Achieving High Temporal Resolution in Single‐Molecule Fluorescence Techniques Using Plasmonic Nanoantennas

Abstract

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 are proposed as a solution, but despite huge fluorescence enhancement having been obtained with these structures, the brightness generally remains below 1 million photons/s/molecule. Therefore, the improvement of temporal resolution is overlooked. This article presents a method for achieving high temporal resolution in single‐molecule fluorescence techniques using plasmonic nanoantennas, specifically optical horn antennas. This work demonstrates 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 measurements. This work expands the applications of plasmonic antennas and zero‐mode waveguides in the fluorescence saturation regime toward brighter single‐molecule signal, faster temporal resolutions, and improved detection rates to advance fluorescence sensing, DNA sequencing, and dynamic studies of molecular interactions.

Single-Molecule FRET at 10 MHz Count Rates

“…Crucially, although methods enabling access to faster time scales through correlation techniques exist, , time-resolving individual rare events, such as the rapid jumps across the barriers between two conformational states (e.g., protein or nucleic acid folding), require very high count rates from single molecules. , These transition paths have gained increasing interest, and smFRET experiments have been playing a major role in revealing the nature and time scales of these paths. , However, the photon count rate (PCR) required for accessing the relevant microsecond time scale mandates the use of high excitation intensities, leading to saturation and increased photobleaching, which makes the task of measuring these transition paths very challenging. Plasmonic hotspots have been shown to increase the photostability and brightness of fluorescent labels , and offer a potentially complementary strategy to the more commonly used chemical photostabilization. − Coupling the emitters to plasmonic hotspots not only increases the electric field between the two plasmonic nanoparticles but also increases the emission rates of the fluorophore, leading to a shorter time that the molecule spends in the reactive excited states as well as reducing the time until the fluorophore is available for re-excitation. , This, in turn, results in improved photostability of fluorescent labels and also enables higher fluorescence intensities without saturation. ,, First examples employing similar strategies for diffusing molecules have appeared and showed promising results, , but they are limited to the submillisecond time scales of molecular diffusion through the excitation volume, which makes the complete observation of complex biomolecular pathways unlikely. DNA origami nanoantennas , have overcome the challenge of selective immobilization of biomolecules in plasmonic hotspots.…”

“…14,15 This, in turn, results in improved photostability of fluorescent labels and also enables higher fluorescence intensities without saturation. 9,16,17 First examples employing similar strategies for diffusing molecules have appeared and showed promising results, 18,19 but they are limited to the submillisecond time scales of molecular diffusion through the excitation volume, which makes the complete observation of complex biomolecular pathways unlikely. DNA origami nanoantennas 20,21 have overcome the challenge of selective immobilization of biomolecules in plasmonic hotspots.…”

Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices

“…Plasmonic nanoparticles are revolutionizing the field because they can enhance a dye’s fluorescence intensity >10 3 -fold resulting in a vastly improved signal-to-background ratio. − These have been employed to probe single biomolecules in highly concentrated samples , and to improve the overall brightness of single fluorophores. − Plasmon-enhanced fluorescence has recently been applied to the field of diagnostics, , where multiple assay designs were used to detect analyte in point-of-care devices. The strong plasmon-enhanced signals generated by a dimeric nanogap antenna enabled miniaturization of the optical setup to a point-of-care device, thereby highlighting the promise of the technology for biosensing.…”

Single-Molecule FRET at 10 MHz Count Rates

“…This, in turn, results in improved photostability of fluorescent labels and also enables higher fluorescence intensities without saturation [9, 16, 17]. First examples employing similar strategies for diffusing molecules have appeared and showed promising results [18, 19], but they are limited to the submillisecond timescales of molecular diffusion through the excitation volume, which makes the complete observation of complex biomolecular pathways unlikely. DNA origami nanoantennas [20, 21] have overcome the challenge of selective immobilization of biomolecules in plasmonic hotspots.…”