Mapping microscope object polarized emission to the back focal plane pattern

Thomas P. Burghardt; Katalin Ajtai

doi:10.1117/1.3155520

1 May 2009 Mapping microscope object polarized emission to the back focal plane pattern

Thomas P. Burghardt, Katalin Ajtai

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Journal of Biomedical Optics, Vol. 14, Issue 3, 034036 (May 2009). https://doi.org/10.1117/1.3155520

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.

©(2009) Society of Photo-Optical Instrumentation Engineers (SPIE)

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Thomas P. Burghardt and Katalin Ajtai "Mapping microscope object polarized emission to the back focal plane pattern," Journal of Biomedical Optics 14(3), 034036 (1 May 2009). https://doi.org/10.1117/1.3155520

Published: 1 May 2009

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