Outline of steps involved in embryo orientation.

<p>(<b>A</b>) Extended depth image of 6 dpi ZF embryo. (<b>B</b>) Grey value image from combination of green and red channels. (<b>C</b>) Blurred grey image after applying closing filter to optimize determination of outline. (<b>D</b>) Embryo segmented after applying intensity threshold and area filter. Arrowhead indicates a red object outside the outline that is excluded from segmentation. (<b>E</b>) Cropped image with only selected object. (<b>F</b>) Embryo rotated by x° for horizontal reorientation. (<b>G</b> and <b>H</b>) Determination of the x position value of the center of mass (<i>cm</i>) and center of centroid (<i>cc</i>). (<b>I</b>) Horizontal flip of the image only if <i>cm</i> is on the left side of <i>cc</i>, resulting in images with the head of the embryo always to the right side. (<b>J</b>) Image after applying closed filter to the combined green and red channel to get the outline of the embryo. Point lying at 75% distance from the extreme left of the embryo outline is calculated. Y-axis is drawn at this X-position from upper to lower outline. Upper rectangle 1 is drawn. (<b>K</b>) Lower rectangle 2 is drawn. (<b>L</b>) Vertical flip of the image only if red intensity in rectangle 1 is higher than in rectangle 2. (<b>M</b>) Schematic representation of calculations for steps <b>E–I</b>. Altogether, this procedure results in images where the head is on the right and the yolk sac is on the bottom of the image. Scale bar = 200 µm in <b>E</b> and <b>I</b>.