Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.

DNA Self-Assembly of Single Molecules with Deterministic Position and Orientation.

An ideal nanofabrication method should allow the organization of nanoparticles and molecules with nanometric positional precision, stoichiometric control, and well-defined orientation. The DNA origami technique has evolved into a highly versatile bottom-up nanofabrication methodology that fulfils almost all of these features. It enables the nanometric positioning of molecules and nanoparticles with stoichiometric control, and even the orientation of asymmetrical nanoparticles along predefined directions. However, orienting individual molecules has been a standing challenge. Here, we show how single molecules, namely, Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami with controlled orientation by doubly linking them to oligonucleotide strands that are hybridized while leaving unpaired bases in the scaffold. Increasing the number of bases unpaired induces a stretching of the fluorophore linkers, reducing its mobility freedom, and leaves more space for the fluorophore to accommodate and find different sites for interaction with the DNA. Particularly, we explore the effects of leaving 0, 2, 4, 6, and 8 bases unpaired and find extreme orientations for 0 and 8 unpaired bases, corresponding to the molecules being perpendicular and parallel to the DNA double-helix, respectively. We foresee that these results will expand the application field of DNA origami toward the fabrication of nanodevices involving a wide range of orientation-dependent molecular interactions, such as energy transfer, intermolecular electron transport, catalysis, exciton delocalization, or the electromagnetic coupling of a molecule to specific resonant nanoantenna modes.

Optical Ultracompact Directional Antennas Based on a Dimer Nanorod Structure.

Controlling directionality of optical emitters is of utmost importance for their application in communication and biosensing devices. Metallic nanoantennas have been proven to affect both excitation and emission properties of nearby emitters, including the directionality of their emission. In this regard, optical directional nanoantennas based on a Yagi-Uda design have been demonstrated in the visible range. Despite this impressive proof of concept, their overall size (~λ2/4) and considerable number of elements represent obstacles for the exploitation of these antennas in nanophotonic applications and for their incorporation onto photonic chips. In order to address these challenges, we investigate an alternative design. In particular, we numerically study the performance of a recently demonstrated "ultracompact" optical antenna based on two parallel gold nanorods arranged as a side-to-side dimer. Our results confirm that the excitation of the antiphase mode of the antenna by a nanoemitter placed in its near-field can lead to directional emission. Furthermore, in order to verify the feasibility of this design and maximize the functionality, we study the effect on the directionality of several parameters, such as the shape of the nanorods, possible defects in the dimer assembly, and different positions and orientations of the nanoemitter. We conclude that this design is robust to structural variations, making it suitable for experimental upscaling.

DNA-Templated Ultracompact Optical Antennas for Unidirectional Single-Molecule Emission.

Optical antennas are nanostructures designed to manipulate light-matter interactions by interfacing propagating light with localized optical fields. In recent years, numerous devices have been realized to efficiently tailor the absorption and/or emission rates of fluorophores. By contrast, modifying the spatial characteristics of their radiation fields remains challenging. Successful phased array nanoantenna designs have required the organization of several elements over a footprint comparable to the operating wavelength. Here, we report unidirectional emission of a single fluorophore using an ultracompact optical antenna. The design consists of two side-by-side gold nanorods self-assembled via DNA origami, which also controls the positioning of the single-fluorophore. Our results show that when a single fluorescent molecule is positioned at the tip of one nanorod and emits at a frequency capable of driving the antenna in the antiphase mode, unidirectional emission with a forward to backward ratio of up to 9.9 dB can be achieved.

An alternative to MINFLUX that enables nanometer resolution in a confocal microscope.

Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1-2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

In Situ Photothermal Response of Single Gold Nanoparticles through Hyperspectral Imaging Anti-Stokes Thermometry.

Several fields of applications require a reliable characterization of the photothermal response and heat dissipation of nanoscopic systems, which remains a challenging task for both modeling and experimental measurements. Here, we present an implementation of anti-Stokes thermometry that enables the in situ photothermal characterization of individual nanoparticles (NPs) from a single hyperspectral photoluminescence confocal image. The method is label-free, potentially applicable to any NP with detectable anti-Stokes emission, and does not require any prior information about the NP itself or the surrounding media. With it, we first studied the photothermal response of spherical gold NPs of different sizes on glass substrates, immersed in water, and found that heat dissipation is mainly dominated by the water for NPs larger than 50 nm. Then, the role of the substrate was studied by comparing the photothermal response of 80 nm gold NPs on glass with sapphire and graphene, two materials with high thermal conductivity. For a given irradiance level, the NPs reach temperatures 18% lower on sapphire and 24% higher on graphene than on bare glass. The fact that the presence of a highly conductive material such as graphene leads to a poorer thermal dissipation demonstrates that interfacial thermal resistances play a very significant role in nanoscopic systems and emphasize the need for in situ experimental thermometry techniques. The developed method will allow addressing several open questions about the role of temperature in plasmon-assisted applications, especially ones where NPs of arbitrary shapes are present in complex matrixes and environments.

