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Research Papers

Mapping microscope object polarized emission to the back focal plane pattern

[+] Author Affiliations
Thomas P. Burghardt

Mayo Clinic Rochester, Department of Biochemistry and Molecular Biology and Department of Physiology and Biomedical Engineering, 200 First Street South West, Rochester, Minnesota 55905

Katalin Ajtai

Mayo Clinic Rochester, Department of Biochemistry and Molecular Biology, 200 First Street South West, Rochester, Minnesota 55905

J. Biomed. Opt. 14(3), 034036 (June 30, 2009). doi:10.1117/1.3155520
History: Received December 17, 2008; Revised March 17, 2009; Accepted April 21, 2009; Published June 30, 2009
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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.

Figures in this Article
© 2009 Society of Photo-Optical Instrumentation Engineers

Citation

Thomas P. Burghardt and Katalin Ajtai
"Mapping microscope object polarized emission to the back focal plane pattern", J. Biomed. Opt. 14(3), 034036 (June 30, 2009). ; http://dx.doi.org/10.1117/1.3155520


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