Figure 1.

Steps involved in the imaging, segmentation and reconstruction of fusiform cells and their synaptic features. (A) A block containing the DCN was cut in the transverse (coronal) plane to show the location of the fields selected for imaging in relation to the different layers and medio-lateral extent of the DCN. The block was then trimmed further into a smaller cube, and 500–600 slices were cut and sequentially imaged in each of six subfields (labeled 1–6). (B) The six image stacks were then stitched together into a montage that spanned much of the dorsoventral thickness of the DCN and included the molecular layer (ML), the fusiform cell layer (FCL) and the outermost part of the deep layer (DL). (C) In the segmentation step, features of interest (cell somata, dendrites, spines and synapses) were separately traced and color-coded in each slice. (D–F) The features of interest were merged along the Z-axis of the image stacks, resulting in the 3D reconstruction of the cell (D). A segment of an apical dendrite (E) and an axonal terminal contacting a spine (F) are shown at higher magnifications.