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Acute kidney injury (AKI) is a known risk factor for chronic kidney disease (CKD). The mechanisms for AKI-CKD transition may involve failed tubule repair and progressive interstitial fibrosis but are incompletely understood. Which events lead to failed tubule repair? Can injury spread over time? And are these processes steered by myofibroblasts and fibrotic tissue remodeling?Here, we combined serial intravital 2-photon microscopy and a novel model of partial ischemia-reperfusion injury (partial IRI), which allowed us to investigate injured tissue alongside uninjured tissue in the same kidney and over time. Thus, we occluded only one branch of the left renal artery for 21 min, rendering half the kidney ischemic. We then tracked tubule necrosis and remodeling alongside myofibroblast dynamics for up to 3 weeks. Necrotic cell death was detected by in vivo propidium iodide staining. PDGFRβ-CreERT2-Salsa6F mice (N=5) identified main myofibroblast precursor cells, by tdTomato-expression. Myofibroblast-differentiation was confirmed by ex vivo αSMA staining. Proximal tubule (PT) function was assessed by in vivo uptake of fluorescent albumin.Necrotic cell death was solely detectable upon reperfusion. Border areas in between post-ischemic and non-ischemic tissue presented severely necrotic PTs alongside uninjured PTs. From 1 day after partial IRI, we observed formation of granular casts in the lumen of necrotic tubules, which 1-2 days later also appeared in initially non-necrotic PTs downstream. Tubule necrosis and granular cast accumulation preceded thinned epithelium that lacked albumin endocytosis (day 3-7) and recruitment of myofibroblasts (peaking D4-10). Of note, non-necrotic tubules, without granular cast accumulation, didn’t remodel nor recruit myofibroblasts.Among all remodeling tubule segments, 24% turned atrophic. Importantly, preceding necrotic cell death was higher in atrophic than in recovering tubules (55±13% vs. 39±18% necrotic cells/total segment cell number, (mean±SD), p Novo Nordisk Foundation, Augustinus Foundation, Aarhus University Research Foundation (AUFF) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD-transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here we present a novel model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death, demonstrated pronounced injury propagation and expansion into previously uninjured tissue regions, which predicted tubule atrophy with epithelial VCAM-1 expression. In summary, our longitudinal analysis of tubule injury, remodeling, and fate, provided important novel insights into AKI pathology.

A reversible switchable on-demand UV-triggered drug delivery system (DDS) based on interpenetrating polymer networks (IPNs) with silicone as the host polymer and spiropyran (SP)-functionalized guest polymer is designed and demonstrated. The photo-responsive IPNs provide a new triggered drug delivery concept as they exploit the change in intermolecular interactions (work of adhesion) among the drug, matrix, and solvent when the incorporated hydrophobic SP moieties transform into the hydrophilic merocyanine form upon light irradiation without degradation and disruption of the DDS. The change in how the copolymer composition (hydrophilicity and content) and the lipophilicity of the drug (log P) affect the release profile was investigated. A thermodynamic model, based on Hansen solubility parameters, was developed to design and optimize the polymer composition of the IPNs to obtain the most efficient light-triggered drug release and suppression of the premature release. The developed IPNs showed excellent result for dopamine, l-dopa, and prednisone with around 90-95% light-triggered release. The model was applied to study the release behavior of drugs with different log P and to estimate if the light-induced hydrophobic-to-hydrophilic switch can overcome the work of adhesion between polymers and drugs and hence the desorption and release of the drugs. To the best of our knowledge, this is the first time that work of adhesion is used for this aim. Comparing the result obtained from the model and experiment shows that the model is useful for evaluating and estimating the release behavior of specific drugs merocyanine, IPN, DDS, and spiropyran.

