Studying the interactions between bacteria and soil colloidal particles in the environment is important for bioaugmentation purposes. Different factors affect the transport of the bacteria in porous media. For example, the soil type, the ionic strength of the substrate, and biological properties, such as the bacterial cell motility. Since organic materials are present in almost all subsurface media, the presence of natural organic matter (NOM) is considered an important factor influencing bacterial transport in porous media. In this work, a model system was developed to examine the interactions between natural colloidal particles and environmental bacteria using Atomic Force Microscopy (AFM). The natural colloids in the environment were modeled by a surface film of adsorbed NOM onto spherical SiO2 particles. Poly(methacrylic acid) (PMA), a simple linear polyelectrolyte, was used to mimic NOM since both are dominated by carboxylic acid functional groups. Soil Humic Acid (SHA) and Suwannee River Humic Acid (SRHA), two acidic polyelectrolytes, were used in further experiments to represent more complicated NOM. A smooth strain of Pseudomonas aeruginosa (PAO1) that coexpresses A-band and B-band polysaccharides, and its rough mutant (AK1401) that only expresses the A-band polysaccharides, were chosen to represent environmental bacteria. The model system was characterized through analysis of the measured forces between the chemically-modified colloidal probes and the bacterial cells. Interestingly, we found that PMA was not a good model for the more complex NOM substances. Differences were also observed in how each bacterium interacted with the three forms of NOM. For example, P. aeruginosa PAO1 had the highest adhesion with both complex forms of NOM, while P. aeruginosa AK1401 had the lowest adhesion with the complex forms of NOM. Since the lipopolysaccharide (LPS) structure is the only difference between the two strains, we attribute the different interactions to differences in LPS structure. The polymer density on the bacterial surface was found to be the most important factor in controlling the nature of the interaction forces.

This thesis reflects two advances in atomic force microscopy. The first half is a new lateral force calibration procedure, which, in contrast to existing procedures, is independent of sample and cantilever shape, simple, direct, and quick. The second half is a high-throughput method for processing, fitting, and analyzing force curves taken on Pseudomonas aeruginosa bacteria in an effort to inspire better care for statistics and increase measurement precision.

Vascular diseases, such as stroke and heart attacks, account for more than 50% of abnormal death worldwide. The cause of these diseases is linked to malfunctions of vascular endothelial cells, in particular the endothelial glycocalyx. This study investigates the location and stability of the endothelial glycocalyx under different flow conditions in vitro. AFM (Atomic Force Microscopy) micro indentation is carried out on endothelial cell membrane to determine its Young's modulus. The Young's modulus of the glycocalyx layer is then deduced from measurements on cell membranes with, and those without, the glycocalyx layer. Heparan sulphate (HS) is an important component of the glycocalyx and can be removed by the enzyme heparinase-III (Hep-III). Our results show the glycocalyx on cultured Human Umbilical Vein Endothelial Cells (HUVECs) has a Young's modulus of ~0.64Kpa. We further observe how the Young's modulus of the endothelial cell membrane decreases with time, as the glycocalyx layer redevelops, following its removal by Hep-III. Steady and oscillatory shear stimulations are used in flow chamber experiments. Under 24 hours' steady shear stimulation (12.6 dyn/cm2), cells are seen to elongate and reorient parallel to the flow direction. The glycocalyx is seen to shift to the peripheral region of the cell surface. With actin depolymerisation treatment, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This occurs together with the loss of focal adhesions on the basal membrane. When endothelial cells are subjected to 24 hours' oscillating shear stress, the size of the cell increases as the oscillatory reversal time (time between changes in oscillatory flow direction) increases. Measurements are taken with oscillatory flow reversal programmed at 5s, 10s and 15s. The angle (between the long axis of the cell and the flow direction) and the aspect ratio (long axis vs short axis) change from 41.57° and 1.72 : 1 (static) to 40.18° and 3.26 : 1 (5s), 36.71° and 4.17 : 1 (10s), 26.5° and 4.39 : 1 (15s). Both the height and the area of the cell increase. The Young's modulus of the endothelial cell membrane is measured under oscillatory flows with different reversal time and compared to that under static flow conditions. An increase in the Young's modulus is observable under oscillatory flows, with the most significant change occurring at the edge (i.e. periphery) of the cell membrane area. As the oscillatory reversal time increases from 5s to 15s, the Young's modulus of the cell membrane increases. In the apical areas of the cell membrane, the increase is less significant. These results indicate that the thickness of the glycocalyx decreases as cells are exposed to oscillatory flows, and the loss is most significant in the peripheral region of the cell membrane. As the oscillatory reversal time increases from 5s to 15s, so the loss in the glycocalyx increases.

