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. 2022 Oct 28;12:18196. doi: 10.1038/s41598-022-23091-2

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© The Author(s) 2022

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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Photomicrograph manipulation can easily change percent coverage and full photomicrograph skeletal analysis results. (a) Example of a low-quality image. (b) Low-quality photomicrograph displayed in binary with no manipulation. Because of high background staining, microglia are overrepresented as a percent coverage of Iba1 staining. (c) When the threshold in B is adjusted to remove some background, some processes on microglial cells are lost and the cells appear less ramified. (d) A highly ramified microglial cell with 5.6% percent coverage. (e) A sparsely ramified microglial cell with 5.6% percent coverage.