Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging.
Often fluorophores observed by microscopy are located close to a planar surface as in total internal reflection microscopy or single molecule studies. The optical properties of fluorescence excitation and emission near a planar surface (possibly metal film coated) between two dielectrics are well understood from the viewpoint of classical electromagnetic theory. We present an experimental method whereby the angular dependence of the emission pattern of a fluorophore near a bare surface or a metal film coated surface that supports surface plasmon resonance can be measured with microscope optics. The technique involves an alteration of the microscope optics to directly record (on a digital CCD camera) the intensity pattern at the objective's back focal plane, which directly maps the angular emission pattern of fluorescence. The experimental emission profile on both glass and aluminum-coated surfaces is anisotropic with a peak at either the critical angle or both the critical angle and the surface plasmon angle. The observed profiles agree well with computer calculations and suggest some optical modifications that are potentially useful in cell biophysics.
Mattheyses
Alexa L
AL
University of Michigan, Biophysics Research Division, Ann Arbor, Michigan 48109, USA. amatthey@umich.edu
Axelrod
Daniel
D
D020099:Coated Materials, Biocompatible / Q000737:chemistry; D005898:Glass / Q000737:chemistry*; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008027:Light; D008670:Metals / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012031:Refractometry / Q000379:methods*; D012542:Scattering, Radiation; D013059:Spectrum Analysis, Raman / Q000379:methods*
D020099:Coated Materials, Biocompatible; D008670:Metals