Flow cytometry (FC) is a pivotal tool for studying the physical and chemical properties of particles. State-of-the-art FC systems are highly advanced, yet they are expensive, bulky, and require high sample volume, qualified operators, and periodic maintenance. The manipulation of particles suspended in viscoelastic fluids has received increasing attention, especially for miniaturized flow cytometry technologies. This study presents a miniaturized optical capillary FC device using the viscoelastic focusing technique. A straight, one inlet/outlet microcapillary device is precisely aligned to a fiber-coupled laser source and detectors. Forward scattered, side scattered, and fluorescently emitted light signals are collected and analyzed in a real-time environment. The developed platform fits onto an inverted microscope stage enabling real-time microscopy imaging of the particles of interest together with the flow cytometry analysis. We achieved stable viscoelastic focusing and performed FC measurements for rigid polystyrene beads (diameters: 2 - 15 μm), non-spherical human erythrocytes, and canonical shape metaphase human chromosomes. We performed cytometry measurements with a throughput of 100 events/s yielding a coefficient of variation of 2%. This newly developed FC device is a versatile tool and can be operated with any inverted microscope to get the mutual benefits of optical and imaging FC measurements. Furthermore, it is possible to extend these benefits by adding more back-end tools, such as optical trapping and Raman spectroscopy.

A compact microfluidic flow cytometer is demonstrated, comprising viscoelastic flow focusing in fused silica capillaries and a fiber optical interface. Viscoelastic flow focusing enables simple device design and operation with a single-inlet/outlet fluidic configuration. Fused silica capillaries with different inner diameters are effortlessly interchanged to eliminate blockage ratio limitations and enable single-train particle focusing for a wide range of particle sizes and geometries. The compact system is mounted on an inverted microscope for easy integration with optical imaging and other optofluidic modalities, such as optical trapping and particle sorting. A real-time cytometric analysis of three channels, forward scattering, side scattering, and fluorescence detection, is performed on LABVIEW. A throughput of 3500 events s(-1) is performed on particles of sizes ranging from 2 to 20 mu m, using capillaries of different inner diameters ranging from 30 to 75 mu m. The outer diameter of all capillaries is identical to the cladding diameter of the applied optical fibers. This enables easy exchange and precise optical alignment of fibers and capillaries on a microfabricated jig. The performance of the microfluidic flow cytometer is benchmarked using polystyrene calibration beads, poly(lactic-co-glycolic acid) particles, erythrocytes, THP-1 leukemic monocytes, and human metaphase chromosomes.

Blood plasma evaluation has high significance in clinical diagnostics. Current schemes involve the preparation of blood plasma by centrifugation of whole blood followed by electrochemical or spectroscopic analysis. However, centrifugation is often too time-consuming for application in clinical emergency and point-of-care settings. We propose to combine microfluidic, instantaneous plasma fractionation with localized spectroscopic methods for in-line analysis. As an example, we present confocal Raman spectroscopy in fractionated plasma domains at two different Raman excitation wavelengths. Resonance Raman spectroscopy with laser excitation at 408 nm allows the specific detection of free hemoglobin in blood plasma at concentrations above 22 mg dl-1 (level of detection). Consequently, we are able to accurately resolve the range of clinical relevance regarding hemolysis. At near-infrared excitation (785 nm) we furthermore demonstrate the acquisition of characteristic Raman spectra of fractionated blood plasma in the microfluidic setting. These spectra can serve as starting point for a multi-parameter regression analysis to quantify a set of blood plasma parameters from a single Raman spectrum. The combined microfluidics and Raman spectroscopy method is non-destructive and has a whole blood consumption of less than 100 μl per hour. It thus allows for continuous in-line blood plasma monitoring.

Plasmonic color metasurfaces enable high resolution, ink-free color decoration and sensing devices. This work presents a full solution from design of plasmonic color elements suitable for master origination by 248 nm Deep Ultraviolet (DUV) lithography to production grade manufacture of plasmonic color metasurfaces on plastic substrates. Design of hybrid aluminum disk-hole structures with hybridized multiple resonances, which are controlled by the disk-hole distance, enables plasmonic color generation across the visible spectrum with larger sized nanostructures, above 160 nm. Such larger structures can be defined by 248 nm DUV lithography as a cost-efficient and high throughput alternative to, e.g., electron beam lithography.

The advent of resonant dielectric nanomaterials has provided a new path for concentrating and manipulating light on the nanoscale. Such high-refractive-index materials support a diverse set of low-loss optical resonances, including Mie resonances, anapole states, and bound states in the continuum. Through these resonances, high-refractive-index materials can be used to engineer the optical near field, both inside and outside the nanostructures, which opens up new opportunities for Raman spectroscopy. In this review, we discuss the impact of high-refractive-index nano-optics on Raman spectroscopy. In particular, we consider the intrinsic Raman enhancement produced by different dielectric resonances and their theoretical description. Using the optical reciprocity theorem, we derive an expression which links the Raman enhancement to the enhancement of the stored electric energy. We also address recent results on surface-enhanced Raman spectroscopy based on high-refractive-index dielectric materials along with applications in stimulated Raman scattering and nanothermometry. Finally, we discuss the potential of Raman spectroscopy as a tool for detecting the optical near-fields produced by dielectric resonances, complementing reflection and transmission measurements.

