ImageJ SurfCut: a user-friendly pipeline for high-throughput extraction of cell contours from 3D image stacks

Background: Many methods have been developed to quantify cell shape in 2D in tissues. For instance, the analysis of epithelial cells in Drosophila embryogenesis or jigsaw puzzle-shaped pavement cells in plant epidermis has led to the development of numerous quantification methods that are applied to 2D images. However, proper extraction of 2D cell contours from 3D confocal stacks for such analysis can be problematic. Results: We developed a macro in ImageJ, SurfCut, with the goal to provide a user-friendly pipeline specifically designed to extract epidermal cell contour signals, segment cells in 2D and analyze cell shape. As a reference point, we compared our output to that obtained with MorphoGraphX (MGX). While both methods differ in the approach used to extract the layer of signal, they output comparable results for tissues with shallow curvature, such as pavement cell shape in cotyledon epidermis (as quantified with PaCeQuant). SurfCut was however not appropriate for cell or tissue samples with high curvature, as evidenced by a significant bias in shape and area quantification. Conclusion: We provide a new ImageJ pipeline, SurfCut, that allows the extraction of cell contours from 3D confocal stacks. SurfCut and MGX have complementary advantages: MGX is well suited for curvy samples and more complex analyses, up to computational cell-based modeling on real templates; SurfCut is well suited for rather flat samples, is simple to use, and has the advantage to be easily automated for batch analysis of images in ImageJ. The combination of these two methods thus provides an ideal suite of tools for cell contour extraction in most biological samples, whether 3D precision or high-throughput analysis is the main priority

Mechanical control of morphogenesis at the shoot apex

Morphogenesis does not just require the correct expression of patterning genes; these genes must induce the precise mechanical changes necessary to produce a new form. Mechanical characterization of plant growth is not new; however, in recent years, new technologies and interdisciplinary collaborations have made it feasible in young tissues such as the shoot apex. Analysis of tissues where active growth and developmental patterning are taking place has revealed biologically significant variability in mechanical properties and has even suggested that mechanical changes in the tissue can feed back to direct morphogenesis. Here, an overview is given of the current understanding of the mechanical dynamics and its influence on cellular and developmental processes in the shoot apex. We are only starting to uncover the mechanical basis of morphogenesis, and many exciting questions remain to be answere

Accurate and versatile 3D segmentation of plant tissues at cellular resolution

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface

Rôle des contraintes mécaniques dans l'orientation du plan de division des cellules du méristème apical caulinaire d'Arabidopsis thaliana

Morphogenesis during primary plant growth is driven by cell division and elongation. In turn, growth generates mechanical stress, which impacts cellular events and channels morphogenesis. Mechanical stress impacts the orientation of division plane in single animal cells; this remains to be fully demonstrated in plants. Currently, cell geometry is proposed to be the main factor determining plane orientation in symmetric divisions: cell divide along one the shortest paths. This geometrical rule was tested on tissues with rather isotropic shapes or growth and the corresponding molecular mechanism remains unknown, although it could involve tension within the cytoskeleton. To address these shortcomings, we developed a pipeline to analyze cell divisions in the different domains of the shoot apical meristem of Arabidopsis thaliana. We computed the probability of each possible planes according to cell geometry and compared the output to observed orientations. A quarter of the cells did not follow the geometrical rule. Boundary domain was enriched in long planes aligned with supracellular maximal tension lines. Computer simulations of a growing tissue following a division rule that relies on tension gave the most realistic outputs. Mechanical perturbations of local stress pattern, by laser ablations, further confirmed the importance of mechanical stress in cell division. To explore the role of microtubules in this process, we developed a microindenter-based protocol to quantify the cytoskeletal response to mechanical stress. This protocol was tested and validated in the katanin and spiral2 mutants, in which the response to stress is delayed or promoted respectively.La morphogenèse des plantes repose sur deux mécanismes cellulaires : la division et l'élongation. Par ailleurs, la croissance est source de contraintes mécaniques qui affectent les cellules et guident la morphogenèse. Si les contraintes mécaniques influencent l'orientation du plan de division dans les cellules animales, rien n'est prouvé pour les cellules végétales. À l'heure actuelle, la forme de la cellule est proposée comme le facteur principal gouvernant l'orientation du plan dans les divisions symétriques : les cellules se divisent selon un des plans les plus courts. Cette règle géométrique a été validée dans des tissus à croissance ou courbure isotropes, mais les mécanismes moléculaires sous-jacents demeurent inconnus. Dans cette thèse, un pipeline a été mis au point pour analyser les divisions cellulaires dans les différents domaines du méristème apical caulinaire d'Arabidopsis thaliana et questionner l'application de la règle géométrique dans ce tissu. La zone frontière du méristème présente une proportion anormalement basse de plans de division très courts. Des simulations de tissus en croissance, dans lesquelles une règle de division mécanique a été implémentée, ont montrées le même biais sur les orientation des plans, comparé à la règle géométrique. Des ablations laser de quelques cellules de l'épiderme ont également été effectuées afin de perturber localement le patron de contraintes mécaniques. Les résultats montrent que l'orientation du plan des divisions postérieures à cette perturbation suit le nouveau patron de contraintes. Enfin, une nouvelle méthode quantitative, basée sur l'utilisation d'un micro-indenteur, a été mise au point pour quantifier la réponse du cytosquelette, et en particulier des microtubules, aux contraintes mécaniques. Le protocole de compression a été testé et validé sur les mutants katanin et spiral2, dans lesquels la réponse aux contraintes est respectivement faible ou amplifiée

