Non-rigid Image Registration for Zebrafish Neuroscience

Non-rigid image registration was performed using the Computational Morphometry Toolkit (CMTK, http://www.nitrc.org/projects/cmtk/), and user interface written by Jefferis and colleagues⁷² with the command string (-awr 010203 -T 8 -X 52 -C 8 -G 80 -R 3 -A '--accuracy 0.4' -W '--accuracy 1.6'). This setup performed registrations at <15min/fish on a 2 × 3.2 GHz Quad-Core Mac Pro (Apple). Our template brain is a 6dpf nacre mutant (mifta−/−) larvae⁷³ , stained with anti-tERK. Staining the fish to be registered with anti-tERK allows for direct registrations to the template. However, in cases where another anti-Mouse IgG1 antibody was used as a cell-type label, anti-tERK could not be easily co-stained. In these cases fish were indirectly registered into the template brain by staining with anti-Syt2/Znp1 and using the anti-Znp1 mean-stack as a template. To register the live 2-photon calcium imaging data during right/left OMR stimulation (Fig. 3D) we used a single registered Tg(Elavl3:GCaMP5G) fish as the template, and command string (-awr 010203 -T 2 -X 52 -C 8 -G 80 -R 4 -A '--accuracy 0.4' -W '--accuracy 0.8'). To register in the live-imaging 2-photon data of Tg(Elavl3:GCaMP5G) fish and the post-fixation data from the same fish to re-identify cells (Supplementary Fig. 2), the GCaMP5G channel imaged by confocal microscopy after fixation was registered into the anatomy stack taken live by 2-photon microscope was registered using command string (-awr 010203 -T 4 -X 200 -C 4 -G 160 -R 5 -A '--accuracy 0.4' -W '--accuracy 0.4')