Interactive simulation and visualization of point spread functions in single molecule imaging.

The point spread function (PSF) is fundamental to any type of microscopy, most importantly so for single-molecule localization techniques, where the exact PSF shape is crucial for precise molecule localization at the nanoscale. Optical aberrations and fixed fluorophore dipoles often result in non-isotropic and distorted PSFs, impairing and biasing conventional fitting approaches. Further, PSF shapes are deliberately modified in PSF engineering approaches for providing improved sensitivity, e.g., for 3D localization or determination of dipole orientation. As this can lead to highly complex PSF shapes, a tool for visualizing expected PSFs would facilitate the interpretation of obtained data and the design of experimental approaches. To this end, we introduce a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. Our tool incorporates a wide range of microscope and fluorophore parameters, including orientationally constrained fluorophores, as well as custom aberrations, transmission and phase masks, thus enabling an accurate representation of various imaging conditions. An additional feature is the simulation of crowded molecular environments with overlapping PSFs. Further, our app directly provides the Cramér–Rao bound for assessing the best achievable localization precision under given conditions. Finally, our software allows for the fitting of custom aberrations directly from experimental data, as well as the generation of a large dataset with randomized simulation parameters, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation.