Detecção de leucócitos em imagens de vídeo de microscopia intravital usando a técnica de congruência de fase

A quantificação do número de leucócitos rolantes e aderidos presentes na microcirculação de pequenos animais é uma tarefa importante para elucidar os mecanismos de inflamações e avaliar os efeitos terapêuticos de novas drogas. Em geral, a contagem de leucócitos é realizada de maneira visual por um observador (técnico laboratorial ou especialista) usando uma sequência de imagens de microscopia intravital (MI). Entretanto, tal tarefa é demorada e suscetível a erros, devido a fadiga visual do observador e a variabilidades inter e intra observadores. Neste trabalho uma nova técnica computacional é proposta para a detecção automática de leucócitos em vídeos de MI. A técnica usa uma medida de blobness, calculada a partir da análise dos autovalores de matrizes locais de momentos de segunda-ordem da medida de congruência de fase, para realçar os leucócitos nas imagens. A detecção dos leucócitos é alcançada pela busca de máximos locais no mapa de medidas de blobness. Usando um conjunto de quadros com os centroides dos leucócitos manualmente marcados, os resultados da técnica proposta foram avaliados usando os valores das métricas medida-F1 e áreas sob as curvas precisão-revocação (AUCPRs) calculadas para cada quadro do vídeo. Uma comparação com a técnica de casamento de padrões também foi realizada. Os resultados obtidos para a técnica proposta (medida-F1=0,791, AUCPR=0,776) foram superiores em comparação com a técnica de casamento de padrões (medida-F1=0,746, AUCPR=0,670)

Modelling and Identification of Immune Cell Migration during the Inflammatory Response

Neutrophils are the white blood cells that play a crucial role in the response of the innate immune system to tissue injuries or infectious threats. Their rapid arrival to the damaged area and timely removal from it define the success of the inflammatory process. Therefore, understanding neutrophil migratory behaviour is essential for the therapeutic regulation of multiple inflammation-mediated diseases. Recent years saw rapid development of various in vivo models of inflammation that provide a remarkable insight into the neutrophil function. The main drawback of the \textit{in vivo} microscopy is that it usually focuses on the moving cells and obscures the external environment that drives their migration. To evaluate the effect of a particular treatment strategy on neutrophil behaviour, it is necessary to recover the information about the cell responsiveness and the complex extracellular environment from the limited experimental data. This thesis addresses the presented inference problem by developing a dynamical modelling and estimation framework that quantifies the relationship between an individual migrating cell and the global environment. \par The first part of the thesis is concerned with the estimation of the hidden chemical environment that modulates the observed cell migration during the inflammatory response in the injured tail fin of zebrafish larvae. First, a dynamical model of the neutrophil responding to the chemoattractant concentration is developed based on the potential field paradigm of object-environment interaction. This representation serves as a foundation for a hybrid model that is proposed to account for heterogeneous behaviour of an individual cell throughout the migration process. An approximate maximum likelihood estimation framework is derived to estimate the hidden environment and the states of multiple hybrid systems simultaneously. The developed framework is then used to analyse the neutrophil tracking data observed in vivo under the assumption that each neutrophil at each time can be in one of three migratory modes: responding to the environment, randomly moving, and stationary. The second part of the thesis examines the process of neutrophil migration at the subcellular scale, focusing on the subcellular mechanism that translates the local environment sensing into the cell shape change. A state space model is formulated based on the hypothesis that links the local protrusions of the cell membrane and the concentration of the intracellular pro-inflammatory signalling protein. The developed model is tested against the local concentration data extracted from the in vivo time-lapse images via the classical expectation-maximisation algorithm

Towards cellular hydrodynamics: collective migration in artificial microstructures