The mechanics behind cell division

International audienceIt is now well established that the orientation of the plane of cell division highly depends on cell geometry in plants. However, the related molecular mechanism remains largely unknown. Recent data in animal systems highlight the role of the cytoskeleton response to mechanical stress in this process. Interestingly, these results are consistent with some data obtained in parallel in plants. Here we review and confront these studies, across kingdoms, and we explore the possibility that the intrinsic mechanical properties of the cytoskeleton play a key role in the nexus between cell division and mechanical stress. This opens many avenues for future research that are also discussed in this review

Time Domain Channel Estimation in OFDM Systems Using limited number of Pilot Tones

This paper deals with the time domain estimation of OFDM channels in the case of a limited number of pilot tones. In such situations, time domain estimation suffers from an ill conditioned sample matrix which render the Least Square (LS) algorithm non efficient. To improve the estimation procedure, two algorithms are proposed. The first algorithm improves the conditioning of the LS matrix by adding a corrective term or penalization factor to the LS pseudo inverse matrix. The second algorithm tries to partially remove the effect of the penalization factor iteratively. Computational results are provided for the case of crosstalk channel estimation in DSL system these results show the effectiveness of the different methods

ImageJ SurfCut: a user-friendly pipeline for high-throughput extraction of cell contours from 3D image stacks

BackgroundMany methods have been developed to quantify cell shape in 2D in tissues. For instance, the analysis of epithelial cells in Drosophila embryogenesis or jigsaw puzzle-shaped pavement cells in plant epidermis has led to the development of numerous quantification methods that are applied to 2D images. However, proper extraction of 2D cell contours from 3D confocal stacks for such analysis can be problematic.ResultsWe developed a macro in ImageJ, SurfCut, with the goal to provide a user-friendly pipeline specifically designed to extract epidermal cell contour signals, segment cells in 2D and analyze cell shape. As a reference point, we compared our output to that obtained with MorphoGraphX (MGX). While both methods differ in the approach used to extract the layer of signal, they output comparable results for tissues with shallow curvature, such as pavement cell shape in cotyledon epidermis (as quantified with PaCeQuant). SurfCut was however not appropriate for cell or tissue samples with high curvature, as evidenced by a significant bias in shape and area quantification.ConclusionWe provide a new ImageJ pipeline, SurfCut, that allows the extraction of cell contours from 3D confocal stacks. SurfCut and MGX have complementary advantages: MGX is well suited for curvy samples and more complex analyses, up to computational cell-based modeling on real templates; SurfCut is well suited for rather flat samples, is simple to use, and has the advantage to be easily automated for batch analysis of images in ImageJ. The combination of these two methods thus provides an ideal suite of tools for cell contour extraction in most biological samples, whether 3D precision or high-throughput analysis is the main priority

Mechanically, the shoot apical meristem of Arabidopsis behaves like a shell inflated by a pressure of about 1 MPa

International audienceIn plants, the shoot apical meristem contains the stem cells and is responsible for the generation of all aerial organs. Mechanistically, organogenesis is associated with an auxin-dependent local softening of the epidermis. This has been proposed to be sufficient to trigger outgrowth, because the epidermis is thought to be under tension and stiffer than internal tissues in all the aerial part of the plant. However, this has not been directly demonstrated in the shoot apical meristem. Here we tested this hypothesis in Arabidopsis using indentation methods and modeling. We considered two possible scenarios: either the epidermis does not have unique properties and the meristem behaves as a homogeneous linearly-elastic tissue, or the epidermis is under tension and the meristem exhibits the response of a shell under pressure. Large indentation depths measurements with a large tip (size of the meristem) were consistent with a shell-like behavior. This also allowed us to deduce a value of turgor pressure, estimated at 0.82 +/- 0.16 MPa. Indentation with atomic force microscopy provided local measurements of pressure in the epidermis, further confirming the range of values obtained from large deformations. Altogether, our data demonstrate that the Arabidopsis shoot apical meristem behaves like a shell under a MPa range pressure and support a key role for the epidermis in shaping the shoot apex