Pointwise error estimates in localization microscopy

• Published in: Nature communications Vol. 8; no. 1; p. 15115
• Main Authors: Lindén, Martin, Ćurić, Vladimir, Amselem, Elias, Elf, Johan
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03.05.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 14/34; 14/35; 14/63; 631/1647/328/2238; 631/57/2265; 639/705/531; Approximation; Cameras; Estimates; Humanities and Social Sciences; Localization; Microscopy; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms15115

Pointwise localization of individual fluorophores is a critical step in super-resolution localization microscopy and single particle tracking. Although the methods are limited by the localization errors of individual fluorophores, the pointwise localization precision has so far been estimated using theoretical best case approximations that disregard, for example, motion blur, defocus effects and variations in fluorescence intensity. Here, we show that pointwise localization precision can be accurately estimated directly from imaging data using the Bayesian posterior density constrained by simple microscope properties. We further demonstrate that the estimated localization precision can be used to improve downstream quantitative analysis, such as estimation of diffusion constants and detection of changes in molecular motion patterns. Finally, the quality of actual point localizations in live cell super-resolution microscopy can be improved beyond the information theoretic lower bound for localization errors in individual images, by modelling the movement of fluorophores and accounting for their pointwise localization uncertainty. Super-resolution localization microscopy produces biophysical information in the form of estimated positions of single molecules. Here, Lindén et al . estimate the uncertainty of single localizations, and show that this additional information can improve data analysis and localization precision.