Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência

Detalhes bibliográficos
Ano de defesa: 2015
Autor(a) principal: Mari, João Fernando
Orientador(a): Saito, José Hiroki lattes
Banca de defesa: Não Informado pela instituição
Tipo de documento: Tese
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal de São Carlos
Programa de Pós-Graduação: Programa de Pós-Graduação em Ciência da Computação - PPGCC
Departamento: Não Informado pela instituição
País: BR
Palavras-chave em Português:
Palavras-chave em Inglês:
Área do conhecimento CNPq:
Link de acesso: https://repositorio.ufscar.br/handle/ufscar/297
Resumo: Microelectrode arrays (MEA) are devices that allow chemical and electrical stimulation and recording of the extracellular electrical activity from entire neuronal cultures over long periods of time, such as several weeks. Some MEA models have transparent substrate, which enables the imaging of culture using optical microscopy. The images are taken from two channels: fluorescence light and transmitted light channels. In the first one, it is possible to visualize the neurons, while in the other one, it is possible to observe the microelectrodes. The objective of this work is to develop methods that enable performing quantitative analysis of the dissociated culture of rat dorsal root ganglion (DRG) neurons plated on MEA by means of the processing of the images, obtained from confocal fluorescence microscopy. We proposed and developed the following methods in order to achieve this objective: (A) A method to automatically identify the microelectrodes in the transmitted light channel using circular Hough Transform and error correction based on the Delaunay triangulation; (B) the registration of a number of images taken at different parts of the MEA in order to generate a unique and high-resolution representation of the whole culture; (C) the segmentation of the neuron in 2D images taken from the fluorescence channel, composed by the steps: preprocessing, thresholding, morphological filtering, neurons occlusion correction, watershed transform and object classification; (D) 2D quantitative analysis based on the identified microelectrodes and on the segmented neurons; (E) a method for generating 3D polygonal models of the neurons from the volumetric images, to be used for visualizing the culture on the MEA by different points of view and zoom levels; and (F) 3D quantitative analysis performed by the processing of the polygonal surfaces in conjunction with the information about the microelectrodes positioning. The results show that the methods are capable to identify the neurons and microelectrodes on the 2D images efficiently. In the 3D images, the preprocessing step which uses information from the 2D segmentation method, showed to be capable to generate correct polygonal models efficiently. Most of the studies involving the analysis of neuron cultures on MEAs consider only qualitative analysis or simple quantitative measures. However, the methods proposed in this thesis enables to obtain important measures related to the neuron culture, such as: the density and morphology of the neurons, and the spatial and topological distribution of the neurons and microelectrodes. The information about neuron morphology is important because they are related to the behavior of this kind of neuron. The spatial and topological distribution of neurons and microelectrodes are used for providing models of the interface between these elements, for supporting the analysis of the electrophysiological signal recorded by the microelectrodes, as well as in the computational simulations of the neuron culture behavior.
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spelling Mari, João FernandoSaito, José Hirokihttp://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4799717Z7http://lattes.cnpq.br/3582704696209050769fde80-edf3-4e2d-97af-ebcdc20c2fa52016-06-02T19:04:00Z2015-05-272016-06-02T19:04:00Z2015-03-09MARI, João Fernando. Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência. 2015. 262 f. Tese (Doutorado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2015.https://repositorio.ufscar.br/handle/ufscar/297Microelectrode arrays (MEA) are devices that allow chemical and electrical stimulation and recording of the extracellular electrical activity from entire neuronal cultures over long periods of time, such as several weeks. Some MEA models have transparent substrate, which enables the imaging of culture using optical microscopy. The images are taken from two channels: fluorescence light and transmitted light channels. In the first one, it is possible to visualize the neurons, while in the other one, it is possible to observe the microelectrodes. The objective of this work is to develop methods that enable performing quantitative analysis of the dissociated culture of rat dorsal root ganglion (DRG) neurons plated on MEA by means of the processing of the images, obtained from confocal fluorescence microscopy. We proposed and developed the following methods in order to achieve this objective: (A) A method to automatically identify the microelectrodes in the transmitted light channel using circular Hough Transform and error correction based on the Delaunay triangulation; (B) the registration of a number of images taken at different parts of the MEA in order to generate a unique and high-resolution representation of the whole culture; (C) the segmentation of the neuron in 2D images taken from the fluorescence channel, composed by the steps: preprocessing, thresholding, morphological filtering, neurons occlusion correction, watershed transform and object classification; (D) 2D quantitative analysis based on the identified microelectrodes and on the segmented neurons; (E) a method for generating 3D