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. 2023 Sep 13;475(11):1283–1300. doi: 10.1007/s00424-023-02855-3

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© The Author(s) 2023

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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Fig. 6 — Comparison of ROI detectors. (A) ROIs detected by individual Ca²⁺ analysis tools. Highlighted areas contain fluorescence activity, similarly detected by all tested applications. Comparing ROIs of the magnified areas reveals segmentation differences, as well as differences in size among Ca²⁺ analysis tools. (B) Fluorescence profiles of magnified ROIs marked with red, yellow, and blue ellipses. Profiles obtained using CHIPS show an increase in background fluorescence over time. Especially the profiles obtained with CHIPS and CaSCaDe differ to those obtained with AQUA and MSparkles. AQuA and MSparkles performed a more accurate segmentation between the ROIs marked with red and yellow ellipses, which is reflected by the corresponding fluorescence profiles. (C) Direct comparison of fluorescence profiles marked by the red ellipse. Profiles are similar around the first event occurring between 90s and 180s. Profiles by CHIPS and CaSCaDe show a third peak and prolonged event, respectively. CHIPS and CaSCaDe show a second fluorescence event at around 270s. AQuA and MSparkles detected this as a separate event, located at the ROI highlighted in yellow