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The authors demonstrate a new microscopy technique called SCAPE 2.0 (Swept, Confocally Aligned Planar Excitation) that can achieve high-resolution volumetric imaging at a high enough framerate to enable realtime imaging. The figures show the system imaging an mCUBIC6, cleared Thy1-GFP mouse brain that was sliced coronally and imaged from below in a glass-bottomed Petri dish. 18 adjacent, overlapping volume strips, 1.10 mm wide in y and 456 microns deep were acquired with 1 micron x-spacing at 520 fps and stitched and cropped into a ~8.37 × 9.06 × 0.385 mm volume with a sampling density of 1 × 1.37 × 1.14 μm x-y-z. Total acquisition took 242 seconds. Color depth-encoding demonstrates how SCAPE 2.0’s near-isotropic sampling enables clear tracing of axons and dendrites across large areas of the intact brain.

(e) Color-depth encoded top-down view of a 385 µm thick volume acquired on a coronal hemi-section of an mCUBIC-cleared Thy1-GFP brain using stage-scanning and stitching. Scale bar: 1 mm. (f) Zoomed view of subregion outlined in (e). (g) YZ plane maximum intensity projection, taken along the X direction of the subregion outlined in (f) demonstrating SCAPE 2.0’s near isotropic sampling and resolution