Application of Surface Plasmon Coupled Emission to Study of Muscle

Borejdo, Julian; Gryczyński, Zygmunt; Calander, Nils; Muthu, Priya; Gryczyński, Ignacy

doi:10.1529/biophysj.106.088369

Cited by 72 publications

(84 citation statements)

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“…It is not clear to us why Olympus coverslips are better than sapphire slips. Olympus would not reveal the composition of their material.We have shown earlier that surface plasmon coupled emission (SPCE) leads to the enhancement of excitation because of the increase in the local field at the metal-buffer interface [6]. The present work demonstrates that SIF leads to an enhancement of emission.…”

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confidence: 64%

Decreasing photobleaching by silver island films: application to muscle

Muthu

Gryczyński

et al. 2007

Analytical Biochemistry

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Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10 −18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-to 5-fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible. Keywords Photobleaching; Silver island films; MicroscopyRecently it has become possible to study single protein molecules in a cell. This avoids problems associated with averaging responses from an assembly of molecules with different kinetics and problems associated with diluting of proteins in vitro. The difficulties of studying large assemblies of molecules are well illustrated by the problem of muscle contraction. Contraction results from the interactions of myosin cross-bridges with actin. Myosin crossbridges act asynchronously, i.e., at any time during muscle contraction each one is in a different part of the mechanochemical cycle. Therefore measurement taken from an assembly of crossbridges at any time during contraction is an average value, likely to obscure details of crossbridge kinetics. Furthermore, it is desirable to detect signal from a working muscle, because contractile proteins may behave differently in solution and in whole muscle, where protein ⋆ Dedicated to Professor Avraham Oplatka on the occasion of his birthday. To be able to obtain information from the individual molecules in muscle, it is necessary to collect data from an extremely small volume, small enough to...

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supporting

confidence: 64%

Decreasing photobleaching by silver island films: application to muscle

Muthu

Gryczyński

et al. 2007

Analytical Biochemistry

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“…Now, the detection volume in an SPCE system will directly depend on the product of two near-field factors: The penetration depth of the excitation field and the distance-dependent coupling of fluorophore radiation to the surface plasmons. The distancedependent coupling of the radiated power of fluorescence to SPCE varies with the distance of the fluorophore from the metal interface as a skewed bell-shaped relation, with its peak at ∼ 25 nm [4,13]. The full-width at half-maximum (FWHM) of the product of evanescent excitation field and the distance-dependent coupling for a typical SPCE structure is ∼ 50 nm [4,46].…”

Section: Smd Using the Proposed Structurementioning

confidence: 99%

“…The distancedependent coupling of the radiated power of fluorescence to SPCE varies with the distance of the fluorophore from the metal interface as a skewed bell-shaped relation, with its peak at ∼ 25 nm [4,13]. The full-width at half-maximum (FWHM) of the product of evanescent excitation field and the distance-dependent coupling for a typical SPCE structure is ∼ 50 nm [4,46]. By contrast, for the proposed structure, the FWHM of the product of evanescent excitation field and the distance-dependent coupling is ∼ 35 nm.…”

Section: Smd Using the Proposed Structurementioning

confidence: 99%

“…By contrast, for the proposed structure, the FWHM of the product of evanescent excitation field and the distance-dependent coupling is ∼ 35 nm. By using techniques as in confocal microscopy, it is possible to limit the lateral dimensions of the detection volume to ∼ 200 nm in each direction [4,46].…”

Section: Smd Using the Proposed Structurementioning

confidence: 99%

“…In this approach, the target molecules, i.e., the sample, are fluorescently labeled and are placed on a thin metal layer, which is deposited on a glass prism [4]. The fluorophore labels are excited by an evanescent light coupled to surface plasmons at the sample-metal layer interface.…”

Section: Introductionmentioning

confidence: 99%

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A proposal and a theoretical analysis of an enhanced surface plasmon coupled emission structure for single molecule detection

Uddin

Tanvir

Talukder

2016

Journal of Applied Physics

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We propose a structure that can be used for enhanced single molecule detection using surface plasmon coupled emission (SPCE). In the proposed structure, instead of a single metal layer on the glass prism of a typical SPCE structure for fluorescence microscopy, a metal-dielectric-metal structure is used. We theoretically show that the proposed structure significantly decreases the excitation volume of the fluorescently labeled sample, and simultaneously increases the peak SPCE intensity and SPCE power. Therefore, the signal-to-noise ratio and sensitivity of an SPCE based fluorescence microscopy system can be significantly increased using the proposed structure, which will be helpful for enhanced single molecule detection, especially, in a less pure biological sample.

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2008

Coupled Bioluminescent Assays

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Application of Surface Plasmon Coupled Emission to Study of Muscle

Cited by 72 publications

References 50 publications

Decreasing photobleaching by silver island films: application to muscle

Decreasing photobleaching by silver island films: application to muscle

A proposal and a theoretical analysis of an enhanced surface plasmon coupled emission structure for single molecule detection

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