Single Molecule Imaging Simulations with Full Fluorophore Photophysics

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. A simulation software named SMIS (Single-Molecule Imaging Simulator) is introduced that simulates a widefield microscope and incorporates fluorophores with arbitrarily complex photophysical properties. With SMIS, intricate data collection schemes combining 3D, multicolor, single-particle-tracking or quantitative SMLM can be implemented virtually and the influence of imaging and environmental parameters evaluated. ### Competing Interest Statement The authors have declared no competing interest.