Amoeboid Shape Dynamics on Flat and Topographically Modified Surfaces

I present an analysis of the shape dynamics of the amoeba Dictyostelium discoideum, a model system for the study of cellular migration. To better understand cellular migration in complicated 3-D environments, cell migration was studied on simple 3-D surfaces, such as cliffs and ridges. D. discoideum interact with surfaces without forming mature focal adhesion complexes. The cellular response to the surface topography was characterized by measuring and looking for patterns in cell shape. Dynamic cell shape is a measure of the interaction between the internal biochemical state of a cell and its external environment. For D. discoideum migrating on flat surfaces, waves of high boundary curvature were observed to travel from the cell front to the cell back. Curvature waves are also easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from the coverslip or cells that are extended over the edge of micro-fabricated cliffs. At the leading edge of adhered cells, these curvature waves are associated with protrusive activity, suggesting that protrusive motion can be thought of as a wave-like process. The wave-like character of protrusions provides a plausible mechanism for the ability of cells to swim in viscous fluids and to navigate complex 3-D topography. Patterning of focal adhesion complexes has previously been implicated in contact guidance (polarization or migration parallel to linear topographical structures). However, significant contact guidance is observed in D. discoideum, which lack focal adhesion complexes. Analyzing the migration of cells on nanogratings of ridges spaced various distances apart, ridges spaced about 1.5 micrometers apart were found to guide cells best. Contact guidance was modeled as an interaction between wave-like processes internal to the cell and the periodicity of the nanograting. The observed wavelength and speed of the oscillations that best couple to the surface are consistent with those of protrusive dynamics. Dynamic sensing via actin or protrusive dynamics might then play a role in contact guidance

Complexity in Developmental Systems: Toward an Integrated Understanding of Organ Formation

During animal development, embryonic cells assemble into intricately structured organs by working together in organized groups capable of implementing tightly coordinated collective behaviors, including patterning, morphogenesis and migration. Although many of the molecular components and basic mechanisms underlying such collective phenomena are known, the complexity emerging from their interplay still represents a major challenge for developmental biology. Here, we first clarify the nature of this challenge and outline three key strategies for addressing it: precision perturbation, synthetic developmental biology, and data-driven inference. We then present the results of our effort to develop a set of tools rooted in two of these strategies and to apply them to uncover new mechanisms and principles underlying the coordination of collective cell behaviors during organogenesis, using the zebrafish posterior lateral line primordium as a model system. To enable precision perturbation of migration and morphogenesis, we sought to adapt optogenetic tools to control chemokine and actin signaling. This endeavor proved far from trivial and we were ultimately unable to derive functional optogenetic constructs. However, our work toward this goal led to a useful new way of perturbing cortical contractility, which in turn revealed a potential role for cell surface tension in lateral line organogenesis. Independently, we hypothesized that the lateral line primordium might employ plithotaxis to coordinate organ formation with collective migration. We tested this hypothesis using a novel optical tool that allows targeted arrest of cell migration, finding that contrary to previous assumptions plithotaxis does not substantially contribute to primordium guidance. Finally, we developed a computational framework for automated single-cell segmentation, latent feature extraction and quantitative analysis of cellular architecture. We identified the key factors defining shape heterogeneity across primordium cells and went on to use this shape space as a reference for mapping the results of multiple experiments into a quantitative atlas of primordium cell architecture. We also propose a number of data-driven approaches to help bridge the gap from big data to mechanistic models. Overall, this study presents several conceptual and methodological advances toward an integrated understanding of complex multi-cellular systems

New algorithms for the analysis of live-cell images acquired in phase contrast microscopy

