Computational protocol: Integration of contractile forces during tissue invagination

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Protocol publication

[…] Embryos were prepared for live imaging and were imaged using an Ultraview spinning disk confocal microscope equipped with a 63×/1.4 NA oil-immersion lens controlled with MetaMorph software (Universal Imaging). An N2 Micropoint laser (Photonics Instruments) tuned to 365 nm was focused on sqh-GFP structures on the ventral surface of the embryo to ablate actomyosin structures. Point ablations were performed by ablating isotropic sqh-GFP spots with a point ∼1 µm in diameter, which took ∼670 ms. Line ablations were performed by making nine sequential point ablations to make a 20-µm incision, which took ∼6.4 s. Z stacks were acquired immediately before and after ablation in order to measure displacement of myosin II structures upon release of tension. [...] Images presented were processed using ImageJ (http://rsb.info.nih.gov/ij/) and Photoshop CS (Adobe Systems, Inc.). A Gaussian smoothing filter with a radius of one pixel was used to reduce noise in published images. Myosin II images presented in all figures are maximum intensity z projections of the apical ∼5 µm of cells in the middle of the image. Because myosin II is almost entirely present on the apical surface of cells, these images represent a surface projection of the embryo. Images of cell outlines are z slices ∼2 µm below the apical surface.Image segmentation was performed using custom MATLAB (MathWorks) software. Raw images were bandpass filtered with effective cutoff wavelengths of ∼1.4 µm (low pass) and ∼16 µm (high pass). Images were then thresholded and skeletonized to reduce the width of the membranes to one pixel. Cells were then indexed and tracked based on the distance between cell centroids at subsequent time points. We manually removed cells with errors in the segmentation to ensure that all cells in the dataset were correctly identified. Aspect ratio and anisotropy were calculated by using the “regionprops” function in MATLAB to measure major axis length, minor axis length, and orientation for individual cells. We measured the intensity of cortical myosin II in individual cells using three-dimensional time-lapse videos that were ∼5 µm in depth. To separate cortical myosin II structures from the diffuse cytoplasmic staining, we smoothed sqh-GFP images using a Gaussian smoothing filter with a three-pixel kernel size, s = 0.5 pixels, and clipped intensity values three standard deviations above the mean. We then made maximum-intensity z projections of myosin II (averaging the two highest-intensity values) and integrated the intensity of all the pixels in a given cell. Data for apical area, myosin intensity, and anisotropy were smoothed using a Gaussian smoothing filter (σ = 18–24 s, three time points) to remove noise.Recoil of sqh-GFP structures after laser ablation was quantified using custom software in which myosin spots were hand selected and tracked from the pre-ablation frame to the post-ablation frame ().To measure the continuity of the supracellular meshwork in control-injected and twiRNAi embryos, we thresholded maximum-intensity projections of sqh-GFP images using the mean pixel intensity as a cutoff. We then used the “bwmorph” function in MATLAB to identify connected objects and identified the largest object at each time point in a time-lapse video. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster