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Laser nano-patterning with near-field optical microscope (NSOM) and the associated energy transport analysis are achieved in this study. Based on combined experimental/theoretical analyses, it is found that laser nano-patterning with a NSOM probes strongly depend on the laser conditions and material properties of the target: the energy transport from the NSOM probes to the targets changes from pure optical to a combination of thermal and optical transport when the pulse duration of laser is increased from femtosecond to nanosecond. As a result, the mechanisms of nano-pattern formation on targets changes from nano-ablation to nano-oxidation/ recrystallization when the laser pulse duration is increased from femtosecond to nanosecond. Also, with the laser nano-patterning experiments, thermal damage of NSOM probes is observed which can be attributed to the low transport efficiency (10-4 – 10-6) and associated heating of the metal cladding of NSOM probes. The heating of NSOM probes are studied with developed time harmonic and transient thermoreflectance (TR) imaging. From time harmonic TR when the NSOM probes are driven with continuous laser, it is found that the location of heating of NSOM probes is ~20-30µm away from the NSOM tip. The strength of the heating is determined by the laser power (linear dependence), wavelength of the laser (stronger with short A), and aperture size of NSOM probes (stronger when aperture size < A/2). From the transient TR imaging when the NSOM probes are driven with pulsed laser, it is found that the peak temperature of the NSOM probe shifts much closer to the tip. The possible reason for the change in the location of peak temperature when continuous laser is changed to pulsed laser can be attributed to the competition between the heat generation and dissipation rates at different location of the probe: the tip experiences highest temperature with pulsed heating as the entire heating processes is adiabatic. The tip also experiences highest heat dissipation rate due to its large surface-to-volume ratio which overcomes the heat generation at the tip under quasi-steady state resulting in shift of the hot spot. The knowledge obtained in this study can be important in the future design of more efficient NSOM probes and other nano-optic devices.

This dissertation aims to further the understanding of the complex communication that occurs as cells interact with topographical and chemical patterns on a biomaterial interface. The research accomplishes this through two aims fabricating cell substrate surface topography and chemical patterns independently using non-cleanroom approaches, and analyzing higher order cellular response to surface features. The work will impact biomaterial surface modification and fabrication which will apply to biomedical implanted devices, tissue engineering scaffolds, and biological analysis devices. The first aim seeks to apply non-traditional topographical and chemical patterning methods in order to create independent topographical and chemical patterns on cell culture substrates. Experiments use the resulting patterned substrates to quantify cellular alignment to surface topography and compare the relative influence of topographical and chemical patterns on cellular response. The combined patterning methods of imprint lithography and micro-contact printing result in a high-throughput technique applicable to a variety of materials and a range of feature sizes from nanoscale through microscale, thereby enabling future analysis of cell response to surface features. The second aim evaluates the impact of topographical and chemical features on cellular differentiation. Experiments use patterned topography overlaid with a characterized chemical model layer to evaluate the effects of topography on myoblast differentiation and alignment. Chemical patterns that independently control available cell spreading area and modulate cell-cell contact are used to investigate the impact of cell-cell contact on differentiation.

Cell adhesion to extracellular matrices (ECM) is critical to many cellular processes including differentiation, proliferation, migration, and apoptosis. Alterations in adhesive mechanisms are central to the behavior of cells in pathological conditions including cancer, atherosclerosis, and defects in wound healing. Although significant progress has been made in identifying molecules involved in adhesion, the mechanisms that dictate the generation of strong adhesive forces remain poorly understood. Specifically, the role of nanoscale geometry of the adhesive interface in integrin recruitment and adhesion forces remains elusive due to limitations in the techniques available for engineering cell adhesion environments. The objective of this project was to analyze the role of nanoscale geometry in cell adhesion strengthening to ECM. Our central hypothesis was that adhesive interactions are regulated by integrin clusters whose recruitment is determined by the nanoscale geometry of the adhesive interface and whose heterogeneity in size, spacing, and orientation modulates adhesion strength. The objective of this project was accomplished by 1) developing an experimental technique capable of producing nanoscale patterns of proteins on surfaces for cell adhesion arrays, 2) assessing the regulation of integrin recruitment by geometry of the adhesive interface, and 3) determining the functional implications of adhesive interface geometry by systematically analyzing the adhesion strengthening response to nanoscale patterns of proteins. A printing technique was developed that patterns proteins into features as small as 90nm with high contrast and high reproducibility. Cell adhesion arrays were produced by directly immobilizing proteins into patterns on mixed-SAMs surfaces with a protein-resistant background. Colocalization analysis of integrin recruitment to FN patterns demonstrated a concentrating effect of bound integrins at pattern sizes with areas equivalent to small nascent focal adhesions. At adhesion areas below 333 × 333 nm2, the frequency of integrin recruitment events decreased significantly indicating a threshold size for integrin clustering. Functionally, pattern sizes below the threshold were unable to participate in generation of adhesion strength. In contrast, patterns between the threshold and micron sizes showed a relationship between adhesion strength and area of individual adhesion points, independent of the total available adhesion area. These studies introduce a robust platform for producing nanoscale patterns of proteins in biologically relevant geometries. Results obtained using this approach yielded new insights on the role of nanoscale organization of the adhesive interface in modulating adhesion strength and integrin recruitment.

