|
Fig. 2
Automated registration and alignment of 3D WISH images.
(A) Alignment accuracy of 3D registration algorithms was verified using 26 wild-type embryos stained with tryptophan hydroxylase 2 (tph2). Stained embryos are randomly divided into three groups and all groups are independently registered to the common reference. The overlay analysis shows two representative embryos from separate registration groups. The position and orientation of each 2D slice within the embryo is indicated on the Nissl-stained two photon reference image to the right. Anterior is to the top. (B) Registration accuracy was quantified by manually segmenting tph2 expression domains in all three independent groups and calculating the average distance between every border voxel in the segmentation. For the hindbrain expression domain in the raphe nuclei (ra), the intra-group registration accuracy is 3.5 μm and the inter-group accuracy is 4.0 µm. For the epiphysis (ep) expression domain, the intra-group accuracy is 2.3 μm and the inter-group accuracy is 2.5 μm. Box-and-whisker plots show results of intra- and inter-group border distance measurements. Tops and bottoms of each box represent the 25th and 75th percentiles of the samples, respectively. Whiskers are drawn from the ends of the interquartile ranges to the furthest observations that fall within ±1.5 times the interquartile range away from the top or bottom of the box. The line in the middle of each box is the sample median. Observations beyond the whisker length are marked as outliers (+ sign). (C) Virtual co-registration of vesicular monoamine transporter 2 (vmat2), tyrosine hydroxylase (th), and tryptophan hydroxylase 2 (tph2) in wild-type embryos at 2 dpf showing patterns of colocalization in the raphe nuclei and dopaminergic (DA) clusters. All 3D reconstructions are generated by averaging eight or more embryos per experimental group. For transverse planes (upper) dorsal is to the top; for frontal planes (lower) anterior is to the top. The position and orientation of each 2D slice within the embryo is indicated on the Nissl-stained two photon reference image to the right of the panel. Scale bar: 100 μm.