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Register a new userPalladium Zero-Mode Waveguides for Optical Single Molecule Detection with Nanopores
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Holes in metal films block any transmitting light if the wavelength is much larger than the hole diameter, establishing such nanopores as so-called Zero Mode Waveguides (ZMWs). Molecules on the other hand, can still passage through these holes. We use this to detect individual fluorophore-labelled molecules as they travel through a ZMW and thereby traverse from the dark region to the illuminated side, upon which they emit fluorescent light. This is beneficial both for background suppression and to prevent premature bleaching. We use palladium as a novel metal-film material for ZMWs, which is advantageous compared to conventionally used metals. We demonstrate that it is possible to simultaneously detect translocations of individual free fluorophores of different colors. Labeled DNA and protein biomolecules can be detected as well at the single-molecule level with a high signal-to-noise ratio and at high bandwidth, which opens the door to a variety of single-molecule biophysics studies.
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We fabricated hybrid metal-dielectric nanoantennas and measured their optical response at three different wavelengths. The nanostructure is fabricated on a bilayer film formed by the sequential deposition of silicon and gold on a transparent substrate. The optical characterization is done via fluorescence measurements. We characterized the fluorescence enhancement, as well as the lifetime and detection volume reduction for each wavelength. We observe that the hybrid metal-dielectric nanoantennas behave as enhanced Zero Mode Waveguides in the near-infrared spectral region. Their detection volume is such that they can perform enhanced single-molecule detection at tens of microM. However, a wavelength blue-shift of 40 nm dramatically decreases the performance of the nanoantennas. We compared their behavior with that of a golden ZMW, and we verified that the dielectric silicon layer improves the design. We interpreted the experimental observations with the help of numerical simulations. In addition, the simulations showed that the field enhancement of the structure highly depends on the incoming beam: tightly focused beams yield lower field enhancements than plane-waves.
Zero-mode waveguides (ZMW) nanoapertures milled in metal films were proposed to improve the FRET efficiency and enable single molecule FRET detection beyond the 10 nm barrier, overcoming the restrictions of diffraction-limited detection in a homogeneous medium. However, the earlier ZMW demonstrations were limited to the Atto 550 - Atto 647N fluorophore pair, asking the question whether the FRET enhancement observation was an artefact related to this specific set of fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to investigate single molecule FRET at large donor-acceptor separations exceeding 10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different chemical structure, surface charge and hydrophobicity as compared to their Atto counterparts. Our single molecule data on Alexa 546 - Alexa 647 demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm, extending the spatial range available for FRET and confirming the earlier conclusions. By showing that the FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, this report is an important step to establish the relevance of ZMWs to extend the sensitivity and detection range of FRET, while preserving its ability to work on regular fluorescent dye pairs.
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 beta-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.
We report an optical technique that yields an enhancement of single-molecule photostability, by greatly suppressing photobleaching pathways which involve photoexcitation from the triplet state. This is accomplished by dynamically switching off the excitation laser when a quantum-jump of the molecule to the triplet state is optically detected. This procedure leads to a lengthened single-molecule observation time and an increased total number of detected photons. The resulting improvement in photostability unambiguously confirms the importance of photoexcitation from the triplet state in photobleaching dynamics, and may allow the investigation of new phenomena at the single-molecule level.
Single molecule localization microscopy (SMLM) techniques enable imaging biological samples well beyond the diffraction limit of light, but they vary significantly in their spatial and temporal resolutions. High-order statistical analysis of temporal fluctuations as in superresolution optical fluctuation imaging (SOFI) also enable imaging beyond diffraction limit, but usually at a lower resolution as compared to SMLM. Since the same data format is acquired for both methods, their algorithms can be applied to the same data set, and thus may be combined synergistically to improve overall imaging performance. Here, we find that SOFI converges much faster than SMLM, provides additive information to SMLM, and can efficiently reject background. We then show how SOFI-assisted SMLM imaging can improve SMLM image reconstruction by rejecting common sources of background, especially under low signal-to-background conditions. The performance of our approach was evaluated using a realistic simulation of fluorescence imaging we developed and further demonstrated on experimental SMLM images of the plasma membrane of activated fixed and live T cells. Our approach significantly enhances SMLM performance under demanding imaging conditions and could set an example for synergizing additional imaging techniques.
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