Improving zero-mode waveguide structure for enhancing signal-to-noise ratio of real-time single-molecule fluorescence imaging: A computational study

Takenaka, Takashi; Akahori, Rena; Higano, Shun; Okubo, Kazuaki; Yamamoto, Hideaki; Ueno, Taro; Funatsu, Takashi

doi:10.1103/physreve.88.012727

Cited by 15 publications

(21 citation statements)

References 20 publications

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“…FIB milling then accurately controls the final geometry of the ZMW allowing to optimize both the diameter and the 50 nm undercut in the glass below the aperture. [67][68][69][70] The sketch of our experiment is depicted on Fig. 1b.…”

Section: Resultsmentioning

confidence: 99%

“…Ensuring the best optical performance for the ZMWs requires specific attention during the metal coating process, as aluminum is highly sensitive to the residual trace amount of oxygen found in the evaporation chamber. , Depending on the deposition parameters, the amount of oxide found within the bulk of the aluminum layer can dramatically change, affecting the dielectric permittivity and the plasmonic losses. , We have observed similar trends, and in order to reach the highest enhancement factors, we found that the aluminum deposition parameters critically need to reach the lowest chamber pressure (<10 –6 mbar) together with fast deposition rates (>10 nm/s). FIB milling then accurately controls the final geometry of the ZMW, allowing both the diameter and the 50 nm undercut in the glass below the aperture to be optimized. − …”

Section: Resultsmentioning

confidence: 99%

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Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

et al. 2019

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Single molecule Förster resonance energy transfer (smFRET) is widely used to monitor conformations and interactions dynamics at the molecular level. However, conventional smFRET measurements are ineffective at donor-acceptor distances exceeding 10 nm, impeding the studies on biomolecules of larger size. Here, we show that zero-mode waveguide (ZMW) apertures can be used to overcome the 10 nm barrier in smFRET. Using an optimized ZMW structure, we demonstrate smFRET between standard commercial fluorophores up to 13.6 nm distance with a significantly improved FRET efficiency. To further break into the classical FRET range limit, ZMWs are combined with molecular constructs featuring multiple acceptor dyes to achieve high FRET efficiencies together with high fluorescence count rates. As we discuss general guidelines for quantitative smFRET measurements inside ZMWs, the technique can be readily applied for monitoring conformations and interactions on large molecular complexes with enhanced brightness.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

et al. 2019

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“…28,66 Focused ion beam (FIB) milling then directly carves the nanoaperture into the aluminum layer, enabling an accurate control on the diameter and the 50 nm undercut in the quartz substrate to optimize the signal to noise ratio. [67][68][69] Figure 1b shows typical SEM images of our ZMW samples with different diameters. Circular apertures milled in a perfect electrical conductor have a theoretical cut-off diameter given by 0.59 𝜆/𝑛 where 𝑛 is the refractive index of the medium filling the aperture.…”

mentioning

confidence: 99%

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

et al. 2019

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Single molecule detection provides detailed information about molecular structures and functions, but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labelling.However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguides nanoapertures enable observing the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. While the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

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“…The milling included a 80 nm deep undercut into the borosilicate glass substrate to maximize the signal enhancement. 39,73 To protect the aluminum surface against corrosion from the salt buffer, a 10 nm-thick SiO2 layer was deposited by plasma-enhanced chemical vapor protection (PECVD, PlasmaPro NGP80 from Oxford Instruments). 74,75 DNA samples.…”

Section: Methodsmentioning

confidence: 99%

Fluorescence Brightness, Photostability, and Energy Transfer Enhancement of Immobilized Single Molecules in Zero-Mode Waveguide Nanoapertures

2022

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Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor single molecules beyond the range accessible to normal microscopes. However, several aspects of the ZMW influence on the photophysics of fluorophores remain inadequately documented and sometimes controversial. Here, we thoroughly investigate the ZMW influence on the fluorescence of single immobilized Cy3B and Alexa 647 molecules, detailing the interplays between brightness, lifetime, photobleaching time, total number of emitted photons and Förster resonance energy transfer (FRET). Despite the plasmonicenhanced excitation intensity in the ZMW, we find that the photostability is preserved with similar photobleaching times as on the glass reference. Both the fluorescence brightness and the total numbers of photons detected before photobleaching are increased, with an impressive gain near five times found for Alexa 647 dyes. Finally, the single-molecule data importantly allow a loophole-free characterization of the ZMW influence on the FRET process. We show that the FRET rate constant is enhanced by 50%, demonstrating that nanophotonics can mediate the energy transfer. These results deepen our understanding of the fluorescence enhancement in ZMWs and are of immediate relevance for single-molecule biophysical applications.

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Improving zero-mode waveguide structure for enhancing signal-to-noise ratio of real-time single-molecule fluorescence imaging: A computational study

Cited by 15 publications

References 20 publications

Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

Fluorescence Brightness, Photostability, and Energy Transfer Enhancement of Immobilized Single Molecules in Zero-Mode Waveguide Nanoapertures

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