Nanoporous materials have been widely used in the fields of biological and chemical sensing, chemical separation, heterogeneous catalysis and biomedicine due to their merits of high surface area-to-volume ratio, chemical and thermal stabilities, and flexible surface modification. However, as the nature of nanoporous materials, they are inherently heterogeneous in the micro- and nanoenvironments. The environmental heterogeneity plays a decisive role in determining the performance of various applications of nanoporous materials. In order to provide an in-depth understanding of the nanoporous materials, it is of great interest to investigate the environmental heterogeneity in them. Single molecule spectroscopy, combined the quantitative confocal fluorescence imaging which possesses the capability of optical sectioning, has demonstrated to be a powerful tool to approach the environmental heterogeneity inside nanoporous materials. Single molecule spectroscopy is an ultrasensitive technique for probing molecular transport and properties of individual molecules. This technique has been extensively used in the research of environmental heterogeneity in nanoporous materials since it removes the issues of ensemble averaging and directly approaches detailed information that is obscured in ensemble measurements. In order to proficiently interpret single molecule data, we developed a comprehensive methodology – single molecule counting – for characterizing molecular transport in nanoporous silica. With this methodology as a tool, the nanoenvironmental heterogeneity inside the nanopores of C18-derivatized silica particles was explored by probing single molecular diffusion inside the pores. By employing single molecule ratiometric spectroscopy and a solvatochromic fluorophore as viii reporter of local environment, the gradient in nanopolarity as well as the nanoviscosity along the C18 layer after the inclusion of solvent was uncovered. The chemical properties of solute molecules at the nanopore surface are ultimately controlled by the energetics of the solute-interface interactions. The imaging of distribution of energies would be a decisive approach to assess the fundamental heterogeneity of the interface. To this end, we investigated the ΔG distribution of C18-derivatized nanoporous silica particles with quantitative confocal imaging. The pixel-to-pixel and particle-to-particle analysis showed the existence of ΔG heterogeneity between particles as well as within individual particles. The heterogeneity in ΔG could be partially responsible for band broadening in chemical separations and significantly affect overall reaction yield when using nanoporous materials as solid support for heterogeneous catalysis.

The fabrication and characterization of surface-anchored hydrogel microstructures are described. The hydrogel structures are constructed from poly(N-isopropylacrylamide), or poly(NIPAAm), which is a well-known thermoresponsive polymer that swells and contracts with changes in temperature. When patterned on a surface, these structures can experience a variety of shape changes induced by nonuniform swelling. Depending on the aspect ratio, patterns can, for instance buckle upon swelling and form wave-like patterns. Such structural changes replicate oscillatory motion of the smooth muscle cells and can be used to transport objects in microfluidics. The work, herein, investigates methods of pattern production and introduces a new technique for characterizing local swelling in the patterns. In order to achieve the latter, fluorescent microspheres were embedded in hydrogel patterns and their positions were mapped in three-dimensions using confocal microscopy. The measurements permit, for the first time, swelling maps of the structures based on relative movements of the microspheres. This information will ultimately aid in understanding how swollen macroscopic structures are related to gradients in localized swelling.

Le domaine des nanomatériaux joue un rôle de plus en plus important dans de nombreuses applications, qu'elles soient de natures biologique, médicales électroniques etc... Dans ce travail, nous présenterons des résultats concernant deux types de nanoparticules, le premier genre traite de nanoparticules hybrides confectionnées chimiquement pour des fins biologiques, le deuxième concerne des nanocristaux de silicium fabriqués par pyrolise laser pour des applications potentielles en optoélectronique. Les études sont menées en mettant en œuvre deux différentes techniques optiques, l'une en champ lointain, l'autre en champ proche. Dans le cas des nanohybrides, nous nous intéresserons à une caractérisation par microscopie en champ proche, qu'elle soit de nature spectroscopique ou d'imagerie simple, en utilisant en particulier une configuration optique guidante. Nous ferons un premier point à propos de l'émission de ses nanoparticules, puis discuterons des problèmes d'artefacts et de la résolution des images que nous pouvons atteindre avec notre montage. Nous prouverons l'importance essentielle du rôle des nanohybrides en tant que marqueur biologiques, et ceci dans deux différentes types de configuration de capteurs biologiques. Les nanoparticules de silicium de petites tailles (< 3 nm) seront étudiées essentiellement par microscopie confocale. Plus précisément, nous nous intéressons aux différents procédés de luminescence qui ont lieu lors de l'excitation d'une nanoparticule unique, en tenant compte des effets de taille et de surface. Nous chercherons à étudier l'influence de l'environnement des nanoparticules sur leurs propriétés spectrales en les plaçant dans des couches minces de natures diélectriques différentes. Nous conclurons enfin sur une brève description des différents effets Sark qui prennent lieu dans un tel système.