Wang Yuhao = 利用X光电子谱和导电原子力显微镜对聚3, 4-乙烯二氧噻酚 : 聚苯磺酸改性的研究 / 王宇昊. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / Text in English; abstracts in English and Chinese. / Wang Yuhao = Li yong X guang dian zi pu he dao dian yuan zi li xian wei jing dui ju 3, 4-yi xi er yang sai fen : ju ben huang suan gai xing de yan jiu / Wang Yuhao. / Abstract --- p.ii / 論文摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.ix / List of Tables --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Review of conducting conjugated polymers --- p.1 / Chapter 1.1.1 --- Development of conjugated polymer --- p.1 / Chapter 1.1.2 --- Basic concepts in independent-electron theories of conducting conjugated polymers --- p.2 / Chapter 1.1.2.1 --- "Huckel model and its difficulty, the importance of election-phonon" --- p.2 / Chapter 1.1.2.2 --- The SSH model and dimerization --- p.3 / Chapter 1.1.2.3 --- "Charge carriers in conducting conjugated polymers: soliton, polaron and bipolaron" --- p.5 / Chapter 1.1.3 --- "Poly(3,4-ethylenedioxythiophene) or PEDT" --- p.5 / Chapter 1.1.4 --- Derivatives of PEDT --- p.6 / Chapter 1.1.5 --- Application of PEDT and its derivatives --- p.7 / Chapter 1.2 --- Polymeric light emitting diodes (PLED) --- p.7 / Chapter 1.2.1 --- Invention Polymeric light emitting diodes (PLED) --- p.7 / Chapter 1.2.2 --- Electric structure of PLEDs --- p.7 / Chapter 1.2.3 --- Transition from excitons to photons --- p.8 / Chapter 1.2.4 --- Controlling electron and hole injection --- p.8 / Chapter 1.2.5 --- Application of PEDT-PSS as hole transporting layer in PLED --- p.9 / Chapter 1.2.6 --- "Phase separating in PEDT-PSS blend, removing the PSS rich layer" --- p.9 / Chapter 1.3 --- Motivations of the thesis work --- p.10 / References --- p.10 / Chapter CHAPTER 2 --- INSTRUMENTATION --- p.27 / Chapter 2.1 --- X-ray Photoelectron Spectroscopy --- p.27 / Chapter 2.1.1 --- History of XPS techniques --- p.27 / Chapter 2.1.2 --- Physical Basis --- p.28 / Chapter 2.1.3 --- Chemical Shift of Binding Energy in XPS --- p.29 / Chapter 2.1.4 --- Binding Energy Referencing in XPS --- p.29 / Chapter 2.1.5 --- Sampling Depth of XPS --- p.30 / Chapter 2.1.6 --- Instrumental Setup of XPS --- p.30 / Chapter 2.2 --- Scanning Probe Microscopy --- p.31 / Chapter 2.2.1 --- Introduction --- p.31 / Chapter 2.2.2 --- Atomic Force Microscopy and Conductive Atomic Force Microscopy --- p.31 / Chapter 2.2.3 --- Instrumental Setup for Conductive AFM --- p.32 / Chapter 2.3 --- The Low Energy Ion Beam (LEIB) system at CUHK --- p.32 / Chapter 2.3.1 --- Introduction --- p.32 / Chapter 2.3.2 --- Principle --- p.33 / Chapter 2.3.3 --- Instrumentation Setup --- p.33 / References --- p.33 / Chapter CHAPTER 3 --- Effects of Ar+ bombardment at 500 and 100eV --- p.42 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Sample Preparation --- p.42 / Chapter 3.3 --- Ar+ sputtering and XPS measurement of the sputtered sample. --- p.43 / Chapter 3.4 --- Results and Discussion --- p.44 / References --- p.49 / Chapter CHAPTER 4 --- Effects of annealing on PEDT-PSS thin films studied by XPS and AFM --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Sample Preparation --- p.60 / Chapter 4.3 --- XPS measurements and results --- p.61 / Chapter 4.3.1 --- XPS of C 1s core level of PEDT-PSS --- p.61 / Chapter 4.3.2 --- XPS of O 1s core level of PEDT-PSS --- p.62 / Chapter 4.3.3 --- XPS of S 2p core level of PEDT-PSS --- p.62 / Chapter 4.3.4 --- XPS of Valence Band of PEDT-PSS --- p.64 / Chapter 4.4 --- C-AFM measurements and results --- p.65 / Chapter 4.4.1 --- C-AFM measurements on PEDT-PSS --- p.65 / Chapter 4.5 --- Measurements and results about film insolubility and conductivity --- p.65 / Chapter 4.5.1 --- Insolubility measurements --- p.66 / Chapter 4.5.2 --- Conductivity measurements --- p.66 / Chapter 4.5.3 --- Results from the film insolubility and conductivity measurements --- p.66 / Chapter 4.6 --- Conclusion --- p.67 / References --- p.68 / Chapter CHAPTER 5 --- Effects of low energy proton bombardment of PEDT-PSS films studied by XPS and AFM --- p.90 / Chapter 5.1 --- Introduction --- p.90 / Chapter 5.2 --- XPS and c-AFM studies of PEDT-PSS films bombarded by H+ --- p.90 / Chapter 5.2.1 --- Sample preparation --- p.90 / Chapter 5.2.2 --- Results and discussion --- p.90 / Chapter 5.3 --- Conductivity measurements --- p.92 / Chapter 5.3.1 --- Sample preparation for conductivity measurements --- p.92 / Chapter 5.3.2 --- Results and discussion --- p.93 / Chapter 5.4 --- Conclusion --- p.93 / References --- p.93 / Chapter CHAPTER 6 --- Concluding Remarks and Future Works --- p.106 / Chapter 6.1 --- Concluding Remarks --- p.106 / Chapter 6.2 --- Future Work --- p.106 / Chapter APPENDIX --- The SSH model in describing polyacetylene --- p.108 / Chapter Part 1 --- Assumptions of the SSH model --- p.108 / Chapter Part 2 --- Bloch model and SSH model. --- p.113 / Reference --- p.117

nÃo hà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo. / IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo. / Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process. / Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process.

L'étude de la dynamique interfaciale des liquides à l'échelle du nanomètre est cruciale pour la compréhension de nombreux phénomènes biologiques et industriels. Pour aborder cette question, nous étudions l'interaction en champ proche d'une sonde et de liquides peu visqueux. La thèse s'articule autour de deux grands axes : le premier s'intéresse à la déformation de l'interface liquide lorsqu'une pointe est approchée et à l'instabilité hydrodynamique du "jump-to-contact" qui en résulte. Le second, plus intrusif, décrit la réponse hydrodynamique d'un liquide soumis à l'oscillation d'un nanocylindre (R ~ 20-100 nm) partiellement immergé. Les mesures sont réalisées par microscope à force atomique (AFM), en mode modulation de fréquence (FM), qui permet de mesurer la force exercée sur la sonde ainsi que les composantes conservatives et dissipatives de l'interaction pointe-liquide. Une première série de mesure est réalisée sur différents liquides modèles avec un AFM couplé à une caméra rapide via un microscope optique inversé. Avant le mouillage de la sonde, les courbes de spectroscopie de force et FM mettent en évidence la déformation de l'interface liquide sur des échelles nanométriques, pour une grande gamme de tailles de sonde (de 10 nm à 30 µm). L'analyse des mesures expérimentales avec le modèle théorique récemment développé par René Ledesma-Alonso permet de déterminer la distance critique dmin en dessous de laquelle l'interface se déstabilise et mouille irréversiblement la pointe ("jump-to-contact"). Un excellent accord est trouvé entre le modèle théorique et les mesures FM. La deuxième série de mesure s'intéresse à l'immersion partielle de pointes AFM cylindriques. Les courbes de spectroscopie FM montrent qu'une certaine quantité de liquide, située dans la couche visqueuse, est entraînée par l'oscillation de la pointe. On mesure simultanément la friction exercée sur la pointe et la masse de liquide ajoutée au système, qui est directement reliée à l'extension du champ de vitesse. Un modèle analytique basé sur la résolution de l'équation de Stokes rend compte quantitativement de l'ensemble des résultats expérimentaux. La dernière série de mesure est réalisée avec des sondes cylindriques spécialement conçues