Disclosed is a method for self-organization of a nanostructure, comprising the steps of, providing a narrow-band light source having a center wavelength; providing a film on a substrate, said thin film having a film thickness, said film thickness being in the range from 0.9 to 1.1 times a Fabry Perot resonance condition thickness for said center wavelength; and irradiating said film, the irradiating comprises using said narrow-band light source to induce self-organization of said film, thereby obtaining nanostructures. A product comprising a substrate and nanostructures produced by said method is further disclosed.

Viscoelastic flow-based particle manipulation techniques enable bio-particle focusing, separation, and enrichment by precisely tuning the rheological parameters, flow conditions, and microchannel geometry. In this study, we fabricated a PDMS-based single inlet/outlet microchannel to separate bio-particles by their size ranging from 1-10 μm. Flow conditions and rheological properties are optimized using 2 μm and 4 μm Polystyrene beads to reach the best particle separation condition. We demonstrated the size-based separation of human chromosomes by separating 1-2 μm size small chromosomes from 8-10 μm size large chromosomes. Thanks to its miniaturized size and simplicity, the isolation chip and unique viscoelastic separation method have great potential to be used as a future pioneering tool for genetic applications to study chromosome abnormalities such as fragile-X and trisomy.

The replacement of plastic with eco-friendly and biodegradable materials is one of the most stringent environmental challenges. In this respect, cellulose stands out as a biodegradable polymer. However, a significant challenge is to obtain biodegradable materials for high-end photonics that are robust in humid environments. Here, we demonstrate the fabrication of high-quality micro- and nanoscale photonic and plasmonic structures via replica molding using pure cellulose and a blended version with nonedible agro-wastes. Both materials are biodegradable in soil and seawater according to the ISO 17556 standard. The pure cellulose films are transparent in the vis–NIR spectrum, having a refractive index similar to glass. The microstructured photonic crystals show high-quality diffractive properties that are maintained under extended exposure to water. Nanostructuring the cellulose transforms it to a biodegradable metasurface manifesting bright structural colors. A subsequent deposition of Ag endowed the metasurface with plasmonic properties used to produce plasmonic colors and for surface-enhanced Raman scattering.

The ongoing effort to implement compact and cheap optical systems is the main driving force for the recent flourishing research in the field of optical metalenses. Metalenses are a type of metasurface, used for focusing and imaging applications, and are implemented based on the nanopatterning of an optical surface. The challenge faced by metalens research is to reach high levels of performance using simple fabrication methods suitable for mass production. In this paper, we present a Huygens nanoantenna-based metalens, designed for outdoor photographic/surveillance applications in the near infrared. We show that good imaging quality can be obtained over a field of view as large as ±15°. This first successful implementation of metalenses for outdoor imaging applications is expected to provide insight and inspiration for future metalens imaging applications.

Plasmonic color generation from metal nanostructures has attracted intensive attention because of their excellence in achieving high spatial resolution, strong color contrast, and long‐term durability. The limited area of plasmonic patterns anchored on substrates and produced by current top‐down methods, however, severely restricts the advanced developments and potential applications in structural color display. Herein a robust method for realizing the laser printing of plasmonic colors inside transparent gold nanodisk‐embedded poly(dimethylsiloxane) matrices is presented. It is found that various colors can not only be easily generated by embedding gold nanodisks of different sizes, but also finely varied by adjusting the laser pulse intensity during printing. It is further demonstrated that multiple color layers can be laser‐printed at different depths. Stereoscopic images in the 3D matrices are laser‐printed with sizes as large as 12 × 15 mm2 and a resolution of 4600 dots per inch.

