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

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).

Differentiating gold nanorod samples using particle size and shape distributions from transmission electron microscope images.

Size and shape distributions of gold nanorod samples are critical to their physico-chemical properties, especially their longitudinal surface plasmon resonance. This interlaboratory comparison study developed methods for measuring and evaluating size and shape distributions for gold nanorod samples using transmission electron microscopy (TEM) images. The objective was to determine whether two different samples, which had different performance attributes in their application, were different with respect to their size and/or shape descriptor distributions. Touching particles in the captured images were identified using a ruggedness shape descriptor. Nanorods could be distinguished from nanocubes using an elongational shape descriptor. A non-parametric statistical test showed that cumulative distributions of an elongational shape descriptor, that is, the aspect ratio, were statistically different between the two samples for all laboratories. While the scale parameters of size and shape distributions were similar for both samples, the width parameters of size and shape distributions were statistically different. This protocol fulfills an important need for a standardized approach to measure gold nanorod size and shape distributions for applications in which quantitative measurements and comparisons are important. Furthermore, the validated protocol workflow can be automated, thus providing consistent and rapid measurements of nanorod size and shape distributions for researchers, regulatory agencies, and industry.

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

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.

Comparison of edge analysis techniques for the determination of the MTF of digital radiographic systems.

The modulation transfer function (MTF) is well established as a metric to characterize the resolution performance of a digital radiographic system. Implemented by various laboratories, the edge technique is currently the most widespread approach to measure the MTF. However, there can be differences in the results attributed to differences in the analysis technique employed. The objective of this study was to determine whether comparable results can be obtained from different algorithms processing identical images representative of those of current digital radiographic systems. Five laboratories participated in a round-robin evaluation of six different algorithms including one prescribed in the International Electrotechnical Commission (IEC) 62220-1 standard. The algorithms were applied to two synthetic and 12 real edge images from different digital radiographic systems including CR, and direct- and indirect-conversion detector systems. The results were analysed in terms of variability as well as accuracy of the resulting presampled MTFs. The results indicated that differences between the individual MTFs and the mean MTF were largely below 0.02. In the case of the two simulated edge images, all algorithms yielded similar results within 0.01 of the expected true MTF. The findings indicated that all algorithms tested in this round-robin evaluation, including the IEC-prescribed algorithm, were suitable for accurate MTF determination from edge images, provided the images are not excessively noisy. The agreement of the MTF results was judged sufficient for the measurement of the MTF necessary for the determination of the DQE.

Measurement of the detective quantum efficiency (DQE) of digital X-ray detectors according to the novel standard IEC 62220-1.

A mobile measurement facility which complies with IEC 62220-1 has been set up to determine the detective quantum efficiency (DQE) of digital X-ray detector systems. Exemplary measurements were performed for two similar CR detector systems, a CsI-based indirect detector and an Se-based direct detector. The standardised radiation quality RQA 5 was applied for measurement and for three of these systems RQA 9 was also applied. A pronounced dependence of DQE on radiation quality was observed for the direct detector, where the DQEs for RQA 5 and RQA 9 differ by a factor of approximately 2. The uncertainty (95% confidence interval) associated with the measured DQE values is within 0.01 and 0.04 depending on, for example, the spatial frequency. Thus, it has been demonstrated that the DQE can be measured accurately and reliably with the accuracy required by the international standard IEC 62220-1. It is now possible to objectively measure and compare DQE values of digital X-ray detector systems.

Reduction of anatomical noise in medical X-ray images.

The X-ray pattern of a mass of very fine non-distinguishable anatomical structures alters completely from radiograph to radiograph due to the unavoidable movements of the patient during the exposure. The corresponding image component shows noise-like behaviour and is therefore referred to as the anatomical noise. Reducing this component would enhance the quality of the clinical X-ray image and increase the detectability of radiological signal. We have found that by comparing two X-ray images of the same anatomy acquired under slightly different imaging geometry, it is possible to reduce the anatomical noise in one of the image pair. The proposed method, which allows this, is based on the appropriate attenuation in the wavelet domain. The values of attenuating factors for the wavelet coefficients are proportional to the correlation between the corresponding features of both images. This method was tested for different changes in the imaging geometry. In the case of no geometrical changes, only the quantum and the electronic noise are reduced. An effect of de-noising for the investigated images is obvious.

Comparison of technical and anatomical noise in digital thorax X-ray images.

