Fluorescence microscopy with 6 nm resolution on DNA origami.

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 nanopillars as standards for three-dimensional superresolution microscopy.

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.

Counting fluorescent dye molecules on DNA origami by means of photon statistics.

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.

Super-resolution imaging of C-type lectin and influenza hemagglutinin nanodomains on plasma membranes using blink microscopy.

Dendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts.

Quantum yield and excitation rate of single molecules close to metallic nanostructures.

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.

DNA origami-based standards for quantitative fluorescence microscopy.

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.

Experimental observations of Soret-driven convection in the transient diffusive boundary layer.

The onset of transient Soret-driven convection is investigated experimentally in a colloidal suspension of thermosensitive nanoparticles by the shadowgraph technique and by particle tracking observations. From the shadowgraph images, the concentration profile is reconstructed, giving evidence of a convective motion inside the transient boundary layer. Furthermore, the latency times for the convection onset are extracted from the measurements. The results point out that particle tracking is superior to the shadowgraph method for detecting the onset of convection. The onset latency times obtained from these experiments obey scaling laws which are in accordance with the predictions from theoretical treatments.