This thesis seeks to investigate how Psalm 139, and biblical texts in general, can be interpreted to facilitate a meaningful reading by bereaved parents after the early loss of a child. By using in-depth interviews with bereaved parents, this study emphasises the reader’s perspective in its exegesis to produce a responsible, contemporary interpretation. I applied insights on the existential challenges of bereaved parents to a reader-oriented exegetical approach to Psalm 139, imagining a hypothetical reader’s creative engagement with the biblical text. To investigate the merit of this approach, the resulting interpretation was then compared to findings from interviews conducted with bereaved parents about their experiences of reading the psalm. This study finds that the individual experience of the reader is hugely influential in the creative process of interpretation. Depending on the reader’s past life and personal convictions, Psalm 139 can be immensely comforting or disconcerting. I conclude that the reader-oriented approach to exegesis is a valuable tool for any biblical scholar and minister, as it can unearth a wealth of relevant, contemporary interpretations inaccessible to the historical approach to exegesis. This thesis contends that scholarly biblical studies, homiletics and pastoral would all benefit greatly from an increased focus on the contemporary reader’s perspective.

Optical materials with a high refractive index enable effective manipulation of light at the nanoscale through strong light confinement. However, the optical near field, which is mainly confined inside such high-index nanostructures, is difficult to probe with existing measurement techniques. Here, we exploit the connection between Raman scattering and the stored electric energy to detect resonance-induced near-field enhancements in silicon nanostructures. We introduce a Raman setup with a wavelength-tunable laser, which allows us to tune the Raman excitation wavelength and thereby identify Fabry-Perot and Mie type resonances in silicon thin films and nanodisk arrays, respectively. We measure the optical near-field enhancement by comparing the Raman response on and off resonance. Our results show that tunable-excitation Raman spectroscopy can be used as a complimentary far-field technique to reflection measurements for nanoscale characterization and quality control. As proof-of-principle for the latter, we demonstrate that Raman spectroscopy captures fabrication imperfections in the silicon nanodisk arrays, enabling an all-optical quality control of metasurfaces.

Hemolysis is the rupture of red blood cells and constitutes the most common reason for unsuitable blood samples in the clinic. To detect hemolysis, one has to separate the hemoglobin in blood plasma from that in red blood cells. However, current methods entail centrifugation for cell-plasma separation, which is complex, time-consuming, and not easy to integrate into point-of-care (PoC) systems. Here, we demonstrate an optofluidic sensor composed of nanofilters on an optical waveguide, which enables evanescent-wave absorption measurement of hemoglobin in plasma with the capability of real-time inline detection on whole blood without extra sample preparation like centrifugation. Long-term testing with inline integration in a modified, commercial blood gas analyzer shows high reliability and repeatability of the measurements even with the presence of interference from bilirubin. We envision that the present work has large potential in improving diagnosis quality by enabling PoC hemolysis detection in blood gas analyzers and can also lend unique sensing capabilities to other applications dealing with complex turbid media.

Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.

Pyrolytic carbon microelectrodes (PCMEs) are a promising alternative to their conventional metallic counterparts for various applications. Thus, methods for the simple and inexpensive patterning of PCMEs are highly sought after. Here, we demonstrate the fabrication of PCMEs through the selective pyrolysis of SU-8 photoresist by irradiation with a low-power, 806 nm, continuous wave, semiconductor-diode laser. The SU-8 was modified by adding Pro-Jet 800NP (FujiFilm) in order to ensure absorbance in the 800 nm range. The SU-8 precursor with absorber was successfully converted into pyrolytic carbon upon laser irradiation, which was not possible without an absorber. We demonstrated that the local laser pyrolysis (LLP) process in an inert nitrogen atmosphere with higher laser power and lower scan speed resulted in higher electrical conductance. The maximum conductivity achieved for a laser-pyrolyzed line was 14.2 ± 3.3 S/cm, with a line width and thickness of 28.3 ± 2.9 µm and 6.0 ± 1.0 µm, respectively, while the narrowest conductive line was just 13.5 ± 0.4 µm wide and 4.9 ± 0.5 µm thick. The LLP process seemed to be self-limiting, as multiple repetitive laser scans did not alter the properties of the carbonized lines. The direct laser writing of adjacent lines with an insulating gap down to ≤5 µm was achieved. Finally, multiple lines were seamlessly joined and intersected, enabling the writing of more complex designs with branching electrodes and the porosity of the carbon lines could be controlled by the scan speed.

We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells.

