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DNA origami can be applied as a "ruler" for nanoscale calibration or super-resolution fluorescence microscopy with an ideal structure for defining fluorophore arrangement, allowing the distance between fluorophores to be precisely controlled at the nanometer scale. DNA origami can also be used as a nanotag with arbitrary programmable shapes for topological identification. In this study, we formed a hexagonal origami structure embedded with three different fluorescent dyes on the surface. The distance between each fluorescent block was ~120 nm, which is below the diffraction limit of light, allowing for its application as a nano-ruler for super-resolution fluorescence microscopy. The outside edge of the hexagonal structure was redesigned to form three different substructures as topological labels. Atomic and scanning force microscopy demonstrated the consistent nanoscale distance between morphological and fluorescent labels. Therefore, this fluorophore-embedded hexagonal origami platform can be used as a dual nanoruler for optical and topological calibration.

Nanosized lipid vesicles are ubiquitous in living systems (e.g. cellular compartments or extracellular vesicles, EVs) and in formulations for nanomedicine (e.g. liposomes for RNA vaccine formulations). The mechanical properties of such vesicles are crucial in several physicochemical and biological processes, ranging from cellular uptake to stability in aerosols. However, their accurate determination remains challenging and requires sophisticated instruments and data analysis. Here we report the first evidence that the surface plasmon resonance (SPR) of citrated gold nanoparticles (AuNPs) adsorbed on synthetic vesicles is finely sensitive to the vesicles’ mechanical properties. We then leverage this finding to show that the SPR tracking provides quantitative access to the stiffness of vesicles of synthetic and natural origin, such as EVs. The demonstration of this plasmon-based ‘‘stiffness nanoruler’’ paves the way for developing a facile, cost-effective and high-throughput method to assay the mechanical properties of dispersions of vesicles of nanometric size and unknown composition at a collective level.

If a macromolecule with an attached single donor–acceptor (D–A) pair jumps from one conformation to another, the distance R in the D–A pair is changed and this fact manifests itself in trajectory E(t) of Forster resonance energy transfer (FRET) efficiency. Earlier influence of conformational changes in such a macromolecule has been studied with the help of a theoretical expression for the distribution P(E) of FRET efficiency. The distribution P(E) did not take into account the existence of triplet levels in the D–A pair. Therefore, we were facing two problems. The first problem was in the investigation of the influence of triplet states existing in real D–A pairs on FRET efficiency. The second problem was in finding the distribution PR(R) with the help of the measured distribution function P(E). Singlet–triplet transitions in the D–A pair create additional fluctuations in the fluorescence of the D–A pair attached to a protein molecule. The method based on statistical treatment of FRET efficiency trajector...

In recent years, DNA origami nanorulers for superresolution (SR) fluorescence microscopy have been developed from fundamental proof-of-principle experiments to commercially available test structures. The self-assembled nanostructures allow placing a defined number of fluorescent dye molecules in defined geometries in the nanometer range. Besides the unprecedented control over matter on the nanoscale, robust DNA origami nanorulers are reproducibly obtained in high yields. The distances between their fluorescent marks can be easily analysed yielding intermark distance histograms from many identical structures. Thus, DNA origami nanorulers have become excellent reference and training structures for superresolution microscopy. In this work, we go one step further and develop a calibration process for the measured distances between the fluorescent marks on DNA origami nanorulers. The superresolution technique DNA-PAINT is used to achieve nanometrological traceability of nanoruler distances following the guide to the expression of uncertainty in measurement (GUM). We further show two examples how these nanorulers are used to evaluate the performance of TIRF microscopes that are capable of single-molecule localization microscopy (SMLM).

Nanoplasmonics has been rapidly developing for the last 30 years and it finds applications in many areas, including medicine, biology, and chemistry. Plasmon nanoparticles are used in biosensing and genomics, for bioimaging of cell structures, for targeted delivery of drugs and photothermolysis of cancer cells. All these applications often require real-time detection of nanoparticle position at the nanoscale making this problem highly relevant. We propose new far-field method of real-time detection of plasmon particles location by detecting the magnitude of localized surface plasmon resonance splitting. In contrast to the existing methods based on the shift of plasmon resonance central line, the suggested technique is sensitive to the surface, and allows detecting metal nanoparticle position relatively to internal interfaces such as cell walls or chemical phase boundaries. The presented numerical simulations are aimed on modelling of biological tissues environment and demonstrate the possibility of high accuracy detection of nanoparticle position. (© 2015 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

Attainment of spatial resolutions far below diffraction limits by means of optical methods constitutes a challenging task. Here, we design nonlinear nanorulers that are capable of accomplishing approximately 1 nm resolutions by utilizing the mechanism of plasmon-enhanced second-harmonic generation (PESHG). Through introducing Au@SiO2 (core@shell) shell-isolated nanoparticles, we strive to maneuver electric-field-related gap modes such that a reliable relationship between PESHG responses and gap sizes, represented by "PESHG nanoruler equation", can be obtained. Additionally validated by both experiments and simulations, we have transferred "hot spots" to the film-nanoparticle-gap region, ensuring that retrieved PESHG emissions nearly exclusively originate from this region and are significantly amplified. The PESHG nanoruler can be potentially developed as an ultrasensitive optical method for measuring nanoscale distances with higher spectral accuracies and signal-to-noise ratios.

