Advanced fluorescence imaging techniques such as single-molecule localization microscopy (SMLM) fundamentally rely on the photophysical behavior of the employed fluorophores. This behavior is generally complex and impacts data quality in a subtle manner. SMIS (Single-Molecule Imaging Simulator) is a simulation software that simulates a widefield microscope and incorporates fluorophores with their spectral and photophysical properties. With SMIS, data collection schemes combining 3D, multicolor, single-particle-tracking or quantitative SMLM can be implemented. The influence of advanced fluorophore characteristics, imaging conditions and environmental parameters can be evaluated. In this workshop, participants will learn how to use SMIS. This will help them to design real experiments and to properly interpret them. The workshop stands at the frontier between 3 modules: Labeling strategies and probes; Nanoscale quantification; Molecular dynamics and interactions. Reference: Bourgeois, D. Single molecule imaging simulations with advanced fluorophore photophysics. Commun Biol 6, 53 (2023). https://doi.org/10.1038/s42003-023-04432-x