The ability to handle single, free molecules in lab-on-a-chip systems is key to the development of advanced biotechnologies. Entropic confinement offers passive control of polymers in nanofluidic systems by locally asserting a molecule's number of available conformation states through structured landscapes. Separately, a range of plasmonic configurations have demonstrated active manipulation of nano-objects by harnessing concentrated electric fields. The integration of these two independent techniques promises a range of sophisticated and complementary functions to handle, for example, DNA, but numerous difficulties, in particular, conflicting requirements of channel size, have prevented progress. Here, we show that metallic V-groove waveguides, embedded in fluidic nanoslits, form entropic potentials that trap and guide DNA molecules over well-defined routes while simultaneously promoting photothermal transport of DNA through the losses of plasmonic modes. The propulsive forces, assisted by in-coupling to propagating channel plasmon polaritons, extend along the V-grooves with a directed motion up to ≈0.5 μm·mW-1 away from the input beam and λ-DNA velocities reaching ≈0.2 μm·s-1·mW-1. The entropic trapping enables the V-grooves to be flexibly loaded and unloaded with DNA by variation of transverse fluid flow, a process that is selective to biopolymers versus fixed-shape objects and also allows the technique to address the challenges of nanoscale interaction volumes. Our self-aligning, light-driven actuator provides a convenient platform to filter, route, and manipulate individual molecules and may be realized wholly by wafer-scale fabrication suitable for parallelized investigation.

An automated method for determining whether dairy cows with subclinical mammary infections recover after antibiotic treatment would be a useful tool in dairy production. For that purpose, inline L-lactate dehydrogenase (LDH) measurements was modeled using a dynamic linear model; the variance parameters were estimated using the expectation-maximization algorithm. The method used to classify cows as infected or uninfected was based on a multiprocess Kalman filter. Two learning data sets were created: infected and uninfected. The infected data set consisted of records from 48 cows with subclinical Staphylococcus aureus infection from 4 herds collected in 2010. The uninfected data set came from 35 uninfected cows collected during 2013 from 2 herds. Bacteriological culturing was used as gold standard. To test the model, we collected data from the 48 infected cows 50 d after antibiotic treatment. As a result of the treatment, this test data set consisted of 25 cows that still had a subclinical infection and 23 cows that were recovered. Model sensitivity was 36.0% and specificity was 82.6%. To a large extent, L-lactate dehydrogenase reflected the cow's immune response to the presence of pathogens in the udder. However, cows that were classified correctly before treatment had a better chance of correct classification after treatment. This indicated a variation between cows in immune response to subclinical mammary infection that may complicate the detection of subclinically infected cows and determination of recovery.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 +/- 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

The absorption in amorphous silicon nano antennas is engineered for the purpose of post process structural change via resonant laser printing by illumination, thus shifting transmission and reflection spectrum in a controlled manner.

Conventional three-dimensional optics requires curvature to control the wave front of light thus making it difficult to reduce the size of the optical systems. Furthermore, for correction of optical aberrations, complex optical systems comprising more than one lens are used. This adds additional bulk, mass and complexity to the optical systems. Recent development in diffractive optics has enabled new thin lightweight optical elements such as metalenses. We introduce resonant laser printing technique as a flexible photo-thermal technology for metalens fabrication with the ability to control the light with microscale precision. Our laser printed metalenses can be integrated in bio-sensors, bio-imaging systems, and optofluidical devices.

Spatially offset Raman spectroscopy (SORS) enables one to distinguish chemical fingerprints of top and subsurface layers. In this paper, we apply SORS to a microfluidic two-layer system consisting of transparent liquid in a microchannel as the surface layer and microfluidic PDMS chip material as the sublayer. By using an imaging spectrograph connected to a microscope, we perform hyperspectral SORS acquisitions. Furthermore, the focus position z is translated. Thus, we combine the two methods of hyperspectral SORS and defocusing micro-SORS, which leads to an integral characterization of the layered system. The collected top and subsurface layers of Raman scattering at the optical axis (zero spatial offset) largely depends on the focus position z. However, the spatially offset Raman scattered intensity from the subsurface layer is constant for a large range of focus positions z. We claim that there is potential for internal referencing and alignment reproducibility. We demonstrate these findings experimentally in a microfluidic scenario where a 16 mu m deep channel is filled with an aqueous hemoglobin solution. Our observation enables consistent concentration measurements in small-volume liquid samples. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

