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. 2024 Sep 30;2:37. doi: 10.1038/s44303-024-00040-4

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — a Schematic of the HiLoTRUST system. b Workflow of the 3D imaging with HiLoTRUST, which contains six steps: (1) fluorescent staining with DAPI and PI for ~150 s, (2) widefield imaging with raster-scanning under speckle illumination by LD, (3) widefield imaging with raster-scanning under uniform illumination by two UV-LEDs, (4) shaving off the imaged layer with the vibratome to expose a layer underneath, (5) HiLo image processing, (6) 3D reconstruction by direct image stacking without image spatial registration. During imaging, the previous four steps will be repeated until the entire sample has been imaged and the whole procedure is fully automated with lab-built hardware and control programs. Note: TRUST and HiLoTRUST images have been normalized with min-max normalization for better visualization.