pour l'étude de la dynamique de nanoménisques. Ces sondes comportent des défauts topographiques annulaires dont l'épaisseur varie entre 10 et 50 nm. Les mesures montrent une divergence du coefficient de friction aux petits angles de contact qui est bien reproduite par un modèle théorique basé sur l'approximation de lubrification. La localisation de la dissipation d'énergie au voisinage de la ligne de contact et les propriétés d'ancrage du ménisque sont également discutées. Les expériences originales développées dans cette thèse démontrent ainsi la capacité de l'AFM à étudier quantitativement les liquides à l'échelle nanométrique et ouvrent la voie à une étude systématique des processus de dissipation au sein de liquides confinés, et notamment au voisinage d'une ligne de contact en mouvement. / The study of the interfacial dynamics of liquids, down to the nanometer-scale, is of primary importance in many domains including biological and industrial phenomena. To address those questions, we study the near-field interaction between a probe and low viscous liquids. The present thesis focuses on two aspects. In the first one, we investigate the liquid interface deformation that occurs when a tip is approached and the resulting "jump-to-contact" hydrodynamic instability. The second part is more intrusive as it describes the hydrodynamic response of a liquid under the oscillation of a partly-immerse nanocylinder (R ~20-100 nm). Our measurements are performed with an Atomic Force Microscope (AFM) in the frequency modulation (FM) mode, which allows to measure the force exerted on the probe along with the conservative and dissipative components of the tip-liquid interaction. A first set of measurements is performed on several model liquids with an AFM coupled with a high-speed camera via an inverse optical microscope. Before the probe wetting, the force and FM spectroscopy curves highlight the liquid interface deformation on nanometer scales for a large range of probe size (from 10 nm to 30 µm). The fitting of our experimental measurements with the theoretical model recently developed by René Ledesma-Alonso, enables to determinate the critical distance dmin below which the interface is destabilized and irreversibly wets the tip (jump-to-contact). The theoretical model and the FM measurements were found to be in good agreement. The second set of measurements focuses on the partial immersion of cylindrical AFM tips. The FM spectroscopy curves show that a certain quantity of liquid, located in the viscous layer, is carried off with the tip oscillation. The friction exerted on the tip and the liquid mass added to the system, which is directly linked to the velocity-field extension, were measured simultaneously. An analytical model based on the Stokes equation quantitatively reproduces our experimental results. The last set of measurements is performed with cylindrical probes specially designed for the study of nanomeniscus dynamics. Those probes possess annular topographic defects, whose thickness varies between 10 nm and 50 nm. The measurements show that he measured friction coefficient surges as the contact angle is decreased. This behavior is well described by a developed theoretical model based on the lubrication approximation. Furthermore, the dissipation pattern in the vicinity of the contact line and the anchoring properties are also discussed. The original experiments developed in this thesis demonstrate thus that AFM is a relevant tool for the quantitative study of liquids at the nanoscale. This work paves the way for systematic studies of dissipation processes in confined liquids, and in particular in the vicinity of moving contact lines.

