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. 2019 Apr 30;9:6644. doi: 10.1038/s41598-019-43137-2

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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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Processing for obtaining the initial threshold for different images of the stack with different high variation of intensity (PHAL layer). Starting from the red layer of the RGB image, the PWM_CURVE is estimated from its grayscale histogram. Then, candidate thresholds are evaluated as inflection points of the curve (red dotted lines). The standard deviation of detected objects intensity using candidate thresholds is calculated and the initial threshold is determined as the one with the lowest standard deviation. In the last column, the application of the initial threshold on the RGB image is shown.