The collective migration of cells governs many biological processes, including embryonic development, wound healing and cancer progression. Observed phenomena are not simply the sum of the individual motion of many isolated cells, but rather emerge as a consequence of their interactions. The movements in epithelial cell sheets display rich phenomenology, such as the occurrence of vortices spanning several cell diameters and the transition from fluid-like behavior at low densities to glass-like behavior at high densities. In this thesis, collective invasion of epithelial cell sheets into microchannels was studied on a phenomenological level within the scope of theoretical approaches to active fluids. In a first project, the motion profile of a cell layer in straight channels was investigated using single cell tracking and particle image velocimetry (PIV) on timelapse microscopy data. A defined plug-flow like velocity profile was observed across the channels. The cell density profile is well-described by the Fisher-Kolmogorov reaction-diffusion equation, which includes active migration and the contribution of proliferation. This study revealed a change in the short scale noise behavior in the presence of this global invasion into a channel. For a closer look at the system’s proliferation component, the effect of an underlying global migration direction on the orientation of the cells’ division axes was examined. We found strong alignment of the axes’ orientation with the imposed movement direction. Specifically, the strongest correlations were observed between the orientation of the cells’ division axes and the local strain rate tensor’s main axis. This is in agreement with the notion that stresses in the migrating cell sheet orient the cell divisions. Expanding the assay of invasion into straight channels, we introduced a constriction, which the cell sheet needs to pass through in order to progress. A plateau of low velocities was observed in the region ahead of the constriction, which was attributed to an increase in local cell density accompanied by jamming. These results were compared to an active isotropic-nematic mixture model. The suitability of this model to describe this assay could be ruled out, however, as it showed qualitatively very different behavior than the experiments. Finally, the frequency of topological nearest-neighbor T1 transitions within a cell sheet was investigated in minimal model systems. In order to study the smallest possible fundamental unit for such transitions, groups of four cells were confined to cloverleaf patterns, which could be shown to inhibit the onset of collective rotation states. Results showed that T1 transitions occurred more frequently for groups of cells with a lower average length of the cell-cell junction that shrinks in the process of this transition. These results are consistent with the notion that the energy barrier which needs to be overcome by the cells in order to perform this transition, scales with the original length of the shrinking junction. Taken together, the results of this thesis contribute to a better understanding of the flow fields for collective cell migration processes in confined geometries. In addition to the insights the phenomenological observations in this work could provide directly, they will also continue to prove useful as a standard for validating detailed theoretical models

Towards cellular hydrodynamics: collective migration in artificial microstructures

The collective migration of cells governs many biological processes, including embryonic development, wound healing and cancer progression. Observed phenomena are not simply the sum of the individual motion of many isolated cells, but rather emerge as a consequence of their interactions. The movements in epithelial cell sheets display rich phenomenology, such as the occurrence of vortices spanning several cell diameters and the transition from fluid-like behavior at low densities to glass-like behavior at high densities. In this thesis, collective invasion of epithelial cell sheets into microchannels was studied on a phenomenological level within the scope of theoretical approaches to active fluids. In a first project, the motion profile of a cell layer in straight channels was investigated using single cell tracking and particle image velocimetry (PIV) on timelapse microscopy data. A defined plug-flow like velocity profile was observed across the channels. The cell density profile is well-described by the Fisher-Kolmogorov reaction-diffusion equation, which includes active migration and the contribution of proliferation. This study revealed a change in the short scale noise behavior in the presence of this global invasion into a channel. For a closer look at the system’s proliferation component, the effect of an underlying global migration direction on the orientation of the cells’ division axes was examined. We found strong alignment of the axes’ orientation with the imposed movement direction. Specifically, the strongest correlations were observed between the orientation of the cells’ division axes and the local strain rate tensor’s main axis. This is in agreement with the notion that stresses in the migrating cell sheet orient the cell divisions. Expanding the assay of invasion into straight channels, we introduced a constriction, which the cell sheet needs to pass through in order to progress. A plateau of low velocities was observed in the region ahead of the constriction, which was attributed to an increase in local cell density accompanied by jamming. These results were compared to an active isotropic-nematic mixture model. The suitability of this model to describe this assay could be ruled out, however, as it showed qualitatively very different behavior than the experiments. Finally, the frequency of topological nearest-neighbor T1 transitions within a cell sheet was investigated in minimal model systems. In order to study the smallest possible fundamental unit for such transitions, groups of four cells were confined to cloverleaf patterns, which could be shown to inhibit the onset of collective rotation states. Results showed that T1 transitions occurred more frequently for groups of cells with a lower average length of the cell-cell junction that shrinks in the process of this transition. These results are consistent with the notion that the energy barrier which needs to be overcome by the cells in order to perform this transition, scales with the original length of the shrinking junction. Taken together, the results of this thesis contribute to a better understanding of the flow fields for collective cell migration processes in confined geometries. In addition to the insights the phenomenological observations in this work could provide directly, they will also continue to prove useful as a standard for validating detailed theoretical models

