Our measurements reveal that electrical and thermal interfaces dominate the operation of such devices, uncovering a thermal boundary resistance of 28 ± 8 m2K/GW at GST-SiO2 interfaces and an effective thermopower 350 ± 50 µV/K at GST-Pt interfaces. We also discuss possible pathways to apply Raman thermometry and SThM techniques to nanoscale and vertical resistive memory devices.The operation of resistive and phase-change memory (RRAM and PCM) is controlled by highly localized self-heating effects, yet detailed studies of their temperature are rare due to challenges of nanoscale thermometry. Here we show that the combination of Raman thermometry and scanning thermal microscopy (SThM) can enable such measurements with high spatial resolution. We report temperature-dependent Raman spectra of HfO Memristive devices are important not only for memory and storage applications; they are also being extensively studied as computing elements for neuromorphic architectures8.Information storage and memory devices based on the change of resistance (i.e. resistive memories or memristors) hold several key advantages over contemporary charge-based memory devices. Such memory devices are two-terminal resistors that retain their resistance state as a function of the applied voltage or current. Several technologies can be included under the general term of “resistive memory”2. In PCM, a nanoscale volume of chalcogenide material (like Ge2Sb2Te5) can be SET to a crystalline (low resistive) state and RESET to an amorphous (high resistive) state using electrical pulses. Self-heating and the local temperature play a major role in the principle of operation of both PCM4 and RRAM10. Many of the advantages 12 and shortcomings (e.g. reliability)13 of these technologies stem from their inherent dependence on self-heating. Therefore, understanding the energy and heat dissipation mechanism is vital for the evaluation, design and optimization of all such future technologies. However, experimental techniques to measure nanoscale device temperature are challenging and scarce15. In particular, spatially resolved measurements revealing energy dissipation mechanism are required for better understanding of the device physics17.The resistive switching in RRAM devices is based on the formation and rupture of conductive filaments in thin metal oxides, like HfO18, but it is applied here to resistive memory devices for the first time. We present temperature-dependent Raman spectroscopy of thin films for two of the most commonly used RRAM oxides: HfO2 and TiO2, and for the PCM material Ge2Sb2Te5 (GST). We then show an experimental measurement of the temperature profile in a Joule-heated PCM device, providing important insights into its operation. Finally, we discuss how Raman and SThM can be used to measure the local temperature rise in vertical and other nanoscale RRAM and PCM device geometries.Here we combine Raman thermometry and scanning thermal microscopy (SThM) to measure the spatially resolved temperature rise in resistive memory devices. This powerful combination of temperature mapping techniques has been previously used on GaN nanowiresℏω) and temperature (T). Stokes (anti-Stokes) lines are due to photons scattered at lower (higher) energy than the incident laser, due to phonon emission and absorption, respectively.  TiO2. The 50 nm thick films were sputtered onto Pt/sapphire and SiO2/Si substrates, respectively, and did not show any measurable Raman features in their (as-deposited) amorphous state. After annealing (see Methods) the films crystallized, exhibiting Raman signals of the monoclinic (HfO2) and anatase (TiO2) phases. The insets show the temperature dependence of a selected mode. The Bg1 monoclinic HfO2 mode24 (~134 cm−1 at 25 °C) shows typical frequency downshift with temperature at rate of ~ −0.011 cm−1/C. The Eg anatase TiO2 mode (~141 cm−1 at 25 °C) however shows anomalous frequency increase with temperature. This trend was previously reported and explained via strong contribution of the quartic anaharmonicity23. For the practical purpose of device thermometry it is sufficient to have a well-defined temperature response of the Raman mode, either positive or negative.Figure devices is challenging, but could be addressed using signal enhancement techniques such as surface- or tip- enhanced Raman spectroscopy (SERS25 or TERS27). In addition, an optically transparent electrode such as graphene28, indium tin oxide (ITO), or a very thin (e.g. sub-10 nm) metal can be used to measure vertical device structures. In the next section we focus on Raman thermometry of PCM, which exhibits a sufficiently strong signal to demonstrate  device thermometry.We note that temperature-dependent Raman data are presented in this section for nanoscale oxide films for the first time. However, obtaining sufficient signal in nanoscale RRAM 31. Unlike oxides, GST absorbs photons in the range of visible excitation lasers (~500–650 nm) due to its smaller band gap (~0.6 eV). In addition, much of the GST volume in a typical PCM cell is crystalline, thus Raman thermometry can be readily applied to PCM devices. Moreover, Raman spectroscopy can be used to identify phase