Generation of simulated data To systematically evaluate the performance of Spot-On as well as other common analysis tools such as MSDi and vbSPT, we considered a comprehensive set of 3480 realistic SPT simulations spanning the range of plausible dynamics. The simulations were performed using simSPT, which is freely available at GitLab: https://gitlab.com/tjian-darzacq-lab/simSPT. The simulation methods are described in detail at GitLab. A full description of the parameters which allows exact reproduction of the simulations is available together with the data (see Data Availability section). Briefly, we parameterized simSPT to consider that particles diffuse inside a sphere (the nucleus) of 8 µm diameter illuminated using HiLo illumination (assuming a HiLo beam width of 4 µm), with an axial detection range of ~700 nm, centered at the middle of the HiLo beam. Molecules are assumed to have a half-life of 4 frames (when inside the HiLo beam) and of 40 frames when outside the HiLo beam. The localization error was set to 25 nm and the simulation was run until 100000 in-focus trajectories were recorded. More specifically, the effect of the exposure time (1 ms, 4 ms, 7 ms, 13 ms, 20 ms), the free diffusion constant (from 0.5 µm²/s to 14.5 µm²/s in 0.5 µm²/s increments) and the fraction bound (from 0 % to 95 % in 5 % increments) were investigated, yielding a dataset consisting of 3480 simulations. The advantage of simulations is that the ground truth is known. This allows a quantitative assessment of which method works the best. Content of the archives: 170718_simSPT_simulations.zip the code and instructions to reproduce the simulations 4um.tar.bz2 simulated data inside a 4 µm nucleus 20um.tar.bz2 simulated data inside a 20 µm nucleus, in which virtually no confinement occurs. subsampled.tar.bz2 is a set of subsampled datasets, containing either 99999, 30000, 10000, 3000, 1000, 300, 100 or 30 trajectories. Each subsampling was done 50 times, yielding 50 files per subsmpling. Formats: The data is provided both in CSV and .mat formats. .mat files are provided in the following dataset: 10.5281/zenodo.835541