An optical nanoruler system based on a conjugated polyelectrolyte-silver nanoprism pair is developed for label-free protein detection by taking advantage of the metal-enhanced fluorescence effect of silver nanostructures. Antibody-antigen interactions induce a change in the metal-fluorophore distance, followed by the response of a fluorescent signal of the conjugated polyelectrolyte. The system is used to detect target antigens sensitively and selectively.

In this study we use nanophotonic effects of graphene to study DNA hybridization: the z−4 nanoscale distance-dependence of the fluorescence lifetime for fluorophores located in the vicinity of graphene is for the first time used to track a DNA hybridization reaction with nanoscale resolution in real time. First, a nanostaircase with ≈2 nm steps from 0 to a total height of 48 nm is used as a nanoruler to confirm the distance dependence law. We find that the axial sensitivity is suited to determine the nanoscale surface roughness of these samples. The proof-of-concept DNA experiments in aqueous medium involve the hybridization of fluorescently labelled DNA beacons attached to CVD grown graphene with complementary (target) DNA added in solution. We track the conformational changes of the beacons statistically by determining the fluorescence lifetimes of the labelling dye and converting them into nanoscale distances from the graphene. In this way, we are able to monitor the vertical displacement of the label during DNA-beacon unfolding with an axial resolution reaching down to 1 nm. The measured distance increase during the DNA hybridization reaction of about 10 nm matches the length of the target DNA strand. Furthermore, the width of the fluorescence lifetime distributions could be used to estimate the molecular tilt angle of the hybridized ds-DNA configuration. The achieved nanoscale sensitivity opens innovation opportunities in material engineering, genetics, biochemistry and medicine., INL received support for this project from the CCDR-N via the project 'Nanotechnology based functional solutions' (Grant No. NORTE-01-0145-FEDER-000019) and from the Portuguese Foundation for Science and Technology (FCT) via the project 'ON4SupremeSens' PTDC/NAN-OPT/29417/2017. Edite Figueiras received a Marie Curie fellowship via the EU-EC COFUND program 'NanoTRAINforGrowth' (Grant No. 600375). U Minho research was partially supported by the FCT in the framework of the Strategic Funding UID/FIS/04650/2013.

International audience; Förster resonance energy transfer (FRET) plays a key role in biochemistry, organic photovoltaics, and lighting sources. FRET is commonly used as a nanoruler for the short (nanometer) distance between donor and acceptor dyes, yet FRET is equally sensitive to the mutual dipole orientation. The orientation dependence complicates the FRET analysis in biological samples and may even lead to the absence of FRET for perpendicularly oriented donor and acceptor dipoles. Here, we exploit the strongly inhomogeneous and localized fields in plasmonic nanoantennas to open new energy transfer routes, overcoming the limitations from the mutual dipole orientation to ultimately enhance the FRET efficiency. We demonstrate that the simultaneous presence of perpendicular near-field components in the nanoantenna sets favorable energy transfer routes that increase the FRET efficiency up to 50% for nearly perpendicular donor and acceptor dipoles. This new facet of plasmonic nanoantennas enables dipole–dipole energy transfer that would otherwise be forbidden in a homogeneous environment. As such, our approach further increases the applicability of single-molecule FRET over diffraction-limited approaches, with the additional benefits of higher sensitivities and higher concentration ranges toward physiological levels.

We demonstrate a plasmon nanoruler using a coupled film-nanoparticle (film-NP) format that is well suited for investigating the sensitivity extremes of plasmonic coupling. Because it is relatively straightforward to functionalize bulk, surface plasmon supporting films such as gold, we are able to precisely control plasmonic gap dimensions by creating ultra-thin molecular spacer layers on the gold films, on top of which we immobilize plasmon resonant nanoparticles (NPs). Each immobilized NP becomes coupled to the underlying film and functions as a plasmon nanoruler, exhibiting a distance-dependent resonance red-shift in its peak plasmon wavelength as it approaches the film. Due to the uniformity of response from the film-NPs to separation distance, we are able to use extinction and scattering measurements from ensembles of film-NPs to characterize the coupling effect over a series of very short separation distances – ranging from 5 – 20 Å – and combine these measurements with similar data from larger separation distances extending out to 27 nm. We find that the film-NP plasmon nanoruler is extremely sensitive at very short film-NP separation distances, yielding spectral shifts as large as 5 nm for every 1 Å change in separation distance. The film-NP coupling at extremely small spacings is so uniform and reliable that we are able to usefully probe gap dimensions where the classical Drude model of the conducting electrons in the metals is no longer descriptive; for gap sizes smaller than a few nanometers, either quantum or semi-classical models of the carrier response must be employed to predict the observed wavelength shifts. We find that, despite the limitations, large field enhancements and extreme sensitivity persist down to even the smallest gap sizes.

