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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).

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At Physikalisch-Technische Bundesanstalt, the National Metrology Institute of Germany, a new type of deep ultraviolet scatterometer has been developed and set up. The concept of the system is very variable and versatile, so that many different types of measurements, e.g., classical scatterometry, ellipsometric scatterometry, polarization-dependent reflectometry, and ellipsometry can be performed. The main application is the characterization of linewidth/critical dimension (CD), grating period (pitch), and edge profile of periodically nanostructured surfaces mainly, but not only, on photomasks. Different operation wavelength between 840 and 193 nm can be used, giving also access to a variety of different at-wavelength metrology connected with state-of-the-art photolithography. It allows to adapt and to vary the measurand and measurement geometry to optimize the sensitivity and the unambiguity for the measurement problem. In this paper the concept, design, and performance of the system is described in detail. First measurement examples are shown and current and future applications are discussed.

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