Application of surface plasmon coupled emission to study of muscle

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 approximately 10 nm), which further limits the thickness of the fluorescent volume to approximately 50 nm. The fluorescence is detected through a confocal aperture, which limits the lateral dimensions of the detection volume to approximately 200 nm. The resulting volume is approximately 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.

FIGURE 1

Concept behind confocal SPCE microscope. The fluorophores are placed on a metal-coated coverslip and excited with green light at an SPR angle. The excitation energy couples to the surface plasmons and radiates to the glass prism (red) as a surface of a cone with half-angle equal to the SPCE angle. Metal can be a thin layer of Al (20 nm thick) or Ag or Au (50 nm thick). The picture of the directional emission is taken from a real experiment.

FIGURE 2

Prismless confocal SPCE microscope. Not to scale.

FIGURE 3

SPCE signal from skeletal myofibrils as a function of the illumination angle. The top panel shows photographs taken from the microscope for various illumination angles. Myofibrils labeled with 0.1 μM fluorescein-phalloidin. λ_ex = 488 nm. Images analyzed by ImageJ.

FIGURE 4

SPCE image of myofibril in rigor. Myofibril labeled with 0.1 μM fluorescein-phalloidin on gold coverslips. The image was contrast enhanced to emphasize superior resolution of the method: Z is the Z-line, O is the overlap zone, I is the I-band. Bar is 10 μm.

FIGURE 5

Electric field of the evanescent wave at the surface. It is normalized to the electric field of the incident wave. Gold layer of 48 nm; excitation wavelength = 633 nm; maximum field at 57.86°; critical angle is 50.32°; integral from critical angle to 90° is 63.27 (solid line, SPCE; broken line, TIRF).

FIGURE 6

(Top) Definition of angles. (Bottom) Calculated power flow to the objective in the SPCE experiments for (left) θ = 0°, i.e., p-orientation and (right) θ = 90°, i.e., s-orientation of the transition moment. The time between excitation of the fluorophores is assumed much longer than the emission time. Note that the power of the p-polarization is ∼10 times greater than that of s-polarization. Gold layer of 48 nm, excitation wavelength = 633 nm, at maximum field (57.86°), emission at 670 nm (solid line, SPCE; broken line, TIRF). The strong dissipation of energy into the metal layer for short distances lowers the power in SPCE but not in TIRF.

FIGURE 7

Coupling of fluorescent dipole moments to surface plasmons (top) and comparison of the dependence of the transition moment angle for TIRF and SPCE (bottom). Gold layer 48 nm; λ_ex= 633 nm; maximum field (57.86°); emission at 670 nm; d = 50 nm.

FIGURE 8

(Left) Geometric arrangements. (A) Reverse Kretschmann (RK) configuration with SPCE observation; (B) reverse Kretschmann configuration with free-space (FS) observation; (C) Kretschmann (KR) configuration with SPCE observation; and (D) Kretschmann configuration with SPCE observation. (Right) Fluorescence spectra of the Rh6G in the presence of a background (DCM in ethanol) measured at various observation/excitation configurations. (Top) Emission spectrum observed at a small angle from the excitation in RK configuration. This free-space (FS) spectrum is dominated by a background DCM emission. The Rh6G emission at 560 nm is minimal. (Middle) In the same (as in a top panel) RK configuration, the observation was made from the prism side at the SPCE angle. In this case the dominant emission is from the Rh6G, and DCM background is greatly suppressed. (Bottom) The sample was rotated to the KR configuration, and the excitation was at a SPR angle. The observation was adjusted to the SPCE angle. Now, essentially only Rh6G emission is present in the spectrum. Note also that the intensity of the SPCE signal in KR configuration is an order of magnitude greater than the intensity in RK configuration.

FIGURE 9

Fluorescent image of the overlap zone. The black dot is a projection of the confocal pinhole on the image plane. (Right) Schematic diagram of the voxel. The fluorescently labeled actin monomers are gray. The observational volume is ∼50 nm thick and has a diameter of ∼200 nm, corresponding to the diffraction limit of the 1.65 NA objective. The resulting detection volume is ∼2 al. Bar = 1 μm.

FIGURE 10

Confocal SPCE signal from rigor myofibril. The data were collected from the overlap zone. Myofibril labeled with 0.1 μM rhodamine-phalloidin on gold coverslips. The exciting light was perpendicular to the plane of the coverslip (p-polarization). (A) Fluorescence intensity. (B) Intensity on expanded scale: gray, original signal; dark gray, low-pass filtered.