Automatic Analysis of Moving Particles by Total Internal Reflection Fluorescence Microscopy Olga Nedzved 1,2 , Luhong Jin 3 , Alexander Nedzved 1,4(&) , Wanni Lin 3 , Sergey Ablameyko 1,4 , and Yingke Xu 3 1 Belarusian State University, Minsk, Belarus olga_nedzved@tut.by 2 Belarusian State Medical University, Minsk, Belarus 3 Department of Biomedical Engineering, MOE Key Laboratory of Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, China 4 United Institute of Informatics Problems, Minsk, Belarus Abstract. Using of TIRF microscopy videosequences allows to study the propagation of intracellular protein vesicles to the cell surface upon insulin stimulation with high spatio-temporal resolution. Traditional tracking algorithms make mistakes for overlapping points at the intersection trajectories. Therefore, a particle registration algorithm based on background correction has shown optimal results for detecting moving particles or particles of variable size. In this paper we test traditional tracking methods and check performance for tracking protein particle in cell from video sequence TIRFM microscopy. We test four algorithms for detection vesicles location: Laplacian of Gaussian (LoG), Dif- ference of Gaussian (DoG), Determinant of Hessian and algorithm on base continues brightness analysis. In our opinion for determination of position of protein vesicles the best algorithm is based on continues brightness analysis. There are many algorithms for tracking single objects and multitracking algo- rithms based on them. We used a function that takes a tracker type as input and creates a tracker object. We analyzed seven different multitracking algorithms with realization in OpenCV (BOOSTING, MIL, KCF, TLD, MEDIANFLOW, MOSSE, CSRT) and additional tracker on base of optical flow. In such tests, MEDIANFLOW tracker gives best results for predictable motion and small displacements. This tracker gives an error message in case of incorrect tracking, unlike other trackers that continue to work even the tracking failed. Keywords: Particles tracking Á TIRFM Á Super resolution microscopy 1 Introduction Many vital life activities occur in the cell level, such as cell signaling transduction or protein transport. Tracking and analyzing of subcellular structures are very important for biochemical and cell biology research. One of the problems of cell biochemistry is propagation of intracellular GLUT4 storage vesicles to the cell surface upon insulin stimulation. GLUT4 is facilitative © Springer Nature Switzerland AG 2019 S. V. Ablameyko et al. (Eds.): PRIP 2019, CCIS 1055, pp. 228–239, 2019. https://doi.org/10.1007/978-3-030-35430-5_19