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A single chromophore can only emit a maximum of one single photon per excitation cycle. This limitation results in a phenomenon commonly referred to as photon antibunching (pAB). When multiple chromophores contribute to the fluorescence measured, the degree of pAB has been used as a metric to "count" the number of chromophores. But the fact that chromophores can switch randomly between bright and dark states also impacts pAB and can lead to incorrect chromophore numbers being determined from pAB measurements. By both simulations and experiment, we demonstrate how pAB is affected by independent and collective chromophore blinking, enabling us to formulate universal guidelines for correct interpretation of pAB measurements. We use DNA-origami nanostructures to design multichromophoric model systems that exhibit either independent or collective chromophore blinking. Two approaches are presented that can distinguish experimentally between these two blinking mechanisms. The first one utilizes the different excitation intensity dependence on the blinking mechanisms. The second approach exploits the fact that collective blinking implies energy transfer to a quenching moiety, which is a time-dependent process. In pulsed-excitation experiments, the degree of collective blinking can therefore be altered by time gating the fluorescence photon stream, enabling us to extract the energy-transfer rate to a quencher. The ability to distinguish between different blinking mechanisms is valuable in materials science, such as for multichromophoric nanoparticles like conjugated-polymer chains as well as in biophysics, for example, for quantitative analysis of protein assemblies by counting chromophores.

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.

Forster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

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

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

Fluorogenic nucleic acid hybridization probes are widely used for detecting and quantifying nucleic acids. The achieved sensitivity strongly depends on the contrast between a quenched closed form and an unquenched opened form with liberated fluorescence. So far, this contrast was improved by improving the quenching efficiency of the closed form. In this study, we modularly combine these probes with optical antennas used for plasmonic fluorescence enhancement and study the effect of the nanophotonic structure on the fluorescence of the quenched and the opened form. As quenched fluorescent dyes are usually enhanced more by fluorescence enhancement, a detrimental reduction of the contrast between closed and opened form was anticipated. In contrast, we could achieve a surprising increase of the contrast with full additivity of quenching of the dark form and fluorescence enhancement of the bright form. Using single-molecule experiments, we demonstrate that the additivity of the two mechanisms depends on the perfect quenching in the quenched form, and we delineate the rules for new nucleic acid probes for enhanced contrast and absolute brightness. Fluorogenic hybridization probes optimized not only for quenching but also for the brightness of the open form might find application in nucleic acid assays with PCR avoiding detection schemes.

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.

Most measurements of fluorescence lifetimes on the single-molecule level are carried out using avalanche photon diodes (APDs). These single-photon counters are inherently slow, and their response shows a strong dependence on photon energy, which can make reconvolution of the instrument response function (IRF) challenging. An ultrafast time resolution in single-molecule fluorescence is crucial, e.g., in determining donor lifetimes in donor-acceptor couples which undergo energy transfer, or in plasmonic antenna structures, where the radiative rate and non-radiative rates are enhanced. We introduce a femtosecond double-excitation (FeDEx) photon correlation technique, which measures the degree of photon antibunching as a function of time delay between two excitation pulses. In this boxcar integration, the time resolution of fluorescence transients is limited solely by the laser pulse length and is independent of the detector IRF. The versatility of the technique is demonstrated with a custom-made donor-acceptor complex with one donor and two acceptors and with single dye molecules positioned accurately between two gold nanoparticles using DNA origami. The latter structures show ∼75-fold radiative-rate enhancement and fluorescence lifetimes down to 19 ps, which is measured without the need for any reconvolution. With the potential of measuring subpicosecond fluorescence lifetimes, plasmonic antenna structures can now be optimized further.

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.

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution.

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.

Single-molecule Forster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between +/- 0.02 and +/- 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods. This work was supported by the European Research Council (ERC; grant agreement nos. 261227 (to A.N.K.), 646451 (to E.L.), 638536 (to T.C.), 671208 (to C.A.M.S.), and 681891 (to T. Hugel)), the Deutsche Forschungsgemeinschaft (DFG) (grant MI 749/4-1 to J.M., grant TI 329/10-1 to P.T., and grant SCHL 1896/3-1 to M.S.), the Swiss National Science Foundation (to B.S.), the German Federal Ministry of Education and Research (BMBF; 03Z2EN11 to M.S.), Research Foundation Flanders (FWO; grant G0B4915N to J. Hendrix), the Agency for Innovation by Science and Technology (IWT Flanders; doctoral scholarship to N.V.), the Danish Council for Independent Research (Sapere Aude grant 0602-01670B to V.B.), the Novo Nordisk Foundation (NNF15OC0017956 to V.B.), the UK BBSRC (grant BB/H01795X/1 to A.N.K.),the National Institute of Mental Health (grant MH081923 to M.E.B.), Clemson University (start-up funds to H. Sanabria, S.R.A., and I.S.Y.-O.), the NIH (grants GM109832 and GM118508 to K.R.W.; grant GM112659 to T. Ha), the NSF (Career Award MCS1749778 to H. Sanabria), the Carl-Zeiss-Stiftung (doctoral fellowship to E.K.), the Stipendienstiftung Rheinland-Pfalz (doctoral scholarship to G.K.), the Braunschweig International Graduate School of Metrology (B-IGSM; to B.W.), the DFG Research Training Group (GrK1952/1 "Metrology for Complex Nanosystems" to B.W.), the University of Sheffield (start-up funds to T.D.C.), and the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2017R1A2B3010309 to N.K.L.).

