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Application of Surface Plasmon Coupled Emission to Study of Muscle

J. Borejdo ^*, Z. Gryczynski ^*, N. Calander , P. Muthu ^* and I. Gryczynski ^* 

^* Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas 76107; Physical Electronics & Photonics, Department of Physics, Chalmers University of Technology, S-412 96 Göteborg, Sweden; and Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas 76107

Correspondence: Address reprint requests to Julian Borejdo, Dept. of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Fax: 817-735-2118; E-mail: jborejdo{at}hsc.unt.edu.

Muscle contraction results from interactions between actin and myosin cross-bridges. Dynamics of this interaction may be quite different in contracting muscle than in vitro because of the molecular crowding. In addition, each cross-bridge of contracting muscle is in a different stage of its mechanochemical cycle, and so temporal measurements are time averages. To avoid complications related to crowding and averaging, it is necessary to follow time behavior of a single cross-bridge in muscle. To be able to do so, it is necessary to collect data from an extremely small volume (an attoliter, 10^–18 liter). We report here on a novel microscopic application of surface plasmon-coupled emission (SPCE), which provides such a volume in a live sample. Muscle is fluorescently labeled and placed on a coverslip coated with a thin layer of noble metal. The laser beam is incident at a surface plasmon resonance (SPR) angle, at which it penetrates the metal layer and illuminates muscle by evanescent wave. The volume from which fluorescence emanates is a product of two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The fluorescence is quenched at the metal interface (up to 10 nm), which further limits the thickness of the fluorescent volume to 50 nm. The fluorescence is detected through a confocal aperture, which limits the lateral dimensions of the detection volume to 200 nm. The resulting volume is 2 x 10^–18 liter. The method is particularly sensitive to rotational motions because of the strong dependence of the plasmon coupling on the orientation of excited transition dipole. We show that by using a high-numerical-aperture objective (1.65) and high-refractive-index coverslips coated with gold, it is possible to follow rotational motion of 12 actin molecules in muscle with millisecond time resolution.