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Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches including optical voltage-sensing dyes and genetically encoded voltage indicators have evolved. Here, we present a DNA nanotechnology-based system. Using DNA origami, we incorporate and optimize different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we use a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA connecting the DNA origami and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit is read out by changes of Fluorescence Resonance Energy Transfer (FRET) of single sensors attached to liposomes. They show a FRET change of ∼5% for ΔΨ=100 mV and allow adapting the potential range of highest sensitivity. Further, the working mechanism is rationalized by molecular dynamics simulations. Our approach holds potential for the application as non-genetically encoded sensors at membranes.

Despite the thorough investigation of graphene since 2004, altering its surface chemistry and reproducible functionalization remain challenging. This hinders fabrication of more complex hybrid materials with controlled architectures, and as a consequence the development of sensitive and reliable sensors and biological assays. In this contribution, we introduce DNA origami structures as nanopositioners for placing single dye molecules at controlled distances from graphene. The measurements of fluorescence intensity and lifetime of single emitters carried out for distances ranging from 3 to 58 nm confirmed the d–4 dependence of the excitation energy transfer to graphene. Moreover, we determined the characteristic distance for 50% efficiency of the energy transfer from single dyes to graphene to be 17.7 nm. Using pyrene molecules as a glue to immobilize DNA origami nanostructures of various shape on graphene opens new possibilities to develop graphene-based biophysics and biosensing.

Smartphone-based fluorescence microscopy has been rapidly developing over the last few years, enabling point-of-need detection of cells, bacteria, viruses, and biomarkers. These mobile microscopy devices are cost-effective, field-portable, and easy to use, and benefit from economies of scale. Recent developments in smartphone camera technology have improved their performance, getting closer to that of lab microscopes. Here, we report the use of DNA origami nanobeads with predefined numbers of fluorophores to quantify the sensitivity of a smartphone-based fluorescence microscope in terms of the minimum number of detectable molecules per diffraction-limited spot. With the brightness of a single dye molecule as a reference, we compare the performance of color and monochrome sensors embedded in state- of-the-art smartphones. Our results show that the monochrome sensor of a smartphone can achieve better sensitivity, with a detection limit of ∼10 fluorophores per spot. The use of DNA origami nanobeads to quantify the minimum number of detectable molecules of a sensor is broadly applicable to evaluate the sensitivity of various optical instruments.

Graphene exhibits outstanding fluorescence quenching properties that can become useful for biophysics and biosensing applications, but it remains challenging to harness these advantages due to the complex transfer procedure of chemical vapor deposition-grown graphene to glass coverslips and the low yield of usable samples. Here, we screen 10 graphene-on-glass preparation methods and present an optimized protocol. To obtain the required quality for single-molecule and super-resolution imaging on graphene, we introduce a graphene screening method that avoids consuming the investigated sample. We apply DNA origami nanostructures to place fluorescent probes at a defined distance on top of graphene-on-glass coverslips. Subsequent fluorescence lifetime imaging directly reports on the graphene quality, as deviations from the expected fluorescence lifetime indicate imperfections. We compare the DNA origami probes with conventional techniques for graphene characterization, including light microscopy, atomic force microscopy, and Raman spectroscopy. For the latter, we observe a discrepancy between the graphene quality implied by Raman spectra in comparison to the quality probed by fluorescence lifetime quenching measured at the same position. We attribute this discrepancy to the difference in the effective area that is probed by Raman spectroscopy and fluorescence quenching. Moreover, we demonstrate the applicability of already screened and positively evaluated graphene for studying single-molecule conformational dynamics on a second DNA origami structure. Our results constitute the basis for graphene-based biophysics and super-resolution microscopy.

We present a technique to determine the orientation of single fluorophores attached to DNA origami structures based on two measurements. First, the orientation of the absorption transition dipole of the molecule is determined through a polarization-resolved excitation measurement. Second, the orientation of the DNA origami structure is obtained from a DNA-PAINT nanoscopy measurement. Both measurements are performed consecutively on a fluorescence wide-field microscope. We employed this approach to study the orientation of single ATTO 647N, ATTO 643, and Cy5 fluorophores covalently attached to a 2D rectangular DNA origami structure with different nanoenvironments, achieved by changing both the fluorophores' binding position and immediate vicinity. Our results show that when fluorophores are incorporated with additional space, for example, by omitting nucleotides in an elsewise double-stranded environment, they tend to stick to the DNA and to adopt a preferred orientation that depends more on the specific molecular environment than on the fluorophore type. With the aid of all-atom molecular dynamics simulations, we rationalized our observations and provide insight into the fluorophores' probable binding modes. We believe this work constitutes an important step toward manipulating the orientation of single fluorophores in DNA origami structures, which is vital for the development of more efficient and reproducible self-assembled nanophotonic devices.