A waveguide Young interferometer is presented with simultaneous detection of complex refractive index of a liquid sample. The real part of the refractive index change (refraction) is detected by tracing phase shifts of the interferogram generated by a sensing and reference waveguide. The imaginary part of the refractive index (absorption) is determined by the attenuation of the transmitted signal at certain wavelength. Furthermore, nano-filters are fabricated atop the sensing waveguide, which enables size-exclusion filtering of species to the evanescent field. It shows capability of distinguishing small and large particles from 100 nm to 500 nm in diameter, which is further confirmed by fluorescent excitation experiments. The present sensor could find broad application in optical characterization of complex turbid media with regard to their complex refractive index.

International audience; The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received 12 million s funding under the European Union Framework Programme 7 from 1st June 2008 to 30th November 2012. The 19 project partners from both academia and industry from in total 7 countries had a project budget of 16 Ms with which they have discovered, created and developed a huge body of insights into nucleic acid analysis. Results have been presented widely in publications and in innumerous public presentations. Results have been moved to spin-offs such as the Olink enrichment kits (now sold by Agilent as Haloplex) and are findingtheir way to the market, such as the Oxford Nanopore MinIon sequencer that was first released to early-access user sites in 2014.

A metric for evaluation of overall metalens performance is presented. It is applied to determination of optimal operating spectral range of a metalens, both theoretically and experimentally. This metric is quite general and can be applied to the design and evaluation of future metalenses, particularly achromatic metalenses.

A photonic crystal slab (PCS) sensor is a universal refractive index sensor with possibilities and performance very similar to surface plasmon resonance (SPR), which represents the gold standard of biosensing. Cheap PCS sensors can be made vacuum-free entirely out of polymers, but come with additional challenges, besides those relating to temperature-variations, which must be considered in any refractive index based method: The polymeric waveguide core was found to swell by ∼0.3% as water absorbed into the waveguide core over ∼1.5 h. This was investigated by monitoring the wavelength of resonant reflection during absorption, by monitoring the release of water using ellipsometry, and by rigorous coupled-wave analysis (RCWA). The approach presented here enables monitoring of water uptake and thermal fluctuations, for drift-free, high-performance operation of a polymeric PCS sensor.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cell (LS174T, LS180 and RKO) lines and fresh colorectal cancers are individually trapped, their genomes extracted and prepared for sequencing, using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06 %) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as extraction is in sub-nanoliter volumes. Our data also demonstrates that high quality single cell sequencing can be achieved using a relatively simple, inexpensive and scalable device.

Laser printing with a spatial light modulator (SLM) has several advantages over conventional raster-writing and dot-matrix display (DMD) writing: multiple pixel exposure, high power endurance and existing software for computer generated holograms (CGH). We present a technique for the design and manufacturing of plasmonic metasurfaces based on ultrafast laser printing with an SLM. As a proof of principle, we have used this technique to laser print a plasmonic metalens as well as high resolution plasmonic color decorations. The high throughput holographic resonant laser printing (HRLP) approach enables on-demand mass-production of customized metasurfaces., Comment: Supplementary information is available upon request to authors

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

According to one aspect, an optoelectric scaffold for accommodating photo- responsive biological components is provided. The scaffold comprises an optical waveguide configured for confining light propagating in a longitudinal direction thereof. The optical waveguide comprises at least one leaky section with enhanced emission of light in a direction transverse or lateral to the longitudinal direction. The scaffold further comprises an electrically conductive layer arranged on an outer surface of the optical waveguide, wherein the electrically conductive layer has an immobilisation or growth support surface for the immobilisation or cultivation of photo-responsive biological components thereon. The electrically conductive layer comprises transparent regions at least partially overlapping the leaky section. The transparent region is configured so as to transmit light from the leaky section of the waveguide to the immobilisation and/or growth support surface. According to a further aspect, an optoelectric device comprises an optoelectric scaffold and a photo-responsive biological component arranged on the immobilisation/growth support surface. The growth support surface is arranged so as to transmit light received from the leaky section of the optical waveguide to the biological component placed thereon.

