Waterpixels

International audience— Many approaches for image segmentation rely on a 1 first low-level segmentation step, where an image is partitioned 2 into homogeneous regions with enforced regularity and adherence 3 to object boundaries. Methods to generate these superpixels have 4 gained substantial interest in the last few years, but only a few 5 have made it into applications in practice, in particular because 6 the requirements on the processing time are essential but are not 7 met by most of them. Here, we propose waterpixels as a general 8 strategy for generating superpixels which relies on the marker 9 controlled watershed transformation. We introduce a spatially 10 regularized gradient to achieve a tunable tradeoff between the 11 superpixel regularity and the adherence to object boundaries. 12 The complexity of the resulting methods is linear with respect 13 to the number of image pixels. We quantitatively evaluate our 14 approach on the Berkeley segmentation database and compare 15 it against the state-of-the-art

Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics

International audienceThe assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects

Second Harmonic Generation imaging of collagen fibrillogenesis

International audienceDevelopment of nonlinear optical microscopy has significantly improved three-dimensional (3D) imaging of biological tissues in recent years. In particular, collagen has been shown to exhibit endogenous Second Harmonic Generation (SHG) signals and SHG microscopy has proved to enable the visualization of collagen architecture in tissues with unequalled contrast and specificity [1, 2]. Type I collagen is a major structural protein in mammals and shows highly structured macromolecular organizations specific to each tissue. It is synthesized by cells as triple helices, which self-assemble outside the cells into fibrils that further form fibers, lamellae or other three-dimensional (3D) networks. This assembly mechanism depends critically on the collagen concentration, as well as on the temperature, pH and ionic strength of the solution in vitro. Thorough characterization of collagen fibrillogenesis is crucial to understand the biological mechanisms of tissue formation and tissue remodeling in response to a variety of pathologies. Booming of tissue engineering furthermore requires advanced in situ quantitative imaging techniques to verify whether the tissue substitutes display appropriate biomimetic 3D organization for cell culture scaffolds or functional implants. In this study, we continuously monitored the formation of collagen fibrils by time-lapse SHG microscopy [3]. Fibrillogenesis was triggered in a controlled way by increasing the pH in a dilute solution of collagen I. The fibril density was measured every 10 to 20 minutes as the number of voxels with significant SHG signal in 3D image stacks [1]. Our results showed reproducible dynamics of fibrillar collagen formation that could be changed by tuning the pH (see figure 1). We also monitored the growth of single fibrils and measured the length increase over time, which had never been reported before using an optical technique. We then correlated these SHG images to TEM images at nanometer-scale resolution by blocking the fibrillogenesis at early stages and drying the samples. It showed that SHG microscopy allows imaging of fibrils with a diameter down to 30-50 nm in our experimental conditions. We finally investigated surface-mediated fibrillogenesis by adding silica nanoparticles to the solution [4]. We used Two-Photon excited fluorescence (2PEF) microscopy to visualize the fluorescently-died nanoparticles and quantify the self-assembly of collagen around these nanoparticles. In conclusion, SHG microscopy enabled sensitive and well contrasted 3D visualization of collagen fibrillogenesis in a non invasive way. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials. 0 200 400 600 800 0.0 0.5 1.0 1.5 2.0 2.5 Pixel fraction (%) Time (min) (d) (a) (c) (b) Fig 1. 3D reconstruction of SHG images of collagen fibrillogenesis (at pH=6.5) after a) 170 b) 410 and c) 730 minutes; d) Experimental kinetics of fibril density in the SHG images (black dots) with exponential fitting (red line). References [1] M

Waterpixels: Superpixels based on the watershed transformation

International audienceMany sophisticated segmentation algorithms rely on a first low-level segmentation step where an image is partitioned into homogeneous regions with enforced compactness and adherence to object boundaries. These regions are called " su-perpixels ". While the marker controlled watershed transformation should in principle be well suited for this type of application , it has never been seriously tested in this setup, and comparisons to other methods were not made with the best possible settings. Here, we provide a scheme for applying the watershed transform for superpixel generation, where we use a spatially regularized gradient to achieve a tunable trade-off between superpixel regularity and adherence to object boundaries. We quantitatively evaluate our method on the Berkeley segmenta-tion database and show that we achieve comparable results to a previously published state-of-the art algorithm, while avoiding some of the arbitrary postprocessing steps the latter requires

Spatial Repulsion Between Markers Improves Watershed Performance

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