Mapping microscope object polarized emission to the back focal plane pattern.
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.
Burghardt
Thomas P
TP
Mayo Clinic Rochester, Department of Biochemistry, 200 First Street South West, Rochester, Minnesota 55905, USA. burghardt@mayo.edu
Ajtai
Katalin
K
D000818:Animals; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D009218:Myosins / Q000737:chemistry; D055668:Photochemical Processes; D011817:Rabbits; D020349:Surface Plasmon Resonance / Q000379:methods*
D049452:Green Fluorescent Proteins; D009218:Myosins