The Ovuscule

We propose an active contour (a.k.a. snake) that takes the shape of an ellipse. Its evolution is driven by surface terms made of two contributions: the integral of the data over an inner ellipse, counterbalanced by the integral of the data over an outer elliptical shell. We iteratively adapt the active contour to maximize the contrast between the two domains, which results in a snake that seeks elliptical bright blobs. We provide analytic expressions for the gradient of the snake with respect to its defining parameters, which allows for the use of efficient optimizers. An important contribution here is the parameterization of the ellipse which we define in such a way that all parameters have equal importance; this creates a favorable landscape for the proceedings of the optimizer. We validate our construct with synthetic data and illustrate its use on real data as well

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication)

Differential human mesenchymal stem cell responses across multi-compartment scaffolds for tendon-bone regeneration

The TBJ is a unique anatomical zone which transmits high tensile loads from aligned, fibrous tendon to stiff bone. Injuries to the rotator cuff account for 4.5 million physician visits per year and an annual 250,000 surgeries in the United States alone. However, current surgical techniques do not provide regeneration at the tendon bone junction (TBJ) and the re-failure rate is extremely high (>90%). This thesis explores approaches to develop a porous collagen-glycosaminoglycan (CG) biomaterial containing overlapping patterns of structural and biomolecular properties to promote TBJ regeneration. Notably, we describe a scaffold containing a gradient interfacial zone between mineralized and non-mineralized CG scaffold compartments. Fabricated via lyophilization, we explore use of diffusion-based approaches to form the gradient interface. We also describe an approach to create an interdigitated interface, and showed composite elastic modulus and failure load increased with increasing interdigitation. Next, we examined cellular response after tensile strain across two multi-compartment scaffold variants, one containing only a mineral gradient (layered) and the other incorporating microstructural alignment characteristic of the native osteotendinous interface (osteotendinous). We found that layered scaffolds induce very little change in nuclear elongation (aspect ratio) or nuclear orientation of human mesenchymal stem cells, while the osteotendinous scaffolds induces an increase in nuclear aspect ratio and alignment with strain. Most notably, cell nuclei and actin fibers were more aligned and aspect ratio was increased at 0% strain in the non-mineralized compartment of the osteotendinous scaffold. This suggests that pore architecture alone was responsible for the cellular response. Finally, we demonstrated orthogonal approaches to both define the elastic modulus and patterned biomolecules (PDGF-BB for proliferation; BMP-2 for osteogenesis) on CG membranes. Using this approach to explore how biomolecular and biophysical cues may work synergistically to direct MSC fate, we saw an increase in proliferation with covalently-bound PDGF-BB, while BMP-2 did not impact proliferation. In the PDGF-BB patterned membranes, we saw that osteogenesis was positively correlated with stiffness but inversely correlated with proliferation. In the BMP-2 patterned scaffolds, osteogenesis was positively correlated stiffness and adipogenesis was inversely correlated with stiffness. These results suggest that mechanical and biomolecular cues play an integral role in cellular response. These platforms each contain the ability to spatially manipulate biomolecular and biophysical cues to elicit cellular response, and will eventually be integrated into a single construct to provide basic cues to enhance TBJ healing and regeneration after injury

Quantitative single-cell analysis of S. cerevisiae using a microfluidic live-cell imaging platform

