Extended Data Fig. 4: CCF registration and QC of reconstructed morphologies.

a, CCF registration workflow. Pipeline of 3D registration from fMOST image (subject) to average mouse brain template of CCFv3 (target). Numbers below each panel indicate the pixel sizes in the order of X*Y*Z. See Methods for explanation of each step. b, Workflow of post-processing process for QC of reconstruction SWC files: 1. automatic detection and correction of basic reconstruction errors including loops, gaps and incorrect node types. 2. Corrections are sent back for manual verification. 3. Automatic detection and correction of trifurcations, which are usually overlapping neurites, instead of branching points. 4. Refinement of SWC files, including pruning of over-traced terminals and shifting skeleton to fit the center of image signals. 5. Resampling of SWC to achieve evenly distributed nodes. 6. SWC registration to the standard CCFv3 mouse brain template. c, Examples of trifurcation before (middle) and after (right) correction (blue and red branches do not cross), and examples of refinement before and after pruning (lower left panels) and shifting (lower right panels). d, Refinement leads to more precisely defined axon termination. Upper, distribution of terminal relocation distance by pruning. Lower, radius-decay curve of terminal signals shows that after refinement the axon ends at a brighter spot (indicating a bouton) rather than tapering off. e, Examples of axon terminals that end with or without a bouton. We established a stringent QC process that includes ensuring the completeness of reconstructed morphologies. A conventional way to assess the completeness of axon labeling and reconstruction is whether an axon ends at a bouton, as indicated by an enlargement with more intense signal (see arrowheads in Extended Data Fig. 2), or gradually tapers off, the former suggesting a complete labeling²³. We implemented this assessment in our reconstruction refinement process to identify potential inaccuracies. In our final QC-passed reconstructions we found that the ratio between terminal axon branches with and without a terminal bouton was about 10:1, indicating a high degree of completeness of our reconstructed morphologies.