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Previous issue date: 2015-07-31 / As prote?nas fluorescentes constituem uma ferramenta fundamental em v?rios campos da biologia, pois permitem enxergar o desenvolvimento de estruturas e processos din?micos de c?lulas em tecido vivo, com o aux?lio da microscopia de fluoresc?ncia. A Optogen?tica ? outra t?cnica que atualmente ganha destaque na Neuroci?ncias e que, de forma geral, permite ativar/desativar neur?nios a partir da irradia??o luminosa de certos comprimentos de onda sobre as c?lulas que possuem canais i?nicos sens?veis ? luz, e ainda pode ser utilizada concomitantemente com as prote?nas fluorescentes. Esta tese possui dois objetivos principais. Inicialmente, s?o estudados os efeitos da intera??o da luz e o c?rebro de camundongos para aplica??es em experimentos de Optogen?tica. Nesta etapa, s?o modelados, a partir de caracter?sticas do c?rebro de camundongos e utilizando a teoria de Kulbelka-Munk, os efeitos de absor??o e espalhamento da luz, em comprimentos de onda espec?ficos, em fun??o da dist?ncia de penetra??o no tecido cerebral. Al?m disso, s?o modeladas as varia??es de temperatura utilizando o m?todo dos elementos finitos na resolu??o da equa??o de bioaquecimento de Pennes, com o aux?lio do COMSOL Multiphysics Modeling Software 4.4, onde s?o simulados protocolos de estimula??o luminosa, tipicamente utilizados em Optogen?tica. Posteriormente, s?o desenvolvidos algoritmos computacionais capazes de reduzir a exposi??o das c?lulas nervosas ? irradia??o luminosa necess?ria ? visualiza??o da fluoresc?ncia emitida por elas. Nesta etapa, s?o descritas as t?cnicas de processamento digital de imagens desenvolvidas para uso em microscopia de fluoresc?ncia, com o intuito de reduzir a exposi??o das amostras de c?rebro ? luz cont?nua, respons?vel pela excita??o dos fluorocromos. As t?cnicas de processamento de imagens desenvolvidas e utilizadas s?o capazes de rastrear, em tempo real, uma regi?o de interesse (ROI) e substituir a fluoresc?ncia emitida pelas c?lulas por uma m?scara virtual, como resultado da sobreposi??o da ROI que est? sendo rastreada e a informa??o de fluoresc?ncia previamente armazenada, mantendo a localiza??o das c?lulas independentemente do tempo de exposi??o ? luz fluorescente. Em resumo, esta tese pretende entender os efeitos da irradia??o luminosa no c?rebro, no contexto da Optogen?tica, al?m de fornecer uma ferramenta computacional que possa auxiliar certos experimentos em microscopia de fluoresc?ncia na redu??o do desvanecimento (bleaching) das amostras e dos danos (photodamage) causados ao tecido devido ? intensa exposi??o das estruturas fluorescentes ? luz. / The fluorescent proteins are an essential tool in many fields of biology, since they allow us to watch the development of structures and dynamic processes of cells in living tissue, with the aid of fluorescence microscopy. Optogenectics is another technique that is currently widely used in Neuroscience. In general, this technique allows to activate/deactivate neurons with the radiation of certain wavelengths on the cells that have ion channels sensitive to light, at the same time that can be used with fluorescent proteins. This dissertation has two main objectives. Initially, we study the interaction of light radiation and mice brain tissue to be applied in optogenetic experiments. In this step, we model absorption and scattering effects using mice brain tissue characteristics and Kubelka-Munk theory, for specific wavelengths, as a function of light penetration depth (distance) within the tissue. Furthermore, we model temperature variations using the finite element method to solve Pennes? bioheat equation, with the aid of COMSOL Multiphysics Modeling Software 4.4, where we simulate protocols of light stimulation tipically used in optogenetics. Subsequently, we develop some computational algorithms to reduce the exposure of neuron cells to the light radiation necessary for the visualization of their emitted fluorescence. At this stage, we describe the image processing techniques developed to be used in fluorescence microscopy to reduce the exposure of the brain samples to continuous light, which is responsible for fluorochrome excitation. The developed techniques are able to track, in real time, a region of interest (ROI) and replace the fluorescence emitted by the cells by a virtual mask, as a result of the overlay of the tracked ROI and the fluorescence information previously stored, preserving cell location, independently of the time exposure to fluorescent light. In summary, this dissertation intends to investigate and describe the effects of light radiation in brain tissue, within the context of Optogenetics, in addition to providing a computational tool to be used in fluorescence microscopy experiments to reduce image bleaching and photodamage due to the intense exposure of fluorescent cells to light radiation.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/20395 |
| Date | 31 July 2015 |
| Creators | Peixoto, Helton Maia |
| Contributors | 01207607703, http://lattes.cnpq.br/8556144121380013, D?ria Neto, Adri?o Duarte, 10749896434, http://lattes.cnpq.br/1987295209521433, Leite, Cicilia Raquel Maia, 03777857416, http://lattes.cnpq.br/9378258073324535, Soares, Heliana Bezerra, 80674585453, http://lattes.cnpq.br/5057165446370629, Le?o, Richardson Naves, 99944251615, http://lattes.cnpq.br/0683942077872227, Figuerola, Wilfredo Blanco, 05579557779, http://lattes.cnpq.br/9912829629195282, Guerreiro, Ana Maria Guimar?es |
| Publisher | Universidade Federal do Rio Grande do Norte, PROGRAMA DE P?S-GRADUA??O EM ENGENHARIA EL?TRICA E DE COMPUTA??O, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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