Our ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.

Our ability to analyze the microstructure of biological tissue in three dimensions (3D) has proven invaluable in modeling its functionality, and therefore providing a better understanding of the basic mechanisms of life. Volumetric imaging of tissue at the cellular level, using serial imaging of consecutive tissue sections, provides such ability to acquire microstructure in 3D. Three-dimensional light microscopy in biology can be broadly classified as using either optical sectioning or physical sectioning. Due to the inherent limitations on the depth resolution in optical sectioning, and the recent introduction of novel techniques, physical sectioning has become the sought-out method to obtain high-resolution volumetric tissue structure data. To meet this demand with increased processing speed in 3D biological imaging, this thesis provides an engineering study and formulation of the tissue sectioning process. The knife-edge scanning microscopy (KESM), a novel physical sectioning and imaging instrument developed in the Brain Networks Laboratory at Texas A&M University, has been used for the purpose of this study. However, the modes of characterizing chatter and its measurement are equally applicable to all current variants of 3D biological microscopy using physical sectioning. We focus on chatter in the physical sectioning process, principally characterizing it by its geometric and optical attributes. Some important nonlinear dynamical models of chatter in the sectioning process, drawn from the metal machining literature, are introduced and compared with observed measurements of chatter in the tissue cutting process. To understand the effects of the embedding polymer on tissue sectioning, we discuss methods to characterize the polymer material and present polymer measurements. Image processing techniques are introduced as a method to abate chatter artifacts in the volumetric data that has already been obtained. Ultra-precise machining techniques, using (1) free-form nanomachining and (2) an oscillating knife, are introduced as potential ways to acquire chatter-free higher-resolution volumetric data in less time. Finally, conclusions of our study and future work conclude the thesis. In this thesis, we conclude that to achieve ultrathin sectioning and high-resolution imaging, embedded plastic should be soft. To overcome the machining defects of soft plastics, we suggested free-form nanomachining and sectioning with an oscillating knife.

Made available in DSpace on 2014-06-11T19:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-14Bitstream added on 2014-06-13T19:09:55Z : No. of bitstreams: 1 rossetto_vjv_me_botfmvz_parcial.pdf: 44582 bytes, checksum: 3c3725afe05c19146568796a0ee21ac3 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As indicações para a cirurgia reparadora da bexiga abrangem traumas graves com perda tecidual extensa, neoplasias, cistites intersticiais, disfunções neurológicas e anormalidades congênitas. Devido a inúmeras complicações associadas às técnicas de cistoplastia com o emprego de segmentos gastrointestinais, o presente estudo teve como objetivo analisar comparativamente a reparação anatômica da bexiga de cães utilizandose a submucosa intestinal suína (SIS) acelular e semeada com células musculares lisas homólogas (CMLH), bem como avaliar as possíveis complicações e efeitos colaterais inerentes a cada um dos tipos de enxerto empregados. Para tal foram utilizados 10 cães adultos, fêmeas, subdivididos em dois grupos, sendo: Grupo Controle (GC), constituído por cinco animais submetidos à cistoplastia com SIS; e Grupo Tratado (GT), constituído por cinco animais submetidos à cistoplastia com SIS semeada com CMLH. As células foram obtidas a partir de fragmentos de espessura completa, provenientes de bexigas de cães que vieram a óbito por motivos não infecciosos ou neoplásicos, cultivadas e expandidas in vitro. Em todos os grupos foi criado um defeito de 3 cm x 2 cm, preservando-se a região do trígono vesical, o qual foi reparado pelos respectivos implantes. Os cães de ambos os grupos foram submetidos à biópsia cirúrgica com 60 dias de pós-operatório, sendo essas obtidas da área central do tecido neoformado e da adjacência entre o mesmo e a bexiga original. Após a implantação dos enxertos, os animais foram avaliados por meio de exames clínico, ultrassonográfico abdominal e laboratoriais. Todas as amostras foram destinadas à avaliação histológica pelas colorações de H/E e Tricromio de Masson, analisadas com um fotomicroscópio Nikon conectado ao sistema de análise de imagem ImageJ. Não foram... / Not available

Orientador: Cláudia Valéria Seullner Brandão / Coorientador: Lígia Souza Lima Silveira da Mota / Banca: Juliany Gomes Quitzan / Banca: Bruno Watanabe Minto / Resumo: As indicações para a cirurgia reparadora da bexiga abrangem traumas graves com perda tecidual extensa, neoplasias, cistites intersticiais, disfunções neurológicas e anormalidades congênitas. Devido a inúmeras complicações associadas às técnicas de cistoplastia com o emprego de segmentos gastrointestinais, o presente estudo teve como objetivo analisar comparativamente a reparação anatômica da bexiga de cães utilizandose a submucosa intestinal suína (SIS) acelular e semeada com células musculares lisas homólogas (CMLH), bem como avaliar as possíveis complicações e efeitos colaterais inerentes a cada um dos tipos de enxerto empregados. Para tal foram utilizados 10 cães adultos, fêmeas, subdivididos em dois grupos, sendo: Grupo Controle (GC), constituído por cinco animais submetidos à cistoplastia com SIS; e Grupo Tratado (GT), constituído por cinco animais submetidos à cistoplastia com SIS semeada com CMLH. As células foram obtidas a partir de fragmentos de espessura completa, provenientes de bexigas de cães que vieram a óbito por motivos não infecciosos ou neoplásicos, cultivadas e expandidas in vitro. Em todos os grupos foi criado um defeito de 3 cm x 2 cm, preservando-se a região do trígono vesical, o qual foi reparado pelos respectivos implantes. Os cães de ambos os grupos foram submetidos à biópsia cirúrgica com 60 dias de pós-operatório, sendo essas obtidas da área central do tecido neoformado e da adjacência entre o mesmo e a bexiga original. Após a implantação dos enxertos, os animais foram avaliados por meio de exames clínico, ultrassonográfico abdominal e laboratoriais. Todas as amostras foram destinadas à avaliação histológica pelas colorações de H/E e Tricromio de Masson, analisadas com um fotomicroscópio Nikon conectado ao sistema de análise de imagem ImageJ. Não foram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Not available / Mestre