and presence of defects in GST as outlined below.Raman spectroscopy has previously been used to characterize nanoscale PCM films34 but we are unaware of temperature-dependent Raman data for GST films of the thickness studied here (~20 nm), which are more relevant for modern memory devices. Figure 29. Upon cooling to room temperature and heating back up to 450 °C the film remains in its stable hcp phase, as shown in Fig. −1) and 400 °C  and Fig. Figure devices exhibit Raman spectra that are different from blanket deposited GST films. Figure Thanks to its material and phase selectivity, Raman spectroscopy can be used to map GST films and PCM devices for phase analysis. However, here we uncover that patterned and processed GST −1, which are not present in the GST film spectra  are known from previous studies as Te peaks35. We have also measured similar peaks in uncapped GST films after exposure to high laser power and formation of dark spots in the film (Supplementary Information Section 36. Such oxide-capped devices switched for many cycles (~105) with good on/off ratio (~102) and low switching energy (<20 pJ). These Te peaks are likely associated with surface oxidation of the GST during processing, resulting in formation of GeOx and SbOx, and precipitation of Te. The former is also evident from X-Ray photoelectron spectroscopy (XPS) of the dark spots at the GST surface (Supplementary Information Section These intense peaks (~120 and ~140 cm−1) Te peak has strong temperature dependence, as shown in Fig. 38 and the other directly from the anti-Stokes to Stokes (AS/S) intensity ratio39:T is the phonon temperature (in K), ωph and ωL are the phonon shift and laser frequency, respectively, IAS/IS the anti-Stokes to Stokes intensity ratio, and kB is the Boltzmann constant. The advantage of the AS/S method is that in principle it does not require a calibration procedure and that the temperature can be obtained with a single measurement. This is attractive for spatial temperature mapping of devices since the temperature map can be obtained in a single map scan, whereas for the peak shift method a calibration measurement is needed and at least two map scans must be obtained38 (e.g. one with and the other without bias). Nonetheless, the uncertainty in the measured temperature of our GST devices using the AS/S method was large compared with the peak shift method, as described below.The position of the lower (~120 cmT = 25 °C). The blue line represents eq. (area under the peak instead of the peak intensity.) Moreover, the absolute temperature T (≥300 K) is obtained from the intensity ratio with relative error ~15% in our case. Since we are interested in measuring the change in temperature (ΔT ~ 100 K above room temperature) the relative error in ΔT is three times larger, or nearly ~ 50%. At the same time, the relative error in ΔT for the peak shift method is less than ~15% [e.g. see Fig. 38 for aligning the two maps. We note that temperature sensitivity of the AS/S method should improve for phonon modes at higher frequencies  measurements on the same devices. SThM is an atomic force microscopy (AFM) based technique that uses a thermo-resistive probe to acquire nanoscale topographic and thermal images simultaneously42, yet it measures the temperature at the top surface of the PMMA capping layer which spreads the heat. Simulations therefore predict slightly different temperature profiles for the Raman and SThM measurements. We note that the SThM is operated here in DC mode and is not calibrated to output temperature directly, rather it outputs a relative signal. We then use Raman thermometry (with same power input conditions) to calibrate the SThM to the temperature in the middle of the PCM device, where the GST temperature (measured by Raman) and the top PMMA temperature (measured by SThM) are similar [see cross-sectional temperature rise in Fig. Figure 43. However, not all materials have a usable Raman signal and the spatial resolution is diffraction-limited . SThM on the other hand has nearly AFM-like spatial resolution, but measures the temperature at the top surface rather than the direct temperature of the material of interest. The temperature sensitivity of the SThM can be better than that of the Raman method, but in order to convert the SThM signal (voltage) to temperature, the Raman method was used for calibration, as outlined above. Both techniques are limited in measuring vertical RRAM devices; the top electrode might block the Raman signal and laterally spread the temperature profile measured by SThM. Nonetheless, the combination of both techniques could become a very powerful tool to study the power dissipation in cases where temperature plays a major role in device operation10. A practical solution is to measure lateral devices or vertical devices having transparently thin top electrode.The main advantage of the Raman measurement is its material selectivity which allows a differential measurement of materials in the laser path, thus enabling even atomic scale resolution in the cross-plane directionFigure 44. Temperature artifacts due to the presence of step heights at the edge of the GST-Pt contacts are negligible