Supplementary Figure 11: SCAPE 2.0 depth-stitched image of a ~2mm thick expanded mouse spinal cord section.

a. 3D volume rendering (Imaris) of a dual color depth-stitched 2 x 1 x 1.8 mm (X, Y, Z) volume of a 4x expanded spinal cord slice from a ChAT-Tomato/Thy1-YFP double transgenic mouse expressing fluorescence in motor neurons. For better visualization of 3D details, magenta and green channels were gamma corrected with values of 1.1 and 1.3, respectively. The volume is composed of 11 successive volumes stitched together over a 1.8 mm depth range, with a total image acquisition time of 44 seconds. See Online Methods for experimental and analysis details, configuration Dz. Volumes were imaged from below through a glass-bottom dish, and acquired by moving the sample gradually closer to the primary objective lens (here a 20x 1.0 NA water immersion objective lens with a 2mm working distance). Top-down (X-Y) MIPs of 100 slices centered around depths b(i) 480µm and b(ii) 1690µm. c. A side-facing (Y-Z) MIP of 1000 slices in the same volume. See Supplementary Video 17 for visualization of 3D rendering. d. A 3D volume rendering (Imaris) of a 0.5 x 1 x 1.73 mm (X,Y,Z) region in the sample at slightly higher (~11.4x) magnification (configuration E_z) showing Thy1-YFP labeling with a total image acquisition time of 45 seconds. A gamma correction of 1.1 was applied for better 3D visualization. Top-down (X-Y) MIPs of 100 slices centered at depths e(i) 440 µm and e(ii) 1445 µm with insets showing zoomed in regions where spines, small processes and intracellular structures are clearly visible. White arrows indicate spines. Scale bars in b-c are 200 μm and in e are 100 µm. Note that the distance acquired along X is easily increased, and limited only by the range of the motorized stage. Additional 1mm wide volume strips along the Y direction can also be acquired and stitched together.