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. 2023 Oct 11;3:e18. doi: 10.1017/S2633903X23000181

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© The Author(s) 2023

This is an Open Access article, distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives licence (http://creativecommons.org/licenses/by-nc-nd/4.0), which permits non-commercial re-use, distribution, and reproduction in any medium, provided that no alterations are made and the original article is properly cited. The written permission of Cambridge University Press must be obtained prior to any commercial use and/or adaptation of the article.

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Figure 1. — Illustration of the workflow to perform single particle tracking together with subsequent analysis of diffusion using TrackAnalyzer software which consists of several processes. (1) Extended Trajectory Analysis. (a) After Acquisition time series of multi-movie data sets. (b) Localization, detection, and subsequent identification of single particles frame by frame. A wide range of features is extracted based on the location, radius, and image data. (c) Single particles are linked to building trajectories over time (single-particle tracking). (2) Motion Type Analysis. The resulting trajectories and links are analyzed after the tracking step to characterize them and evaluate the type of motion by applying quantitative analysis of diffusion, mean square displacement (MSD), and moment scaling spectrum (MSS) slope. (3) Cluster Size Analysis. The number of receptors per spot is calculated by applying Gaussian Mixture Model fitting and Single-step Photobleaching Analyses. (4) Chemotaxis and Migration Analysis. Several quantitative and statistical features (center of mass, forward migration indices, velocity, …) are calculated to characterize trajectories. (5) Spot and Trajectory Filtering, (6) Manual Spot and Track Classification, and (7) Basic Statistical Analysis. Features extracted from spots and tracks will be used to either filter or classify them depending on user-defined conditions.