The development of ultrasound contrast agents, or microbubbles, over the past 40 years has increased the possibilities for diagnostic imaging, although, more recently they have been proposed as a new vehicle for delivery of drugs and genes. However, there yet remains a considerable lack of fundamental understanding of microbubble behaviour under ultrasound excitation which has restricted their translation to therapeutic use. This project focussed on three key areas relating to the generation, observation, and bioeffects of microbubbles and the ultrasound used in their excitation. The experimental endeavour involved first, a full characterisation of the performance of a rotating mirror high-speed camera (Cordin 550-62) that was previously used by our group [and others] to investigate microbubble dynamics. Specifically, the investigation begins with an assessment of the frame-rate reporting accuracy of the system, a key aspect to the robustness of quantitative measurements extracted from recorded image sequences. This is then followed by the demonstration of a novel method of analysis for examining the image formation process in this type of camera, which facilitates a sensor-by-sensor assessment of performance that was not previously realised. Consolidating with previous work from within the group, this new analysis method was used to clarify previous data, and in the process suggested the presence of a temporal anomaly embedded within recorded images. In addition, the analysis also revealed empirical evidence for the mechanisms leading to this anomaly. Following on, a holographic optical tweezer system was developed for the purpose of exercising precise spatial control over microbubbles within their experimental environment. By positioning microbubbles in specific arrangements, interesting behaviours that were not previously achieved experimentally in the context of shelled microbubbles, were observed. Furthermore, by careful positioning of microbubbles within the imaging plane, it was possible to exploit the temporal anomaly present in the camera to greatly improve the integrity of data recorded, and to also operate in an enhanced imaging mode. Group aspirations to accelerate the development of therapeutic microbubbles had previously generated some early work on the in-house generation of bespoke bubble populations using microfluidic lab-on-a-chip techniques. In order to facilitate further development in this area, a finite-element computational model was herein developed to aid next generation chip design. Finally, in a slightly different context, considering not only the mechanical effect a microbubble may effect in a therapeutic treatment, a single biological cell assay was developed in order to probe any mechanical effects that were induced by the excitation ultrasound itself. Capitalising on the precise force control possible with atomic force spectroscopy, the elastic moduli of cells pre- and post-ultrasound insonation (sans microbubbles) were recorded. These new developments have extended the group capability and expertise in the areas of high-speed imaging, experimental observations of microbubble dynamics and with microfluidic generation of microbubbles. Additionally, the insights garnered have both served to consolidate the group's previous and as yet unpublished data, opening the way for circulation with absolute confidence in the integrity of that data.

Esta tese aborda a aplicação de técnicas avançadas de processamento e análise de imagens em problemas originais envolvendo imagens de microscopia de força atômica. Para isso, foi desenvolvida uma série de algoritmos para a caracterização e o entendimento do processo de formação de novos materiais poliméricos com perspectivas de inúmeras aplicações tecnológicas. As análises envolveram a determinação da orientação da morfologia de substratos para alinhamento de cristais líquidos, contagem e estimativa dos raios de domínios em filmes automontados POMA/PVS, análise do aumento da fotoluminescência em filmes PPV e estudos da curvatura espontânea de macromoléculas de polímeros. Dentre os algoritmos principais podemos citar a determinação da inclinação dos autovalores da matriz de covariância das coordenadas dos pontos da forma, aplicação da técnica dos máximos regionais e diagramas de Voronoi, filtros passabanda 2D através da transformada de Fourier e extração da curvatura multiescala. A implementação destes algoritmos envolveu algoritmos básicos de análise de imagens tais como esqueletização, dilatações exatas e extração do contorno de formas. A principal contribuição deste trabalho foi a implementação do software denominado SPIA (Scanning Probe Image Analysis) que possui ferramentas para análise e processamento de imagens incluindo todas as que foram utilizadas no decorrer deste trabalho além de outras ferramentas. Este software foi desenvolvido em ambiente Delphi sob o paradigma da orientação a objetos para plataformas Windows NT/9X/2000/XP. Possui uma interface amigável e semelhante a outros softwares dedicados a processamento de imagens. Todas as técnicas aplicadas foram testadas