Defining a universal coordinate system for alignment of test and reference embryos.

<p>Green, <i>unc-130</i>::GFP segmentation. Red, mCherry segmentation. <b>A</b>) Raw segmented test embryo. <b>B</b>) Axes of the embryo determined using convex hull of mCherry segmentation. Long axis, orange. <b>C</b>) Using information in <b>B</b>, embryo is centered, aligned to the camera axis, and rescaled. <b>D-F</b>) An axis perpendicular to the long axis is defined by generating a vector (yellow arrow) pointing between clusters of <i>unc-130</i>::GFP expression (<b>D</b>), and projecting onto a plane perpendicular to the long axis (<b>E</b>). Solid olive-green arrow, perpendicularly projected vector. Dashed arrow, direction of projection. <b>F</b>) Raw overlays of <i>unc-130</i>::GFP segmentation of a test embryo (gray) and the reference (green), prior to establishing axes perpendicular to the long axis. This step uses a relaxed segmentation threshold. <b>G</b>) Overlay of same segmentation as in <b>F</b>, after rotation to align the projection vectors found in <b>E</b>. <b>H-I</b>) Fine correction for rotation transform in <b>G</b>. <b>H</b>) Segmentation as in <b>G</b>, with a tighter segmentation threshold, at a time point twenty minutes earlier. <b>I</b>) Point clouds from <b>H</b> are matched using CPD-register, to provide fine corrections.