Former studies by Hoeschen and Buhr indicated a higher total noise in a thorax image than expected from technical noise, i.e. quantum and detector noise. This difference results from the overlay of many small anatomical structures along the X-ray beam, which leads to a noise-like appearance without distinguishable structures in the projected image. A method is proposed to quantitatively determine this 'anatomical noise' component, which is not to be confused with the anatomical background (e.g. ribs). This specific anatomical noise pattern in a radiograph changes completely when the imaging geometry changes because different small anatomical structures contribute to the projected image. Therefore, two images are taken using slightly different exposure geometry, and a correlation analysis based on wavelet transforms allows to determining the uncorrelated noise components. Since the technical noise also differs from image to image, which makes it difficult to separate the anatomical noise, images of a lung phantom were produced on a low-sensitive industrial X-ray film using high-exposure levels. From these results, the anatomical noise level in real clinical thorax radiographs using realistic exposure levels is predicted using the general dose dependence described in the paper text and compared with the quantum and detector noise level of an indirect flat-panel detector system. For consistency testing, the same lung phantom was imaged with the same digital flat-panel detector and the total image noise including anatomical noise is determined. The results show that the relative portion of anatomical noise may exceed the technical noise level. Anatomical noise is an important contributor to the total image noise and, therefore, impedes the recognition of anatomical structures.

Measurement of the modulation transfer function of digital X-ray detectors with an opaque edge-test device.

A variant of the edge method for the determination of the pre-sampled modulation transfer function (MTF) of digital X-ray imaging devices has been developed and accepted as the standard method in the novel DQE measurement standard IEC 62220-1. An opaque tungsten edge-test device accomplishes the ideal step-like profile of the incident X rays. The edge spread function is measured over a large region across the edge transition that enables an accurate MTF measurement including the 'low-frequency drop'. The method has been applied to different state-of-the-art X-ray imaging detectors, a computed radiography, a CsI-based indirect and an Se-based direct flat-panel detector. The MTF measurement results will be presented. In contrast to the opaque edge device, the commonly used semi-transparent edge-test devices produce scatter radiation that deteriorates the incident X-ray profile, which leads to a systematic overestimation of the MTF.

Determination of the modulation transfer function using the edge method: influence of scattered radiation.

The edge method for measuring the modulation transfer function (MTF) has recently gained popularity due to its simplicity and appropriateness particularly for digital imaging systems. Often edge test devices made of rather thin metal sheets are used, which are semitransparent to x rays and may generate scattered radiation. The effect of this scattered radiation on the determined MTF was investigated both theoretically (assuming an ideal detector) and experimentally using a CsI-based digital detector. It was found that the MTF increases due to the scattered radiation for all spatial frequencies larger than 0 mm(-1). The theoretical model developed in this study predicts that the maximum error compared to the true detector MTF is given by S/A, where A is the attenuated fraction and S is the scattered fraction reaching the detector, relative to the incident radiation. Theoretical and experimental results are in good agreement for radiation qualities corresponding to general radiography (RQA3, RQA5, and RQA7), whereas for chest beam quality (RQA9) the experimentally observed MTF error is larger than predicted by the simple model, possibly because the energy response of the CsI-based detector differs from that of an ideal one. The theoretical MTF error reaches a value of 18% for a 0.25 mm thick lead edge of RQA9. Since the MTF enters squared into the determination of the detective quantum efficiency (DQE), an error of at least 36% in DQE may result when using this edge test device. In conclusion, the use of fully absorbing edge material is advised for MTF determination with the edge method.

Accuracy of a simple method for deriving the presampled modulation transfer function of a digital radiographic system from an edge image.

Several methods for accurately deriving the presampled modulation transfer function (MTF) of a pixelated detector from the image of a slightly slanted edge have been described in the literature. In this paper we report on a simple variant of the edge method that produces sufficiently accurate MTF values for frequencies up to the Nyquist frequency limit of the detector with little effort in edge alignment and computation. The oversampled ESF is constructed in a very simple manner by rearranging the pixel data of N consecutive lines corresponding to a lateral shift of the edge by one pixel. A regular subsampling pitch is assumed for the oversampled ESF, which is given by the original pixel sampling distance divided by the integer number N. This allows the original data to be used for further computational analysis (differentiation and Fourier transform) without data preprocessing. Since the number of lines leading to an edge shift by one pixel generally is a fractional number rather than an integer, a systematic error may be introduced into the presampled MTF. Simulations and theoretical investigations show that this error is proportional to 1/N and increases with spatial frequency. For all frequencies up to the Nyquist limit, the relative error delta MTF/MTF is smaller than 1/(2N). It can thus be kept below a given threshold by suitably selecting N, which furnishes a certain maximum edge angle. The method is especially useful for applications where the presampled MTF is needed only for frequencies up to the Nyquist frequency limit, such as the determination of the detective quantum efficiency (DQE).