12 research outputs found

    Using DNA origami nanorulers as traceable distance measurement standards and nanoscopic benchmark structures

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    Abstract 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)

    Das Jahr des Lichtes

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    PTB-Mitteilungen. Band 125 (2015), Heft 4. ISSN 0030-834X1.: Stefan Kück und Armin Sperling: Einführung in die Metrologie des Lichtes. doi: 10.7795/310.20150401 http://dx.doi.org/10.7795/310.20150401 2.: Armin Sperling: Die Candela als Maßeinheit des Lichtes. doi: 10.7795/310.20150402 http://dx.doi.org/10.7795/310.20150402 3.: Detlef Lindner und Stefan Winter: Rückführung in der Photometrie - die Realisierung und Rückführung der Candela . doi: 10.7795/310.20150403 http://dx.doi.org/10.7795/310.20150403 4.: Matthias Lindemann: Kurze Geschichte der Goniophotometrie in der PTB. doi: 10.7795/310.20150404 http://dx.doi.org/10.7795/310.20150404 5.: Thorsten Gerloff: Metrologie für moderne Lichtquellen: LEDs und OLEDs. doi: 10.7795/310.20150405 http://dx.doi.org/10.7795/310.20150405 6.: Egbert Buhr, Alfred Schirmacher und Andreas Höpe: Effektpigmente: Eine Herausforderung für die optische Messtechnik. doi: 10.7795/310.20150406 http://dx.doi.org/10.7795/310.20150406 7.: Impressum

    Handling and storage of human body fluids for analysis of extracellular vesicles

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    Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze–thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze–thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100–200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze–thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for comparative EV studies and to create reliable biorepositories

    Traceable size determination of nanoparticles, a comparison among European metrology institutes

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    Within the European iMERA-Plus project ‘Traceable Characterisation of Nanoparticles’ various particle measurement procedures were developed and finally a measurement comparison for particle size was carried out among seven laboratories across six national metrology institutes. Seven high quality particle samples made from three different materials and having nominal sizes in the range from 10 to 200 nm were used. The participants applied five fundamentally different measurement methods, atomic force microscopy, dynamic light scattering (DLS), small-angle x-ray scattering, scanning electron microscopy and scanning electron microscopy in transmission mode, and provided a total of 48 independent, traceable results. The comparison reference values were determined as weighted means based on the estimated measurement uncertainties of the participants. The comparison reference values have combined standard uncertainties smaller than 1.4 nm for particles with sizes up to 100 nm. All methods, except DLS, provided consistent results

    Measuring soot particles from automotive exhaust emissions

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    The European Metrology Research Programme participating countries and the European Union jointly fund a three year project to address the need of the automotive industry for a metrological sound base for exhaust measurements. The collaborative work on particle emissions involves five European National Metrology Institutes, the Tampere University of Technology, the Joint Research Centre for Energy and Transport and the Leibniz Institute for Tropospheric Research. On one hand, a particle number and size standard for soot particles is aimed for. Eventually this will allow the partners to provide accurate and comparable calibrations of measurement instruments for the type approval of Euro 5b and Euro 6 vehicles. Calibration aerosols of combustion particles, silver and graphite proof partially suitable. Yet, a consensus choice together with instrument manufactures is pending as the aerosol choice considerably affects the number concentration measurement. Furthermore, the consortium issued consistent requirements for novel measuring instruments foreseen to replace today’s opacimeters in regulatory periodic emission controls of soot and compared them with European legislative requirements. Four partners are conducting a metrological validation of prototype measurement instruments. The novel instruments base on light scattering, electrical, ionisation chamber and diffusion charging sensors and will be tested at low and high particle concentrations. Results shall allow manufacturers to further improve their instruments to comply with legal requirements
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