DNA-PAINT Probe Modifications Support High-Resolution Imaging with Shorter Binding Domains.

DNA-based Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is an effective super resolution microscopy technique, and its optimization is key to improve nanoscale detection. The state-of-the-art improvements that are at the base of this optimization have been first routinely validated on DNA nanostructure devices before being tested on biological samples. This allows researchers to finely tune DNA-PAINT imaging features in a more controllable in vitro environment. Dye-labeled oligonucleotide probes with short hybridization domains can expand DNA-PAINT's detection by targeting short nucleotide sequences and improving resolution, speed, and multiplexing. However, developing these probes is challenging as their brief bound state makes them difficult to capture under routine imaging conditions. To extend dwell binding times and promote duplex stability, we introduced structural and chemical modifications to our imager probes. The modifications included mini-hairpins and/or Bridged Nucleic Acids (BNA); both of which increase the thermomechanical stability of a DNA duplex. Using this approach we demonstrate DNA-PAINT imaging with approximately 5 nm resolution using a 4-nucleotide hybridization domain that is 43% shorter than previously reported probes. Imager probes with such short hybridization domains are key for improving detection on DNA nanostructure devices because they have the capability to target a larger number of binding domains per localization unit. This is essential for metrology applications such as Nucleic Acid Memory (NAM) where the information density is dependent on the binding site length. The selected imager probes reported here present imaging resolution equivalent to current state-of-the-art DNA-PAINT probes, creating a strategy to image shorter DNA domains for nanoscience and nanotechnology alike.

DNA nanostructure decoration: a how-to tutorial.

DNA Nanotechnology is being applied to multiple research fields. The functionality of DNA nanostructures is significantly enhanced by decorating them with nanoscale moieties including: proteins, metallic nanoparticles, quantum dots, and chromophores. Decoration is a complex process and developing protocols for reliable attachment routinely requires extensive trial and error. Additionally, the granular nature of scientific communication makes it difficult to discern general principles in DNA nanostructure decoration. This tutorial is a guidebook designed to minimize experimental bottlenecks and avoid dead-ends for those wishing to decorate DNA nanostructures. We supplement the reference material on available technical tools and procedures with a conceptual framework required to make efficient and effective decisions in the lab. Together these resources should aid both the novice and the expert to develop and execute a rapid, reliable decoration protocols.

Binding, brightness, or noise? Extracting temperature-dependent properties of dye bound to DNA.

We present a method for extracting temperature-dependent thermodynamic and photophysical properties of SYTO-13 dye bound to DNA from fluorescence measurements. Together, mathematical modeling, control experiments, and numerical optimization enable dye binding strength, dye brightness, and experimental noise (or error) to be discriminated from one another. By focusing on the low-dye-coverage regime, the model avoids bias and can simplify quantification. Utilizing the temperature-cycling capabilities and multi-reaction chambers of a real-time PCR machine increases throughput. Significant well-to-well and plate-to-plate variation is quantified by using total least squares to account for error in both fluorescence and nominal dye concentration. Properties computed independently for single-stranded DNA and double-stranded DNA by numerical optimization are consistent with intuition and explain the advantageous performance of SYTO-13 in high-resolution melting and real-time PCR assays. Distinguishing between binding, brightness, and noise also clarifies the mechanism for the increased fluorescence of dye in a solution of double-stranded DNA compared to single-stranded DNA; in fact, the explanation changes with temperature.

Interventions for Driving Disruption in Community Rehabilitation: A Chart Audit.