Genome-wide manipulations and measurements have made huge progress over the last decades. In Saccharomyces cerevisiae, a well-studied eukaryotic model organism, homologous recombination allows for systematic deletion or alteration of a majority of its genes. Important products of these manipulation techniques are two libraries of modified strains: A deletion library consisting of all viable knockout mutants, and a GFP library in which 4159 proteins are successfully tagged with GFP. In addition, the development of a method that allows for the systematic construction of double mutants led to a virtually infinite number of potential strains of interest. These advancements in combinatorial biology need to be matched by methods of data measurement and analysis. In order to simultaneously observe the spatio-temporal dynamics of thousands of strains from the GFP library, Dénervaud et al. developed a microfluidic platform that allows for parallel imaging of 1152 strains in a single experiment. On this platform, strains can be grown and monitored in a controllable environment for several days, which results in the imaging of several millions of cells during one experiment. To objectively and quantitatively analyze this immense amount of information, we implemented an image analysis pipeline, which can extract experiment-wide information on single-cell protein abundance and subcellular localization. The construction of a supervised classifier to quantify localization information on a single cell level is a new approach and was invaluable to detect dynamic localization changes within the proteome. Using five different stress conditions, we gained insight into temporal changes of abundance and localization of multiple proteins. For example, we found that while localization changes can often be fast and transient, long-term response of a cell is usually enabled by changes in abundance. This shows a well-orchestrated response of a cell to external stimuli. To extend knowledge about cellular mechanisms, we used our microfluidic platform for two separate screens, combining GFP-reporter with additional deletion mutants. The advantage of our platform in comparison to more common approaches lies in its simultaneous measurement of fluorescence and phenotypic information on cell size and growth. For each deletion, we can quantify not only its influence onto the respective GFP-reporter under changing conditions, but also its effect on cell growth and size. We showed that it is advantageous to combine this information, as it allows pointing out possible underlying mechanisms of gene network regulations. In a first screen we investigated the behavior of several gene networks upon UV irradiation damage. We were able to show that four gene deletions influenced the localization of ribonucleotide-diphosphate reductase (Rnr4p). A second screen was designed to find genes that influence the induction of the galactose network. This screen uses more than 500 deletions of genes mostly related to chromatin in combination with two different reporter strains. A main focus of this study was the inheritance of memory during galactose reinduction. We found several previously unknown genes that potentially influence either induction or reinduction and were picked as candidates for further inheritance studies. Our microfluidic platform allows for unprecedented studies of proteomes in flux. [...

The Effect of Gradations in Mineral Content, Matrix Alignment, and Applied Strain on Human Mesenchymal Stem Cell Morphology within Collagen Biomaterials

The tendon-bone junction (TBJ) is a unique, mechanically dynamic, structurally graded anatomical zone which transmits tensile loads between tendon and bone. Current surgical repair techniques rely on mechanical fixation and can result in high re-failure rates. We have recently described a new class of collagen biomaterial that contains discrete mineralized and structurally aligned regions linked by a continuous interface to mimic the graded osteotendinous insertion. Here we report the combined influence of graded biomaterial environment and increasing levels of applied strain (0 – 20%) on MSC orientation and alignment. In osteotendinous scaffolds, which contain opposing gradients of mineral content and structural alignment characteristic of the native osteotendinous interface, MSC nuclear and actin alignment was initially dictated by the local pore architecture, while applied tensile strain enhanced cell alignment in the direction of strain. Comparatively, in layered scaffolds that did not contain any structural alignment cues, MSCs were randomly oriented in the unstrained condition, then became oriented in a direction perpendicular to applied strain. These findings provide an initial understanding of how scaffold architecture can provide significant, potentially competitive, feedback influencing MSC orientation under applied strain, and forms the basis for future tissue engineering efforts to regenerate the osteotendinous enthesis

A Fully Automated Approach to Segmentation of Irregularly Shaped Cellular Structures in EM Images

While there has been substantial progress in segmenting natural im-ages, state-of-the-art methods that perform well in such tasks unfortunately tend to underperform when confronted with the different challenges posed by electron microscope (EM) data. For example, in EM imagery of neural tissue, numerous cells and subcellular structures appear within a single image, they exhibit irregular shapes that cannot be easily modeled by standard techniques, and confusing textures clutter the background. We propose a fully automated approach that handles these challenges by using sophisticated cues that capture global shape and texture information, and by learning the specific appearance of object boundaries. We demonstrate that our approach significantly outperforms state-of-the-art techniques and closely matches the performance of human annotators