Summary: DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes. Fil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Steiner, Florian. Ludwig Maximilians Universitat; Alemania Fil: Zähringer, Jonas. Ludwig Maximilians Universitat; Alemania Fil: Lopez, Lucía Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Bohlen, Johann. Ludwig Maximilians Universitat; Alemania Fil: Richter, Lars. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Cole, Fiona. Ludwig Maximilians Universitat; Alemania Fil: Tinnefeld, Philip. Ludwig Maximilians Universitat; Alemania Fil: Stefani, Fernando Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina

DNA nanotechnology and advances in the DNA origami technique have enabled facile design and synthesis of complex and functional nanostructures. Molecular devices are, however, prone to rapid functional and structural degradation due to the high proportion of surface atoms at the nanoscale and due to complex working environments. Besides stabilizing mechanisms, approaches for the self‐repair of functional molecular devices are desirable. Here we exploit the self‐assembly and reconfigurability of DNA origami nanostructures to induce the self‐repair of defects of photoinduced and enzymatic damage. We provide examples of repair in DNA nanostructures showing the difference between unspecific self‐regeneration and damage specific self‐healing mechanisms. Using DNA origami nanorulers studied by atomic force and superresolution DNA PAINT microscopy, quantitative preservation of fluorescence properties is demonstrated with direct potential for improving nanoscale calibration samples., Self‐assembly and reconfigurability of DNA origami is exploited to realize self‐repairing functional nanodevices. A self‐regenerating brightness label is realized by constant exchange of labeling strands from solution. Structural self‐healing of a DNA origami nanoruler is achieved by excess of staple strands that exchange with enzymatically or photo‐induced damaged strands.

Photobleaching is an effect terminating the photon output of fluorophores, limiting the duration of fluorescence-based experiments. Plasmonic nanoparticles (NPs) can increase the overall fluorophore photostability through an enhancement of the radiative rate. In this work, we use the DNA origami technique to arrange a single fluorophore in the 12-nm gap of a silver NP dimer and study the number of emitted photons at the single molecule level. Our findings yielded a 30× enhancement in the average number of photons emitted before photobleaching. Numerical simulations are employed to rationalize our results. They reveal the effect of silver oxidation on decreasing the radiative rate enhancement., We acknowledge funding by a starting grant (SiMBA, EU 261162) of the European Research Council (ERC) and the Deutsche Forschungsgesellschaft (AC 279/2-1 and TI 329/9-1). IK is grateful for the support by the Mobility Plus grant 1269/MOB/IV/2015/0 from the Polish Ministry of Science and Higher Education (MNiSW). CV thanks a scholarship of the Studienstiftung des deutschen Volkes. AIF-D and AC-G acknowledge funding from the Spanish MINECO under Contracts FIS2015- 64951-R and MDM-2014-0377-16-4, respectively. AIF-D also acknowledges funding from EU Seventh Framework Programme under Grant Agreement FP7-PEOPLE- 2013-CIG-630996. GA and PT acknowledge funding of the state ministry for research of lower saxony in the frame of the “Quantum- and Nanometrology” (QUANOMET) strategic research area. Quanomet is part of the LUH-TUBS research alliance

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).

Graphene is considered a game-changing material, especially for its mechanical and electrical properties. This work exploits that graphene is almost transparent but quenches fluorescence in a range up to ≈40 nm. Graphene as a broadband and unbleachable energy-transfer acceptor without labeling, is used to precisely determine the height of molecules with respect to graphene, to visualize the dynamics of DNA nanostructures, and to determine the orientation of Forster-type resonance energy transfer (FRET) pairs. Using DNA origami nanopositioners, biosensing, single-molecule tracking, and DNA PAINT super-resolution with

