| 1. |
- Borejdo, J., et al.
(författare)
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Application of surface plasmon coupled emission to study of muscle
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 91:7, s. 2626-2635
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Tidskriftsartikel (refereegranskat)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. © 2006 by the Biophysical Society.
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| 2. |
- Borejdo, J., et al.
(författare)
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Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope
- 2006
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Ingår i: Optics Express. - 1094-4087. ; 14:17, s. 7878-7888
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Tidskriftsartikel (refereegranskat)abstract
- Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (PCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. PCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ∼35 nm.
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| 3. |
- Borejdo, J., et al.
(författare)
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Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils
- 2008
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Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1560-2281 .- 1083-3668. ; 13:3, s. 10-
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Tidskriftsartikel (refereegranskat)abstract
- Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.
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| 4. |
- Calander, Nils, 1953, et al.
(författare)
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Interference of surface plasmon resonances causes enhanced depolarized light scattering from metal nanoparticles
- 2007
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Ingår i: Chemical Physics Letters. - : Elsevier BV. - 0009-2614. ; 434:4-6, s. 326-330
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Tidskriftsartikel (refereegranskat)abstract
- We show that the strongly depolarized light scattering from noble metal particles is a result of interference of two surface plasmon resonances on the same particle. The maximum depolarization occurs between two resonances. Under favorable conditions the anisotropy of the scattering light can be much lower than what is possible for dielectric particles. This explanation is discussed in relation to earlier published experimental measurements. Comparison of experimental results with theoretical calculations provides information on the shape distribution of metallic particles in the suspension. (c) 2006 Elsevier B.V. All rights reserved.
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| 5. |
- Gryczynski, I., et al.
(författare)
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Directional two-photon induced surface plasmon-coupled emission
- 2005
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Ingår i: Thin Solid Films. - : Elsevier BV. - 0040-6090. ; 491:1-2, s. 173-176
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Tidskriftsartikel (refereegranskat)abstract
- We measured a directional surface plasmon-coupled emission (SPCE) induced by a two-photon absorption. A 60 nm thick layer of poly(vinyl alcohol) film doped with rhodamine 123 was deposited on a silvered (50 nm Ag) glass slide, which was attached to a hemicylindrical glass prism. The 820 nm excitation from a femtosecond Ti:Sapphire laser was used either in reverse Kretschmann or Kretschmann configuration. The angular distribution of two-photon induced SPCE does not depend on the used configuration. The two-photon induced SPCE can be applied to improve immunoassays and deoxyribonucleic acid detection. © 2005 Elsevier B.V. All rights reserved.
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| 6. |
- Gryczynski, Z., et al.
(författare)
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Minimization of detection volume by surface-plasmon-coupled emission
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 356:1, s. 125-131
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Tidskriftsartikel (refereegranskat)abstract
- We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.
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| 7. |
- Klitgaard, S., et al.
(författare)
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Long wavelength depolarized light scattering from silver nanoparticles
- 2007
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Ingår i: Chemical Physics Letters. - : Elsevier BV. - 0009-2614. ; 443:1-3, s. 1-5
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Tidskriftsartikel (refereegranskat)abstract
- We report the depolarized light scattering from heterodisperse silver nanoparticles. The profile of the wavelength dependent anisotropy of the colloidal solution of silver nanoparticles extends to the red and near-infrared (NIR) spectral region. For long wavelengths, above 600 nm, the anisotropy drops below 0.5. The presence of such a strong orthogonal component in the scattering opens new opportunities for imaging in dispersive media when polarizers can be used to suppress the background. The anisotropy profile of the scattering of heterodisperse silver nanoparticles can be satisfactorily explained by a theory based on interference between two surface plasmon resonances. (c) 2007 Elsevier B.V. All rights reserved.
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| 8. |
- Matveeva, E. G., et al.
(författare)
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Red blood cells do not attenuate the SPCE fluorescence in surface assays
- 2007
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 388:5-6, s. 1127-1135
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Tidskriftsartikel (refereegranskat)abstract
- We describe the positive effect of surface plasmon-coupled fluorescence emission (SPCE) on the detection of a signal from a surface immunoassay in highly absorbing or/and scattering samples. A model immunoassay using fluorescently labeled anti-rabbit antibodies that bind to rabbit immunoglobulin on a silver surface was performed, and the signal was detected in the presence of various highly absorbing and/or scattering solutions or suspensions, such as hemoglobin solution, plastic beads, and red blood cells. The results showed that a highly absorbing solution consisting of small molecules (dye, hemoglobin) attenuates the SPCE signal approximately 2-3-fold. In contrast, suspensions with the same absorption containing large particles (large beads, red blood cell suspension) attenuate the SPCE signal only slightly, approximately 5-10%. Also, a suspension of large undyed, highly scattering beads does not reduce the SPCE signal. The effects on the immunoassay signal of the sample background absorption and scattering, the size of the background particles, and the geometry of the experimental set-up are discussed. We believe that SPCE is a promising technique in the development of biosensors utilized for surface-based assays, as well as any assays performed directly in highly absorbing and/or scattering solutions without washing or separation procedures.
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