Mapping microscope object polarized emission to the back focal plane pattern

Thomas P. Burghardt, Katalin Ajtai

Research output: Contribution to journalArticle

7 Scopus citations

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.

Original languageEnglish (US)
Article number034036
JournalJournal of Biomedical Optics
Volume14
Issue number3
DOIs
StatePublished - 2009

Keywords

  • dipole orientation
  • evanescent emission
  • muscle fiber myosin cross-bridge
  • near-field
  • photoactivated green fluorescent protein
  • single-molecule imaging
  • surface plasmon resonance
  • total internal reflection

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Biomedical Engineering

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