polygonal models of the neurons from the volumetric images, to be used for visualizing the culture on the MEA by different points of view and zoom levels; and (F) 3D quantitative analysis performed by the processing of the polygonal surfaces in conjunction with the information about the microelectrodes positioning. The results show that the methods are capable to identify the neurons and microelectrodes on the 2D images efficiently. In the 3D images, the preprocessing step which uses information from the 2D segmentation method, showed to be capable to generate correct polygonal models efficiently. Most of the studies involving the analysis of neuron cultures on MEAs consider only qualitative analysis or simple quantitative measures. However, the methods proposed in this thesis enables to obtain important measures related to the neuron culture, such as: the density and morphology of the neurons, and the spatial and topological distribution of the neurons and microelectrodes. The information about neuron morphology is important because they are related to the behavior of this kind of neuron. The spatial and topological distribution of neurons and microelectrodes are used for providing models of the interface between these elements, for supporting the analysis of the electrophysiological signal recorded by the microelectrodes, as well as in the computational simulations of the neuron culture behavior.Matrizes de Microeletrodos (MEAs) são dispositivos que permitem estimular quimicamente ou eletricamente e registrar a atividade elétrica extracelular de culturas de neurônios durante um longo período de tempo, da ordem de várias semanas. Modelos de MEAs com o substrato transparente permitem imagear a cultura por meio de microscopia óptica. As imagens são obtidas em dois canais: um de luz de fluorescência e outro de luz de transmissão. O primeiro permite visualizar os neurônios, enquanto o segundo os microeletrodos. O objetivo deste trabalho é desenvolver métodos que permitam realizar análises quantitativas de culturas dissociadas de neurônios de gânglio da raiz dorsal (Dorsal Root Ganglion DRG) de ratos em MEAs por meio do processamento de imagens obtidas por microscopia confocal de fluorescência. Os seguintes métodos foram propostos e desenvolvidos para atingir este objetivo: (A) Identificação automática dos microeletrodos nas imagens do canal de luz de transmissão utilizando a transformada de Hough circular e correção de erros baseado na triangulação de Delaunay; (B) Registro de várias imagens tomadas de diferentes regiões da MEA para gerar uma única imagem em alta resolução que contemple a cultura toda; (C) Segmentação dos neurônios em imagens 2D obtidas a partir do canal de fluorescência, composto por etapas de pré-processamento, segmentação, filtragem morfológica, correção da oclusão de neurônios, transformada watershed e classificação de objetos; (D) Análise quantitativa 2D baseada nos microeletrodos identificados e nos neurônios segmentados; (E) Método para geração de modelos poligonais 3D dos neurônios a partir de imagens volumétricas, modelos os quais são utilizados para visualização da cultura na MEA por diferentes pontos de vista e níveis de zoom; e (F) Análise quantitativa 3D realizada por meio do processamento das superfícies poligonais juntamente com as informações sobre a posição dos microeletrodos. Os resultados mostram que os métodos são capazes de identificar com eficiência os neurônios e microeletrodos presentes nas imagens 2D. Nas imagens 3D, a etapa de pré-processamento utilizando informações resultantes do método de segmentação 2D se mostrou eficiente na geração dos modelos poligonais corretos. Enquanto a maioria das análises de imagens de culturas de neurônios em MEA consideram apenas análises quantitativas simples, os métodos aqui propostos permitem obter importantes medidas quantitativas relacionadas às culturas, tais como: a densidade e morfologia dos neurônios, assim como a distribuição espacial e topológica dos neurônios em relação aos microeletrodos. As informações sobre a morfologia são importantes, pois estão relacionadas com o comportamento desse tipo de neurônio. A distribuição espacial e topológica dos neurônios e microeletrodos permitem modelar a interface entre neurônios e microeletrodos e auxiliar nos estudos dos sinais eletrofisiológicos capturados pelos microeletrodos, assim como em simulações computacionais do comportamento dessas culturas.application/pdfporUniversidade Federal de São CarlosPrograma de Pós-Graduação em Ciência da Computação - PPGCCUFSCarBRProcessamento de imagensMicroeletrodosMicroscopia confocalNeurôniosSegmentação de imagemAnálise quantitativaMicroelectrode arraysConfocal fluorescence microscopyCultured DRG neuronsSegmentation3D visualizationQuantitative analysisCIENCIAS EXATAS E DA TERRA::CIENCIA DA COMPUTACAOAnálise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescênciainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis-1-151b9675e-5744-4345-98e1-2c5caead4a56info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINAL6814.pdfapplication/pdf27157124https://repositorio.ufscar.br/bitstream/ufscar/297/1/6814.pdfccc98f69d1fc4cdc487ac2e9917edfc0MD51TEXT6814.pdf.txt6814.pdf.txtExtracted texttext/plain0https://repositorio.ufscar.br/bitstream/ufscar/297/2/6814.pdf.txtd41d8cd98f00b204e9800998ecf8427eMD52THUMBNAIL6814.pdf.jpg6814.pdf.jpgIM Thumbnailimage/jpeg9283https://repositorio.ufscar.br/bitstream/ufscar/297/3/6814.pdf.jpgc8077b8b469e48e8a4528994609b7c7cMD53ufscar/2972023-09-18 18:31:27.332oai:repositorio.ufscar.br:ufscar/297Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:27Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false
dc.title.por.fl_str_mv Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
title Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
spellingShingle Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
Mari, João Fernando
Processamento de imagens
Microeletrodos
Microscopia confocal
Neurônios
Segmentação de imagem
Análise quantitativa
Microelectrode arrays
Confocal fluorescence microscopy
Cultured DRG neurons
Segmentation
3D visualization
Quantitative analysis
CIENCIAS EXATAS E DA TERRA::CIENCIA DA COMPUTACAO
title_short Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
title_full Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
title_fullStr Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
title_full_unstemmed Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
title_sort Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência
author Mari, João Fernando
author_facet Mari, João Fernando
author_role author
dc.contributor.authorlattes.por.fl_str_mv http://lattes.cnpq.br/3582704696209050
dc.contributor.author.fl_str_mv Mari, João Fernando
dc.contributor.advisor1.fl_str_mv Saito, José Hiroki
dc.contributor.advisor1Lattes.fl_str_mv http://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4799717Z7
dc.contributor.authorID.fl_str_mv 769fde80-edf3-4e2d-97af-ebcdc20c2fa5
contributor_str_mv Saito, José Hiroki
dc.subject.por.fl_str_mv Processamento de imagens
Microeletrodos
Microscopia confocal
Neurônios
Segmentação de imagem
Análise quantitativa
topic Processamento de imagens
Microeletrodos
Microscopia confocal
Neurônios
Segmentação de imagem
Análise quantitativa
Microelectrode arrays
Confocal fluorescence microscopy
Cultured DRG neurons
Segmentation
3D visualization
Quantitative analysis
CIENCIAS EXATAS E DA TERRA::CIENCIA DA COMPUTACAO
dc.subject.eng.fl_str_mv Microelectrode arrays
Confocal fluorescence microscopy
Cultured DRG neurons
Segmentation
3D visualization
Quantitative analysis
dc.subject.cnpq.fl_str_mv CIENCIAS EXATAS E DA TERRA::CIENCIA DA COMPUTACAO
description Microelectrode arrays (MEA) are devices that allow chemical and electrical stimulation and recording of the extracellular electrical activity from entire neuronal cultures over long periods of time, such as several weeks. Some MEA models have transparent substrate, which enables the imaging of culture using optical microscopy. The images are taken from two channels: fluorescence light and transmitted light channels. In the first one, it is possible to visualize the neurons, while in the other one, it is possible to observe the microelectrodes. The objective of this work is to develop methods that enable performing quantitative analysis of the dissociated culture of rat dorsal root ganglion (DRG) neurons plated on MEA by means of the processing of the images, obtained from confocal fluorescence microscopy. We proposed and developed the following methods in order to achieve this objective: (A) A method to automatically identify the microelectrodes in the transmitted light channel using circular Hough Transform and error correction based on the Delaunay triangulation; (B) the registration of a number of images taken at different parts of the MEA in order to generate a unique and high-resolution representation of the whole culture; (C) the segmentation of the neuron in 2D images taken from the fluorescence channel, composed by the steps: preprocessing, thresholding, morphological filtering, neurons occlusion correction, watershed transform and object classification; (D) 2D quantitative analysis based on the identified microelectrodes and on the segmented neurons; (E) a method for generating 3D polygonal models of the neurons from the volumetric images, to be used for visualizing the culture on the MEA by different points of view and zoom levels; and (F) 3D quantitative analysis performed by the processing of the polygonal surfaces in conjunction with the information about the microelectrodes positioning. The results show that the methods are capable to identify the neurons and microelectrodes on the 2D images efficiently. In the 3D images, the preprocessing step which uses information from the 2D segmentation method, showed to be capable to generate correct polygonal models efficiently. Most of the studies involving the analysis of neuron cultures on MEAs consider only qualitative analysis or simple quantitative measures. However, the methods proposed in this thesis enables to obtain important measures related to the neuron culture, such as: the density and morphology of the neurons, and the spatial and topological distribution of the neurons and microelectrodes. The information about neuron morphology is important because they are related to the behavior of this kind of neuron. The spatial and topological distribution of neurons and microelectrodes are used for providing models of the interface between these elements, for supporting the analysis of the electrophysiological signal recorded by the microelectrodes, as well as in the computational simulations of the neuron culture behavior.
publishDate 2015
dc.date.available.fl_str_mv 2015-05-27
2016-06-02T19:04:00Z
dc.date.issued.fl_str_mv 2015-03-09
dc.date.accessioned.fl_str_mv 2016-06-02T19:04:00Z
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dc.identifier.citation.fl_str_mv MARI, João Fernando. Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência. 2015. 262 f. Tese (Doutorado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2015.
dc.identifier.uri.fl_str_mv https://repositorio.ufscar.br/handle/ufscar/297
identifier_str_mv MARI, João Fernando. Análise quantitativa de culturas de neurônios em matrizes de microeletrodos por meio do processamento de imagens de microscopia confocal de fluorescência. 2015. 262 f. Tese (Doutorado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2015.
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