here compared with the observed interfacial heating as discussed in Supplementary Information Section density there.First, it is evident that significant heat is generated at the contacts2 and GST-Pt interfaces to be 28 ± 8 and 25 ± 9 m2K/GW respectively. Since the Si and SiO2 thermal properties are well known, and given the insensitivity to the bulk GST thermal conductivity  technique, but it is extracted here within a functioning PCM device for the first time. The measured TBR of the GST-SiO2 interface is equivalent to a Kapitza length of ~50 nm of SiO2, which accounts for more than 25% of the total thermal resistance in our device. Importantly, the relative contribution of the TBR is expected to increase and dominate as devices are scaled down in size46.Second, given the power input and the measured temperature rise we can extract the thermal boundary resistance (TBR) of the GST-SiO47 as confirmed by reversing the current flow direction. We extract an effective thermopower S = 350 ± 50 µV/K, which includes both thermoelectric and thermionic effects at the GST-Pt contact. Similar values were reported for GST in the mixed amorphous and fcc phase with Pt48 and with TiW49 contacts slightly above room temperature, after annealing up to ~ 150 °C. Following anneal at higher temperature (>200 °C) and higher degree of crystallization, the thermopower is expected to drop significantly (below 50 µV/K)49. The higher temperature at the grounded contact is also consistent with the location of damage after device breakdown, shown in Supplementary Information Section 50. We note that our test devices are lateral and larger than state-of-the-art PCM devices54; however the physical insights are valid and highly valuable: the power generation is dominated by electrical contacts and the heat dissipation is limited by thermal interfaces in nanoscale devices44.Third, we also observe a clear asymmetry in heating at the two contacts, with the higher temperature at the edge of the grounded electrode. This contact heating asymmetry is due to a combination of thermoelectric and thermionic effects2 and TiO2, and we used spatial mapping of temperature (with both Raman and SThM) to provide physical insight into the operation of PCM devices. Our approach takes advantage of the benefits of each technique, e.g. selectivity (Raman) and high spatial resolution (SThM), and can be extended to a wide variety of devices. We uncover significant heating at the contacts, suggesting that power dissipation is often dominated by electrical contact resistance. Contact heating is asymmetric, depending on current flow direction, showing that thermoelectric effects must be taken into account when designing PCM programming pulses. We also extract the TBR of the GST-SiO2 interface and find that it is equivalent to ~ 50 nm thick SiO2, contributing much of the device thermal resistance. The role of both electrical contacts and thermal interfaces will only become more dominant as devices are scaled to sub-50 nm dimensions. Uncovering the spatial distribution of temperature rise in such self-heated memristive devices is essential for their understanding, and their future design and integration.In summary, in this study we have laid the fundamental basis for thermometry of RRAM and PCM data storage devices. We presented the first measurements of thermal Raman signatures in nanoscale films of HfORaman measurements were carried out on a Horiba Labram Evolution HR using a 633 nm laser with an 1800 l/mm grating. The red laser is chosen here since it provides Raman signal comparable to other laser lines in our system (e.g. 532 nm) and allows the measurement of anti-Stokes signal by using a volume Bragg grating optical filter at 633 nm. All measurements were done in air.2 films were deposited by reactive sputtering from an Hf target in an Ar:O2 (7:3) plasma at 4 mTorr, with a forward RF power of 150 W, at room temperature onto thin Pt films (50 nm) on a sapphire substrate. The Pt/sapphire substrate was used to enhance the Raman signal of the HfO2 film. TiO2 films were sputtered from a Ti target in an Ar:O2 (14:1) plasma at 5 mTorr onto SiO2 (90 nm) on Si substrates. To obtain Raman signal the films were crystallized by annealing: the TiO2 for 1 hour at 400 °C and the HfO2 for 2 hours at 600 °C, both in air.HfO2 (90 nm) on Si substrates, immediately followed (without breaking chamber vacuum) by sputtering 20 nm SiO2 to prevent oxidation when later exposed to air. GST devices (section “Raman Spectroscopy of GST Devices”) were prepared as follows. First, contacts and pads were defined by photolithography. Contact separation defined channel lengths L = 2 to 20 µm. The 40 nm Pt contacts (with 2 nm Ti adhesion layer) were then deposited by e-beam evaporation followed by patterning of channels (of widths W = 1 to 10 µm) with e-beam lithography, sputtering of 20 nm GST and lift-off. Devices were capped by spin coating ~150 nm poly(methyl methacrylate) (PMMA) to prevent oxidation, then baked on a hot plate at 180 °C for 10 minutes (in air) to crystallize the GST film.Blanket GST films (20 nm) discussed in the section “Raman Spectroscopy of GST Films” were sputtered onto SiOThe SThM measurements were carried out in passive mode and under DC bias. Measured PCM devices were Joule heated electrically by applying constant voltage to the contact pads for ~10 minutes during the SThM scan. The SThM thermal probe model used in this work is a DM-GLA-5 from Anasys®, which is made of a thin Pd layer on SiN.Supplementary Information