Traffic accidents are a result of the convergence of hazards, malfunctioning of vehicles and human negligence that have adverse economic and health impacts and effects. Unfortunately, avoiding them completely is very difficult, but with quick response to rescue and first aid, the mortality rate of inflicted persons can be reduced significantly. Smart and innovative technologies can play a pivotal role to respond faster to traffic crash emergencies comparing conventional means of transportation. For instance, Rescue Emergency Drone (RED) can provide faster and real-time crash site risk assessment to emergency medical services, thereby helping them to quickly and accurately assess a situation, dispatch the right equipment and assist bystanders to treat inflicted person properly. To conduct a research in this regard, the case of a traffic roundabout that is prone to frequent traffic accidents on the outskirts of Esbjerg, a town located on western coast of Denmark is hypothetically considered. Along with manual calculations, Emergency Disaster Management Simulation (EDMSIM) has been used to verify the response time of RED from a fire station of the town to the presumed crash site. The results of the study demonstrate the robustness of RED into emergency services to help save lives., {"references":["World Health Organization (2017) \"Road Traffic Injuries\". (Accessed on 29/08/2017), Available at: http://www.who.int/mediacentre/factsheets/fs358/en/.","Frederick B. Rogers (2014) 'The Golden Hour in Trauma: Dogma or Medical Folklore?' MD, MS, FACS Medical Director, Trauma Program, LGH and Katelyn Rittenhouse, BS.","Alec Momont (2016) \"Ambulance Drone\", Delft University of Technology (2016) (Accessed on 07/08/2017). 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Colors of materials are precisely laser-controlled using new manufacturing technology with nanopatterned semiconductor surfaces., Man-made structural colors, which originate from resonant interactions between visible light and manufactured nanostructures, are emerging as a solution for ink-free color printing. We show that non-iridescent structural colors can be conveniently produced by nanostructures made from high-index dielectric materials. Compared to plasmonic analogs, color surfaces with high-index dielectrics, such as germanium (Ge), have a lower reflectance, yielding a superior color contrast. Taking advantage of band-to-band absorption in Ge, we laser-postprocess Ge color metasurfaces with morphology-dependent resonances. Strong on-resonance energy absorption under pulsed laser irradiation locally elevates the lattice temperature (exceeding 1200 K) in an ultrashort time scale (1 ns). This forms the basis for resonant laser printing, where rapid melting allows for surface energy–driven morphology changes with associated modification of color appearance. Laser-printable high-index dielectric color metasurfaces are scalable to a large area and open a new paradigm for printing and decoration with nonfading and vibrant colors.

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

We demonstrate the optothermal actuation of individual capillary burst valves in an all-polymer microfluidic device. The capillary burst valves are realised in a planar design by introducing a fluidic constriction in a microfluidic channel of constant depth. We show that a capillary burst valve can be burst by raising the temperature due to the temperature dependence of the fluid surface tension. We address individual valves by using a local heating platform based on a thin film of near infrared absorber dye embedded in the lid used to seal the microfluidic device [L. H. Thamdrup et al., Nano Lett. 10, 826-832 (2010)]. An individual valve is burst by focusing the laser in its vicinity. We demonstrate the capture of single polystyrene 7 μm beads in the constriction triggered by the bursting of the valve.

Mastitis is a serious production-limiting disease, with effects on milk yield, milk quality, and conception rate, and an increase in the risk of mortality and culling. The objective of this study was 2-fold: (1) to develop an economic optimization model that incorporates all the different types of pathogens that cause clinical mastitis (CM) categorized into 8 classes of culture results, and account for whether the CM was a first, second, or third case in the current lactation and whether the cow had a previous case or cases of CM in the preceding lactation; and (2) to develop this decision model to be versatile enough to add additional pathogens, diseases, or other cow characteristics as more information becomes available without significant alterations to the basic structure of the model. The model provides economically optimal decisions depending on the individual characteristics of the cow and the specific pathogen causing CM. The net returns for the basic herd scenario (with all CM included) were $507/cow per year, where the incidence of CM (cases per 100 cow-years) was 35.6, of which 91.8% of cases were recommended for treatment under an optimal replacement policy. The cost per case of CM was $216.11. The CM cases comprised (incidences, %) Staphylococcus spp. (1.6), Staphylococcus aureus (1.8), Streptococcus spp. (6.9), Escherichia coli (8.1), Klebsiella spp. (2.2), other treated cases (e.g., Pseudomonas; 1.1), other not treated cases (e.g., Trueperella pyogenes; 1.2), and negative culture cases (12.7). The average cost per case, even under optimal decisions, was greatest for Klebsiella spp. ($477), followed by E. coli ($361), other treated cases ($297), and other not treated cases ($280). This was followed by the gram-positive pathogens; among these, the greatest cost per case was due to Staph. aureus ($266), followed by Streptococcus spp. ($174) and Staphylococcus spp. ($135); negative culture had the lowest cost ($115). The model recommended treatment for most CM cases (>85%); the range was 86.2% (Klebsiella spp.) to 98.5% (Staphylococcus spp.). In general, the optimal recommended time for replacement was up to 5 mo earlier for cows with CM compared with cows without CM. Furthermore, although the parameter estimates implemented in this model are applicable to the dairy farms in this study, the parameters may be altered to be specific to other dairy farms. Cow rankings and values based on disease status, pregnancy status, and milk production can be extracted; these provide guidance when determining which cows to keep or cull.