PURPOSE: After injury or illness, a person's ability to drive may be impacted and they may experience a period of "driving disruption," a period during which they cannot drive although they have not permanently ceased driving. They may require additional information and supports from treating rehabilitation services; however, this process is less understood than others related to driving. MATERIALS AND METHODS: This study aimed to document the prevalence of driving-related issues and the current practices of a community rehabilitation service, regarding driving interventions. An audit of 80 medical records was conducted in a multidisciplinary community rehabilitation service in Brisbane, Australia. RESULTS: In total, 61% of clients were "driving-disrupted" on admission and 35% remained driving-disrupted on discharge. Majority of driving-disrupted clients had an acquired brain injury (ABI). Driving-related interventions were not routinely provided, with 29% receiving no information or supports. Clients with ABI more frequently received information; provision of psychosocial support and community access training was infrequent. CONCLUSIONS: This study highlights that return to driving is a common issue and goal for people undergoing community rehabilitation, with the period of driving disruption extending beyond rehabilitation discharge. It also highlights gaps in community rehabilitation practice, and opportunities to better support these clients.IMPLICATIONS FOR REHABILITATIONMany clients of community rehabilitation services experience driving disruption, often beyond discharge.Driving disruption should be recognised and documented by community rehabilitation services.Current practices may not adequately address the practical and psychological needs of clients experiencing driving disruption.

Synthesizing the biochemical and semiconductor worlds: the future of nucleic acid nanotechnology.

Since its inception nearly 40 years ago [Kallenbach, et al., Nature, 1983, 305, 829; N. C. Seeman, J. Theoretical Biology, 1982, 99, 237], Nucleic Acid Nanotechnology (NAN) has matured and is beginning to find commercial applications. For the last 20 years, it has been suggested that NAN might be an effective replacement for parts of the semiconductor lithography or protein engineering workflows. However, in that time, these incumbent technologies have made significant advances, and our understanding of NAN's strengths and weaknesses has progressed, suggesting that the greatest opportunities in fact lie elsewhere. Given the commitment of resources necessary to bring new technologies to the market and the desire to use those resources as wisely as possible, we conduct a critical examination of where NAN may benefit from, and provide benefit to, adjacent technologies and compete least with market incumbents. While the accuracy of our conclusions may be limited by our ability to extrapolate from the current state of NAN to its future commercial success, we conclude that the next promising direction is to create a bridge between biology and semiconductor technology. We also hope to stimulate a robust conversation around this technology's capabilities with the goal of building consensus on those research and development efforts that would advance NAN to the greatest effect in real-world applications.

Best practice for improved accuracy: A critical reassessment of van't Hoff analysis of melt curves.

Biomolecular thermodynamics, particularly for DNA, are frequently determined via van't Hoff analysis of optically measured melt curves. Accurate and precise values of thermodynamic parameters are essential for the modeling of complex systems involving cooperative effects, such as RNA tertiary structure and DNA origami, because the uncertainties associated with each motif in a folding energy landscape can compound, significantly reducing the power of predictive models. We follow the sources of uncertainty as they propagate through a typical van't Hoff analysis to derive best practices for melt experiments and subsequent data analysis, assuming perfect signal baseline correction. With appropriately designed experiments and analysis, a van't Hoff approach can provide surprisingly high precision, e.g., enthalpies may be determined with a precision as low as 10-2 kJ ⋅ mol-1 for an 8-base DNA oligomer.

Unmasking the Resolution-Throughput Tradespace of Focused-Ion-Beam Machining.

Focused-ion-beam machining is a powerful process to fabricate complex nanostructures, often through a sacrificial mask that enables milling beyond the resolution limit of the ion beam. However, current understanding of this super-resolution effect is empirical in the spatial domain and nonexistent in the temporal domain. This article reports the primary study of this fundamental tradespace of resolution and throughput. Chromia functions well as a masking material due to its smooth, uniform, and amorphous structure. An efficient method of in-line metrology enables characterization of ion-beam focus by scanning electron microscopy. Fabrication and characterization of complex test structures through chromia and into silica probe the response of the bilayer to a focused beam of gallium cations, demonstrating super-resolution factors of up to 6 ± 2 and improvements to volume throughput of at least factors of 42 ± 2, with uncertainties denoting 95% coverage intervals. Tractable theory models the essential aspects of the super-resolution effect for various nanostructures. Application of the new tradespace increases the volume throughput of machining Fresnel lenses by a factor of 75, enabling the introduction of projection standards for optical microscopy. These results enable paradigm shifts of sacrificial masking from empirical to engineering design and from prototyping to manufacturing.