<p>Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle.</p><p>Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles.</p><p><i>Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.</i></p>

Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle. Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles. Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.

Physical forces in the cellular microenvironment play an important role in governing cell function. Forces transmitted through the cell cause distinct deformation of the nucleus, and possibly play a role in force-mediated gene expression. The work presented in this thesis drew upon innovative strategies employing simultaneous atomic force and laser-scanning confocal microscopy, as well as parallel optical stretching experiments, to gain unique insights into the response of eukaryotic cell nuclei to external force. Non-destructive approaches confirmed the existence of a clear anisotropy in nuclear mechanical properties, and showed that the nucleus' mechanical response to extracellular forces is differentially governed by both nuclear and cytoskeletal prestress: nuclear prestress regulates shape and anisotropic deformation, whereas cytoskeletal prestress modulates the magnitude and degree of deformation. Importantly, the anisotropic mechanical response was conserved among diverse differentiated cell types from multiple species, suggesting that nuclear mechanical anisotropy plays an important role in cell function.

Cold acclimation is a complex process where plants acquire increased freezing tolerance following exposure to low, non-freezing temperatures. Microtubules are dynamic components of the cytoskeleton that are essential for plant growth and development, and there are multiple lines of evidence indicating microtubules are involved in the acquisition of freezing tolerance. <p>The organization of microtubules (MTs) was tracked over the course of a cold acclimation period using GFP:TUB6 and fluorescent imaging tools. Experiments found that MTs undergo incomplete, transient disassembly following exposure to acclimating temperatures, which is accompanied by intranuclear tubulin accumulation and followed by MT reassembly. The importance of the observed changes to MT organization was examined with MT disrupting chemicals that caused reduced MT dynamics or induced transient MT disassembly similar to that of cold acclimation. Results of these experiments suggest that MT reorganization is important for cold acclimation, but the disassembly and reassembly do not directly control cold acclimation.<p>MT binding proteins are likely to play a key role in the low temperature response because they control MT activity and organization, participate in low temperature signal transduction pathways, and mediate interactions between various elements of this pathway. By employing a number of proteomics techniques we were able to identify 96 tubulin-binding proteins from untreated and short term cold acclimated Arabidopsis plants. Proteins both known to and predicted to bind to MTs and unexpected MT binding proteins were identified. The identified tubulin binding proteins have a range of cellular functions, including RNA transport and protein translation, stress responses, and functions related to various metabolic pathways, and cell growth and organization. <p>Exposure to low temperatures affected the binding of some of these proteins to MTs with the identified tubulin binding proteins potentially involved in the cold acclimation process and stress response through a number of possible pathways.<p>This study represents the first live cell imaging of MT reorganization in response to low temperatures and the first time microtubule binding proteins from whole plant protein extracts were identified using 1D gel LC-MS/MS analysis.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

Cold acclimation is a complex process where plants acquire increased freezing tolerance following exposure to low, non-freezing temperatures. Microtubules are dynamic components of the cytoskeleton that are essential for plant growth and development, and there are multiple lines of evidence indicating microtubules are involved in the acquisition of freezing tolerance. <p>The organization of microtubules (MTs) was tracked over the course of a cold acclimation period using GFP:TUB6 and fluorescent imaging tools. Experiments found that MTs undergo incomplete, transient disassembly following exposure to acclimating temperatures, which is accompanied by intranuclear tubulin accumulation and followed by MT reassembly. The importance of the observed changes to MT organization was examined with MT disrupting chemicals that caused reduced MT dynamics or induced transient MT disassembly similar to that of cold acclimation. Results of these experiments suggest that MT reorganization is important for cold acclimation, but the disassembly and reassembly do not directly control cold acclimation.<p>MT binding proteins are likely to play a key role in the low temperature response because they control MT activity and organization, participate in low temperature signal transduction pathways, and mediate interactions between various elements of this pathway. By employing a number of proteomics techniques we were able to identify 96 tubulin-binding proteins from untreated and short term cold acclimated Arabidopsis plants. Proteins both known to and predicted to bind to MTs and unexpected MT binding proteins were identified. The identified tubulin binding proteins have a range of cellular functions, including RNA transport and protein translation, stress responses, and functions related to various metabolic pathways, and cell growth and organization. <p>Exposure to low temperatures affected the binding of some of these proteins to MTs with the identified tubulin binding proteins potentially involved in the cold acclimation process and stress response through a number of possible pathways.<p>This study represents the first live cell imaging of MT reorganization in response to low temperatures and the first time microtubule binding proteins from whole plant protein extracts were identified using 1D gel LC-MS/MS analysis.