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 language | English (US) |
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Article number | 034036 |
Journal | Journal of Biomedical Optics |
Volume | 14 |
Issue number | 3 |
DOIs | |
State | Published - 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