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. 2018 Mar 9;8:4311. doi: 10.1038/s41598-018-22489-1

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© The Author(s) 2018

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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Intensify3D normalization of 2-Photon imaging data corrects and facilitates the reconstruction of CChIs (a). Orthogonal projections of 3D rendered image stacks before (left) and after (right) normalization. Images are shown together with mean (red) and median (green) relative fluorescent units values across all imaging planes. Note the homogeneous average and median intensity levels post-normalization. Horizontal and vertical scale bars represent 25 µm. For presentation purposes, the z dimension is smaller than x and y dimensions (b). Semi-automated reconstructions of the 2 CChIs (A and B) image stacks above, before and after normalization. Side boxes present the directions of view (c). Top view of the deeper portion (red and green frame) of reconstructed CChIs. Red arrows mark deep dendrites that were detected in the normalized, but not the pre-normalized reconstruction; orange arrow represents the reciprocal dendrites (d). Comparison of the diameter of deep to superficial (blue to red gradient represents Z depth) neurites as a function of distance from the soma. This analysis is based on the reconstructions of both CChIs. Notice that pre-normalization deep dendrites (black rectangles) seem smaller in diameter compared to superficial ones of similar somatic distance. In comparison, post-normalization dendrites show similar diameters to superficial dendrites of similar somatic distance. Illustration of lens and red excitation light illustrates the direction of illumination and decay of light as a function of depth.