La détection et la caractérisation automatisée des cellules constituent un enjeu important dans de nombreux domaines de recherche tels que la cicatrisation, le développement de l'embryon et des cellules souches, l’immunologie, l’oncologie, l'ingénierie tissulaire et la découverte de nouveaux médicaments. Étudier le comportement cellulaire in vitro par imagerie des cellules vivantes et par le criblage à haut débit implique des milliers d'images et de vastes quantités de données. Des outils d'analyse automatisés reposant sur la vision numérique et les méthodes non-intrusives telles que la microscopie à contraste de phase (PCM) sont nécessaires. Comme les images PCM sont difficiles à analyser en raison du halo lumineux entourant les cellules et de la difficulté à distinguer les cellules individuelles, le but de ce projet était de développer des algorithmes de traitement d'image PCM dans Matlab® afin d’en tirer de l’information reliée à la morphologie cellulaire de manière automatisée. Pour développer ces algorithmes, des séries d’images de myoblastes acquises en PCM ont été générées, en faisant croître les cellules dans un milieu avec sérum bovin (SSM) ou dans un milieu sans sérum (SFM) sur plusieurs passages. La surface recouverte par les cellules a été estimée en utilisant un filtre de plage de valeurs, un seuil et une taille minimale de coupe afin d'examiner la cinétique de croissance cellulaire. Les résultats ont montré que les cellules avaient des taux de croissance similaires pour les deux milieux de culture, mais que celui-ci diminue de façon linéaire avec le nombre de passages. La méthode de transformée par ondelette continue combinée à l’analyse d'image multivariée (UWT-MIA) a été élaborée afin d’estimer la distribution de caractéristiques morphologiques des cellules (axe majeur, axe mineur, orientation et rondeur). Une analyse multivariée réalisée sur l’ensemble de la base de données (environ 1 million d’images PCM) a montré d'une manière quantitative que les myoblastes cultivés dans le milieu SFM étaient plus allongés et plus petits que ceux cultivés dans le milieu SSM. Les algorithmes développés grâce à ce projet pourraient être utilisés sur d'autres phénotypes cellulaires pour des applications de criblage à haut débit et de contrôle de cultures cellulaires.Automated cell detection and characterization is important in many research fields such as wound healing, embryo development, immune system studies, cancer research, parasite spreading, tissue engineering, stem cell research and drug research and testing. Studying in vitro cellular behavior via live-cell imaging and high-throughput screening involves thousands of images and vast amounts of data, and automated analysis tools relying on machine vision methods and non-intrusive methods such as phase contrast microscopy (PCM) are a necessity. However, there are still some challenges to overcome, since PCM images are difficult to analyze because of the bright halo surrounding the cells and blurry cell-cell boundaries when they are touching. The goal of this project was to develop image processing algorithms to analyze PCM images in an automated fashion, capable of processing large datasets of images to extract information related to cellular viability and morphology. To develop these algorithms, a large dataset of myoblasts images acquired in live-cell imaging (in PCM) was created, growing the cells in either a serum-supplemented (SSM) or a serum-free (SFM) medium over several passages. As a result, algorithms capable of computing the cell-covered surface and cellular morphological features were programmed in Matlab®. The cell-covered surface was estimated using a range filter, a threshold and a minimum cut size in order to look at the cellular growth kinetics. Results showed that the cells were growing at similar paces for both media, but their growth rate was decreasing linearly with passage number. The undecimated wavelet transform multivariate image analysis (UWT-MIA) method was developed, and was used to estimate cellular morphological features distributions (major axis, minor axis, orientation and roundness distributions) on a very large PCM image dataset using the Gabor continuous wavelet transform. Multivariate data analysis performed on the whole database (around 1 million PCM images) showed in a quantitative manner that myoblasts grown in SFM were more elongated and smaller than cells grown in SSM. The algorithms developed through this project could be used in the future on other cellular phenotypes for high-throughput screening and cell culture control applications

Multi-Temporal Remote-Sensing-based Mapping and Characterization of Landscape Evolution of a Meandering River Floodplain