Plasmonic structures are known to affect the fluorescence properties of dyes placed in close proximity. This effect has been exploited in combination with single-molecule techniques for several applications in the field of biosensing. Among these plasmonic structures, top-down zero-mode waveguides stand out due to their broadband capabilities. In contrast, optical antennas based on gold nanostructures exhibit fluorescence enhancement on a narrow fraction of the visible spectrum typically restricted to the red to near-infrared region. In this contribution, we exploit the DNA origami technique to self-assemble optical antennas based on large (80 nm) silver nanoparticles. We have studied the performance of these antennas with far- and near-field simulations and characterized them experimentally with single-molecule fluorescence measurements. We demonstrate that silver-based optical antennas can yield a fluorescence enhancement of more than 2 orders of magnitude throughout the visible spectral range for high intrinsic quantum yield dyes. Additionally, a comparison between the performance of gold and silver-based antennas is included. The results indicate that silver-based antennas strongly outperform their gold counterparts in the blue and green ranges and exhibit marginal differences in the red range. These characteristics render silver-based optical antennas ready for applications involving several fluorescently labeled species across the visible spectrum.

An ideal point light source is as small and as bright as possible. For fluorescent point light sources, homogeneity of the light sources is important as well as that the fluorescent units inside the light source maintain their photophysical properties, which is compromised by dye aggregation. Here we propose DNA origami as a rigid scaffold to arrange dye molecules in a dense pixel array with high control of stoichiometry and dye-dye interactions. In order to find the highest labeling density in a DNA origami structure without influencing dye photophysics, we alter the distance of two ATTO647N dyes in single base pair steps and probe the dye-dye interactions on the single-molecule level. For small distances strong quenching in terms of intensity and fluorescence lifetime is observed. With increasing distance, we observe reduced quenching and molecular dynamics. However, energy transfer processes in the weak coupling regime still have a significant impact and can lead to quenching by singlet-dark-state-annihilation. Our study fills a gap of studying the interactions of dyes relevant for superresolution microscopy with dense labeling and for single-molecule biophysics. Incorporating these findings in a 3D DNA origami object will pave the way to bright and homogeneous DNA origami nanobeads.

We demonstrate the capability of DNA selfassembledoptical antennas to direct the emission of anindividual fluorophore, which is free to rotate. DNA origami isused to fabricate optical antennas composed of two colloidalgold nanoparticles separated by a predefined gap and to place asingle Cy5 fluorophore near the gap center. Although thefluorophore is able to rotate, its excitation and far-field emissionis mediated by the antenna, with the emission directionalityfollowing a dipolar pattern according to the antenna mainresonant mode. This work is intended to set out the basis formanipulating the emission pattern of single molecules with selfassembledoptical antennas based on colloidal nanoparticles. Fil: Hübner, Kristina. Universidad de Múnich; Alemania Fil: Pilo-Pais, Mauricio. Fribourg University; Suiza Fil: Selbach, Florian. Universidad de Múnich; Alemania Fil: Liedl, Tim. Universidad de Múnich; Alemania Fil: Tinnefeld, Philip. Universidad de Múnich; Alemania Fil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Acuna, Guillermo P.. Fribourg University; Suiza

In recent years, DNA nanotechnology has matured to enable robust production of complex nanostructures and hybrid materials. We have combined DNA nanotechnology with sensitive optical detection to create functional single-molecule devices that enable new applications in single-molecule biosensing and superresolution microscopy. Starting with superresolution nanorulers and brightness reference samples we determined the resolving power of superresolution microscopes and evaluated the sensitivity of smartphone cameras. To improve the sensitivity we created DNA origami optical antennas for metal enhanced fluorescence. The unique ability of our DNA origami nanoantennas to place molecular assays specifically in the plasmonic hotspot is used for detecting Zika-virus nucleic acids. The fluorescence enhancement is further exploited to achieve ultra-sensitive detection on low-cost devices such as a modified smartphone leading to the democratization of single-molecule detection.

Utilizing self-assembled DNA structures in the development of nanoelectronic circuits requires transforming the DNA strands into highly conducting wires. Toward this end, we investigate the use of DNA self-assembled nanowires as templates for the deposition of a superconducting material. Nanowires formed by the deposition of superconducting NbN exhibit thermally activated and quantum phase slips as well as exceptionally large negative magnetoresistance. The latter effect can be utilized to suppress a significant part of the low temperature resistance caused by the quantum phase slips.

The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami‐based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw‐like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays.

The DNA origami technique itself is considered a milestone of DNA nanotechnology and DNA origami nanorulers represent the first widespread application of this technique. DNA origami nanorulers are used to demonstrate the capabilities of techniques and are valuable training samples. They have meanwhile been developed for a multitude of microscopy methods including optical microscopy, atomic force microscopy, and electron microscopy, and their unique properties are further exploited to develop point-light sources, brightness references, nanophotonic test structures, and alignment tools for correlative microscopy. In this perspective, we provide an overview of the basics of DNA origami nanorulers and their increasing applications in fields of optical and especially super-resolution fluorescence microscopy. In addition, emerging applications of reference structures based on DNA origami are discussed together with recent developments.

