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. 2020 Dec 11;10:21774. doi: 10.1038/s41598-020-78640-4

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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 licence, and indicate if changes were made. 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/4.0/.

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CUBIC Acid Fast (CAF) staining. CUBIC Acid Fast staining procedure demonstrated in (A) J774 macrophages infected with or without BCG, and (B) in a single CLSM optical section of Mtb infected tissue with a non-thresholded image presented. Multiple thresholded optical sections are then constructed into a Z stack (2 µm step size), with a maximum intensity projection presented (C) with the AurO/RhodB (yellow) and DAPI (blue) signals visible. (D) shows the isosurfaces generated from the AurO/RhodB signal (turquoise) and the DAPI signal (grey), and (E) illustrates just the AurO/RhodB signal which the volumes of the bacterial aggregates can be measured, as demonstrated in (F) with colony volume colour coded (in µm³). Scale bar = 50 µm (A, B) and 100 µm (C–F).