Thermal nanoimprint lithography (T-NIL) is a cheap and fast technique to produce nanopatterns in polymeric materials. It creates these patterns by pressing a stamp down into a polymer film that has been heated above its glass transition temperature. These nanopatterned polymer films can be used in a wide variety of scientific fields, not the least the organic semiconductor industry. There the nanopatterned films have, among else, been used to improve the efficiency of organic light-emitting diodes (OLEDs). The light-emitting electrochemical cell (LEC), which is similar in structure to an OLED, also uses polymer films in their device structure but the light emitting layer also contains an electrolyte. However, it has not been shown if nanopatterns can improve LECs as well or if it is even possible to make an imprint in their polymer films that are mixed with an electrolyte. This thesis shows that T-NIL can be used to imprint nanopatterns in films made of poly(ethylene oxide) and the conjugated polymer Super Yellow. The best nanopatterns were produced by setting the imprint parameters to  85 °C, 10 bar, 1800 s for poly(ethylene oxide) and 115 °C, 20 bar, 1800 s for Super Yellow. Imprints were also performed on polystyrene but no nanopatterns could be produced. This was most likely because the stamp could not handle the high temperature that is required to make a nanopattern in polystyrene. The best imprint parameters of Super Yellow were then used to produce a pattern in a film made of Super Yellow mixed with the salt tetrahexylammonium tetrafluoroborate (THABF4) in order to be able to produce one imprinted and one reference LEC. The imprinted LEC had a luminosity of 139 cd/m2, an improvement of 20% compared to the reference’s 115 cd/m2 when operated under identical conditions.  The forward direction and the angular dependent electroluminescence spectrum of the imprinted LEC clearly showed an effect not observed in the reference. These findings show that the polymer films used in a LEC can be imprinted with a nanopattern by using T-NIL. The imprinted films can be used to create functional LECs that show different behavior and a higher luminosity compared to a non-imprinted reference. If these results can be repeated it might be the starting point of a brighter future.

<p>Extracellular matrix (ECM) creates high-resolution chemical patterns, by assembling simple molecules with nm-scale features (e.g., carbohydrates, nucleotides, amino acids) into complex structures up to micrometers and extending to even larger scales across tissues (e.g., glycans, DNA, proteins), capable of carrying out the diverse and complex cellular functions. Mimicking the complexity of such biological systems requires precise control over the chemical patterning on substrates that exhibit physiochemical properties similar to biological systems (such as hydrogels). Although hydrogels provide tunable physiochemical properties suitable for biological applications; it is a porous material where pore sizes can range from 30 nm to greater than 1000 nm. Due to this structural heterogeneity, chemical patterning below the length scale of this heterogeneity is very challenging.</p> <p>Here, we demonstrate a new assembly system for generating a nanostructured presentation of carbohydrates on the hydrogel surface. This approach is based on the striped phases assembly of functional alkanes where 1-nm resolution functional patterns are readily assembled on substrates such as highly ordered pyrolytic graphite (HOPG). In this assembly, molecules are stabilized by noncovalent interactions, including alkyl-pi interactions underlying the HOPG, van der Waals interaction between the adjacent alkyl chains, and hydrogen bonding between polar head groups. Topochemical polymerization converts internal diynes into conjugated polydiacetylenes (PDAs). PDAs can also be utilized to covalently attach the striped pattern to polyacrylamide hydrogels through free radical chemistry.</p> <p>Here, we synthesize new amphiphiles with carbohydrate headgroups (N-acetyl-D-glucosamine (GlcNAc), and D-glucuronic acid (GlcA)), assembled into striped phases on HOPG and covalently transfer to polyacrylamide hydrogels. GlcNAc binds to wheat germ agglutinin (WGA), a lectin that binds specifically in a multivalent fashion (dissociation constant KD in nm range) to GlcNAc. We show that GlcNAc striped phases generate highly selective interactions with wheat germ agglutinin (WGA) but do not induce specific binding with concanavalin A (another lectin molecule that does not target GlcNAc). We further demonstrate that WGA binding affinity can be modulated by shifting the position of diacetylenes that bring the polymer backbone closer to the GlcNAc, increasing the effecting local concentration of carbohydrates.</p> <p>We investigated the possibility of using sPDA for secondary functionalization with complex biological molecules (such as biotin and cRGD) to mimic the ECM composition closely. The unusual reactivity of the sPDA backbones during the covalent transfer of the striped phase monolayer to hydrogels illustrates the potential of sPDA reactivity azides. In this work, we show that the addition of substituted azide molecules to sPDA-functionalized hydrogels produces a decrease in the fluorescence of the sPDA monolayer. Since these reactions are occurring on porous hydrogel surfaces characterization using techniques such as IR or NMR is difficult. We carried out further solution-phase reactions using a soluble PDA where PDA UV-vis absorption spectra red-shift after the reaction between the PDA backbone and azide. These experiments support the hypothesis of sPDA and azide click reaction.</p>