title: Characterisation of the effects of optical aberrations in single molecule techniques.
abstract: 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.
authors.affiliation: Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0FA, UK.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Coles
authors.affiliation: Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0FA, UK.
authors.forename: Stephen E D
authors.initials: SE
authors.lastname: Webb
authors.affiliation: UK ATC, Royal Observatory Edinburgh, Blackford Hill, Edinburgh, EH9 3HJ, UK.
authors.forename: Noah
authors.initials: N
authors.lastname: Schwartz
authors.affiliation: Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0FA, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rolfe
authors.affiliation: Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0FA, UK.
authors.forename: Marisa
authors.initials: M
authors.lastname: Martin-Fernandez
authors.affiliation: Central Laser Facility, Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0FA, UK.
authors.forename: Valentina
authors.initials: V
authors.lastname: Lo Schiavo
keywords: (010.1080) Active or adaptive optics; (170.2520) Fluorescence microscopy