Large meandering river floodplains are critical components of the Earth ecosystems for their high biodiversity and productivity. However, it is challenging to study these regions because of their complex land-covers and dynamic surface processes. This study applies soft classification and change-detection analysis to five Landsat 5 Thematic Mapper (TM) satellite images to examine long-term surface-cover composition and configuration change of the Rio Beni floodplain in Bolivia from 1987 to 2006. One hard/crisp classification algorithm (i.e., ISODATA) and two soft classification algorithms (i.e., Bayes classification and fuzzy classification) were applied to the study-area satellite images to examine the performances of classifying and mapping meandering river-floodplain environments between hard and soft classification approaches. In all five scenes, three algorithms achieved ~90% classification accuracy via hard classification outputs. However, the two soft algorithms were of more utility in this study because their results were less affected by “salt-and-pepper” noise and provided extra land-cover probability/membership layers. A novel change-detection algorithm was proposed in this study, namely Modified Change Vector Analysis (MCVA). The MCVA operated in fuzzy-membership space, considered change uncertainty during the thresholding stage, and utilized change-vector directions to modify the determination of change/no-change status for each pixel. A fuzzy Markov Random Field (FMRF) model was applied to further refine the change maps by incorporating spatial change uncertainty. A second thresholding stage was also applied to separate a type of change referred to as “transitional change,” which preserved fuzzy membership information and provided a concise map output. Compared with three traditional change-detection algorithms, the MCVA achieved higher change-detection accuracy and provided more detailed change dynamics regarding the land-surface change. Dynamics of major floodplain cover types (i.e., oxbow lakes, river, sand, forest, non-forest vegetation, and dry and wet soil) were investigated via multi-temporal analysis. Over the observing period of 1987 to 2006, 74.4% of pixels remained the same land-cover, 20% experienced clear land-cover change and 5.6% experienced transitional land-cover change. The riparian area experienced more dramatic change than other parts of the Rio Beni floodplain during this period. Additional analysis of landscape metrics provided information regarding the spatial patterns of the land-cover, but future work would be needed to further examine its utility in understanding floodplain dynamics. This study provides information on remote-sensing-based mapping and quantitative characterization methods for meandering river floodplains. The spatiotemporal patterns of landscape on Rio Beni floodplain can be used in sustainable management and protection of floodplain ecosystems

Structure, Dynamics, and Regulation of Collective Cell Migration

Collective migration is the process by which cells organize individual motions to productively migrate as a group and plays a fundamental role in organism development, tissue regeneration, and cancer invasion. In development, coordinated migration facilitates the formation of complex organ structures and is required for proper dissemination of neural crest cells throughout an organism. After injury, this process allows breaches in epithelial layers to be repaired while maintaining tissue integrity, and in cancer, collective behavior enhances invasion of tumor cells into the surrounding tissue. Chapter 1 provides an introduction for the role of collective migration across an organism’s lifespan, the mechanisms used by cells to generate motile force, and the emergence of collective behavior. Chapter 2 dissects the intertwined roles of three fundamental parameters often altered in collective migration processes: cell density, cell adhesion, and cell-cell contractility through the Rho-ROCK-Myosin II signaling axis. Through quantitative analysis of large-scale time-lapse imaging and mathematical modeling, I identify force-sensitive contractility and cell packing as mediators of two distinct classes of collective migration. From these results, I formulate a phase-diagram of collective cell migration and test predictions in an in-vivo epithelium using genetic manipulations to drive collective motion between predicted migratory phases. In Chapter 3, the effect of phenotypic heterogeneity on the organization of cells is examined, providing insight into the effects of early cancer progression on epithelial dynamics. I find that mutant cells within an otherwise wild-type tissue impact organization through local and field-effects, disrupting normal dynamics and leading to cell-type segregation. Chapter 4 provides a theoretical framework for quantitatively understanding and predicting the dynamics of protein interactions underlying biological processes including collective migration. Traditional chemical kinetics approaches break down in situations where components are slow diffusing or in countable numbers, requiring the formulation of new models that take into account this level of complexity. Here I develop an event-driven algorithm that bridges well-mixed and unmixed systems and use it to predict the effect of apparent changes in enzymatic efficiency due to alterations in mobility that may be caused by protein complex formation. Overall the work in this dissertation advances our understanding of the structure and dynamics of collective migration and the parameters governing this process by combining quantitative statistical analysis, mathematical modeling, and in-vivo live imaging

Wildlife Communication

This report contains a progress report for the ph.d. project titled “Wildlife Communication”. The project focuses on investigating how signal processing and pattern recognition can be used to improve wildlife management in agriculture. Wildlife management systems used today experience habituation from wild animals which makes them ineffective. An intelligent wildlife management system could monitor its own effectiveness and alter its scaring strategy based on this

Automatic analysis of malaria infected red blood cell digitized microscope images

