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. 2021 Oct 21;11:20782. doi: 10.1038/s41598-021-00301-x

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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 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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Increasing number of scans average per line. (a) Representative images of U937-PR9 cells upon staining of DNA replication foci. Each image is obtained by combining and averaging STED images after each acquisition step of increasing number of line-averages (see “Methods” for a detailed description of acquisition settings) Scale bar represents 3 µm. (b–d) Quantification of Resolution, Brightness and Noise parameters in function of the number of line-averages by application of the QuICS algorithm. At least ten images for each condition have been acquired. (e) Quantification of Photobleaching in function of the number of line-averages. Photobleaching was calculated as the percentage reduction of average fluorescence intensity with respect to the initial value. (f) Representation of the Brightness variation in function of the Photobleaching. At least ten images for each condition have been quantified. Error bars represent the SEM and ***p < 0.001, **p < 0.01, *p < 0.05.