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A ‘pocket guide’ to total internal reflection fluorescence
Author(s) -
MARTINFERNANDEZ M.L.,
TYNAN C.J.,
WEBB S.E.D.
Publication year - 2013
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/jmi.12070
Subject(s) - total internal reflection fluorescence microscope , context (archaeology) , evanescent wave , reflection (computer programming) , total internal reflection , microscopy , field (mathematics) , nanotechnology , electromagnetic field , optics , physics , computer science , materials science , biology , paleontology , mathematics , quantum mechanics , pure mathematics , programming language
Summary The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single‐molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a ‘one stop shop’ explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.