Extended Data Fig. 1: Improved visualization of tissue architecture with STED microscopy in coCATS.

a, coCATS in adult mouse hippocampus (DG hilus) with labeling by in vivo microinjection into the lateral ventricle. Overviews and magnified views of the same region in confocal (top) and xy-STED mode (bottom) with lateral resolution increase. Tissue structure is visible more clearly in STED mode. For example, individual axons are discernible as fine rings only in STED mode. b, Line profile (width = 3 pixels) in a region of axon bundles as indicated by the lines in a, revealing modulation by individual axons in STED but not in confocal mode. c, Confocal and STED images of a single 40 nm Crimson bead with line profiles and full width at half maximum (FWHM). STED power was the same as in a. d, Single axial sections in the neuropil of an organotypic hippocampal slice with overviews and magnified views of the boxed region. Same region imaged in confocal (top) and z-STED mode (bottom). Resolution increase is stronger in the axial direction, yielding near-isotropic resolution. STED performance is high in the central ~10–15 µm of the axial range, for which correction of spherical aberrations was set by the objective’s correction collar. Decreasing STED performance above and below reflects the well-known sensitivity of the z-STED pattern to spherical aberration. e, Line profile (width = 3 pixels) as indicated in c. f, Confocal and STED images of a single 40 nm Crimson bead with corresponding line profiles. STED power was the same as in d. All data acquired with the same high-numerical aperture (NA = 1.35) silicone oil immersion objective. Comparison of confocal vs. STED performance in coCATS-labeled specimens was performed in n = 3 biological specimens. Imaging of fluorescent beads is representative of typical microscope performance and was acquired in one imaging session.