Single-molecule fluorescence techniques are essential for investigating the molecular mechanisms in biological processes. However, achieving sub-millisecond temporal resolution to monitor fast ...molecular dynamics remains a significant challenge. The fluorescence brightness is the key parameter that generally defines the temporal resolution for these techniques. Conventional microscopes and standard fluorescent emitters fall short in achieving the high brightness required for sub-millisecond monitoring. Plasmonic nanoantennas have been proposed as a solution, but despite huge fluorescence enhancement have been obtained with these structures, the brightness generally remains below 1 million photons/s/molecule. Therefore, the improvement of temporal resolution has been overlooked. In this article, we present a method for achieving high temporal resolution in single-molecule fluorescence techniques using plasmonic nanoantennas, specifically optical horn antennas. We demonstrate about 90% collection efficiency of the total emitted light, reaching a high fluorescence brightness of 2 million photons/s/molecule in the saturation regime. This enables observations of single molecules with microsecond binning time and fast fluorescence correlation spectroscopy (FCS) measurements. This work expands the applications of plasmonic antennas and zero-mode waveguides in the fluorescence saturation regime towards brighter single-molecule signal, faster temporal resolutions and improved detection rates to advance fluorescence sensing, DNA sequencing and dynamic studies of molecular interactions.
Using the ultraviolet autofluorescence of tryptophan aminoacids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labelling. However, the low ...autofluorescence signals have so far limited the UV detection to large proteins containing several tens of tryptophan residues. This limit is not compatible with the vast majority of proteins which contain only a few tryptophans. Here we push the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Our results show how the combination of nanophotonic plasmonic antennas, antioxidants and background reduction techniques can improve the signal-to-background ratio by over an order of magnitude and enable UV-FCS on thermonuclease proteins with a single tryptophan residue. This sensitivity breakthrough unlocks the applicability of UV-FCS technique to a broad library of label-free proteins.
Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with ...other species. To avoid the need for a fluorescent label, the intrinsic autofluorescence of proteins in the ultraviolet offers the benefits of fluorescence techniques without introducing the labelling drawbacks. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. Here we introduce optical horn antennas, a dedicated nanophotonic platform enabling the label-free detection of single proteins in the UV. This design combines fluorescence plasmonic enhancement, efficient collection up to 85{\textdegree} angle and background screening. We detect the UV autofluorescence from immobilized and diffusing single proteins, and monitor protein unfolding and dissociation upon denaturation. Optical horn antennas open up a unique and promising form of fluorescence spectroscopy to investigate single proteins in their native states in real time.
Single photon sources with high brightness and subnanosecond lifetimes are key components for quantum technologies. Optical nanoantennas can enhance the emission properties of single quantum ...emitters, but this approach requires accurate nanoscale positioning of the source at the plasmonic hotspot. Here, we use plasmonic nanoantennas to simultaneously trap single colloidal quantum dots and enhance their photoluminescence. The nano-optical trapping automatically locates the quantum emitter at the nanoantenna hotspot without further processing. Our dedicated nanoantenna design achieves a high trap stiffness of 0.6 fN/nm/mW for quantum dot trapping, together with a relatively low trapping power of 2 mW/\(\mu\)m\(^2\). The emission from the nanoantenna-trapped single quantum dot shows 7x increased brightness, 50x reduced blinking, 2x shortened lifetime and a clear antibunching below 0.5 demonstrating true single photon emission. Combining nano-optical tweezers with plasmonic enhancement is a promising route for quantum technologies and spectroscopy of single nano-objects.
The vast majority of proteins are intrinsically fluorescent in the ultraviolet, thanks to the emission from their tryptophan and tyrosine amino-acid constituents. However, the protein ...autofluorescence quantum yields are generally very low due to the prevailing quenching mechanisms by other amino acids inside the protein. This motivates the interest to enhance the radiative emission rate of proteins using nanophotonic structures. Although there have been numerous reports of Purcell effect and local density of optical states (LDOS) control in the visible range using single dipole quantum emitters, the question remains open to apply these concepts in the UV on real proteins containing several tryptophan and tyrosine amino acids arranged in a highly complex manner. Here, we report the first complete characterization of the Purcell effect and radiative rate enhancement for the UV intrinsic fluorescence of label-free \b{eta}-galactosidase and streptavidin proteins in plasmonic aluminum nanoapertures. We find an excellent agreement with a calibration performed using a high quantum yield UV fluorescent dye. Demonstrating and intensifying the Purcell effect is essential for the applications of UV plasmonics and the label-free detection of single proteins.