Fluorescence resonance energy transfer (FRET) has been instrumental in determining the structure and dynamics of biomolecules but distances above 8 nanometers are not accessible. However, with the advent and rapid development of super-resolution (SR) microscopy, distances between two fluorescent dyes below 20 nanometers can be resolved, which hitherto has been inaccessible for fluorescence microscopy approaches due to the limited resolving power of an optical imaging system that is determined by the fundamental laws of light diffraction (referred to as the diffraction limit). Therefore, the question arises whether SR microscopy can ultimately close the resolution gap between FRET and the diffraction limit and whether SR microscopy can be employed for the structural interrogation of proteins in the sub-20 nm range? Here, we show that the combination of DNA nanotechnology and single-molecule biochemistry allows the first step towards the investigation of the structural organization of a protein via SR microscopy. Limiting factors and possible future directions for the full implementation of SR microscopy as a structural tool are discussed.

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.

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.

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

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.

Despite common presumption due to fast photodestruction pathways through higher excited states, we show that further improvement of photostability is still achievable with diffusion-limited photoprotection formulas. Single-molecule fluorescence spectroscopy reveals that thiolate ions effectively quench triplet states of dyes by photoinduced electron transfer. Interestingly, this reaction rarely yields a radical anion of the dye, but direct return to the ground state is promoted by an almost instantaneous back electron transfer (geminate recombination). This type of mechanism is not detected for commonly used reductants such as ascorbic acid and trolox. The mechanism avoids the formation of radical cations and improves the photostability of single fluorophores. We find that a combination of β-mercaptoethanol and classical reducing and oxidizing systems yields the best results for several dyes including Atto532 and Alexa568.

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.

While nanophotonic devices are unfolding their potential for single-molecule fluorescence studies, metallic quenching and steric hindrance, occurring within these structures, raise the desire for site-specific immobilization of the molecule of interest. Here, we refine the single-molecule cut-and-paste technique by optical superresolution routines to immobilize single fluorescent molecules in the center of nanoapertures. By comparing their fluorescence lifetime and intensity to stochastically immobilized fluorophores, we characterize the electrodynamic environment in these nanoapertures and proof the nanometer precision of our loading method.

Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine-thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation. By carefully investigating the sequence and length of DS DNA, we present the optimal design of the template. Benefiting from the smart design and facile synthesis, a simple, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection is proposed. Through the systematic comparison, it is found that the strategy based on CuNPs formation is more sensitive for ATP assay than that based on AgNCs synthesis, and the detection limitation was found to be 81 pM. What's more, the CuNPs formation-based method is successfully applied in the detection of ATP in human serum as well as the determination of cellular ATP. In addition to small target molecule, the sensing strategy was also extended to the detection of biomacromolecule (DNA), which illustrates the generality of this biosensor.

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

In recent years different implementations of superresolution microscopy based on targeted switching (STED, GSD, and SSIM) have been demonstrated. The key elements to break the diffraction barrier are two distinct molecular states that generate a saturable nonlinear fluorescence response with respect to the excitation intensity. In this paper, we demonstrate that a nonlinearity can even be encoded in fluorescent probes, which then increase the resolution of a standard confocal microscope. This nonlinearity is achieved by an intensity dependent blocking of the resonance energy transfer between a donor and one or more acceptor fluorophores, utilizing radical anion states of the acceptor. In proof-of-principle experiments, we demonstrate a significant resolution increase using probes with different numbers of acceptor fluorophores. Quantitative description by a theoretical model paves the way for the development of fluorescent probes that can more than double the resolution of essentially any confocal microscope in all three dimensions.

In this paper we experimentally combine a recently developed AFM-based molecule-by-molecule assembly (single-molecule cut-and-paste, SMCP) with subdiffraction resolution fluorescence imaging. Using “Blink-Microscopy”, which exploits the fluctuating emission of single molecules for the reconstruction of superresolution images, we resolved SMCP assembled structures with features below the diffraction limit. Artificial line patterns then served as calibration structures to characterize parameters, such as the labeling density, that can influence resolution of Blink-Microscopy besides the localization precision of a single molecule. Finally, we experimentally utilized the adjustability of blink parameters to demonstrate the general connection of photophysical parameters with spatial resolution and acquisition time in superresolution microscopy.

