Description

Estimating the localization spread function of static single molecule localization microscopy images. Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method to estimate the localization spread function (LSF) of a static SMLM dataset directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.