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

Abstract

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. However, optical aberrations and fixed fluorophore dipoles can lead to non-isotropic and distorted PSFs, thereby complicating and biasing conventional fitting approaches. In addition, some researchers deliberately modify the PSF by introducing specific phase shifts in order to provide improved sensitivity, e.g., for localizing molecules in 3D, or for determining the dipole orientation. For devising an experimental approach, but also for interpreting obtained data it would be helpful to have a simple visualization tool which calculates the expected PSF for the experiment in mind. To address this need, we have developed a comprehensive and accessible computer application that allows for the simulation of realistic PSFs based on the full vectorial PSF model. It incorporates a wide range of microscope and fluorophore parameters, enabling an accurate representation of various imaging conditions. Further, our app directly provides the Cramer-Rao bound for assessing the best achievable localization precision under given conditions. In addition to facilitating the simulation of PSFs of isotropic emitters, our application provides simulations of fixed dipole orientations as encountered, e.g., in cryogenic single-molecule localization microscopy applications. Moreover, it supports the incorporation of optical aberrations and phase manipulations for PSF engineering, as well as the simulation of crowded environments with overlapping molecules. Importantly, our software allows for the fitting of custom aberrations directly from experimental data, effectively bridging the gap between simulated and experimental scenarios, and enhancing experimental design and result validation.