Characterisation of the effects of optical aberrations in single molecule techniques

Optical aberrations degrade image quality in fluorescence microscopy, including for single-molecule based techniques. These depend on post-processing to localize individual molecules in an image series. Using simulated data, we show the impact of optical aberrations on localization success, accuracy and precision. The peak intensity and the proportion of successful localizations strongly reduces when the aberration strength is greater than 1.0 rad RMS, while the precision of each of those localisations is halved. The number of false-positive localisations exceeded 10% of the number of true-positive localisations at an aberration strength of only ~0.6 rad RMS when using the ThunderSTORM package, but at greater than 1.0 rad RMS with the Radial Symmetry package. In the presence of coma, the localization error reaches 100 nm at ~0.6 rad RMS of aberration strength. The impact of noise and of astigmatism for axial resolution are also considered. Understanding the effect of aberrations is crucial when deciding whether the addition of adaptive optics to a single-molecule microscope could significantly increase the information obtainable from an image series.