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The Role of Molecular Dipole Orientation in Single‐Molecule Fluorescence Microscopy and Implications for Super‐Resolution Imaging
Author(s) -
Backlund Mikael P.,
Lew Matthew D.,
Backer Adam S.,
Sahl Steffen J.,
Moerner W. E.
Publication year - 2014
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201300880
Subject(s) - fluorophore , microscopy , super resolution microscopy , resolution (logic) , orientation (vector space) , fluorescence microscope , optics , fluorescence lifetime imaging microscopy , microscope , molecular imaging , materials science , chemistry , fluorescence , physics , scanning confocal electron microscopy , computer science , artificial intelligence , geometry , mathematics , microbiology and biotechnology , biology , in vivo
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.