Failure Mechanisms in DNA Self-Assembly: Barriers to Single-Fold Yield.

Understanding the folding process of DNA origami is a critical stepping stone to the broader implementation of nucleic acid nanofabrication technology but is notably nontrivial. Origami are formed by several hundred cooperative hybridization events-folds-between spatially separate domains of a scaffold, derived from a viral genome, and oligomeric staples. Individual events are difficult to detect. Here, we present a real-time probe of the unit operation of origami assembly, a single fold, across the scaffold as a function of hybridization domain separation-fold distance-and staple/scaffold ratio. This approach to the folding problem elucidates a predicted but previously unobserved blocked state that acts as a limit on yield for single folds, which may manifest as a barrier in whole origami assembly.

Analysis and uncertainty quantification of DNA fluorescence melt data: Applications of affine transformations.

Fluorescence-based measurements are a standard tool for characterizing the thermodynamic properties of DNA systems. Nonetheless, experimental melt data obtained from polymerase chain-reaction (PCR) machines (for example) often leads to signals that vary significantly between datasets. In many cases, this lack of reproducibility has led to difficulties in analyzing results and computing reasonable uncertainty estimates. To address this problem, we propose a data analysis procedure based on constrained, convex optimization of affine transformations, which can determine when and how melt curves collapse onto one another. A key aspect of this approach is its ability to provide a reproducible and more objective measure of whether a collection of datasets yields a consistent "universal" signal according to an appropriate model of the raw signals. Importantly, integrating this validation step into the analysis hardens the measurement protocol by allowing one to identify experimental conditions and/or modeling assumptions that may corrupt a measurement. Moreover, this robustness facilitates extraction of thermodynamic information at no additional cost in experimental time. We illustrate and test our approach on experiments of Förster resonance energy transfer (FRET) pairs used study the thermodynamics of DNA loops.

Revealing thermodynamics of DNA origami folding via affine transformations.

Structural DNA nanotechnology, as exemplified by DNA origami, has enabled the design and construction of molecularly-precise objects for a myriad of applications. However, limitations in imaging, and other characterization approaches, make a quantitative understanding of the folding process challenging. Such an understanding is necessary to determine the origins of structural defects, which constrain the practical use of these nanostructures. Here, we combine careful fluorescent reporter design with a novel affine transformation technique that, together, permit the rigorous measurement of folding thermodynamics. This method removes sources of systematic uncertainty and resolves problems with typical background-correction schemes. This in turn allows us to examine entropic corrections associated with folding and potential secondary and tertiary structure of the scaffold. Our approach also highlights the importance of heat-capacity changes during DNA melting. In addition to yielding insight into DNA origami folding, it is well-suited to probing fundamental processes in related self-assembling systems.

So, You Want to Have a Nanofab? Shared-Use Nanofabrication and Characterization Facilities: Cost-of-Ownership, Toolset, Utilization, and Lessons Learned.

Nanofabrication/characterization facilities enable research and development activities across a host of science and engineering disciplines. The collection of tools and supporting infrastructure necessary to construct, image, and measure micro- and nanoscale materials, devices, and systems is complex and expensive to establish, and it is costly to maintain and optimize. As a result, these facilities are typically operated in a shared-use mode. We discuss the key factors that must be considered to successfully create and sustain such facilities. These include the need for long-term vision and institutional commitment, and the hands-on involvement of managers in facility operations. We consider startup, operating, and recapitalization costs, together with algorithms for cost recovery and tool-time allocation. The acquisition of detailed and comprehensive project and tool-utilization data is essential for understanding and optimizing facility operations. Only such a data-driven decision-making approach can maximize facility impact on institutional goals. We illustrate these concepts using the National Institute of Standards and Technology (NIST) NanoFab as our test case, but the methodologies and resources presented here should be useful to all those faced with this challenging task.

Managing the transition to non-driving in patients with dementia in primary care settings: facilitators and barriers reported by primary care physicians.

