PMC

(A) Regions displayed (cyan patches) in B, C and D. (B) Spinal projection of an early-born V2a neuron (dorsal MiV1). (i) Coronal view of a dorsal MiV1 neuron (white arrowhead) electroporated with a dextran dye (magenta) in the background of V2a neurons (green). Dotted circles indicate dorsal MiV1. The number near the white arrowhead indicates normalized dorso-ventral position of the labeled cell (see Materials and methods). (ii) Side view of the spinal projection from dorsal MiV1. (iii) Same as in (ii) but overlaid on V2a neurons shown in green. (iv-vii) Sagittal optical slices showing the projection (iv) Projection relative to spinal mnx1+ neurons. Primary motoneurons (PMN) are indicated with asterisks. Processes juxtaposed to the cell body of PMN are highlighted with filled arrows. (v) Projection relative to spinal islet1+ neurons. Secondary motoneurons (SMN) are indicated with asterisks. Processes close to the cell body of SMN are highlighted with filled arrows. (vi-vi’) Projection relative to spinal V2a neurons. (vi) Optical slice showing the processes. (vi’) Optical slice medial to vi. (vii-vii’) Projection relative to a backfilled multipolar commissural descending neuron (MCoD). (vii) Optical slice showing the projection. (vii’) Optical slice lateral to vii showing the location of MCoD. (viii-xi) Coronal views of the projection of dorsal MiV1 in the rostral spinal cord (viii, x) and in the middle spinal cord (ix, xi) with V2a neurons labeled in EGFP (viii, ix) or with islet+ neurons labeled in EGFP (x, xi). (C) Spinal projection of an intermediate V2a neuron (middle MiV1). Images are organized similarly to B. (vii) Circumferential ipsilateral descending neuron (CiD) is indicated with asterisks. (D) Spinal projection of a late-born V2a neuron (ventral MiV1). Images are organized similarly to B. (iv-iv’) Projection relative to spinal mnx+ neurons. (iv) Optical slice showing the processes. (iv’) Optical slice medial to iv. (v–v’) Projection relative to spinal islet+ neurons. (v) Optical slice showing the processes. (v’) Optical slice medial to v. (vi-vi’) Projection relative to spinal V2a neurons. (vi) Optical slice showing the processes. (vi’) Optical slice medial to v. (vii) Projection relative to a backfilled MCoD. (vii-xi) Coronal views are organized as in B. (E) Dorsal extent of axon innervation of V2a reticulospinal neurons in rhombomere 4. The dorsal extent of axon innervation is normalized to the thickness of spinal cord in dorsoventral axis. The main effect of hindbrain cell type (dorsal, middle, and ventral MiV1) was significant (p<0.001). (F) Somatic innervations of rhombomere 4 V2a reticulospinal neurons to spinal cell types. Percentage of cells showing somatic innervation to specific spinal cell types (PMN, SMN, vsx2, MCoD) are shown. The number of neurons showing innervation to a given class and the number of neurons examined are indicated at the bottom of each bar. The main effect of hindbrain cell type (dorsal, middle and ventral) was significant (p<0.001). The interaction of hindbrain cell type and spinal cell type was also significant (p<0.001).
10.7554/eLife.42135.024Figure 8—source data 1.Contains numerical data plotted in .

Projection (optical) and planar (film) PDD and CBP plots comparing the results of the three modalities, optical imaging, TPS dose prediction, and film measurement. a) Registered film (magenta/gray) and TPS (green) planar dose images, with colored line plots designating the physical locations of the subsequent plots (c-h); b) gamma index image between the registered film dose image and a TPS dose plane adjacent to the film; c) projection (optical) and planar (film) CBPs at 2.5cm phantom depth (green horizontal line); d) projection (optical) and planar (film) CBPs intersecting all three cavities at 5.5cm phantom depth (blue horizontal line); e) projection (optical) and planar (film) PDDs directly to the right of the metal phantom alignment pin (white line); f) projection (optical) and planar (film) PDDs 2mm to the left of the 3cm cavity (yellow line); g) projection (optical) and planar (film) PDDs 2mm to the left of the 2cm cavity, intersecting the metal pin (magenta line); f) projection (optical) and planar (film) PDDs 2mm to the left of the 1cm cavity (cyan line).

Dotted lines outline the neuropil area of the ganglion in all parts of the figure. A1. Volume-rendered view of LY dye-filled AGR with single projection, branching further into four sub-branches in the STG neuropil. The AGR soma is located in the stn outside the lower-left corner of the frame. Blend mode projection of 8 merged confocal image stacks, each consisting of 84 optical slices (acquired at resolution of 0.067µm x 0.067µm x 0.378µm). Scale bar is 30µm. A2 shows close-up of the boxed area in A1, showing the widened end of the AGR projection within the STG. Scale bar is 5µm. B1. Volume-rendered view of LY dye-filled AGR with single projection, branching further into two short sub-branches in the STG neuropil. The AGR soma is located in the stn outside the lower-left corner of the frame. Blend mode projection of 4 merged confocal image stacks, each consisting of 226 optical slices (acquired at resolution of 0.174µm x 0.174µm x 0.294µm). Scale bar is 30µm. B2 shows a volume-rendered surface projection of the boxed area in B1 from a different angle, emphasizing the big balloon-like widening (not the soma) of the axonal AGR projection. Scale bar is 10µm. C. Volume-rendered view of LY dye-filled AGR with single projection, branching further into two sub-branches in the STG neuropil. The AGR soma is located in the stn outside the lower-left corner of the frame. Blend mode projection of 5 merged confocal image stacks, each consisting of 169 optical slices (acquired at resolution of 0.068µm x 0.068µm x 0.462µm). Scale bar is 30µm. D. Volume-rendered view of LY dye-filled AGR with two simple projections in the STG neuropil. The AGR soma is located in the stn outside the lower left corner of the frame. Blend mode projection of 18 merged confocal image stacks, each consisting of 115 optical slices (acquired at resolution of 0.183µm x 0.183µm x 0.504µm). Scale bar is 30µm. E. Volume-rendered view of LY dye-filled AGR with two projections, each branching further into two or three sub-branches in the STG neuropil. The AGR soma is located in the stn outside the lower-left corner of the frame. Blend mode projection of 10 merged confocal image stacks, each consisting of 264 optical slices (acquired at resolution of 0.179µm x 0.179µm x 0.38µm), ventral view of same preparation as . Scale bar is 30µm. F. Volume-rendered view of LY dye-filled AGR with three projections, one of them branching further into two sub-branches in the neuropil. The AGR soma is located in the stn outside the lower-left corner of the frame. Blend mode projection of 9 merged confocal image stacks, each consisting of 229 optical slices (acquired at resolution of 0.168µm x 0.168µm x 0.252µm). Scale bar is 50µm.

(a) The 3D CAD model of projection optics along with the optical parameters (this figure was created by SOLIDWORKS 2016 (www.solidworks.com)). (b) The 3D CAD optical design of the projection optics. In this design, the ray tracing (optical paths) of five optical fields are shown (Optical design was done by Zemax OpticStudio 2016 (www.zemax.com), and then 3D CAD optical model imported to SOLIDWORKS for creating the figure). (c) The PSF function and the 2D cross-section of PSF for the designed projection optics (this figure was created by Origin 2017 (www.originlab.com)).