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. 2021 Dec 28;102(5):545–553. doi: 10.1038/s41374-021-00714-2

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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. 6 — Monochrome images of transmitted light at 513, 620, 390, and 770 nm where eosin (A), hematoxylin (B), HCC CDC (D), and Cy7 (E) primarily absorb light, respectively, staining the two components of the H&E stain (A & B), CD3 (D) and CD8 (E). Spectrally unmixed images of CD3 (G), CD8 (H), and eosin (I). C Color composite image formed from the eosin (A) and hematoxylin (B) monochrome images. F Color composite image formed from the CD3 image (D), pseudo-colored magenta, and the CD8 image (E), pseudo-colored cyan. Dashed circles in (D) and (G) emphasize regions with faint hematoxylin crosstalk (D) that is removed after unmixing (G). Images were recorded using a ×20 objective.