OBJECTIVES: This research addresses dementia and driving cessation, a major life event for affected individuals, and an immense challenge in primary care. In Australia, as with many other countries, it is primarily general practitioners (GPs) who identify changes in cognitive functioning and monitor driving issues with their patients with dementia. Qualitative evidence from studies with family members and other health professionals shows it is a complicated area of practice. However we still know little from GPs about how they manage the challenges with their patients and the strategies that they use to facilitate driving cessation. METHODS: Data were collected through five focus groups with 29 GPs at their primary care practices in metropolitan and regional Queensland, Australia. A semi-structured topic guide was used to direct questions addressing decision factors and management strategies. Discussions were audio recorded, transcribed verbatim and thematically analyzed. RESULTS: Regarding the challenges of raising driving cessation, four key themes emerged. These included: (i) Considering the individual; (ii) GP-patient relationships may hinder or help; (iii) Resources to support raising driver retirement; and (iv) Ethical dilemmas and ethical considerations. The impact of discussing driving cessation on GPs is discussed. CONCLUSIONS: The findings of this study contribute to further understanding the experiences and needs of primary care physicians related to managing driving retirement with their patients with dementia. Results support a need for programs regarding identification and assessment of fitness to drive, to upskill health professionals and particularly GPs to manage the complex issues around dementia and driving cessation, and explore cost-effective and timely delivery of such support to patients.

Ultrasonic cement removal in cement-in-cement revision total hip arthroplasty: What is the effect on the final cement-in-cement bond?

OBJECTIVES: Previous studies have evidenced cement-in-cement techniques as reliable in revision arthroplasty. Commonly, the original cement mantle is reshaped, aiding accurate placement of the new stem. Ultrasonic devices selectively remove cement, preserve host bone, and have lower cortical perforation rates than other techniques. As far as the authors are aware, the impact of ultrasonic devices on final cement-in-cement bonds has not been investigated. This study assessed the impact of cement removal using the Orthosonics System for Cemented Arthroplasty Revision (OSCAR; Orthosonics) on final cement-in-cement bonds. METHODS: A total of 24 specimens were manufactured by pouring cement (Simplex P Bone Cement; Stryker) into stainless steel moulds, with a central rod polished to Stryker Exeter V40 specifications. After cement curing, the rods were removed and eight specimens were allocated to each of three internal surface preparation groups: 1) burr; 2) OSCAR; and 3) no treatment. Internal holes were recemented, and each specimen was cut into 5 mm discs. Shear testing of discs was completed by a technician blinded to the original grouping, recording ultimate shear strengths. Scanning electron microscopy (SEM) was completed, inspecting surfaces of shear-tested specimens. RESULTS: The mean shear strength for OSCAR-prepared specimens (33.6 MPa) was significantly lower than for the control (46.3 MPa) and burr (45.8 MPa) groups (p < 0.001; one-way analysis of variance (ANOVA) with Tukey's post hoc analysis). There was no significant difference in shear strengths between control and burr groups (p = 0.57). Scanning electron microscopy of OSCAR specimens revealed evidence of porosity undiscovered in previous studies. CONCLUSION: Results show that the cement removal technique impacts on final cement-in-cement bonds. This in vitro study demonstrates significantly weaker bonds when using OSCAR prior to recementation into an old cement mantle compared with cement prepared with a burr or no treatment. This infers that care must be taken in surgical decision-making regarding cement removal techniques used during cement-in-cement revision arthroplasty, suggesting that the risks and benefits of ultrasonic cement removal need consideration.Cite this article: A. Liddle, M. Webb, N. Clement, S. Green, J. Liddle, M. German, J. Holland. Ultrasonic cement removal in cement-in-cement revision total hip arthroplasty: What is the effect on the final cement-in-cement bond? Bone Joint Res 2019;8:246-252. DOI: 10.1302/2046-3758.86.BJR-2018-0313.R1.

Aligned carbon nanotube morphogenesis predicts physical properties of their polymer nanocomposites.