Cells are complex systems in which dynamic gene expression and protein-interaction networks adapt to changes in the environment. Seeding and subsequent spreading of cells on substrates represents an example of adaptation to a major perturbation. The formation of adhesive interactions and self-organisation of the cytoskeleton during initial spreading might prime future cell behaviour. To elucidate the role of these events on later cellular behaviour, we mapped the trajectories by which cells respond to seeding on substrates with different physical properties. Our experiments on cell spreading dynamics on collagen- or fibrin-coated polyacrylamide gels and collagen or fibrin hydrogels show that on each substrate, cells follow distinct trajectories of morphological changes, culminating in fundamentally different cell states as quantified by RNA-expression levels, YAP/TAZ localisation, proliferation and differentiation propensities. The continuous adaptation of the cell to environmental cues leaves traces due to differential cellular organisation and gene expression profiles, blurring correlations between a particular physical property and cellular phenotype. The cellular state at a particular time can be characterised by the combined abundance and organisation of all its components. A key challenge is to understand how cells reach a particular state upon a response to changes in their environment2. As a first step, one can study the isolated components, including gene expression levels and protein localisation at steady. However, cell development is a dynamic process, following trajectories across a metaphorical ‘landscape’ of gene expression profiles that involve multiple so-called attractors4. Changes in the environment will affect this ‘landscape’, as the cell adapts by altering cellular organisation and changing gene expression profiles, thus potentially altering cell state and cell fate. An important example of such an adaptation process is the spreading of cells on a substrate, the dynamics of which have been studied in detail10. It is clear that adaptation to the substrate, and the forces experienced during spreading, lead to different dynamic changes in cell shape12. The balance of forces, the development of focal adhesions, and the build-up of tension in the cytoskeleton on substrates with different mechanical characteristics, have all been captured in impressive studies15. With time, cells reach a steady state, and numerous studies have demonstrated correlations between a wide range of mechanical characteristics and steady state properties such as cell adhesion, spreading area, proliferation and differentiation23. The question we address here, is whether the adaptation of cellular shape and organisation during the spreading of cells on substrates with different mechanical properties, impacts on future cellular phenotypes and cell fate. We therefore developed a time-resolved, systems level study, which would allow us to follow both invariant and divergent characteristics of cells while they spread on different substrates, and provide a direct window on the cellular processes that integrate the multitude of mechanical cues over time.Cells are complex systems, where the interplay of many (simple) components leads to the emergence of highly sophisticated behaviourHere, we follow how hMSCs adapt, upon seeding, to different substrates (PAAm hydrogels coated with collagen and fibrin vs. collagen and fibrin hydrogels) over 24 hours. On each substrate, cells follow distinct trajectories of morphological changes, culminating in fundamentally different cell states, as reflected in significant differences in gene expression profiles and protein localisation characteristics. These results challenge the view that characterisation of cellular phenotypes at apparent steady states without knowledge of the prior events can provide us with a complete picture of how cells sense the mechanical properties of their environment.Human mesenchymal stem cells (hMSCs) were cultured on polyacrylamide (PAAm) gels of medium (≈3 kPa) and high (≈23 kPa) stiffness, coated with either collagen filaments or fibrin monomers and compared to hMSCs cultured on collagen type I (<1 kPa) or fibrin (<1 kPa) gels, respectively  using live cell imaging techniques. Low cell densities were used in order to observe single cells and eliminate cell-cell communication. Figure pes Fig.  showing ing Fig. . An evidThe evolution of cell morphology on all six substrates is shown in Fig. 26, and our findings are in agreement with previous literature reports30. We performed bead displacement studies to quantitatively compare substrate deformation on PAAm and protein substrates  was approx. 