occurred via removal of the polymeric material. Wear mechanism observed for the monobrushes was similar to that observed for the spin-coated thick films of the same polymeric material. However, extent of the wear on the brush surfaces significantly differed from that on the spin-coated films. In case of PS+P2VP and PS+PAA binary brush samples, change in the wear mode was observed on treatment with the different selective solvents. On treatment with toluene (PS on the top), both of these binary brushes showed the wear by formation of the ripples. On the other hand, when these binary brushes were treated with selective solvent for P2VP or PAA, wear occurred mainly via removal of the polymeric material. The amount of wear increased with the number of scans for all the polymer brush samples. Moreover, wear on the polymer brush surfaces was also increased on increase in the applied load and decrease in the scan speed. Wear behavior on macroscale was averaged due to contact between surfaces at large number of asperities. Our results show that adhesion, friction, and wear of polymer surfaces can be controlled/tuned by the use of binary polymer brushes.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 107-109). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 107-109). Also available on the Internet.

The high degree of modification through chemical substitution afforded by the perovskite crystal structure and its related counterparts allows a systematic study of structure-property relationships critical to understand the wide variety of exotic phenomena observed in these materials where the spin, charge, orbital, and lattice degrees of freedom are highly correlated. From the multiple phenomena observed in these materials, which includes multiferroicity, catalytic activity, and high temperature superconductivity, this study is concerned with a material that displays colossal magnetoresistance (CMR), La₂₋₂xSr₁₊₂xMn₂O₇; this is a naturally bilayered manganite that exhibits CMR at a paramagnetic to ferromagnetic transition that coincides with an insulator to metal transition. The strong correlation between different degrees of freedom in the material leads to considerable variation in its magnetic properties due to doping even in the small range studied of 0.32 [less than or equal to] x [less than or equal to] 0.4, where the easy axis of magnetization changes from the c-axis to the ab plane. Magnetic force microscopy (MFM) was used for this part of the work, to visualize the local variation of the out of plane (c-axis) magnetization or magnetic microstructure of La₂₋₂xSr₁₊₂xMn₂O₇ for 0.32 [less than or equal to] x [less than or equal to] 0.4 at the exposed ab surface and its evolution due to an applied magnetic field at 4 K. For the x = 0.32 composition, which is close to the out of plane to in plane magnetization transition, a strong preferred magnetization direction within the ab plane or magnetocrystalline anisotropy was observed. The stray magnetic field of the MFM tip perturbs the magnetic microstructure of low coercivity materials like diluted magnetic semiconductors, making it unsuitable for the study of such materials. For this reason, as part of this project a scanning Hall probe microscope (SHPM), a magnetic imaging technique complementary to MFM that uses a Hall sensor that provides a magnetically non-invasive calibrated measurement of the stray fields at the surface of a sample with good resolution (~ 1 [micrometer]), was designed. The construction of a compact cryogenic variable-temperature (77 - 300 K) SHPM, highlighting its features, is described. / text

Carbon nanotubes (CNTs) have extraordinary electrical and mechanical properties, and many potential applications have been proposed, ranging from nanoscale devices to reinforcement of macroscopic structures. However, due to their small sizes, characterization of their mechanical properties and deformation behaviours are major challenges. Theoretical modelling of deformation behaviours has shown that multi-walled carbon nanotubes (MWCNTs) can develop ripples in the walls on the contracted side when bent above a critical curvature. The rippling is reversible and accompanied by a reduction in the bending stiffness of the tubes. This behaviour will have implications for future nanoelectromechanical systems (NEMS). Although rippling has been thoroughly modelled there has been a lack of experimental data thus far. In this study, force measurements have been performed on individual MWCNTs and vertically aligned carbon nanofibers (VACNFs). This was accomplished by using a custom-made atomic force microscope (AFM) inside a scanning electron microscope (SEM). The measurements were done by bending free-standing MWCNTs/VACNFs with the AFM sensor in a cantilever-to-cantilever fashion, providing force-displacement curves. From such curves and the MWCNT/VACNF dimensions, measured from SEM-images, the critical strain for the very onset of rippling and the Young’s modulus, E, could be obtained. To enable accurate estimations of the nanotube diameter, we have developed a model of the SEM-image formation, such that intrinsic diameters can be retrieved. We have found an increase in the critical strain for smaller diameter tubes, a behaviour that compares well with previous theoretical modelling. VACNFs behaved very differently, as they did not display any rippling and had low bending stiffnesses due to inter-wall shear. We believe that our findings will have implications for the design of future NEMS devices that employ MWCNTs and VACNFs. / <p>Artikel 2 Image formation mechanisms tidigare som manuskript, nu publicerad: urn:nbn:se:kau:diva-16425 (MÅ 150924)</p>