Carbon nanostructure (CNS) based polymer nanocomposites (PNCs) are of interest due to the superior properties of the CNS themselves, scale effects, and the ability to transfer these properties anisotropically to the bulk material. However, measurements of physical properties of such materials are not in agreement with theoretical predictions. Recently, the ability to characterize the 3D morphology of such PNCs at the nanoscale has been significantly improved, with rich, quantitative data extracted from tomographic transmission electron microscopy (TEM). In this work, we use new, nanoscale quantitative 3D morphological information and stochastic modeling to re-interpret experimental measurements of continuous aligned carbon nanotube (A-CNT) PNC properties as a function of A-CNT packing/volume fraction. The 3D tortuosity calculated from tomographic reconstructions and its evolution with volume fraction is used to develop a novel definition of waviness that incorporates the stochastic nature of CNT growth. The importance of using randomly wavy CNTs to model these materials is validated by agreement between simulated and previously-measured PNC elastic moduli. Secondary morphological descriptors such as CNT-CNT junction density and inter-junction distances are measured for transport property predictions. The scaling of the junction density with CNT volume fraction is observed to be non-linear, and this non-linearity is identified as the primary reason behind the previously unexplained scaling of aligned-CNT PNC longitudinal thermal conductivity. By contrast, the measured electrical conductivity scales linearly with volume fraction as it is relatively insensitive to junction density beyond percolation. This result verifies prior hypotheses that electrical conduction in such fully percolated and continuous CNT systems is dominated by the bulk resistivity of the CNTs themselves. This combination of electron tomographic data and stochastic simulations is a powerful method for establishing a predictive capability for nanocomposite structure-property relations, making it an essential aid in understanding and tailoring the next-generation of advanced composites.

Low-Temperature Growth of Carbon Nanotubes Catalyzed by Sodium-Based Ingredients.

Synthesis of low-dimensional carbon nanomaterials such as carbon nanotubes (CNTs) is a key driver for achieving advances in energy storage, computing, and multifunctional composites, among other applications. Here, we report high-yield thermal chemical vapor deposition (CVD) synthesis of CNTs catalyzed by reagent-grade common sodium-containing compounds, including NaCl, NaHCO3 , Na2 CO3 , and NaOH, found in table salt, baking soda, and detergents, respectively. Coupled with an oxidative dehydrogenation reaction to crack acetylene at reduced temperatures, Na-based nanoparticles have been observed to catalyze CNT growth at temperatures below 400 °C. Ex situ and in situ transmission electron microscopy (TEM) reveal unique CNT morphologies and growth characteristics, including a vaporizing Na catalyst phenomenon that we leverage to create CNTs without residual catalyst particles for applications that require metal-free CNTs. Na is shown to synthesize CNTs on numerous substrates, and as the first alkali group metal catalyst demonstrated for CNT growth, holds great promise for expanding the understanding of nanocarbon synthesis.

Masculinity and preventing falls: insights from the fall experiences of men aged 70 years and over.

PURPOSE: To explore men's fall experiences through the lens of masculine identities so as to assist health professionals better engage men in fall prevention programs. METHODS: Twenty-five men, aged 70-93 years who had experienced a recent fall, participated in a qualitative semi-structured interview. Men's willingness to engage in fall prevention programs taking account of individual contexts and expressions of masculinity, were conceptualised using constant comparative methods. RESULTS: Men's willingness to engage in fall prevention programs was related to their perceptions of the preventability of falls; personal relevance of falls; and age, health, and capability as well as problem-solving styles to prevent falls. Fall prevention advice was rarely given when men accessed the health system at the time of a fall. CONCLUSIONS: Contrary to dominant expectations about masculine identity, many men acknowledged fall vulnerability indicating they would attend or consider attending, a fall prevention program. Health professionals can better engage men by providing consistent messages that falls can be prevented; tailoring advice, understanding men are at different stages in their awareness of fall risk and preferences for action; and by being aware of their own assumptions that can act as barriers to speaking with men about fall prevention. Implications for rehabilitation Men accessing the health system at the time of the fall, and during rehabilitation following a fall represent prime opportunities for health professionals to speak with men about preventing falls and make appropriate referrals to community programs. Tailored advice will take account of individual men's perceptions of preventability; personal relevance; perceptions of age, health and capability; and problem-solving styles.

