Single Molecule Imaging of Fluorescently Labeled Proteins on Metal by Surface Plasmons in Aqueous Solution

Yokota, Hiroaki; Saito, Kiwamu; Yanagida, Toshio

doi:10.1103/physrevlett.80.4606

Cited by 88 publications

(81 citation statements)

References 25 publications

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“…The use of aluminum with fluorescence was not obvious because of earlier reports of strong quenching on aluminum surfaces. 15 However, several reports described the transmission of ultraviolet light by thin aluminum films as seen by excitation of fluorescence with total internal reflection, 16 attenuated total internal reflection, 17 or light scattering. 18 Additionally, aluminum has been suggested for use in SPR, 19 and calculations of the spatial distribution have been reported for fluorophores near an aluminum surface.…”

Section: Resultsmentioning

confidence: 99%

Ultraviolet Surface Plasmon-Coupled Emission Using Thin Aluminum Films

et al. 2004

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Surface plasmon-coupled emission (SPCE) is the directional radiation of light into a substrate due to excited fluorophores above a thin metal film. To date, SPCE has only been observed with visible wavelengths using silver or gold films. We now show that SPCE can be observed in the ultraviolet region of the spectrum using thin (20 nm) aluminum films. We observed directional emission in a quartz substrate from the DNA base analogue 2-aminopurine (2-AP). The SPCE radiation occurs within a narrow angle at 59° from the normal to the hemicylindrical prism. The excitation conditions precluded the creation of surface plasmons by the incident light. The directional emission at 59° is almost completely p-polarized, consistent with its origin from surface plasmons due to coupling of excited 2-AP with the aluminum. The emission spectra and lifetimes of the SPCE are those characteristic of 2-AP. Different emission wavelengths radiate at slightly different angles on the prism providing intrinsic spectral resolution from the aluminum film. These results indicate that SPCE can be used with numerous UV-absorbing fluorophores, suggesting biochemical applications with simultaneous surface plasmon resonance and SPCE binding assays.Surface plasmon resonance (SPR) is widely used for detection of bioaffinity reactions on surfaces. [1][2][3] Surface plasmons are oscillating electrical charges on a metallic surface. When a thin metal film is illuminated through a glass prism, and the angle of incidence is appropriate, the surface plasmons in resonance with the incident light occur at the metal-air or water interface. This results in strong absorption, which is measured as a decrease of reflectivity. The SPR angle is sensitive to the refractive index of the sample above the metal, distal from the glass prism. The origin of this sensitivity is the evanescent field from the plasmons, which penetrates approximately λ/3 into the sample.In several recent reports we described a related phenomenon, surface plasmon-coupled emission (SPCE). 4,5 We found that excited fluorophores near a thin metal surface can couple with the surface plasmons, resulting in directional radiation into the glass substrate. The spectral properties of the radiation were found to be essentially identical to those of the fluorophore, except for a highly p-polarized emission, too large to be due to optical photoselection. The angular dependence of the radiation, as well as the p-polarization, are consistent with radiating surface plasmons, the reverse process of surface plasmon absorption.Our previous studies of SPCE used thin silver 5-7 or gold 8 surfaces. This choice of metals restricts the selection of fluorophores to those absorbing and emitting at visible or longer wavelengths. However, many widely used fluorophores absorb or emit at ultraviolet wavelengths. We now report that SPCE at UV wavelengths can be observed using thin *To whom correspondence should be addressed: lakowicz@cfs.umbi.umd.edu. NIH Public Access MATERIALS AND METHODS Sample PreparationQuar...

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Section: Resultsmentioning

confidence: 99%

Ultraviolet Surface Plasmon-Coupled Emission Using Thin Aluminum Films

et al. 2004

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“…[4 -8] Molecules adsorbed onto roughened Ag and Au surfaces or nanostructured Ag and Au colloids produce a millionfold increase in the intensity of Raman, [8,9] absorption, [10 -12] and fluorescence emission. [7,13,14] Recently, it has been reported that even single-molecule spectroscopy can be performed by SERS, suggesting that the enhancement factor (EF) can reach as high as 10 14 -10 15 . [15 -19] Second-harmonic generation at the silver-air interface is also enhanced by surface roughness and it is accompanied by a broad luminescence background extending far beyond the anti-Stokes side of the second harmonic.…”

Section: Introductionmentioning

confidence: 99%

Adjustment and control of SERS activity of metal substrates by pressure

Xia

Jia

Liu

et al. 2009

J Raman Spectroscopy

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Metal pellets of silver and copper for surface-enhanced Raman scattering (SERS) spectroscopy were prepared by compression with different pressures. It was found that the SERS activity of the pellet could be controlled by pressure. Enhanced Raman scattering properties of the metal pellets in the presence of adsorbed 4-mercaptobenzoic acid (4-MBA) with excitation at 632.8 or 514 nm could be obtained by choosing proper pressure of pellatization. The SERS peak intensity of the band at ∼1584 cm −1 of 4-MBA adsorbed on the metal pellets varies as a function of applied pressure, and which is about 1.2-32 times greater than when it is adsorbed on silver and copper particles. The calculated results of three-dimensional finite-difference time-domain method (3D-FDTD) are in good agreement with the experimental data. Moreover, no spurious peaks appear in the SERS spectra of the samples because no other chemicals are involved in the simple preparation process of the metal pellets, which will facilitate its use as an SERS-active substrate for analytical purposes. In summary, SERS-active metal pellets can be produced simply and cost effectively by the method reported here, and this method is expected to be utilized in the development of SERS-based analytical devices.

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“…The main advantage of SMS method is its ability to observe phenomena hidden in ensemble measurements. 10,[24][25][26][27] A single molecule can be a reporter of its local environment. The reporter properties of the fluorophores can be exploited to reveal inherent differences in local environments or to monitor time-dependent changes in the host.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Fluorescence of Cy5-Labeled Oligonucleotides Near Silver Island Films: A Distance Effect Study Using Single Molecule Spectroscopy

Lakowicz

2006

J. Phys. Chem. B

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We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.

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Single Molecule Imaging of Fluorescently Labeled Proteins on Metal by Surface Plasmons in Aqueous Solution

Cited by 88 publications

References 25 publications

Ultraviolet Surface Plasmon-Coupled Emission Using Thin Aluminum Films

Ultraviolet Surface Plasmon-Coupled Emission Using Thin Aluminum Films

Adjustment and control of SERS activity of metal substrates by pressure

Enhanced Fluorescence of Cy5-Labeled Oligonucleotides Near Silver Island Films: A Distance Effect Study Using Single Molecule Spectroscopy

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