Over the past few decades, fluorescence microscopy has proven to become the most widely used imaging technique in the field of life sciences. Unfortunately, all classical optical microscopy techniques have one thing in common: their resolution is limited by the diffraction. Thankfully, due to the very strong interest, development of fluorescent microscopy techniques is very intense, with novel solutions surfacing repeatedly. The major breakthrough came with the appearance of super-resolution microscopy techniques, enabling imaging well below the diffraction barrier and opening the new era of nanoscopy. Among the fluorescent super-resolution techniques, Stimulated Emission Depletion (STED) microscopy has been particularly interesting, as it is a purely optical technique which does not require post image processing. STED microscopy has proven to resolve structures down to the molecular resolution. However, super-resolution microscopy is not a cure to all the problems and it also has its limits. What has shown to be particularly challenging, was the super-resolution imaging of thick samples. With increased thickness of biological structures, the aberrations increase and signal-to-noise (SNR) decreases. This becomes even more evident in the super-resolution imaging, as the nanoscopic techniques are especially sensitive to aberrations and low SNR. The aim of this work is to propose and develop a 3D STED microscope that can successfully image thick biological samples with nanoscopic resolution. In order to achieve that, adaptive optics (AO) has been employed for correcting the aberrations, using the indirect wavefront sensing approach. This thesis presents a custom built 3D STED microscope with the AO correction and the resulting images of thick samples with resolution beyond diffraction barrier. The developed STED microscope achieved the resolution of 60nm in lateral and 160nm in axial direction. What is more, it enabled super-resolution imaging of thick, aberrating samples. HeLa, RPE-1 cells and dopaminergic neuron differentiated from human IPS cells were imaged using the microscope. The results shown in this thesis present 3D STED imaging of thick biological samples and, what is particularly worth to highlight, 3D STED imaging at the 80μm depth, where the excitation and depletion beams have to propagate through the thick layer of tissue. 3D STED images at such depth has not been reported up to date.

Imagens multiespectrais são utilizadas em diferentes aplicações, que vão desde sensoriamento remoto a processos médicos. No caso de imagens multiespectrais oriundas de microscopia confocal de varredura à laser (Confocal Laser Scanning Microscopy-CLSM), a extração da informação se inicia pela conversão das assinaturas espectrais, em uma imagem RGB. Esta imagem é a referência para a seleção da região de interesse, da qual se obtém a assinatura espectral média, originada do arquivo multiespectral (LSM). Mesmo utilizando um padrão muito bem estabelecido de conversão, alguns pontos devem ser considerados: i) o processo de conversão reduz a informação, a uma ordem de 10-145%; ii) a cor é uma experiência sensorial, subjetiva e pessoal, interferindo na seleção da região de interesse e; iii) a assinatura é obtida pela média espectral, da região de interesse, selecionada manualmente.Assim, esta tese de doutorado propõem um método de mapeamento e visualização das informações de imagens multiespectrais, combinando um algoritmo de agrupamento não supervisionado(kmeans) e um algoritmo que define uma paleta de cores coerentes com a informação espectral das regiões mapeadas. Aplicou-se o método em três casos de estudos de tecidos vegetais: i) no pré-tratamento de paredes celulares da cana-de-açúcar; ii) na plasticidade foliar do Jacaranda caroba e; iii) no uso de assinaturas espectrais na classificação de plantas do Cerrado. Os resultados demonstraram que o método é bastante robusto, permitindo de forma inovadora a: visualização, análise e comparação de imagens multiespectrais qualitativa e quantitativamente, e que seu uso é viável em qualquer área de pesquisa que utilize imagens multiespectrais. / Multispectral images are used in different applications, ranging from remote sensing images to medical images. In the case of multispectral images derived from confocal laser scanning microscopy (CLSM), the extraction of information begins with the conversion of spectral signatures in an RGB image. This is the reference for selecting the region of interest, from which it gets the average spectral signature, originated from multispectral file (LSM). Even using a very well established pattern of conversion, some points should be considered: i) the conversion process reduces the information on the order of 10-145%; ii) the color is a sensory experience, subjective and personal, interfering in the selection of the interest region and; the signature is obtained by the spectral average, from interest region which is selected manually. Thus, this doctoral thesis proposes a method of mapping and visualization of multispectral imaging information, combining an unsupervised clustering algorithm (kmeans) and an algorithm that defines a consistent color palette with the spectral information of mapped regions. The proposed method was applied in three cases plant tissue studies: i) in the pre-treating the cell walls of sugarcane; ii) in the leaf plasticity of Jacaranda caroba; iii) in the use of spectral signatures in the Cerrado plant classification. The results showed that the proposed method is quite robust. It presents innovation to the visualization and analysis of multispectral images and makes possible a qualitative and quantitative comparison of a group of multispectral images. Besides that, its use is feasible in any area of research, which are using multispectral images.

Todos os seres vivos compartilham uma característica comum na sua composição estrutural, a célula. No corpo humano, as células vasculares de músculo liso são fundamentais para o bom funcionamento dos vasos arteriais. A principal função dessas células é contrair e regular o calibre desses vasos, a pressão sanguínea e a distribuição do fluxo de sangue. Devido a isto, alterações mecânicas sofridas por estas células acarretam modificações estruturais nos vasos, podendo levar à hipertensão, vasoespasmo e arteriosclerose. O principal objetivo do nosso trabalho foi o de desenvolver uma nova plataforma de análise de imagens de células vasculares para caracterizar suas propriedades estruturais. Em nossa plataforma, analisamos parâmetros estruturais de células vasculares de músculo liso de diferentes leitos arteriais, com as fibras de actina evidenciadas com marcadores fluorescentes, obtidas por microscopia confocal. Estes parâmetros são: o Índice de Alinhamento das fibras de actina da imagem, a distribuição de comprimento dessas fibras e sua dimensão fractal. Mostramos que com esses parâmetros somos capazes de comparar células de leitos arteriais diferentes de forma quantitativa, assim como, correlacionar esses parâmetros com suas propriedades mecânicas. / All living organisms share a unique characteristic in their structural composition, the cell. In the human body, vascular smooth muscle cells are fundamental for the ideal functioning of the arterial vessels. The main function of these cells is to contract and regulate these vessels caliber, as well as the blood pressure and flow distribution of blood. Due to the exposed above, mechanical alterations suffered by these cells cause structural modifications in vessels, which may lead to hypertension, vasospasm and atherosclerosis. The main objective of our research was to develop a new framework of image analysis for vessel cells in order to characterize their structural properties. In our framework we analyzed structural parameters of vascular smooth muscle cells from different arterial sites, with actin fibers labeled with fluorescent markers, obtained by confocal microscopy. These parameters are: the actin fibers alignment index of the image, the length distribution of these fibers and their fractal dimension. We presented that with these parameters we are able to quantitatively compare cells in different arterial sites as well as correlate these parameters with their mechanical properties.

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin binding peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of α5β1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization. Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Pre-incubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha5 and beta1 integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Anti-β2- integrin antibody served as the negative control. Traction force microscopy studies showed that only the force transduced via integrins could potentially trigger cytoskeletal remodeling in cultured VSMCs. Atomic force microscopy revealed a significant increase in surface roughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves. Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.

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.