30 min on collagen and 4 hours on fibrin. After 8.5 h the motion of the fluorescent beads ceased, indicating mechanical equilibrium between the cell traction forces and the elastic resistance of the protein gels, possibily in combination with remodelling of the gels34. Within this time frame, most cells remained within the window of recording. Figure versus fibrin as the onset of deformation is much sooner on collagen and cell area increases earlier on fibrin than on collagen.The combination of distinct spreading trajectories but significant overlap in morphology at 24 h prompted us to explore the spreading trajectories on stiff (≈23 kPa) PAAm and protein gels in more detail. We observed that cell spreading on protein gels is associated with cellular remodelling of the substrates . We chose to compare RNA expression levels on collagen and fibrin gels vs. stiff PAAm gels coated with collagen and fibrin, as these substrates showed different trajectories but closest morphologies after 24 h. See SI for more experimental details. Interestingly, the number of differentially expressed genes increases much faster on protein gels compared to PAAm gels, even though changes in morphology occur much faster on PAAm, see Figure It is evident that cell spreading follows distinct trajectories associated with different materials properties of the substrate. We wished to investigate the intracellular imprints of these trajectories by studying the changes in gene expression over time using genome-wide RNA sequencing to obtain more molecular insight into the dynamics of cellular processes during the observed dynamic spreading behaviour on the different types of substrates. For this, we harvested cells for RNA isolation after 1 h, 5 h, 10 h and 24 h and compared expression levels to the original cell suspension at the moment of seeding as a common reference sample in technical duplicates (t0 (fragments per kilobase of exon per million reads mapped (FPKM) ≥ 2) and were differentially expressed in at least one time-point compared to t0 (p ≤ 10−10 and fold-change ≥5). For a general overview on similarities and differences between samples at identical time points, the top 25 upregulated and downregulated genes were compared. The overlap in the top 25 upregulated genes between the various substrates decreased over time  was applied (top-bottom) for all 18 samples , as well as substrate specific effects . We used principal component analysis (PCA) to identify common underlying structures in the RNA-seq dataset that could help identify the observed differences between the samples Fig. . PrincipTo identify the biological and molecular processes represented in the different clusters, we performed gene ontology overrepresentation (GO) analysis. A selection of the biological processes that were found significantly overrepresented (p < 0.001) are presented in Fig. 38. Figure 3), albeit slightly lower on fibrin gels. Immunostainings showed that activated integrin β1 is present on all substrates and aligned with intracellular stress fibres  and transcriptional coactivator with PDZ-binding motif (TAZ). It is thought that cells growing on stiff substrates can develop stress fibres and cytoskeletal tension, which are known to prevent phosphorylation of YAP/TAZ, resulting in nuclear localisationing Fig.  shows thing Fig. . Controling Fig. . Similard Figure .Figure 644.Interestingly, Cluster 2 is enriched in genes involved in cell cycle progression and proliferation, whereas Cluster 5 is enriched in muscle cell proliferative genes. Thus, our RNA-sequencing analysis suggests that cells seeded on collagen only gels might initiate towards a muscle cell fate and do not follow the same cell proliferation program as cells on the other substrates. Subsequent quantification of hMSC proliferation as determined by EdU incorporation over 24 h shows significantly lower proliferation on collagen than on stiff PAAm-col whereas cell proliferation on fibrin is not significantly different from cells on stiff PAAm-fb Figs  and S10.47. hMSCs were seeded on the four types of gels. After culturing in mixed adipogenic/osteogenic medium for 10 days, Oil red O staining showed no difference between the substrates in the relative presence of adipocytes  impacted on future events, such as the differentiation of hMSCs into osteoblasts and adipocytestes Fig. . Differetes Fig. . These eWe have shown that cellular adaptation to the mechanical environment upon seeding follows distinct trajectories. Differences in phenotypes arise early, as exemplified by the isotropic spreading on stiff PAAm gels, whereas cells on protein gels show matrix deformation, and spreading along deformed regions. However, after strikingly different initial morphologies, cellular phenotypes appear to converge over time, and after 24 h cells can look remarkably similar. In contrast, the morphological trajectories are accompanied by intracellular ones that do not converge: RNA-seq data show on the one hand that cells on all substrates follow a common expression pattern of early response genes, but differences appear in for example integrin expression patterns, especially when comparing PAAm and protein gels, but also between collagen and fibrin substrates. At 24 h, the ‘molecular starting points’ for further development, i.e. the occupancy