Tomografia estendida : do básico até o mapeamento de cérebro de camundongos

Orientador: Mateus Borba CardosoTese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb WataghinResumo: Esta tese apresentará uma introdução a imagens de raios-x e como adquirir e processar imagens usando linhas de luz síncrotron. Apresentará os desafios matemáticos e técnicos para reconstruir amostras em três dimensões usando a reconstrução de Tomografia Computadorizada, uma técnica conhecida como CT. Esta técnica tem seu campo de visão limitado ao tamanho da câmera e ao tamanho da iluminação. Uma técnica para ampliar esse campo de visão vai ser apresentada e os desafios técnicos envolvidos para que isso aconteça. Um \textit{pipeline} é proposto e todos os algoritmos necessários foram empacotados em um pacote python chamado Tomosaic. A abordagem baseia-se em adquirir tomogramas parciais em posiçoes pré definidas e depois mesclar os dados em um novo conjunto de dados. Duas maneiras possíveis são apresentadas para essa mescla, uma no domínio das projeções e uma no domínio dos sinogramas. Experimentos iniciais serão então usadas para mostrar que o método proposto funciona com computadores normais. A técnica será aplicada mais tarde para pesquisar a anatomia de cérebros de camundongo completos. Um estudo será apresentado de como obter informação em diferentes escalas do cérebro completo do rato utilizando raios-xAbstract: This thesis will present an introduction to x-ray images and how to acquire and thread images using synchrotron beamlines. It will present the mathematical and technical challenges to reconstruct samples in three dimensions using Computed Tomography reconstruction, a technique known as CT. This technique has a field of view bounded to the camera size and the illumination size. A technique to extended this field of view is going to be presented and the technical challenges involved in order for that to happen will be described. A pipeline is proposed and all the necessary algorithms are contained into a python packaged called Tomosaic. The approach relies on acquired partial tomogram data in a defined grid and later merging the data into a new dataset. Two possible ways are presented in order to that: in the projection domain, and in the sinogram domain. Initial experiments will then be used to show that the pipeline works with normal computers. The technique will be later applied to survey the whole anatomy of whole mouse brains. A study will be shown of how to get the complete range of scales of the mouse brain using x-ray tomography at different resolutionsDoutoradoFísicaDoutor em Ciências163304/2013-01247445/2013, 1456912/2014CNPQCAPE

Virtual reality for 3D histology: multi-scale visualization of organs with interactive feature exploration

Background Virtual reality (VR) enables data visualization in an immersive and engaging manner, and it can be used for creating ways to explore scientific data. Here, we use VR for visualization of 3D histology data, creating a novel interface for digital pathology to aid cancer research. Methods Our contribution includes 3D modeling of a whole organ and embedded objects of interest, fusing the models with associated quantitative features and full resolution serial section patches, and implementing the virtual reality application. Our VR application is multi-scale in nature, covering two object levels representing different ranges of detail, namely organ level and sub-organ level. In addition, the application includes several data layers, including the measured histology image layer and multiple representations of quantitative features computed from the histology. Results In our interactive VR application, the user can set visualization properties, select different samples and features, and interact with various objects, which is not possible in the traditional 2D-image view used in digital pathology. In this work, we used whole mouse prostates (organ level) with prostate cancer tumors (sub-organ objects of interest) as example cases, and included quantitative histological features relevant for tumor biology in the VR model. Conclusions Our application enables a novel way for exploration of high-resolution, multidimensional data for biomedical research purposes, and can also be used in teaching and researcher training. Due to automated processing of the histology data, our application can be easily adopted to visualize other organs and pathologies from various origins.</p

The study of renal function and toxicity using zebrafish (Danio rerio) larvae as a vertebrate model

Zebrafish (Danio rerio) is a powerful model in biomedical and pharmaceutical sciences. The zebrafish model was introduced to toxicological sciences in 1960, followed by its use in biomedical sciences to investigate vertebrate gene functions. As a consequence of many research projects in this field, the study of human genetic diseases became instantly feasible. Consequently, zebrafish have been intensively used in developmental biology and associated disciplines. Due to the simple administration of medicines and the high number of offspring, zebrafish larvae became widely more popular in pharmacological studies in the following years. In the past decade, zebrafish larvae were further established as a vertebrate model in the field of pharmacokinetics and nanomedicines. In this PhD thesis, zebrafish larvae were investigated as an earlystage in vivo vertebrate model to study renal function, toxicity, and were applied in drug-targeting projects using nanomedicines. The first part focused on the characterization of the renal function of three-to four-dayold zebrafish larvae. Non-renal elimination processes were additionally described. Moreover, injection techniques, imaging parameters, and post-image processing scripts were established to serve as a toolbox for follow-up projects. The second part analyzed the impact of gentamicin (a nephrotoxin) on the morphology of the pronephros of zebrafish larvae. Imaging methodologies such as fluorescent-based laser scanning microscopy and X-ray-based microtomography were applied. A profound comparison study of specimens acquired with different laboratory X-ray-based microtomography devices and a radiation facility was done to promote the use of X-ray-based microtomography for broader biomedical applications. In the third part, the toxicity of nephrotoxins on mitochondria in renal epithelial cells of proximal tubules was assessed using the zebrafish larva model. Findings were compared with other teleost models such as isolated renal tubules of killifish (Fundulus heteroclitus). In view of the usefulness and high predictability of the zebrafish model, it was applied to study the pharmacokinetics of novel nanoparticles in the fourth part. Various in vivo pharmacokinetic parameters such as drug release, transfection of mRNA/pDNA plasmids, macrophage clearance, and the characterization of novel drug carriers that were manipulated with ultrasound were assessed in multiple collaborative projects. Altogether, the presented zebrafish model showed to be a reliable in vivo vertebrate model to assess renal function, toxicity, and pharmacokinetics of nanoparticles. The application of the presented model will hopefully encourage others to reduce animal experiments in preliminary studies by fostering the use of zebrafish larvae

Magnetic Resonance Imaging of the Rat Retina

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2 emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed high blood-volume in the choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layer-specific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway

Magnetic Resonance Imaging of the Rat Retina: a Dissertation

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway

Neuroimaging - Clinical Applications

Modern neuroimaging tools allow unprecedented opportunities for understanding brain neuroanatomy and function in health and disease. Each available technique carries with it a particular balance of strengths and limitations, such that converging evidence based on multiple methods provides the most powerful approach for advancing our knowledge in the fields of clinical and cognitive neuroscience. The scope of this book is not to provide a comprehensive overview of methods and their clinical applications but to provide a "snapshot" of current approaches using well established and newly emerging techniques