To begin, acquire multiview images of early stage zebra fish embryos using a light sheet microscope. Once interest points are detected, select all the views, right click and choose Register using Interest Points. For checking how precisely registration has worked, select two consecutive views.
Go to the overlapping region and toggle between the two views to observe if the beads imaged from different angles are superimposed. After successfully registering, click on Save in the multi-view explorer window to save the updated XML file. Now open the bead.
XML file in a text editor of choice and copy the entire block under view registrations. Open the embryo. XML file and replace the view registrations block with the block copied from the beads file.
Then open the embryo. XML file in Big Stitcher and carefully check if the registration has worked successfully for the embryo. Repeat the same for the beads by checking the overlap of structures of interest in every two consecutive views.
To perform multi-view deconvolution, select all the views in the bead file, right click, and choose point spread functions and extract options. Proceed with the default point spread function sizes and click on OK.Then re-save the XML file. Next, open the embryo.
XML file and assign the point spread function for each view separately. Right click a view, click on Point Spread Functions, choose Assign, and select Advanced, followed by Assign new PSF to all selected views. Click on Browse, go to the XML file path and open the PSF folder.
In the selected view, choose the matching point spread function with the corresponding ID, and click on OK.After the point spread function has been assigned for all the views, right click and choose multi-view deconvolution. Select the bounding box as currently selected views. At 70%epiboly, embryos assumed horizontal, vertical or oblique orientations within the polymer tube with horizontal being the least represented, while the vertical and oblique positions were equally observed.
When samples were prepared before gastrulation, about 25%of the embryos exhibited horizontal orientation. The rest of the embryos exhibited various other orientations at the bud stage. However, many of them reoriented to the horizontal position during early somite formation.
A comparison of cellular scale information infused images showed no significant difference in nuclei information, but cell boundaries were better resolved in the 30 degree set. Compared to the original input images, the segmented images contained errors. The software either failed to detect a cell boundary or drew non-existent cell boundaries.
The number of errors drastically increased infused images obtained from imaging at 45 and 60 degree angular intervals compared to 30 degrees.
