Fluorescence emission at dielectric and metal-film interfaces

Hellen, Edward H.; Axelrod, Daniel

doi:10.1364/josab.4.000337

Cited by 231 publications

(244 citation statements)

References 31 publications

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Section: Resultsmentioning

confidence: 99%

“…The fluorescence collection factor Q(z), as defined by Hellen and Axelrod (29), captures the well established phenomenon that a radiating fluorophore near a high refractive index substrate exhibits altered fluorescence that depends on its distance from the interface (30,31). This phenomenon is attributed to two effects (29,32): (i) interference between emitted light propagating directly from the radiating dipole and light that is reflected from the material interface; and (ii) power dissipation into the high refractive index material as the radiating dipole is closer to the interface. The model developed for the collection factor by Hellen and Axelrod considers a constant-amplitude oscillating dipole at a three-layer interface composed of two semiinfinite, nonattenuating dielectric regions 1 (the aqueous media) and 3 (the lithium niobate) with indices of refraction of n 1 and n 3 , respectively, separated by an intermediate region 2 (the SiO 2 spacer) with index of refraction of n 2 and thickness l. The radiating dipole is located in region 1 at a distance z from the region 2-3 interface, and its transition dipole moment is oriented with polar angle ⌰ relative to the surface normal.…”

Section: Resultsmentioning

confidence: 99%

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High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Ajo‐Franklin

Kam

Boxer

2001

Proc. Natl. Acad. Sci. U.S.A.

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Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO 2 on lithium niobate (LiNbO3, n ‫؍‬ 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead͞ protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Ajo‐Franklin

Kam

Boxer

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Without deposition of the phosphonate polymer, neutravidin bound to both the fused silica and the aluminum surfaces with high densities. Fluorescence from the beads was enhanced by the proximity of the metal (25,26), resulting in higher signal levels from the aluminum surface regions. In contrast, excellent bias of neutravidin adsorption toward the fused silica surface was observed for PVPA-treated samples, translating to very few biotinylated beads detectable by fluorescence microscopy on the aluminum surface.…”

Section: Resultsmentioning

confidence: 99%

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Korlach

Marks

Cicero

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

209

147

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Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of Ϸ30% were obtained for a range of ZMW diameters (70 -100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.fluorescence ͉ metal passivation ͉ microscopy ͉ polyvinyl phosphonic acid ͉ single molecule N anofabrication techniques have enabled new approaches to interrogate individual biomolecules by fluorescence techniques (reviewed in refs. 1 and 2). The extremely small size scale of the associated devices results in a drastic illumination volume reduction, allowing single-molecule investigations to take place at fluorophore concentrations increased to biologically relevant levels. In addition to higher temporal resolution and higher signal-to-noise ratios, they also provide spatial resolution beyond diffraction-limited optics.The zero-mode waveguide (ZMW) is one such nanophotonic confinement structure often consisting of a circular hole in a metal cladding film on a solid transparent substrate (3). In conjunction with laser-excited fluorescence, they provide observation volumes on the order of zeptoliters (10 Ϫ21 l), three to four orders of magnitude smaller than far-field excitation volumes. Applications of circular ZMWs have included the detection of single-molecule DNA polymerase activity by using labeled nucleotides at micromolar concentrations (3), the study of -repressor oligomerization dynamics (4), two-color crosscorrelation to rapidly screen for DNA restriction enzyme activity (5), and diffusion analysis of labeled membrane proteins in lipid bilayers of model membranes and living cells (6-11). C-shaped apertures have been described to study DNA hybridization interactions (12).ZMW technology applications have been limited by the unavailability of selective immobilization methods to position molecules exclusively in the observation volume, immediately above the transparent ZMW floor. One approach to selective immobilization exploits a feature inherent in the ZMW architecture. The transparent substrate and metal cladding are made of different materials, opening the possibility for a selective derivatization that will direct protein adsorption to the floor and not to the nearby metal walls. The nanometer size scale and three-dimensional n...

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“…Where a fluorophore is very close to the cover slip, its emitted near-field can interact with the surface and be converted into propagating light [8,9]. This near-field component is predominantly seen as "supercritical" emission (i.e.…”

Section: Emission Characteristicsmentioning

confidence: 99%

“…-The information contained in the supercritical emission can be used to measure molecular orientations, distance from the surface, and thickness of intermediate layers [8,11]. -Near the surface, the majority of the emitted light is emitted into the glass, further enhancing the efficiency of detection of molecules in an objective TIRF configuration (see Section 3.4) [9].…”

Section: Emission Characteristicsmentioning

confidence: 99%

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Knight

2014

Pure and Applied Chemistry

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Total internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in cell and molecular biology and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical implementation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

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Fluorescence emission at dielectric and metal-film interfaces

Cited by 231 publications

References 31 publications

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

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