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

Development of portable and cost-effective optical imaging devices is essential for quantification of molecular bioassays at the point of care (POC). Mobile phone-based microscopy tools have shown great potential as biomedical reader platforms due to their small size, large distribution volume, and constantly improving optical properties. However, the optical detection sensitivity remains a challenge to further improve the diagnostic capabilities of microscopy and sensing devices based on mobile-phones. Here, we demonstrate a simple strategy to enhance the signal intensity of a smartphone fluorescence microscope by approximately an order of magnitude using surface-enhanced fluorescence (SEF) created by a thin silver film. This plasmonics-enhanced smartphone microscopy platform relies on an opto-mechanical attachment based on the Kretschmann configuration, where the sample is placed on a silver-coated glass coverslip and excited by a laser diode from the backside through a glass hemisphere. The fluorescence enhancement effect was systematically optimized by tuning the metal film thickness, spacer distance, excitation angle, and polarization, and experimentally validated by comparison to theoretical simulations. With this mobile device, single fluorescent beads as small as 50 nm and individual quantum dots (ca. 20 nm dia.) were detected. We further quantified the sensitivity limit of this mobile platform to be around 80 fluorophores per diffraction-limited spot by imaging DNA origami based brightness standards labeled with different numbers of fluorophores. We believe that this SEF-based mobile microscopy platform opens up various new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

In this contribution, we fabricate hybrid constructs based on a natural light-harvesting complex, peridinin–chlorophyll a–protein, coupled to dimer optical antennas self-assembled with the help of the DNA origami technique. This approach enables controlled positioning of individual complexes at the hotspot of the optical antennas based on large, colloidal gold and silver nanoparticles. Our approach allows us to selectively excite the different pigments present in the harvesting complex, reaching a fluorescence enhancement of 500-fold. This work expands the range of self-assembled functional hybrid constructs for harvesting sunlight and can be further developed for other pigment–proteins and proteins.

Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot.

We resolved the organization of subunits in cytoskeletal polymers in cells by light microscopy. Septin GTPases form linear complexes of about 32 nm length that polymerize into filaments. We visualized both termini of septin complexes by single molecule microscopy in vitro. Complexes appeared as 32 nm spaced localization pairs, and filaments appeared as stretches of equidistant localizations. Cellular septins were resolved as localization pairs and thin stretches of equidistant localizations.

Because of the limited signal-to-background ratio, molecular diagnostics requires molecular amplification of the target molecules or molecular signal amplification after target recognition. For direct molecular detection, we demonstrate a purely physical fluorescence enhancement process which can elevate the fluorescence signal of single fluorescent dyes by several orders of magnitude. To this end, DNA origami-based optical antennas with a height of around 125 nm are used, which utilize metallic nanoparticles to create a hotspot where fluorescence signals are enhanced by plasmonic effects. By equipping the hotspot with a molecular beacon-like structure, we combine the plasmonic signal enhancement with a specific signal generation, leading to an enhanced and therefore easy to detect signal only in the presence of the specific target nucleic acid. Exemplified with Zika virus detection, we show the applicability of this approach by detecting Zika-specific artificial DNA and RNA both in buffer and in heat-inactivated human blood serum. We show the sensitivity against small nucleotide variations of this approach, allowing the discrimination of closely related pathogens. Furthermore, we show how the modularity offered by DNA nanotechnology enables multiplexing by incorporating orthogonal fluorescent labels for the simultaneous detection of different sequences. The achieved signal enhancement will allow technically simplified signal detection, paving the way for single molecule-based point-of-care diagnosis.

Sensing nucleic acids typically involves the recognition of a specific sequence and reporting by, for example, a fluorogenic reaction yielding one activated dye molecule per detected nucleic acid. Here, we show that after binding to a DNA origami track a bound DNA target (a “DNA walker”) can release the fluorescence of many molecules by acting as the catalyst of an enzymatic nicking reaction. As the walking kinetics sensitively depends on the walker sequence, the resulting brightness distribution of DNA origamis is a sequence fingerprint with single-nucleotide sensitivity. Using Monte Carlo simulations, we rationalize that the random self-avoiding walk is mainly terminated when steps to nearest neighbors are exhausted. Finally, we demonstrate that the DNA walker is also active in a plasmonic hotspot for fluorescence enhancement, indicating the potential of combining different amplification mechanisms enabled by the modularity of DNA nanotechnology.