extensivamente e os resultados que corroboram sua eficiência são mostrados ao longo da tese / This thesis address the application of advanced techniques of processing and analysisof images in original problems involving images of atomic force microscopy. For this, a series of algorithms for characterization and understanding of process of formation of new polymeric materials was developed and implemented, with perspectives of many technological applications. The analysis was applied to the determination of orientation of the morphology of substrates for alignment of liquid crystals, counting and estimative of radiuses of granules in layer-by-layer polymer films, analysis of enhancement of photoluminescence in PPV cast films, as well as the study of curvature spontaneous of macromolecules. The principal algorithms included are determination of inclination of eigenvectors of matrix of covariance of coordinates of points of shape,application of regional maxima technique and Voronoi diagrams, passband filters 2D through Fourier Transform and curvature multiscale. The implementation of these algorithms involved a series of image analysis algorithms such as squeletonization, exact dilations and extracting of contour of shapes. The principal contribution of this work was to develop a software called SPIA (Scanning Probe Image Analysis) that includes tools for analysis of processing of images including that were used in this work. This software was developed in Delphi under object orientation paradigm to Windows NT/9X/2000/XP. It has a friendly interface similar to other image processing softwares. All this techniques were tested extensively and the results that corroborate the robustness of the algorithms are included throughout the thesis

The mechanisms linking deposits of insoluble fibrils of amyloid proteins to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings suggest that transiently formed intermediate aggregates, and not the prominent neuronal plaques, represent the principal toxic agent. Evaluating the neurotoxicity of intermediate aggregates, however, requires unambiguous characterization of all aggregate structures present, their relative distributions, and how they evolve in time. Hen-egg white lysozyme represents an attractive model for studying intermediate aggregate formation since it is an extensively characterized globular protein, and its human variants can lead to systemic amyloidosis. Combining in-situ dynamic light scattering (DLS) with atomic force microscopy (AFM), we have characterized the morphologies and growth kinetics of intermediate aggregates formed during lysozyme fibrillogenesis. Upon incubation at elevated temperatures, small uniform oligomers form with their numbers increasing for several hours. After a variable lag period protofibrils spontaneously nucleate. The heights and widths of protofibrils closely match those of oligomers. This match in physical dimensions, combined with the delayed onset of protofibril nucleation vs. the continuous formation of oligomers, suggest that protofibrils both nucleate and grow from oligomers. Protofibril morphologies and structures, visualized with AFM, are quite distinct from subsequently emerging mature fibrils. Overall, the evolution of aggregate morphologies during lysozyme fibrillogenesis follows a clear hierarchical pathway: amyloid monomers initially coalesce into oligomers of uniform size. Their steadily increasing numbers eventually induce nucleation and growth of protofibrils. Protofibrils, in turn, nucleate and grow via oligomer addition until they start to self-assemble into micron-sized double-stranded fibrils.

We present the development of a far-field magneto-optical Kerr effect microscope. An inverted optical microscope was constructed to accommodate Kerr imaging and atomic force microscopy. In Kerr microscopy, magnetic structure is investigated by measuring the polarization rotation of light reflected from a sample in the presence of a magnetic field. Atomic force microscopy makes use of a probe which is scanned over a sample surface to map the topography. The design was created virtually in SolidWorks, a three-dimensional computer-aided drafting environment, to ensure compatibility and function of the various components, both commercial and custom-machined, required for the operation of this instrument. The various aspects of the microscope are controlled by custom circuitry and a field programmable gate array data acquisition card at the direction of the control code written in National Instrument LabVIEW. The microscope has proven effective for both Kerr and atomic force microscopy. Kerr images are presented which reveal the bit structure of magneto-optical disks, as are atomic force micrographs of an AFM calibration grid. Also discussed is the future direction of this project, which entails improving the resolution of the instrument beyond the diffraction limit through near-field optical techniques. Preliminary work on fiber probe designs is presented along with probe fabrication work and the system modifications necessary to utilize such probes.