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. 2019 Dec 4;9:18295. doi: 10.1038/s41598-019-54244-5

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© The Author(s) 2019

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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Three-dimensional image collected from cleared kidney sample. (a) 3D rendering of an image volume collected from rat kidney tissue following clearing. Red – anti-vimentin immunofluorescence, Green – fluorescein-labeled Lens culinaris agglutinin, Blue – Hoechst 33342-labeled nuclei. A movie of the volume rendering is shown in Supplementary Video 1. (b) Single plane image from a subvolume of the image of nuclei that was used to derive the synthetic image volume. (c) Single plane image from the synthetic image volume obtained from volume shown in panel B. (d) Binary segmentation of the focal plane shown in panel c. (e) Screen capture of scatterplot from VTEA, in which the mean fluorescence intensity of fluorescein-Lens culinaris is plotted against the mean fluorescence intensity of an anti-vimentin antibody (x and y, respectively). Box indicates gate used to distinguish podocytes in panel f. (f) Screen-capture of image window from VTEA, showing segmented nuclei (blue) and gated podocytes (pink). Image volume shown in panel a is 256 microns across and 144 microns deep. Panels b, c and d represent an image field that is 32 microns across.