Simultaneous, accurate measurement of the 3D position and orientation of single molecules

Mikael P. Backlund, Matthew D. Lew, Adam S. Backer, Steffen J. Sahl, Ginni Grover, Anurag Agrawal, Rafael Piestun, W. E. Moerner

Research output: Contribution to journalArticlepeer-review


Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2x worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4x worse than the photon-limited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

Original languageEnglish (US)
Pages (from-to)19087-19092
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number47
StatePublished - Nov 20 2012
Externally publishedYes

ASJC Scopus subject areas

  • General


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