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. 2023 Aug 31;42(7):1051–1064. doi: 10.1038/s41587-023-01911-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a, Volumetric STED dataset in the mouse hippocampal CA3 stratum lucidum recorded at near-isotropic resolution after in vivo microinjection of coCATS label (gray, STAR RED-NHS) into the lateral ventricle and transcardial fixative perfusion. Glial fibrillary acidic protein (GFAP, magenta), an intermediate filament expressed in astrocytes, was immunolabeled (AF594) and imaged with near-isotropic STED. Data was denoised with N2V. Arrowheads at image edges indicate position of corresponding orthogonal sections. b, 3D-renderings of manual segmentations of an astrocyte in the imaging volume in a, viewed from three different perspectives. Manual segmentation of coCATS data was first performed blinded (magenta) to GFAP. Then the GFAP data was provided to the segmenter, allowing to assign additional structures (violet) in the CATS channel, in particular fine protrusions, to that cell. Note that segmentation was still based on the coCATS channel, as intermediate filaments do not fully trace out cell shape. coCATS in vivo microinjection into the lateral ventricle was performed in n = 10 biological specimens. Astrocyte 3D-reconstruction was performed once.