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. 2018 Jan 9;8:212. doi: 10.1038/s41598-017-18569-3

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© The Author(s) 2017

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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Flow chart and graphical user interface (GUI) of HybTrack. (a) Tracking is started by selecting the image and setting input parameters. The user needs to annotate particles to track in the first image frame. Based on the initial positions, the tracking algorithm proceeds to search for local maxima and calculates the sub-pixel coordinates. If the local maxima is not bright enough, a pop-up window appears for manual detection of the particle. If there are overlapping particles within the scan region, two options are provided, Manual selection or Linear motion. This process is repeated for all annotated particles and image frames. (b) GUI interface of HybTrack. After setting the parameters, tracking process is started, and the result is saved as a text file.