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. 2021 Feb 4;12:789. doi: 10.1038/s41467-020-20753-5

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© The Author(s) 2021

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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Fig. 4 — a Nalm6 cells were incubated with ¹²⁷I-dU for 30 min and then without a label for 30 min or 2 h, followed by a 30 min incubation with ⁸¹Br-dU. The endogenous DNA backbone was visualized in the ³¹P channel (blue) and replication sites were detected in the ¹²⁷I-dU (green) and ⁸¹Br-dU (red) channels. IBT images are shown from the 75^th slice to the 850^th slice of the cell. b Upper images: 3D visualization of ³¹P (blue), replication sites labeled with ¹²⁷I-dU (green), and replication sites labeled with ⁸¹Br-dU (magenta) across 1000 slices with a 30 min chase and 2 h chase. b1 The 3D spatial distributions across 100 tomographic slices of replication sites for Deconvolution-IBT and SILM-IBT are illustrated. b2 In the SILM-IBT, peaks were identified in a single replication site and shown as green circles for ¹²⁷I-dU replication, blue circles for phosphorus, and magenta circles for ⁸¹Br-dU replication. b3 Histogram of pairwise distances between the ¹²⁷I-dU replication peaks, yielding an average 170 nm separation with a wide range extending to 600 nm. b4 Peak counts per slice in the ¹²⁷I-dU channel across 40 slices. Lower images: 3D render of ³¹P (blue) across 800 slices. b5 In the middle and right images, the spatial distributions across 100 slices are presented for chromatin with³¹P (blue), replication sites labeled with ¹²⁷I-dU (green), and ⁸¹Br-dU (magenta) in the form of Deconvolution-IBT and SILM-IBT renders, respectively. b6 Peaks identified in the ⁸¹Br-dU channel overlaid with the original values of both ¹²⁷I-dU and ⁸¹Br-dU replication voxels. c Schematic of the shorter chase time (30 min) for the replication site, leading to higher spatial overlap than the longer chase. d Box plot of co-localization of ¹²⁷I-dU and ⁸¹Br-dU channels for the 2 h (long) and 30 min (short) chase experiments. Median, first and third quartile, and 95% confidence interval of the median are shown. Each data point corresponds to co-localization values per ion beam depth (n = 600 slices over a long IBT scan), providing a significant p-value < 2.2e-16 (***) by the Wilcox test (Two-sided).