The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging

Mikael P. Backlund, Matthew D. Lew, Adam S. Backer, Steffen J. Sahl, W. E. Moerner

Research output: Contribution to journalReview article

Abstract

Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

Original languageEnglish (US)
Pages (from-to)587-599
Number of pages13
JournalChemPhysChem
Volume15
Issue number4
DOIs
StatePublished - Mar 17 2014
Externally publishedYes

Keywords

  • fluorescence microscopy
  • molecular orientation
  • rotational mobility
  • single-molecule studies
  • super-resolution imaging

ASJC Scopus subject areas

  • Atomic and Molecular Physics, and Optics
  • Physical and Theoretical Chemistry

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