of the complex networks that govern cell behaviour and responses, are very different among the different substrates. Gene expression profiles do show that the early differences have resulted in different molecular trajectories, leading cells to integrate the various mechanical cues in different ways. The different trajectories also lead to significant differences in the localisation of YAP/TAZ, proliferation rates, and cellular differentiation on different substrates. It is clear that the different adaptation routes cells take on different substrates leave an imprint, and affect future events such as lineage selection.Our studies make clear that the continuously evolving patterns of morphological changes and gene expression profiles that result from probing the environment, blur the correlations between a particular property of the substrate and cellular phenotype. The impact of different environmental factors such as matrix elasticity or topography might thus depend on how they shape the adaptation trajectory that cells take. Our findings highlight the importance of studying the response of cells over time, and early spreading trajectories could provide a strong indication of future cellular behaviour.6. Glass coverslips  were oxidised using oxygen plasma and then incubated in a 0.3 wt/vol% solution of 3-(trimethoxysilyl)propyl methacrylate (Sigma Aldrich) in dry toluene overnight. The slides were washed thoroughly with ethanol and water.This method was based on a previously described protocolSolutions of acrylamide (AA) at final concentrations of 5, 8 and 20 wt/vol% and bis-acrylamide (BA) at 0.01, 0.02, 0.15 and 0.375 wt/vol% were prepared. Polymerisation was initiated by the addition of 5 µL of 10 wt/vol% ammonium persulfate (Sigma Aldrich) and 1.5 µL TEMED (Sigma Aldrich) to the AA/BA solutions in PBS. 4 µL of the gel precursor solution was immediately pipetted onto de methacrylated glass coverslips and a 20 mm glass coverslip, washed but untreated, was carefully placed on top of the polymerizing solution. After 1.5–2 h, the samples were soaked in PBS buffer overnight to remove the remaining monomer and crosslinker. The top coverslips were peeled off to obtain the PAAm gels adhering to the coverslides.−1 solution in milliQ H2O was pipetted onto the gel surfaces, which were then placed under a 365 nM UV lamp  and irradiated for 5 min. The gels were washed twice with PBS and the procedure was repeated once. After the second round of washing with PBS, the substrates were coated with 50 µg mL−1 of rat type I collagen in PBS for 2 h at room temperature or with 0.1 mg mL−1 of bovine fibrinogen in PBS for 1.5 h followed by 0.5 h incubation with thrombin 1 U mL−1 in PBS. Samples were again washed two times with PBS prior to cell seeding.To facilitate cell adhesion, the hydrogel was covered with a covalently bound protein coating using N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate  as a crosslinker. 2 × 15 µL of a 1 mg mL−1) and then put through a 0.22 µm filter to sterilise the solution. Gelation was achieved by mixing the FBNG solution with CO2-independent medium  in equal volumes, resulting in 2 mg mL−1 fibrin gels after 1 h incubation at 37 °C. For gel deformation studies, fibrin gels were stained directly after gelation with FITC prior to cell seeding.Fibrinogen  was dissolved in PBS  for 1 hour at 37 °C and subsequently with Alexa488-conjugated secondary antibody , prior to cell seeding.Rat tail collagen type I (BD biosciences) gels were prepared similarly to the manufacturer’s protocol. High concentration collagen was diluted to 4 mg mL38. For each gel, indentation force curves at 30 different locations on the gels were acquired. Before and during indentation experiments gels were kept in medium in 37 °C. To address local stiffness changes generated by cells on protein gels and PAAm gel, we applied spatially resolved AFM nanoindentation in live-cell culture by probing the matrix around single cells. To obtain stiffness values from force curves we used the PUNIAS software (http://punias.free.fr). Specifically, we corrected for baseline tilt, and used the linear fitting option for the Hertz model with a Poisson ratio of 0.5 on the indentation curve.Substrate stiffness was measured by nanoindentation under an atomic force microscope (Bruker Nanoscope) using the “point and shoot” procedure . A fluorescent polystyrene bead  was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N/m . The system was calibrated in cell-free medium at 37 °C prior to each experiment by measuring the deflection sensitivity when pressing the cantilever onto a glass coverslip, which allowed the cantilever spring constant to be determined using the thermal noise method−2 for the analysis of spreading, gel deformation and YAP/TAZ expression. Spreading and gel deformation were observed at multiple time points. YAP/TAZ expression was studied after 24 and 48 h in culture.hMSCs were obtained from Lonza and cultured up to passage 6 before seeding them onto gels at a density of circa 1250 cm−7 M dexamethasone, 5 mM β-glycerolphosphate, 0.1 mM ascorbic acid-2-phosphate, 250 µM 3-isobutyl-1-methylxanthine, 5 µg mL−1 insulin (from