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The point spread function (PSF) is fundamental to any type of microscopy,most importantly so for single-molecule localization techniques, where theexact PSF shape is crucial for precise molecule localization at the nanoscale.However, optical aberrations and fixed fluorophore dipoles can lead tonon-isotropic and distorted PSFs, thereby complicating and biasing conventionalfitting approaches. In addition, some researchers deliberately modify the PSFby introducing specific phase shifts in order to provide improved sensitivity,e.g., for localizing molecules in 3D, or for determining the dipoleorientation. For devising an experimental approach, but also for interpretingobtained data it would be helpful to have a simple visualization tool whichcalculates the expected PSF for the experiment in mind. To address this need,we have developed a comprehensive and accessible computer application thatallows for the simulation of realistic PSFs based on the full vectorial PSFmodel. It incorporates a wide range of microscope and fluorophore parameters,enabling an accurate representation of various imaging conditions. Further, ourapp directly provides the Cramer-Rao bound for assessing the best achievablelocalization precision under given conditions. In addition to facilitating thesimulation of PSFs of isotropic emitters, our application provides simulationsof fixed dipole orientations as encountered, e.g., in cryogenic single-moleculelocalization microscopy applications. Moreover, it supports the incorporationof optical aberrations and phase manipulations for PSF engineering, as well asthe simulation of crowded environments with overlapping molecules. Importantly,our software allows for the fitting of custom aberrations directly fromexperimental data, effectively bridging the gap between simulated andexperimental scenarios, and enhancing experimental design and resultvalidation.

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