We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence.

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical 'X' shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.

Knowledge about the Holocene evolution of the Greenland ice sheet (GrIS) is important to put the recent observations of ice loss into a longer-term perspective. In this study, we use six new threshold lake records supplemented with two existing lake records to reconstruct the Holocene ice marginal fluctuations of the Qassimiut lobe (QL) – one of the most dynamic parts of the GrIS in South Greenland. Times when the ice margin was close to present extent are characterized by clastic input from the glacier meltwater, whereas periods when the ice margin was behind its present day extent comprise organic-rich sediments. We find that the overall pattern suggests that the central part of the ice lobe in low-lying areas experienced the most prolonged ice retreat from ~9–0.4 cal. ka BP, whereas the more distal parts of the ice lobe at higher elevation re-advanced and remained close to the present extent during the Neoglacial between ~4.4 and 1.8 cal. ka BP. These results demonstrate that the QL was primarily driven by Holocene climate changes, but also emphasises the role of local topography on the ice marginal fluctuations.

Transition state theory (TST) provides a simple interpretation of many thermally activated processes. It applies successfully on timescales and length scales that differ several orders of magnitude: to chemical reactions, breaking of chemical bonds, unfolding of proteins and RNA structures and polymers crossing entropic barriers. Here we apply TST to out-of-equilibrium transport through confined environments: the thermally activated translocation of single DNA molecules over an entropic barrier helped by an external force field. Reaction pathways are effectively one dimensional and so long that they are observable in a microscope. Reaction rates are so slow that transitions are recorded on video. We find sharp transition states that are independent of the applied force, similar to chemical bond rupture, as well as transition states that change location on the reaction pathway with the strength of the applied force. The states of equilibrium and transition are separated by micrometres as compared with angstroms/nanometres for chemical bonds., Transition state theory has proven to be a powerful tool for the analysis of a number of processes, perhaps most commonly chemical reactions. Here, the authors use transition state theory to model a directly observable, micron scale process—the transport of DNA molecules in a confined environment.

Nitrogen-vacancy (NV) centers in diamonds are interesting due to their remarkable characteristics that are well suited to applications in quantum-information processing and magnetic field sensing, as well as representing stable fluorescent sources. Multiple NV centers in nanodiamonds (NDs) are especially useful as biological fluorophores due to their chemical neutrality, brightness and room-temperature photostability. Furthermore, NDs containing multiple NV centers also have potential in high-precision magnetic field and temperature sensing. Coupling NV centers to propagating surface plasmon polariton (SPP) modes gives a base for lab-on-a-chip sensing devices, allows enhanced fluorescence emission and collection which can further enhance the precision of NV-based sensors. Here, we investigate coupling of multiple NV centers in individual NDs to the SPP modes supported by silver surfaces protected by thin dielectric layers and by gold V-grooves (VGs) produced via the self-terminated silicon etching. In the first case, we concentrate on monitoring differences in fluorescence spectra obtained from a source ND, which is illuminated by a pump laser, and from a scattering ND illuminated only by the fluorescence-excited SPP radiation. In the second case, we observe changes in the average NV lifetime when the same ND is characterized outside and inside a VG. Fluorescence emission from the VG terminations is also observed, which confirms the NV coupling to the VG-supported SPP modes., 22 pages, 13 figures