Different optical and calorimetric techniques were utilized to gain a better understanding of aggregated macromolecules. This research looked at two different macromolecules: poly(9,9'-dioctylfluorene), a conjugated polymer that forms aggregates in organic solvents; and bovine insulin, which forms amyloid fibrils. Conjugated polymers are of increasing interest due to their thermal stability and ease of solution processing for use in devices. A member of the polyfluorene family, poly(9,9'-dioctylfluorene) (PFO), has been studied due to its blue-emitting spectral properties. However, PFO has been found to form aggregates in solution, which is detected by the presence of a red-shifted absorption peak. This peak is caused when a section of the backbone planarizes forming the [beta]-phase. The [beta]-phase can be removed from the solution upon heating and will not return until the solution is cooled, making it a non-equilibrium process. The dissolution and reformation of the -phase were monitored using absorption spectroscopy and differential scanning calorimetry. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were able to probe the aggregates in films. It is important to understand polymer properties in solution in order to understand film morphology. Amyloid fibrils contribute to over 20 different neurodegenerative diseases, in which cures have yet to be found. The fibrils form when a soluble protein misfolds and self-assembles to form insoluble protein aggregates, and the cause of the fibril formation in vivo has still yet to be determined. Spectroscopy studies have been made possible with the use of fluorescent dyes: thioflavin T (ThT), BTA-2, and Congo red (CR). These dyes bind to amyloid fibrils and exhibit changes in their spectral properties. However, the exact mechanism for the binding of these dyes has only recently been studied. Through the use of calorimetry, the forces involved with binding of ThT and CR to amyloid fibrils can be determined. Absorption and fluorescence spectroscopy techniques were employed to study the spectral properties of these dyes. Polarized NSOM was used to determine the ThT or BTA-2's orientation with an individual fibril. Understanding how these dyes bind to fibrils will enable researchers to use spectroscopy to study the early stages of fibril formation. / text

The understanding of the biological processes at the cellular and subcellular level requires the ability to directly visualize them. Fluorescence microscopy played a key role in biomedical imaging because of its high sensitivity and specificity. However, traditional fluorescence microscopy has a limited resolution due to optical diffraction. In recent years, various approaches have been developed to overcome the diffraction limit. Among these techniques, nonlinear structured illumination microscopy (SIM) has been demonstrated a fast and full field superresolution imaging tool, such as Saturated-SIM and Photoswitching-SIM. In this dissertation, I report a new approach that applies nonlinear structured illumination by combining a uniform excitation field and a patterned stimulated emission depletion (STED) field. The nature of STED effect allows fast switching response, negligible stochastic noise during switching, low shot noise and theoretical unlimited resolution, which predicts STED-SIM to be a better nonlinear SIM. After the algorithm development and the feasibility study by simulation, the STED-SIM microscope was tested on fluorescent beads samples and achieved full field imaging over 1 x 10 micron square at the speed of 2s/frame with 4-fold improved resolution. Our STED-SIM technique has been applied on biological samples and superresolution images with tubulin of U2OS cells and granules of neuron cells have been obtained. In this dissertation, an effort to apply a field enhancement mechanism, surface plasmon resonance (SPR), to nonlinear STED-SIM has been made and around 8 time enhancement on STED quenching effect was achieved. Based on this enhancement on STED, 1D SPR enhanced STED-SIM was built and 50 nm resolution of fluorescence beads sample was achieved. Algorithm improvement is required to achieve full field superresolution imaging with SPR enhanced STED-SIM. The application of nonlinear structured illumination in two photon light-sheet microscopy is also studied in this dissertation. Fluorescent cellular imaging of deep internal organs is highly challenging because of the tissue scattering. By combining two photon Bessel beam light-sheet microscopy and nonlinear SIM, 3D live sample imaging at cellular resolution in depth beyond 200 microns has been achieved on live zebrafish. Two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity.

Titanium carbide (TiC) and titanium carbonitride (Ti(C,N)) are both common components in hard, wear resistant ceramic-metal composites, or cermets. In this study the intermetallic nickel aluminide (Ni3Al) has been used as a binder for the production of TiC and Ti(C,N) based cermets. These cermets offer several improved characteristics relative to conventional WC-based ‘hardmetals’, such as lower mass and improved oxidation resistance, which are also combined with high fracture resistance, hardness and wear resistance. The cermets were produced using an in-situ, reaction sintering procedure to form the stoichiometric Ni3Al binder, with the binder contents varied from 20 to 40 vol%. However, for high N content Ti(C,N) cermets, the wettability of molten Ni3Al is relatively poor, which leads to materials with residual porosity. Therefore various amounts of Mo2C (1.25, 2.5, 5 and 10 vol%) were incorporated into the Ti(C0.3,N0.7)-Ni3Al cermets, with the aim of improving the densification behaviour. Mo2C was found to improve upon the wettability during sintering, thus enhancing the densification, especially at the lower binder contents. The tribological behaviour of TiC and Ti(C,N) cermets have been evaluated under reciprocating sliding conditions. The wear tests were conducted using a ball-on-flat sliding geometry, with a WC-Co sphere as the counter-face material, for loads from 20 to 60 N. The wear response was characterised using a combination of scanning electron microscopy, energy dispersive X-ray spectroscopy, and focused ion beam microscopy. Initially, two-body abrasive wear was observed to occur, which transitions to three-body abrasion through the generation of debris from the cermet and counter-face materials. Ultimately, this wear debris is incorporated into a thin tribolayer within the wear track, which indicates a further transition to an adhesive wear mechanism. It was found that Mo2C additions had a positive effect on both the hardness and indentation fracture resistance of the samples, but had a detrimental effect on the sliding wear response of the cermets. This behaviour was attributed to increased microstructural inhomogeneity with Mo2C additions.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.