A DNA Origami Platform for Single-Pair Förster Resonance Energy Transfer Investigation of DNA-DNA Interactions and Ligation.

DNA double-strand breaks (DSBs) pose an everyday threat to the conservation of genetic information and therefore life itself. Several pathways have evolved to repair these cytotoxic lesions by rejoining broken ends, among them the nonhomologous end-joining mechanism that utilizes a DNA ligase. Here, we use a custom-designed DNA origami nanostructure as a model system to specifically mimic a DNA DSB, enabling us to study the end-joining of two fluorescently labeled DNA with the T4 DNA ligase on the single-molecule level. The ligation reaction is monitored by Förster resonance energy transfer (FRET) experiments both in solution and on surface-anchored origamis. Due to the modularity of DNA nanotechnology, DNA double strands with different complementary overhang lengths can be studied using the same DNA origami design. We show that the T4 DNA ligase repairs sticky ends more efficiently than blunt ends and that the ligation efficiency is influenced by both DNA sequence and the incubation conditions. Before ligation, dynamic fluctuations of the FRET signal are observed due to transient binding of the sticky overhangs. After ligation, the FRET signal becomes static. Thus, we can directly monitor the ligation reaction through the transition from dynamic to static FRET signals. Finally, we revert the ligation process using a restriction enzyme digestion and religate the resulting blunt ends. The here-presented DNA origami platform is thus suited to study complex multistep reactions occurring over several cycles of enzymatic treatment.

Directing Single-Molecule Emission with DNA Origami-Assembled Optical Antennas.

We demonstrate the capability of DNA self-assembled optical antennas to direct the emission of an individual fluorophore, which is free to rotate. DNA origami is used to fabricate optical antennas composed of two colloidal gold nanoparticles separated by a predefined gap and to place a single Cy5 fluorophore near the gap center. Although the fluorophore is able to rotate, its excitation and far-field emission is mediated by the antenna, with the emission directionality following a dipolar pattern according to the antenna main resonant mode. This work is intended to set out the basis for manipulating the emission pattern of single molecules with self-assembled optical antennas based on colloidal nanoparticles.

DNA-Mediated Self-Assembly of Plasmonic Antennas with a Single Quantum Dot in the Hot Spot.

DNA self-assembly is a powerful tool to arrange optically active components with high accuracy in a large parallel manner. A facile approach to assemble plasmonic antennas consisting of two metallic nanoparticles (40 nm) with a single colloidal quantum dot positioned at the hot spot is presented here. The design approach is based on DNA complementarity, stoichiometry, and steric hindrance principles. Since no intermediate molecules other than short DNA strands are required, the structures possess a very small gap (≈ 5 nm) which is desired to achieve high Purcell factors and plasmonic enhancement. As a proof-of-concept, the fluorescence emission from antennas assembled with both conventional and ultrasmooth spherical gold particles is measured. An increase in fluorescence is obtained, up to ≈30-fold, compared to quantum dots without antenna.

Sculpting Light by Arranging Optical Components with DNA Nanostructures.

DNA nanotechnology has developed into a state where the design and assembly of complex nanoscale structures has become fast, reliable, cost-effective, and accessible to non-experts. Nanometer-precise positioning of organic (dyes, biomolecules, etc.) and inorganic (metal nanoparticles, colloidal quantum dots, etc.) components on DNA nanostructures is straightforward and modular. In this perspective article, we identify the opportunities and challenges that DNA-assembled devices and materials are facing for optical antennas, metamaterials, and sensing applications. With the abilities of arranging hybrid materials in defined geometries, plasmonic effects will, for example, amplify molecular recognition transduction so that single-molecule events will be measureable with simple devices. On the larger scale, DNA nanotechnology has the potential of breaking the symmetry of common self-assembled functional materials creating pre-defined optical properties such as refractive index tuning, Bragg reflection and topological insulation.

Plasmon-Exciton Coupling Using DNA Templates.

Coherent energy exchange between plasmons and excitons is a phenomenon that arises in the strong coupling regime resulting in distinct hybrid states. The DNA-origami technique provides an ideal framework to custom-tune plasmon-exciton nanostructures. By employing this well controlled self-assembly process, we realized hybrid states by precisely positioning metallic nanoparticles in a defined spatial arrangement with fixed nanometer-sized interparticle spacing. Varying the nanoparticle diameter between 30 nm and 60 nm while keeping their separation distance constant allowed us to precisely adjust the plasmon resonance of the structure to accurately match the energy frequency of a J-aggregate exciton. With this system we obtained strong plasmon-exciton coupling and studied far-field scattering at the single-structure level. The individual structures displayed normal mode splitting up to 170 meV. The plasmon tunability and the strong field confinement attained with nanodimers on DNA-origami renders an ideal tool to bottom-up assembly plasmon-exciton systems operating at room temperature.