Extending plasmonics into the ultraviolet range imposes the use of aluminum to achieve the best optical performance. However, water corrosion is a major limitation for UV aluminum plasmonics, as this ...phenomenon occurs significantly faster in the presence of UV light, even at low laser powers of a few microwatts. Here, we assess the performance of nanometer-thick layers of various metal oxides deposited by atomic layer deposition (ALD) and plasma-enhanced chemical vapor deposition (PECVD) on top of aluminum nanoapertures to protect the metal against UV photocorrosion. The combination of a 5 nm Al2O3 layer covered by a 5 nm TiO2 capping provides the best resistance performance, while a single 10 nm layer of SiO2 or HfO2 is a good alternative. We also report the influence of the laser wavelength, the laser operation mode, and the pH of the solution. Appropriately choosing these conditions significantly extends the range of optical powers for which the aluminum nanostructures can be used. As an application, we demonstrate the label-free detection of streptavidin proteins with an improved signal-to-noise ratio. Our approach is also beneficial to promote the long-term stability of aluminum nanostructures. Finding the appropriate nanoscale protection against aluminum corrosion is the key to enabling the development of UV plasmonic applications in chemistry and biology.
Extending plasmonics into the ultraviolet range imposes the use of aluminum to achieve the best optical performance. However, water corrosion is a major limiting issue for UV aluminum plasmonics, as ...this phenomenon occurs significantly faster in presence of UV light, even at low laser powers of a few microwatts. Here we assess the performance of nanometer-thick layers of various metal oxides deposited by atomic layer deposition (ALD) and plasma-enhanced chemical vapor deposition (PECVD) on top of aluminum nanoapertures to protect the metal against UV photocorrosion. The combination of a 5 nm Al2O3 layer covered by a 5 nm TiO2 capping provides the best resistance performance, while a single 10 nm layer of SiO2 or HfO2 is a good alternative. We also report the influence of the laser wavelength, the laser operation mode and the pH of the solution. Properly choosing these conditions significantly extends the range of optical powers for which the aluminum nanostructures can be used. As application, we demonstrate the label-free detection of streptavidin proteins with improved signal to noise ratio. Our approach is also beneficial to promote the long-term stability of the aluminum nanostructures. Finding the appropriate nanoscale protection against aluminum corrosion is the key to enable the development of UV plasmonic applications in chemistry and biology.
Nanoapertures milled in metallic films called zero-mode waveguides (ZMWs) overcome the limitations of classical confocal microscopes by enabling single molecule analysis at micromolar concentrations ...with improved fluorescence brightness. While the ZMWs have found many applications in single molecule fluorescence studies, their shape has been mainly limited to be circular. Owing to the large parameter space to explore and the lack of guidelines, earlier attempts using more elaborate shapes have led to unclear conclusions whether or not the performance was improved as compared to a circular ZMW. Here, we comparatively analyze the performance of rectangular-shaped nanoapertures milled in aluminum to enhance the fluorescence emission rate of single molecules from the near infrared to the deep ultraviolet. Our new design is based on rational principles taking maximum advantage of the laser linear polarization. While the long edge of the nanorectangle is set to meet the cut-off size for the propagation of light into the nanoaperture, the short edge is reduced to 30 nm to accelerate the photodynamics while maintaining bright fluorescence rates. Our results show that both in the red and in the ultraviolet, the nanorectangles provide 50% brighter photon count rates as compared to the best performing circular ZMWs and achieve fluorescence lifetimes shorter than 300 ps. These findings can be readily used to improve the performance of ZMWs, especially for fast biomolecular dynamics, bright single-photon sources, and ultraviolet plasmonics.
Nanoapertures milled in metallic films called zero-mode waveguides (ZMWs) overcome the limitations of classical confocal microscopes by enabling single molecule analysis at micromolar concentrations with improved fluorescence brightness.
Extending plasmonics into the ultraviolet range imposes the use of aluminum to achieve the best optical performance. However, water corrosion is a major limiting issue for UV aluminum plasmonics, as ...this phenomenon occurs significantly faster in presence of UV light, even at low laser powers of a few microwatts. Here we assess the performance of nanometer-thick layers of various metal oxides deposited by atomic layer deposition (ALD) and plasma-enhanced chemical vapor deposition (PECVD) on top of aluminum nanoapertures to protect the metal against UV photocorrosion. The combination of a 5 nm Al2O3 layer covered by a 5 nm TiO2 capping provides the best resistance performance, while a single 10 nm layer of SiO2 or HfO2 is a good alternative. We also report the influence of the laser wavelength, the laser operation mode and the pH of the solution. Properly choosing these conditions significantly extends the range of optical powers for which the aluminum nanostructures can be used. As application, we demonstrate the label-free detection of streptavidin proteins with improved signal to noise ratio. Our approach is also beneficial to promote the long-term stability of the aluminum nanostructures. Finding the appropriate nanoscale protection against aluminum corrosion is the key to enable the development of UV plasmonic applications in chemistry and biology.
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