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

We develop a new method based on the continuous-fraction approach as applied to the electrostatic boundary problem for determining surface plasmon resonances of coupled nanospheres. The resonance frequency dependence on interparticle gap (plasmon nanoruler effect) is determined by avoiding time-consuming numerical calculations even for very small interparticle separations, where known procedures face serious difficulties. Corrections due to retardation effects are accounted for, allowing description of larger nanopairs. Good agreement is demonstrated both with results of numerical calculations and with experimental data.

Dynamic structural changes of folding protein and biological macromolecules can be monitored using the single-pair fluorescence resonance energy transfer (sp-FRET) technique, which can be used as a nanoscale ruler. Although recent progresses in applying such a state-of-the-art nanoruler to biological systems have been made, this technique still needs to be improved. In order to understand that what kind of information can be extracted from such measurements, we simulate a sp-FRET experiment as an illustrative example, in which single FRET dye pair is attached to the ends of a folding protein chain that undergoes diffusion in water. And we propose novel analysis methods with the definite purpose of extracting dynamic information at nanometer scale and the free-energy profile for protein folding as much as possible. It is suggested that the present analysis method is particularly useful for extracting the single-molecule fluorescence trajectories and free-energy landscape of protein folding. This work establishes a useful analysis technique and guideline for the sp-FRET spectroscopy experiments to monitor the folding free-energy landscape of proteins.

We report on our recent progress in the study of single Dibenzoterrylene (DBT) molecules as single photon sources and nanoscale probes. We consider DBT molecules embedded in thin anthracene films, a system that allows stable single photon emission both at room and at cryogenic temperatures. We investigate the most important optical properties of the DBT:anthracene system as a whole. We then perform a full statistical study of the coupling between single DBT molecules by measuring the lifetimes of DBT both in the coupled and in the uncoupled case. The experimental results are framed into a simple universal scaling model, where the magnitude of coupling depends solely on universal parameters and on the distance d between the single emitter and the graphene monolayer. We apply this model to infer d and provide a proof of principle for a position ruler at the nanoscale [1].

We design a two-dimensional distance and angle location ruler based on surface plasmon resonance (SPR) effect, whose light-scattering spectra shift when either the distance of two gold nanospheres or the angle between their axis and the polarization is changed.

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution.

Let me get my nanoruler: Activatable probes based on radionuclide and quantum dots (QDs) were constructed using DNA as a linker. Cerenkov radiation from (64)Cu was used to excite the QDs in a distance-dependent manner. The luminescence was lowest nearest to the QD and increased with distance.

Simultaneous measurement of surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR) in nanoparticle dimers presents outstanding opportunities in molecular identification and in the elucidation of physical properties, such as the size, distance, and deformation of target species. SERS–LSPR instrumentation exists and has been used under limited conditions, but the extraction of SERS and LSPR readouts from a single measurement is still a challenge. Herein, the extraction of LSPR spectra from SERS signals is reported and a tool for measuring the interparticle distance from Raman enhancement data by the standardization of the SERS signal is proposed. The SERS nanoruler mechanism incorporates two important aspects (the LSPR scattering peak shift and the Raman shift for measuring interparticle distance), and signifies their exact one-to-one correspondence after spectral correction. The developed methodology is applied to calculate the interparticle distance between nanoparticle dimers from SERS signals, to detect and quantify DNA at the single-molecule level in a base-pair-specific manner. It is also shown that the SERS nanoruler concept can be used in structural analysis for the specific detection of the interaction of immunoglobulin G (IgG) with its target from bianalyte Raman signals with identical shaping at single-molecule resolution. The SERS profile shaping approach not only offers a new detection mechanism for single molecules, but also has excellent potential for studying protein interactions and the intracellular detection of mRNA.

The localized surface plasmon resonance (LSPR) spectrum associated with a gold nanoparticle (NP) coupled to a gold film exhibits extreme sensitivity to the nano-gap region where the fields are tightly localized. The LSPR of an ensemble of film-coupled NPs can be observed using an illumination scheme similar to that used to excite the surface plasmon resonance (SPR) of a thin metallic film; however, in the present system, the light is used to probe the highly sensitive distance-dependent LSPR of the gaps between NPs and film rather than the delocalized SPR of the film. We show that the SPR and LSPR spectral contributions can be readily distinguished, and we compare the sensitivities of both modes to displacements in the average gap between a collection of NPs and the gold film. The distance by which the NPs are suspended in solution above the gold film is fixed via a thin molecular spacer layer, and can be further modulated by subjecting the NPs to a quasistatic electric field. The observed LSPR spectral shifts triggered by the applied voltage can be correlated with Angstrom scale displacements of the NPs, suggesting the potential for chip-scale or flow-cell plasmonic nanoruler devices with extreme sensitivity.

