Fig. 1

High-throughput image analysis workflow to quantify CMT arrays in hypocotyl cells. Panels describe each step of the image analysis strategy including the statistical analysis of the data. A 3D view of a raw confocal z-stack from an Arabidopsis thaliana light-grown hypocotyl expressing the GFP-MBD microtubule reporter line. B “Angle2Ablation_Workflow_Toolset” highlighted within the tool menu of ImageJ. C, D Epidermal cell contours (C) and CMTs outer epidermal signal (D) extracted from the raw z-stack and projected in 2D using the SurfCut ImageJ macro. E Average projection image of several cell contour images aligned from the same sample timelapse using the “Cell Preproc” tool. F–H Description of the analysis process of the “ROI Maker” macro. F MorphoLibJ morphological segmentation of the cell contour image. Note that while not all cells are perfectly segmented, those surrounding the ablation and the ablation site are correctly segmented. G 2D image of the outer epidermal CMTs overlayed with the ROIs of each cell region to analyze surrounding the ablation (yellow) and the ablation ROI (red). H Image of the geometry-based proxy for the tensile stress pattern, overlayed with the cell contour image and cell ROIs for context. I–J FibrilTool quantification of I the CMT arrays signal and J the tensile stress proxy. K Image of the outer epidermal CMT arrays overlayed with FibrilTool visual representation (lines) of anisotropy and angle for the CMTs (magenta) and the tensile stress proxy (green) as well as the angle difference (yellow) calculated with the “A2A” macro. L–Q Output of the statistical analysis done with a python script. Plots describing the angle to ablation and anisotropy for each genotype (L, Q), for each sample in a given genotype (M, P) or for each cell in a given sample (N, Q). In L, M, O, and P, the data plotted are the mean and the bootstrapped 95% confidence interval. Scale bars are 50 µm