Detecção e rastreamento de leucócitos em imagens de microscopia intravital via processamento espaçotemporal

Over the last few years, a large number of researchers have directed their efforts and interests for the in vivo study of the cellular and molecular mechanisms of leukocyte-endothelial interactions in the microcirculation of many tissues under different inflammatory conditions. The main goal of these studies is to develop more effective therapeutic strategies for the treatment of inflammatory and autoimmune diseases. Nowadays, analysis of the leukocyte-endothelial interactions in small animals is performed by visual assessment from an intravital microscopy image sequences. Besides being time consuming, this procedure may cause visual fatigue of the observer and, therefore, generate false statistics. In this context, this work aims to study and develop computational techniques for the automatic detection and tracking of leukocytes in intravital video microscopy. For that, results from frame to frame processing (2D – spatial analysis) will be combined with those from the three-dimensional analysis (3D=2D+t – spatio-temporal analysis) of the volume formed by stacking the video frames. The main technique addressed for both processings is based on the analysis of the eigenvalues of the local Hessian matrix. While the 2D image processing aims the leukocyte detection without worrying about their tracking, 2D+t processing is intended to assist on the dynamic analysis of cell movement (tracking), being able to predict cell movements in cases of occlusion, for example. In this work we used intravital video microscopy obtained from a study of Multiple Sclerosis in mice. Noise reduction and registration techniques comprise the preprocessing step. In addition, techniques for the analysis and definition of cellular pathways comprise the post processing step. Results of 2D and 2D+t processing steps, compared with conventional visual analysis, have shown the effectiveness of the proposed approach.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Nos últimos anos, um grande número de pesquisadores tem voltado seus esforços e interesses para o estudo in vivo dos mecanismos celulares e moleculares da interação leucócitoendotélio na microcirculação de vários tecidos e em várias condições inflamatórias. O principal objetivo desses estudos é desenvolver estratégias terapêuticas mais eficazes para o tratamento de doenças inflamatórias e autoimunes. Atualmente, a análise de interações leucócito-endotélio em pequenos animais é realizada de maneira visual a partir de uma sequência de imagens de microscopia intravital. Além de ser demorado, esse procedimento pode levar à fadiga visual do observador e, portanto, gerar falsas estatísticas. Nesse contexto, este trabalho de pesquisa tem como objetivo estudar e desenvolver técnicas computacionais para a detecção e rastreamento automáticos de leucócitos em vídeos de microscopia intravital. Para isso, resultados do processamento quadro a quadro do vídeo (2D – análise espacial) serão combinados com os resultados da análise tridimensional (3D=2D+t – análise espaço-temporal) do volume formado pelo empilhamento dos quadros do vídeo. A principal técnica abordada para ambos os processamentos é baseada na análise local dos autovalores da matriz Hessiana. Enquanto o processamento de imagens 2D tem como objetivo a detecção de leucócitos sem se preocupar com seu rastreamento, o processamento 2D+t pretende auxiliar na análise dinâmica de movimentação das células (rastreamento), sendo capaz de prever movimentos celulares em casos de oclusão, por exemplo. Neste trabalho foram utilizados vídeos de microscopia intravital obtidos a partir de um estudo da Esclerose Múltipla realizado com camundongos. Técnicas de redução de ruído e estabilização do movimento das sequências de imagens compõem a etapa de pré-processamento, assim como técnicas para a definição e análise dos caminhos celulares compõem a etapa de pós-processamento. Resultados das etapas de processamento 2D e 2D+t, comparados com a convencional análise visual, demonstraram a eficácia da abordagem proposta.FAPESP: 2013/26171-