bovine pancreas) and 5 × 10−8 M rosiglitazone maleate). Cells were seeded at circa 2500 cm−2 for osteogenic differentiation and 25000 cm−2 for adipogenic differentiation in MIX medium on the substrates. Medium was changed two times per week. Osteogenic differentiation was analysed after 7 days by staining for alkaline phosphatase, and adipogenic differentiation after 10 days by staining with Oil red O. ALP/Oil red O positive cells were counted manually from at least five different bright field images. The percentage of differentiation towards each lineage was quantified by setting a uniform threshold in each experiment and manually counting the number of positively stained cells in relation to the total number of nuclei (DAPI staining).All adipo/osteo differentiation experiments were performed in a 1:1 mixture of adipogenic and osteogenic induction medium , YAP/TAZ , β1 integrin , MyoD  and Alexa633-conjugated phalloidin (Sigma Aldrich) and subsequently with Alexa488 or -546-conjugated secondary antibodies (Life Technologies) and DAPI. For proliferation studies, EdU labelling was performed following the manufacturer’s protocol . Quantification of nuclear localisation was performed manually. Cells were considered to have nuclear localisation when the level of fluorescence in the nucleus was higher than the level in the cytosol.2 stage incubator and Okolab 2D time lapse software). The initial bead position was determined ~20 min after cell seeding, when cells were attached to the gel surface. A time series of images was collected at a frequency of one image every 10 min. Beads displacement analyses were performed with a custom script in Fiji software.Phase contrast images were acquired at 2 min intervals on an IncuCyte live cell analysis system (Essen BioScience). For bead displacement studies, PAAm pre-gels as well as collagen and fibrin solutions were mixed with fluorescent beads, which served as fiduciary markers for tracking the deformation of the matrix under adherent cells. The bead displacement field around each cell was mapped from a time series of fluorescent images that were collected using an inverted time lapse microscope  by using two separate aliquots of the cell suspension prior to seeding on the substrate.RNAs from 6 subsamples were isolated by an extraction using Trizol reagent (Life Technologies) according to the manufacturer’s protocol. The RNA pellets from these subsamples were resuspended in water and pooled to a total volume of exactly 100 µL. The samples were further purified by means of a Nucleospin RNA clean-up kit (Machery-Nagel), including an on column treatment with rDNAse. Finally the samples were eluted in 60 µL of RNAse-free water. Quality control was performed using an Agilent 6000 nano kit on an Agilent BioAnalyzer 2100. Subsequent RNA sequencing was performed by ServiceXS Leiden, The Netherlands. For the reference samples at tpost hoc correction with *p < 0.05, **p < 0.01 and NS: p > 0.05. Analysis was performed on triplicate independent experiments using cells from the same donor throughout. ANOVA was chosen over t-tests due to its more conservative nature.Significance of differences throughout this study were tested using one-way analysis of variance (ANOVA) with Tukey MovieS1MovieS2MovieS3MovieS4MovieS5MovieS6MovieS7Supplementary Data File (Table S3)Supplementary Figures and Tables


The temporal variation of N2O fluxes showed only low emissions over the vegetable cropping phases, but significantly higher emissions were observed post-harvest accounting for 50–70% of the annual emissions. NIs reduced N2O emissions by 20–60% over the vegetable cropping phases; however, this mitigation was offset by elevated N2O emissions from the NIs treatments over the post-harvest fallow period. Annual N2O emissions from the conventional fertiliser, the DMPP treatment, and the 3MP + TZ treatment were 1.3, 1.1 and 1.6 (sem = 0.2) kg-N ha−1 year−1, respectively. This study highlights that the use of NIs in vegetable systems can lead to elevated N2O emissions by storing N in the soil profile that is available to soil microbes during the decomposition of the vegetable residues. Hence the use of NIs in vegetable systems has to be treated carefully and fertiliser rates need to be adjusted to avoid an oversupply of N during the post-harvest phase.To investigate the effect of nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and 3-methylpyrazole 1,2,4-triazole (3MP + TZ), on N Different studies have shown that NIs can reduce N2O emissions from cropping soils92O emissions and total N losses in cropping systems by 48% and 27%, respectively2O emissions and productivity refer to forage or cereal systems or laboratory experiments and the efficacy of many NIs in vegetable production systems remains unknown. Such data is urgently needed to identify management strategies that maximise the efficient use of fertiliser N while minimising environmental impacts from intensive vegetable production.It has been shown that ammonia oxidation dominates N2O emissions, soil inorganic N and yield in a sub-tropical vegetable production system in Australia over 355 days using a high frequency automated greenhouse gas