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Journal of Biomedical Optics

Mapping microscope object polarized emission to the back focal plane pattern
Author(s): Thomas P. Burghardt; Katalin Ajtai
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Paper Abstract

The back focal plane (BFP) intensity pattern from a high-aperture objective separately maps far- and near-field emission from dipoles near a bare glass or metal-film-coated glass/aqueous interface. Total internal reflection (TIR) excitation of a fluorescent sample gave a BFP pattern interpreted in terms of fluorescent dipole orientation and distance from the interface. Theoretical consideration of this system led to identification of emission characteristics that remove a dipole orientation degeneracy in conventional microscope fluorescence polarization measurements. BFP pattern inspection removes the degeneracy. Alternatively, a BFP mask blocking a small fraction of emitted light in a standard imaging microscope prevents uniform collection of the BFP intensity and also eliminates the degeneracy. The BFP pattern from a single photoactivated photoactivatable green fluorescent protein (PAGFP) tagged myosin in a muscle fiber was observed despite the large background light from the highly concentrated myosin tagged with unphotoactivated PAGFP. This was accomplished by imaging the pattern from a nontelecentric plane, where most of the background intensity's pattern was translated laterally from the single-molecule object's pattern. TIR/BFP pattern imaging requires a simple alteration of the fluorescence microscope and is consistent with single-molecule imaging in a fluorophore dense three-dimensional object like a muscle fiber.

Paper Details

Date Published: 1 May 2009
PDF: 8 pages
J. Biomed. Opt. 14(3) 034036 doi: 10.1117/1.3155520
Published in: Journal of Biomedical Optics Volume 14, Issue 3
Show Author Affiliations
Thomas P. Burghardt, Mayo Clinic (United States)
Katalin Ajtai, Mayo Clinic (United States)

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