Molecular Orientation Affects Localization Accuracy in Superresolution Far-Field Fluorescence Microscopy

• Published in: Nano letters Vol. 11; no. 1; pp. 209 - 213
• Main Authors: Engelhardt, Johann, Keller, Jan, Hoyer, Patrick, Reuss, Matthias, Staudt, Thorsten, Hell, Stefan W
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12.01.2011
• Subjects: Condensed matter: structure, mechanical and thermal properties; Exact sciences and technology; Fluorescent Dyes - analysis; Microscopy, Fluorescence - methods; Nanoscale materials: clusters, nanoparticles, nanotubes, and nanocrystals; Physics; Sensitivity and Specificity; Stochastic Processes; Structure of solids and liquids; crystallography; localization; single molecules; Microscopy; fluorescence; resolution; superresolution; Molecular orientation; Fluorophore; Imaging; Far field; Inclination; Switching; Fluorescence microscopy
• ISSN: 1530-6984; 1530-6992
• DOI: 10.1021/nl103472b

We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.