Extended Data Fig. 4: Granule segmentation and distance measurement.
From: Direct observation of translational activation by a ribonucleoprotein granule
a, Images of germ granules are segmented with the ilastik program. The top shows the original grayscale images of Vasa-mApple at the posterior pole of an embryo (left) and zoomed image of the outline region in the germplasm (right). The bottom shows the black-and-white binary images of segmented germ granules (granules in white). b, Schematic of the distance measurement program. The granule surface is defined after segmentation by ilastik. The coordinates of mRNA smFISH or scFv-GFP/anti-GCN4 spots are determined by FISH-Quant and used to measure the distance to the closest granule surface. The schematic is drawn in 2D but the actual data and measurement are in 3D. c-f, Control and validation experiments of distance measurement. (Left) representative images of germplasm with germ granules marked by Vasa-mApple (blue). Magenta: c suntag mRNA smFISH; d is the same image as c with mRNA channel rotated by 180° to shuffle the mRNA distribution; e has the same Vasa channel image as c with one million simulated points randomly distributed within the image; f smFISH of osk mRNA, which is not a component of germ granules. The distributions of mRNA foci or simulated points are plotted in the relative frequency histograms on the right. The x-axis refers to the distance of foci centroids to the border of the closest granule; the zero marks the granule border; a negative value denotes being inside a granule and a positive value denotes outside. Numbers of foci plotted: c, 415 foci from one embryo; d, 563 foci from one embryo; e, 249672 foci from one embryo; f, 8078 foci from four embryos. g, The distributions of mRNA foci or simulated points in (c-f) are plotted together as a kernel density estimate (KDE) plot. The distributions of shuffled mRNA, random points, and osk mRNA show a shift away from the surface of germ granules when compared to suntag-nanos mRNA. The ticks on the x-axis represent the mean values of the distributions.
