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J Microsc. 2013 Oct;252(1):16-22. doi: 10.1111/jmi.12070. Epub 2013 Jul 24.

A 'pocket guide' to total internal reflection fluorescence.

Author information

1
Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, UK.

Abstract

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.

KEYWORDS:

Evanescent field; TIRF microscopy; polarization; single-molecule fluorescence imaging; total internal reflection

PMID:
23889125
PMCID:
PMC4285862
DOI:
10.1111/jmi.12070
[Indexed for MEDLINE]
Free PMC Article

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