The aim of a nanoruler is to measure distances on a length scale in the order of nanometers. This concept can be realized by two plasmonic nanoparticles (usually spheres), which are placed within a short distance to each other. Since the plasmon modes of both particles are coupled, they influence each other along the interparticle axis (i.e. longitudinal plasmon mode, resulting in a shift of the resonance wavelength compared to the single particle. Because the resulting resonance wavelength depends on the interparticle distance, the spectral investigation of such a dimer allows the determination of the interparticle distance.

Förster resonance energy transfer (FRET) is applied extensively in all fields of biological research and technology, generally as a 'nanoruler' with a dynamic range corresponding to the intramolecular and intermolecular distances characterizing the molecular structures that regulate cellular function. The complex underlying network of interactions reflects elementary reactions operating under strict spatio-temporal control: binding, conformational transition, covalent modification and transport. FRET imaging provides information about all these molecular processes with high specificity and sensitivity via probes expressed by or introduced from the external medium into the cell, tissue or organism. Current approaches and developments in the field are discussed with emphasis on formalism, probes and technical implementation.

WOS:000456749900050; International audience; DNA-nanoparticle conjugates are important tools in nanobiotechnology. Knowing the orientation, function, and length of DNA on nanoparticle surfaces at low nanomolar concentrations under physiological conditions is therefore of great interest. Here, we investigate the conformation of a 31 nucleotides (nt) long DNA attached to a semiconductor quantum dot (QD) via Forster resonance energy transfer (FRET) from Tb-DNA probes hybridized to different positions on the QD-DNA. Precise Tb-to-QD distance determination from 7 to 14 nm along 26 nt of the peptide-appended QD-DNA was realized by time-resolved FRET spectroscopy. The FRET nanoruler measured linear single-stranded (ssDNA) and double-stranded (dsDNA) extensions of similar to 0.15 and similar to 0.31 nm per base, reflecting the different conformations. Comparison with biomolecular modeling confirmed the denser conformation of ssDNA and a possibly more flexible orientation on the QD surface, whereas the dsDNA was fully extended with radial orientation. The temporally distinct photoluminescence decays of the different DNA-FRET configurations were used for prototypical DNA hybridization assays that demonstrated the large potential for extended temporal multiplexing. The extensive experimental and theoretical analysis of 11 different distances/configurations of the same QD-DNA conjugate provided important information on DNA conformation on nanoparticle surfaces and will be an important benchmark for the development and optimization of DNA-nanobiosensors.

The evaluation of distances as small as few nanometers using optical waves Is a very challenging task that can pave the way for the development of new applications in biotechnology and nanotechnology. In this article, we propose a new measurement method based on the control of the nonlinear optical response of plasmonic nanostructures by means of Fano resonances. It is shown that Fano resonances resulting from the coupling between a bright mode and a dark mode at the fundamental wavelength enable unprecedented and direct manipulation of the nonlinear electromagnetic sources at the nanoscale. In the case of second harmonic generation from gold nanodolmens, the different nonlinear sources distributions induced by the different coupling regimes are clearly revealed in the far-field distribution. Hence, the configuration of the nanostructure can be accurately determined In 3-dimensions by recording the wave scattered at the second harmonic wavelength. Indeed, the conformation of the different elements building the system is encoded in the nonlinear far-field distribution, making second harmonic generation a promising tool for reading 3-dimension plasmonic nanorulers. Furthemore, it is shown that 3-dimension plasmonic nanorulers can be implemented with simpler geometries than in the linear regime while providing complete Information on the structure conformation, including the top nanobar position and orientation.

The realization and detection of Majorana fermions in condensed matter systems are of considerable importance and interest. We propose a scheme to detect the Majorana fermions by Fano resonance in hybrid nanostructures made of semiconductor quantum dots and quantum wire in proximity to superconductor. Through detailed theoretical studies of the transport properties of our hybrid nanostructures based on the non-equilibrium Green’s function technique and the equation of motion approach, it is found that the Fano resonance in the current response due to the interference among different transmission paths may give clear signature of the existence of Majorana modes. Moreover, we have found a peculiar relationship between the Fano factor q and the Majorana bound state coupling strength/the length of nanowire, which can be used for a design of an electronic nanoruler. Our method of detection of Majorana fermions based on Fano resonance is related to the global conductance profile, thus is robust to perturbations.