Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration

Monitoring and mathematical modeling of in vitro human megakaryocyte expansion and maturation dynamics

La mégakaryopoïèse est un processus complexe, qui prend naissance à partir des cellules souches hématopoïétiques (HSC). Ces dernières se différencient par étapes successives en mégakaryocytes (MKs) qui, suite à leur maturation, libèrent les plaquettes. Afin de modéliser le sort des HSCs lors de la mégakaryopoïèse en culture, un nouveau modèle mathématique a été développé, basé sur un programme de différenciation tridimensionnelle (3-D) où chaque sous-population est représentée par un compartiment. Dans le but d’évaluer la prolifération, la différenciation des MKs immatures puis matures, la cinétique de mort cellulaire ainsi que le nombre de plaquettes produites, à partir des cellules de sang de cordon (CB) ombilical enrichies en CD34+, un ensemble d'équations différentielles a été déployé. Les cellules CD34+ ont été placées en culture dans un milieu optimisé pour la différenciation mégakaryocytaire. Les paramètres cinétiques ont été estimés pour deux températures d'incubation (37°C versus 39°C). Les résultats des régressions ont été validés par l'évaluation de l'estimabilité des paramètres, en utilisant des analyses de sensibilité locale et globale, puis la détermination d'un intervalle de confiance. Ceux-ci ont été comparés par le biais de tests statistiques et d’analyses en composante principale (ACP). Le modèle proposé pourrait permettre de mieux comprendre les phénomènes complexes observés. Les MKs sont uniques parmi les cellules hématopoïétiques, étant les seules à devenir polyploïdes au cours de leur développement par l’entremise de l’endomitose, un processus mitotique qui se termine prématurément durant la cytocinèse. Pour obtenir une image plus complète et exhaustive de la mégacaryopoïèse, une approche d’imagerie cellulaire à grand champ et à long terme a été développée permettant de suivre individuellement l'évolution des HSCs lors de leur différenciation ex vivo. Cela a permis de démontrer que les MKs polyploïdes sont encore capables de se diviser et de produire des cellules filles polyploïdes, et que ce processus est plus fréquent chez les MKs issues de CB que de moelle osseuse d'adulte. De plus, le processus de formation des proplaquettes semble également réversible. Les phénomènes énoncés plus haut étaient inversement proportionnels au niveau de ploïdie des MKs. En conclusion, cette étude a dévoilé de nouvelles propriétés jusqu’ici inconnues des MKs.Megakaryopoiesis is a complex process, which is initiated with the proliferation and the differentiation of hematopoietic stem cells (HSC) into megakaryocytes (MK), followed by the maturation of MK and ended by platelet release. To describe the fates of HSC during ex vivo megakaryopoiesis, a new mathematical model was developed based on a 3-dimensional kinetic developmental program. To address this, a set of differential equations was applied to analyze the proliferation, differentiation and death kinetic rates of purified cord blood (CB)-CD34+ cells, immature and mature MKs, as well as platelet number and productivity. CB-CD34+ cells were placed in culture optimized for MK differentiation. The kinetic parameters were estimated for two incubation temperatures (37°C vs. 39°C). The regression results have been validated by assessing the parameter identifiability using local and global sensitivity analyses and confidence intervals, and compared using statistical tests and principal component analysis (PCA). Furthermore, PCA was applied on the solution matrix to construct a simplified MK differentiation pathway model, and to reveal dependencies among the model parameters. The proposed model provides insight into phenomena that would be otherwise difficult to interpret. MKs are unique among mammalian marrow cells as they polyploidize during their natural development. It is universally accepted that MK becomes polyploid by repeatedly deviating from normal cell cycling, where it ceases to complete cytokinesis and divide. To challenge this long-standing hypothesis and to obtain a more comprehensive picture of megakaryopoiesis, a long-term and large-field live cell imaging approach of in vitro MK culture was developed. Using CB- and bone marrow (BM)-CD34+ as starting cells, the direct observation of cells undergoing differentiation and maturation over a 5-day culture period is reported for the first time. Herein, direct visual proof that polyploid MKs can complete cytokinesis during its normal development is presented. This phenomenon was found not restricted to CB- as the BM-derived polyploid MK also underwent division. However the latter showed significantly lower proliferation rate. This new finding explains in part the unresolved issue of low ploidy levels observed in CB-MK and contests the notion that polyploid MKs do not divide