Malaria is one of the three most serious diseases worldwide, affecting millions each year, mainly in the tropics where the most serious illnesses are caused by Plasmodium falciparum. This thesis is concerned with the automatic analysis of images of microscope slides of Giemsa stained thin-films of such malaria infected blood so as to segment red-blood cells (RBCs) from the background plasma, to accurately and reliably count the cells, identify those that were infected with a parasite, and thus to determine the degree of infection or parasitemia. Unsupervised techniques were used throughout owing to the difficulty of obtaining large quantities of training data annotated by experts, in particular for total RBC counts. The first two aims were met by optimisation of Fisher discriminants. For RBC segmentation, a well-known iterative thresholding method due originally to Otsu (1979) was used for scalar features such as the image intensity and a novel extension of the algorithm developed for multi-dimensional, colour data. Performance of the algorithms was evaluated and compared via ROC analysis and their convergence properties studied. Ways of characterising the variability of the image data and, if necessary of mitigating it, were discussed in theory. The size distribution of the objects segmented in this way indicated that optimisation of a Fisher discriminant could be further used for classifying objects as small artefacts, singlet RBCs, doublets, or triplets etc. of adjoining cells provided optimisation was via a global search. Application of constraints on the relationships between the sizes of singlet and multiplet RBCs led to a number of tests that enabled clusters of cells to be reliably identified and accurate total RBC counts to be made. Development of an application to make such counts could be very useful both in research laboratories and in improving treatment of malaria. Unfortunately, the very small number of pixels belonging to parasite infections mean that it is difficult to segment parasite objects and thus to identify infected RBCs and to determine the parasitemia. Preliminary attempts to do so by similar, unsupervised means using Fischer discriminants, even when applied in a hierarchical manner, though suggestive that it may ultimately be possible to develop such a system remain on the evidence currently available, inconclusive. Appendices give details of material from old texts no longer easily accessible

An image-based data-driven analysis of cellular architecture in a developing tissue

Quantitative microscopy is becoming increasingly crucial in efforts to disentangle the complexity of organogenesis, yet adoption of the potent new toolbox provided by modern data science has been slow, primarily because it is often not directly applicable to developmental imaging data. We tackle this issue with a newly developed algorithm that uses point cloud-based morphometry to unpack the rich information encoded in 3D image data into a straightforward numerical representation. This enabled us to employ data science tools, including machine learning, to analyze and integrate cell morphology, intracellular organization, gene expression and annotated contextual knowledge. We apply these techniques to construct and explore a quantitative atlas of cellular architecture for the zebrafish posterior lateral line primordium, an experimentally tractable model of complex self-organized organogenesis. In doing so, we are able to retrieve both previously established and novel biologically relevant patterns, demonstrating the potential of our data-driven approach

Computing Interpretable Representations of Cell Morphodynamics

Shape changes (morphodynamics) are one of the principal ways cells interact with their environments and perform key intrinsic behaviours like division. These dynamics arise from a myriad of complex signalling pathways that often organise with emergent simplicity to carry out critical functions including predation, collaboration and migration. A powerful method for analysis can therefore be to quantify this emergent structure, bypassing the low-level complexity. Enormous image datasets are now available to mine. However, it can be difficult to uncover interpretable representations of the global organisation of these heterogeneous dynamic processes. Here, such representations were developed for interpreting morphodynamics in two key areas: mode of action (MoA) comparison for drug discovery (developed using the economically devastating Asian soybean rust crop pathogen) and 3D migration of immune system T cells through extracellular matrices (ECMs). For MoA comparison, population development over a 2D space of shapes (morphospace) was described using two models with condition-dependent parameters: a top-down model of diffusive development over Waddington-type landscapes, and a bottom-up model of tip growth. A variety of landscapes were discovered, describing phenotype transitions during growth, and possible perturbations in the tip growth machinery that cause this variation were identified. For interpreting T cell migration, a new 3D shape descriptor that incorporates key polarisation information was developed, revealing low-dimensionality of shape, and the distinct morphodynamics of run-and-stop modes that emerge at minute timescales were mapped. Periodically oscillating morphodynamics that include retrograde deformation flows were found to underlie active translocation (run mode). Overall, it was found that highly interpretable representations could be uncovered while still leveraging the enormous discovery power of deep learning algorithms. The results show that whole-cell morphodynamics can be a convenient and powerful place to search for structure, with potentially life-saving applications in medicine and biocide discovery as well as immunotherapeutics.Open Acces