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

Resolution of emerging superresolution microscopy is commonly characterized by the width of a point-spread-function or by the localization accuracy of single molecules. In contrast, resolution is defined as the ability to separate two objects. Recently, DNA origamis have been proven as valuable scaffold for self-assembled nanorulers in superresolution microscopy. Here, we use DNA origami nanorulers to overcome the discrepancy of localizing single objects and separating two objects by resolving two docking sites at distances of 18, 12, and 6 nm by using the superresolution technique DNA PAINT(point accumulation for imaging in nanoscale topography). For the smallest distances, we reveal the influence of localization noise on the yield of resolvable structures that we rationalize by Monte Carlo simulations.

DNA origami used as a platform to study the bleaching behavior of four commonly used fluorophores in cw-STED nanoscopy., Superresolution microscopy is currently revolutionizing optical imaging. A key factor for getting images of highest quality is – besides a well-performing imaging system – the labeling of the sample. We compared the fluorescent dyes Abberior Star 488, Alexa 488, Chromeo 488 and Oregon Green 488 for use in continuous wave (cw-)STED microscopy in aqueous buffer and in a durable polymer matrix. To optimize comparability, we designed DNA origami standards labeled with the fluorescent dyes including a bead-like DNA origami with dyes focused on one spot and a DNA origami with two marks at a designed distance of ∼100 nm. Our data show that all dyes are well suited for cw-STED microscopy but that the optimal dye depends on the embedding medium. The precise comparison enabled by DNA origami nanorulers indicates that these structures have matured from the proof-of-concept to easily applicable tools in fluorescence microscopy.

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single-molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1-20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well-defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non-invasively within 150 ms. The method might become a quantifying add-on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT-type microscopy.

Over the last decade, two fields have dominated the attention of sub-diffraction photonics research: plasmonics and fluorescence nanoscopy. Nanoscopy based on single-molecule localization offers a practical way to explore plasmonic interactions with nanometre resolution. However, this seemingly straightforward technique may retrieve false positional information. Here, we make use of the DNA origami technique to both control a nanometric separation between emitters and a gold nanoparticle, and as a platform for super-resolution imaging based on single-molecule localization. This enables a quantitative comparison between the position retrieved from single-molecule localization, the true position of the emitter and full-field simulations. We demonstrate that plasmonic coupling leads to shifted molecular localizations of up to 30 nm: a single-molecule mirage., The near-field interaction of single emitters and plasmonic structures can alter the perceived physical location of the emitter. Here, Raab et al. use DNA origami and far-field super-resolution microscopy to quantitatively evaluate this localization offset for gold nanoparticles.

The remarkable performance and quantum efficiency of biological light-harvesting complexes has prompted a multidisciplinary interest in engineering biologically inspired antenna systems as a possible route to novel solar cell technologies. Key to the effectiveness of biological “nanomachines” in light capture and energy transport is their highly ordered nanoscale architecture of photoactive molecules. Recently, DNA origami has emerged as a powerful tool for organizing multiple chromophores with base-pair accuracy and full geometric freedom. Here, we present a programmable antenna array on a DNA origami platform that enables the implementation of rationally designed antenna structures. We systematically analyze the light-harvesting efficiency with respect to number of donors and interdye distances of a ring-like antenna using ensemble and single-molecule fluorescence spectroscopy and detailed Förster modeling. This comprehensive study demonstrates exquisite and reliable structural control over multichromophoric geometries and points to DNA origami as highly versatile platform for testing design concepts in artificial light-harvesting networks.