Electron beam-based metrology after CMOS.

The magnitudes of the challenges facing electron-based metrology for post-CMOS technology are reviewed. Directed selfassembly, nanophotonics/plasmonics, and resistive switches and selectors, are examined as exemplars of important post-CMOS technologies. Materials, devices, and architectures emerging from these technologies pose new metrology requirements: defect detection, possibly subsurface, in soft materials, accurate measurement of size, shape, and roughness of structures for nanophotonic devices, contamination-free measurement of surface-sensitive structures, and identification of subtle structural, chemical, or electronic changes of state associated with switching in non-volatile memory elements. Electron-beam techniques are examined in the light of these emerging requirements. The strong electron-matter interaction provides measurable signal from small sample features, rendering electron-beam methods more suitable than most for nanometer-scale metrology, but as is to be expected, solutions to many of the measurement challenges are yet to be demonstrated. The seeds of possible solutions are identified when they are available.

Subnanometer localization accuracy in widefield optical microscopy.

The common assumption that precision is the limit of accuracy in localization microscopy and the typical absence of comprehensive calibration of optical microscopes lead to a widespread issue-overconfidence in measurement results with nanoscale statistical uncertainties that can be invalid due to microscale systematic errors. In this article, we report a comprehensive solution to this underappreciated problem. We develop arrays of subresolution apertures into the first reference materials that enable localization errors approaching the atomic scale across a submillimeter field. We present novel methods for calibrating our microscope system using aperture arrays and develop aberration corrections that reach the precision limit of our reference materials. We correct and register localization data from multiple colors and test different sources of light emission with equal accuracy, indicating the general applicability of our reference materials and calibration methods. In a first application of our new measurement capability, we introduce the concept of critical-dimension localization microscopy, facilitating tests of nanofabrication processes and quality control of aperture arrays. In a second application, we apply these stable reference materials to answer open questions about the apparent instability of fluorescent nanoparticles that commonly serve as fiducial markers. Our study establishes a foundation for subnanometer localization accuracy in widefield optical microscopy.

Enhanced durability of carbon nanotube grafted hierarchical ceramic microfiber-reinforced epoxy composites.

As carbon nanotube (CNT) infused hybrid composites are increasingly identified as next-generation aerospace materials, it is vital to evaluate their long-term structural performance under aging environments. In this work, the durability of hierarchical, aligned CNT grafted aluminoborosilicate microfiber-epoxy composites (CNT composites) are compared against baseline aluminoborosilicate composites (baseline composites), before and after immersion in water at 25 °C (hydro) and 60 °C (hydrothermal), for extended durations (90 d and 180 d). The addition of CNTs is found to reduce water diffusivities by approximately 1.5 times. The mechanical properties (bending strength and modulus) and the damage sensing capabilities (DC conductivity) of CNT composites remain intact regardless of exposure conditions. The baseline composites show significant loss of strength (44 %) after only 15 d of hydrothermal aging. This loss of mechanical strength is attributed to fiber-polymer interfacial debonding caused by accumulation of water at high temperatures. In situ acoustic and DC electrical measurements of hydrothermally aged CNT composites identify extensive stress-relieving micro-cracking and crack deflections that are absent in the aged baseline composites. These observations are supported by SEM images of the failed composite cross-sections that highlight secondary matrix toughening mechanisms in the form of CNT pullouts and fractures which enhance the service life of composites and maintain their properties under accelerated aging environments.

Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA-Protein Nanostructures.

Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method. This process simultaneously shapes the structure at the nanoscale and directs the large-scale geometry. The DNA skeleton guides the assembly of RecA protein filaments, which provides rigidity at the micrometer scale. We use our modular design strategy to assemble tetrahedral, rectangular, and linear shapes of defined dimensions. This method enables the robust construction of complex assemblies, greatly extending the range of DNA-based self-assembly methods.