The main purpose of these simulations was the generation of artificial line-scan imaging data with known underlying ground truth. To this end, we integrated the equations of motion and then, at stroboscopic time steps corresponding to the experimental scanning frequency 1,800 Hz = 1/0.556 ms, computed the contribution of every particle to the intensity measured at a specific pixel (with a pixel size of 50 nm) by convolution with a Gaussian PSF (lateral beam waist ω0 = 0.33 µm and axial beam waist ωz = 1.12 µm) (SI Appendix, Fig. S3A). This way, we generate series of pixel graphics (one line per time step, 1,000 lines per image) (SI Appendix, Fig. S3A) suitable for the same analysis as the experimental data with full control about the dynamics of the particles and the geometry they are moving on (composed of prototypical structures such as tubules and planar membranes) as well as the relative orientation and location of the imaging apparatus (SI Appendix, Fig. S3A).

This software has been written in C++ (using Libtiff as well as the gnu scientific library) and is available on GitLab (see Data Availability). Further details on the implementation of these simulations can be found in SI Appendix, Supplementary Materials and Methods.

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