We study the distance-dependent quenching of fluorescence due to a metallic nanoparticle in proximity of a fluorophore. In our single-molecule measurements, we achieve excellent control over structure and stoichiometry by using self-assembled DNA structures (DNA origami) as a breadboard where both the fluorophore and the 10 nm metallic nanoparticle are positioned with nanometer precision. The single-molecule spectroscopy method employed here reports on the co-localization of particle and dye, while fluorescence lifetime imaging is used to directly obtain the correlation of intensity and fluorescence lifetime for varying particle to dye distances. Our data can be well explained by exact calculations that include dipole dipole orientation and distances. Fitting with a more practical model for nanosurface energy transfer yields 10.4 nm as the characteristic distance of 50% energy transfer. The use of DNA nanotechnology together with minimal sample usage by attaching the particles to the DNA origami directly on the microscope coverslip paves the way for more complex experiments exploiting dye nanoparticle interactions. Fil: Acuna, Guillermo P.. Technische Universität Braunschweig; Alemania Fil: Bucher, Martina. Ludwig Maximilians Universitat; Alemania Fil: Stein, Ingo H.. Ludwig Maximilians Universitat; Alemania Fil: Steinhauer, Christian. Ludwig Maximilians Universitat; Alemania Fil: Kuzyk, Anton. Technische Universitat Munchen; Alemania Fil: Holzmeister, Phil. Technische Universität Braunschweig; Alemania Fil: Schreiber, Robert. Ludwig Maximilians Universitat; Alemania Fil: Moroz, Alexander. Fil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Liedl, Tim. Ludwig Maximilians Universitat; Alemania Fil: Simmel, Friedrich C.. Technische Universitat Munchen; Alemania Fil: Tinnefeld, Philip. Technische Universität Braunschweig; Alemania

Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor―acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Forster radius R o could directly be determined from the distance dependence of energy transfer yielding R 0 = 5.3 ± 0.3 nm for the Cy3―Cy5 pair.

Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffolds as well as a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure's surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT.

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with

Fluorescence resonance energy transfer (FRET) represents a mechanism to transport light energy at the nanoscale, as exemplified by nature's light-harvesting complexes. Here we used DNA origami to arrange fluorophores that transport excited-state energy from an input dye to an output dye. We demonstrate that energy-transfer paths can be controlled on the single-molecule level by the presence of a "jumper" dye that directs the excited-state energy either to a red or to an IR output dye. We used single-molecule four-color FRET with alternating laser excitation to sort subpopulations and to visualize the control of energy transfer.

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

Nanotechnology is challenged by the need to connect top-down produced nanostructures with the bottom-up world of chemistry. A nanobiotechnological prime example is the positioning of single polymerase molecules in small holes in metal films, so-called zeromode waveguides (ZMWs), which is required for single-molecule real-time DNA sequencing. In this work, we present nanoadapters made of DNA (DNA origami) that match the size of the holes so that exactly one nanoadapter fits in each hole. By site-selective functionalization of the DNA origami nanoadapters, we placed single dye molecules in the ZMWs, thus optimizing the hole usage and improving the photophysical properties of dyes compared to stochastically immobilized molecules.

The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles? size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching. Fil: Pellegrotti, Jesica Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Acuña, Guillermo. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Puchkova, Anastasiya. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Holzmeister, Phil. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Gietl, Andreas. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Lalkens, Birka. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Tinnefeld, Philip. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania

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.

The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. Here, we show how bottom-up nano-antennas enhance the fluorescence intensity in a reduced hot-spot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle dimers are positioned with nanometer precision. A maximum of almost 100fold intensity enhancement is obtained using 100 nm gold nanoparticles within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nano-antenna, we are able to perform single molecule measurements at concentrations as high as 500 nM which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic nanoparticles with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single molecule measurements at high concentrations where processes like DNA sequencing occur.

Nanopillars are promising nanostructures composed of various materials that bring new functionalities for applications ranging from photovoltaics to analytics. We developed DNA nanopillars with a height of 220 nm and a diameter of ~14 nm using the DNA origami technique. Modifying the base of the nanopillars with biotins allowed selective, upright, and rigid immobilization on solid substrates. With the help of site-selective dye labels, we visualized the structure and determined the orientation of the nanopillars by three-dimensional fluorescence superresolution microscopy. Because of their rigidity and nanometer-precise addressability, DNA origami nanopillars qualify as scaffold for the assembly of plasmonic devices as well as for three-dimensional superresolution standards.

Building a Fluorescent Hotspot When two gold nanoparticles come close together, their overlapping plasmonic fields can create a region that acts as a nanoantenna that can enhance the fluorescent emission of a molecule. Acuna et al. (p. 506 ) used a surface-anchored DNA origami structure to assemble one or two gold nanoparticles next to a dye trapped within the structure. A > 100-fold enhancement in fluorescent emission was observed when the dye molecules were located in a 23-nm gap between two 100-nm gold nanoparticles.

In recent years, a number of approaches have emerged that enable far-field fluorescence imaging beyond the diffraction limit of light, namely super-resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super-resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single-molecule compatible fluorophore can be used for super-resolution microscopy. We present examples for super-resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super-resolution microscopes including AFM-based single-molecule assembly and DNA origami.