Application of surface plasmon coupled emission to study of muscle

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, I. Gryczynski

Research output: Contribution to journalArticle

73 Scopus citations

Abstract

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 × 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.

Original languageEnglish
Pages (from-to)2626-2635
Number of pages10
JournalBiophysical Journal
Volume91
Issue number7
DOIs
StatePublished - Oct 2006

    Fingerprint

Cite this