In fluorescence microscopy, the distribution of the emitting molecule number in space is usually obtained by dividing the measured fluorescence by that of a single emitter. However, the brightness of individual emitters may vary strongly in the sample or be inaccessible. Moreover, with increasing (super-) resolution, fewer molecules are found per pixel, making this approach unreliable. Here we map the distribution of molecules by exploiting the fact that a single molecule emits only a single photon at a time. Thus, by analysing the simultaneous arrival of multiple photons during confocal imaging, we can establish the number and local brightness of typically up to 20 molecules per confocal (diffraction sized) recording volume. Subsequent recording by stimulated emission depletion microscopy provides the distribution of the number of molecules with subdiffraction resolution. The method is applied to mapping the three-dimensional nanoscale organization of internalized transferrin receptors on human HEK293 cells., Mapping the distribution of fluorescence molecules, rather than just their emission intensity, can improve super-resolution fluorescence microscopy. Here, the authors present a general solution for rendering the number of fluorescent molecules recorded by confocal or STED microscopy.

We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of > 90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.

We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.

We demonstrate measurements of the efficiency of competing Förster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.

A bichromophoric model systema short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5embedded in poly(vinyl alcohol) (PVA) was used to investigate energy transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T1 → TN → S1 → S0) in single Cy5 molecules. We observed that even when the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet−triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.

The interaction of dyes and metallic nanostructures strongly affects the fluorescence and can lead to significant fluorescence enhancement at plasmonic hot spots, but also to quenching. Here we present a method to distinguish the individual contributions to the changes of the excitation, radiative and non-radiative rate and use this information to determine the quantum yields for single molecules. The method is validated by precisely placing single fluorescent dyes with respect to gold nanoparticles as well as with respect to the excitation polarization using DNA origami nanostructures. Following validation, measurements in zeromode waveguides reveal that suppression of the radiative rate and enhancement of the non-radiative rate lead to a reduced quantum yield. Because the method exploits the intrinsic blinking of dyes, it can generally be applied to fluorescence measurements in arbitrary nanophotonic environments.

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 radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time.

A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds t...

We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.

Dark quencher chromophores are interesting alternatives to common single-molecule FRET acceptors. Due to their short excited state lifetime, they should be less prone to complex photophysics and bleaching. We find, however, that for common enzymatic oxygen scavenging systems and photoprotection strategies – the gold standard of single-molecule measurements – the quenchers BBQ650 and BHQ-2 induce frequent blinking of the donor molecule. They switch in a photoinduced process to what we identify as a radical anion state and back. We further make use of the broad absorption spectrum for selective bleaching of the quenchers in order to photoactivate the fluorescence of initially completely quenched dye molecules. This represents a general strategy to turn fluorescent dyes into photoactivatable probes.

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.

High quality and facile DNA functionalized quantum dots (QDs) as efficient fluorescence nanomaterials are of great significance for bioimaging both in vitro and in vivo applications. Herein, we offer a strategy to synthesize DNA-functionalized Zn(2+) doped CdTe QDs (DNA-QDs) through a facile one-pot hydrothermal route. DNA is directly attached to the surface of QDs. The as-prepared QDs exhibit small size (3.85 ± 0.53 nm), high quantum yield (up to 80.5%), and excellent photostability. In addition, the toxicity of QDs has dropped considerably because of the Zn-doping and the existence of DNA. Furthermore, DNA has been designed as an aptamer specific for mucin 1 overexpressed in many cancer cells including lung adenocarcinoma. The aptamer-functionalized Zn(2+) doped CdTe QDs (aptamer-QDs) have been successfully applied in active tumor-targeted imaging in vitro and in vivo. A universal design of DNA for synthesis of Zn(2+) doped CdTe QDs could be extended to other target sequences. Owing to the abilities of specific recognition and the simple synthesis route, the applications of QDs will potentially be extended to biosensing and bioimaging.

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.

Recently, a new approach for super-resolution microscopy has emerged which is based on the successive localization of single molecules. The majority of molecules are prepared to reside in a nonfluorescent dark state, leaving only a few single molecules fluorescing. The single molecules can subsequently be localized on the camera image. Successive localization of all molecules allows reconstruction of a super-resolved image of the labeled structure. A variety of ways for limiting the number of locatable molecules have been developed recently which expand this current field of imaging. Here we describe a super-resolution microscopy method that employs the use of reversible, generic dark states, for example radical ion states. This method requires only a single laser source and can be carried out with many fluorescent dyes, in some cases, even in living cells. We provide a step-by-step procedure for this method, which we have called Blink Microscopy.