2,218 research outputs found

    Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis

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    In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis

    Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis

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    The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions

    3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

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    We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms

    Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro

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    Automated or semiautomated time-lapse analysis of early stage embryo images during the cleavage stage can give insight into the timing of mitosis, regularity of both division timing and pattern, as well as cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. The study of live embryos poses not only new requirements on the hardware and embryo-holding equipment but also indirectly on analytical software and data analysis as four-dimensional video sequencing of embryos easily creates high quantities of data. The ability to continuously film and automatically analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was not possible unless by a tedious manual process. In recent years, several methods have been developed that enable this dynamic monitoring of live embryos. Here we describe three methods with variations in hardware and software analysis and give examples of the outcomes. Together, these methods open a window to new information in developmental embryology, as embryo division pattern and lineage are studied in vivo

    Evaluation of the effectiveness of simple nuclei-segmentation methods on Caenorhabditis elegans embryogenesis images

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    BACKGROUND: For the analysis of spatio-temporal dynamics, various automated processing methods have been developed for nuclei segmentation. These methods tend to be complex for segmentation of images with crowded nuclei, preventing the simple reapplication of the methods to other problems. Thus, it is useful to evaluate the ability of simple methods to segment images with various degrees of crowded nuclei. RESULTS: Here, we selected six simple methods from various watershed based and local maxima detection based methods that are frequently used for nuclei segmentation, and evaluated their segmentation accuracy for each developmental stage of the Caenorhabditis elegans. We included a 4D noise filter, in addition to 2D and 3D noise filters, as a pre-processing step to evaluate the potential of simple methods as widely as possible. By applying the methods to image data between the 50- to 500-cell developmental stages at 50-cell intervals, the error rate for nuclei detection could be reduced to ≤ 2.1% at every stage until the 350-cell stage. The fractions of total errors throughout the stages could be reduced to ≤ 2.4%. The error rates improved at most of the stages and the total errors improved when a 4D noise filter was used. The methods with the least errors were two watershed-based methods with 4D noise filters. For all the other methods, the error rate and the fraction of errors could be reduced to ≤ 4.2% and ≤ 4.1%, respectively. The minimum error rate for each stage between the 400- to 500-cell stages ranged from 6.0% to 8.4%. However, similarities between the computational and manual segmentations measured by volume overlap and Hausdorff distance were not good. The methods were also applied to Drosophila and zebrafish embryos and found to be effective. CONCLUSIONS: The simple segmentation methods were found to be useful for detecting nuclei until the 350-cell stage, but not very useful after the 400-cell stage. The incorporation of a 4D noise filter to the simple methods could improve their performances. Error types and the temporal biases of errors were dependent on the methods used. Combining multiple simple methods could also give good segmentations

    Computer vision profiling of neurite outgrowth mordphodynamics reveals spatio-temporal modularity of Rho GTPase signaling

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    Neurite outgrowth is essential to build the neuronal processes that produce axons and dendrites that connect the adult brain. In cultured cells, the neurite outgrowth process is highly dynamic, and consists of a series of repetitive morphogenetic sub-processes (MSPs), such as neurite initiation, elongation, branching, growth cone motility and collapse (da Silva and Dotti 2002). Neurons also actively migrate, which might in part reflect neuronal migration during brain development. Each of the different MSPs inherent to neurite outgrowth and cell migration is likely to be regulated by precise spatio-temporal signaling networks that control cytoskeletal dynamics, trafficking and adhesion events. These MSPs can occur on a range of time and length scales. For example, microtubule bundling in the neurite shaft can be maintained during hours, while growth cone filopodia dynamically explore their surrounding on time scales of seconds and length scales of single microns. This implies that a correct understanding of these processes will require analysis with an adequate spatio-temporal resolution. The Rho family of GTPases are signaling switches that regulate a wide variety of cellular processes, such as actin and adhesion dynamics, gene transcription, and neuronal differentiation (Boguski and McCormick 1993). Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), and are switched off by GTPase activating proteins (GAPs). Upon activation, Rho GTPases can associate with effectors to initiate a downstream response. Current models propose that Rac1 and Cdc42 regulate neurite extension, while RhoA controls growth cone collapse and neurite retraction (da Silva and Dotti 2002). However, until now the effects of Rho GTPases on neurite outgrowth have mostly been assessed using protein mutants in steady-state experiments, most often at late differentiation stages, which do not provide any insight about the different MSPs during neurite outgrowth. However, our proteomic analysis of biochemically-purified neurites from N1E-115 neuronal-like cells (Pertz et al. 2008), has suggested the existence of an unexpectedly complex 220 proteins signaling network consisting of multiple GEFs, GAPs, Rho GTPases, effectors and additional interactors. This is inconsistent with the simplistic view that classical experiments have provided before. In order to gain insight into the complexity of this Rho GTPase signaling network, we performed a siRNA screen that targets each of these 220 proteins individually. We hypothesized that specific spatio-temporal Rho GTPase signaling networks control different MSPs occurring during neurite outgrowth, and therefore designed an integrated approach to capture the whole morphodynamic continuum of this process. Perturbations of candidates that lead to a similar phenotype might be part of a given spatio-temporal signaling network. This approach consisted of: 1) A high content microscopy platform that allowed us to produce 8000 timelapse movies of 660 siRNA perturbations; 2) A custom built, computer vision approach that allowed us to automatically segment and track neurite and soma morphodynamics in the timelapse movies (collaboration with the group of Pascal Fua, EPFL, Lausanne); 3) A sophisticated statistical analysis pipeline that allowed the extraction of morphological and morphodynamic signatures (MDSs) relevant to each siRNA perturbation (collaboration with the group of Francois Fleuret, IDIAP). Analysis of our dataset revealed that each siRNA perturbation led to a quantifiable phenotype, emphasizing the quality of our proteomic dataset. Hierarchical clustering of the MDSs revealed the existence of 24 phenoclusters that provide information about neurite length, branching, number of neurites, soma migration speed, and a panel of additional morphological and morphodynamic features that are more difficult to grasp using visual inspection. This complex phenotypic space can more easily be understood when classified according to the first 4 features. Our screen then suggests the existence of 4 major morphodynamic phenotypes that define distinct stages of the neurite outgrowth process. These consist of phenotypes with short neurites, multiple short neurites, long neurites, and long and branched neurites. Further subdivision using the other features provides more information, with cell migration features being very often affected. This implies a high overlap between the signaling machinery that regulates the neurite outgrowth and cell migration processes. The high phenotypical redundancy (24 clusters for 220 candidate genes) provides only limited information to deduce unambiguous signaling networks regulating distinct MSPs. Further knowledge acquired from other approaches we used to study Rho GTPase signaling (FRET biosensors, and other live cell imaging techniques), made us realize that some morphodynamic phenotypes can only be understood when growth cone dynamics are inspected at a much higher resolution. For this purpose, we decided to further investigate a defined subset of genes using high resolution live cell imaging and a custom built growth cone segmentation and tracking pipeline for accurate quantification (collaboration with the group of Gaudenz Danuser, Harvard Medical School, Boston). These results shed light into how distinct cytoskeletal networks enabling growth cone advance can globally impact the neurite outgrowth process. A clear understanding of spatio-temporal Rho GTPase signaling will therefore require multi-scale approaches. Our results provide the first insight into the complexity of spatio-temporal Rho GTPase signaling during neurite outgrowth. The technologies we devised and our initial results, pave the way for a systems biology understanding of these complex signaling systems
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