Investigation of the numerics of point spread function integration in single molecule localization.

The computation of point spread functions, which are typically used to model the image profile of a single molecule, represents a central task in the analysis of single molecule microscopy data. To determine how the accuracy of the computation affects how well a single molecule can be localized, we investigate how the fineness with which the point spread function is integrated over an image pixel impacts the performance of the maximum likelihood location estimator. We consider both the Airy and the two-dimensional Gaussian point spread functions. Our results show that the point spread function needs to be adequately integrated over a pixel to ensure that the estimator closely recovers the true location of the single molecule with an accuracy that is comparable to the best possible accuracy as determined using the Fisher information formalism. Importantly, if integration with an insufficiently fine step size is carried out, the resulting estimates can be significantly different from the true location, particularly when the image data is acquired at relatively low magnifications. We also present a methodology for determining an adequate step size for integrating the point spread function.