measurement system. The two main objectives of the study were: to investigate N2O emissions (including EFs) and vegetable yield from a typical vegetable rotation in response to different N fertiliser products; and to test the assumption that the use of NI will decrease N2O emissions.We conducted a field experiment to investigate the effect of two NIs on NOver the 1 year observation period 587 mm of rainfall was recorded at the study site. In addition the site received 645 mm of irrigation amounting to a total of 1231 mm of total water input. This rainfall was 25% lower that the long term annual precipitation (770 mm). The mean air temperature during the study period was 20.8 °C; maximum hourly air temperature (44.2 °C) was recorded in January 2013, while minimum hourly air temperature (1.6 °C) was recorded in July 2014 .2O fluxes occurred in response to fertilisation events and post-harvest after the incorporation of vegetable residues into the soil (2O emission peak was observed in early November 2013 after the first fertiliser side dressing (36 kg-N ha−1 Urea) with significantly elevated emissions in the CONV treatment. Overall highest emissions were observed after the incorporation of vegetable residues into the soil in December and January. The incorporation of the green bean residues on December 9, 2013 resulted in approx. 2 weeks of elevated N2O emissions reaching peak emissions of 30–50 g N2O-N ha−1day−1 in the different treatments with significantly lower emissions in the DMPP and 0 N treatment. The incorporation of the forage sorghum on January 17, 2014 resulted in an even higher N2O emission pulse reaching peak emissions of 40–80 g N2O-N ha−1day−1 in the different treatments with significantly elevated emissions in the DMPP and 3MP + TZ treatment  found in the present study were 0.08%, 0.21% and 0.13% for the for DMPP, 3MP + TZ and CONV treatments, respectively  Mg ha−1 and total lettuce yield from 41.0 to 68.0 (se 3.9) Mg ha−1 in the different fertiliser treatments  Mg haeatments . There w4+ and NO3− concentrations in the surface soils varied in response to fertilisation events and there was a significant effect of the NI treatments  have been reported6142O emissions can mainly be attributed to the lower fertiliser N application rates used in our study. Our system received a fertiliser rate of 310 kg-N ha−1, while many intensively managed vegetable systems are characterised by extremely high N inputs, often exceeding 1000 kg-N ha−1 yr−1 6142O losses from denitrification in vegetable systemset al.2O-N ha−1 yr−1) from a vegetable rotation at the same experimental site as in this study using a combination of manure and mineral fertiliser (up to 500 kg N ha−1 yr−1 applied). This shows that the alluvial soils in this vegetable production region have only a limited N2O emission potential, which is further supported by comparably low N2O emissions reported from experimental sites with differing soil texture in the same region52O emission potential from different vegetable systems are not entirely clear. Environmental conditions and management in our study are typically attributed to high N2O emissions: high rainfall/irrigation and soil temperatures in the sub-tropical climate, high N fertiliser rates (310 kg N ha−1 yr−1), incorporation of vegetable residues and frequent tillage. Therefore, we presume that the comparable low N2O losses observed are mainly related to the soil characteristics at our study site. Soil pH is known to be a principal control of soil denitrification and a positive relationship between soil pH and the N2/N2O product ratio has often been reported2/N2O product ratio and the low SOC content (1.5%) limited denitrifier activity, thus explaining the comparably low N2O emissions in this sub-tropical vegetable production system.This is the first study to report on the effect of NIs on Net al.et al.2O emissions in vegetable production systems and demonstrates that regional EFs are better suited to reliably estimate N2O emissions from vegetable crops. It is also noteworthy that the EF presented in this study accounts for direct fertiliser induced emissions only and does not consider emissions stemming from N returned to the soil with the vegetable residues. The IPCC methodology assumes that emissions from unfertilised soils are the equivalent of background emissions; however, it has been acknowledged that emissions from unfertilised soils may be greater than “natural emissions” due to mineral N released from the mineralisation of plant residues and/or soil organic matter2O emission inventories of vegetable cropping systems.Annual emission factors, corrected for background emissions, ranged from 0.08 to 0.21% of total fertiliser N applied for the different treatments. These are in line with the results of De Rosa, 2O fluxes showed only low emissions over the vegetable cropping phases, but significantly elevated emissions were observed post-harvest following vegetable residue incorporation into the soil. Overall 50 to 70% of the total emissions occurred during the post-harvest phase  or decreased (10%) cumulative CO2 fluxes in the DMPP and 3MP + TZ treatment, respectively. The effect of NIs on soil C mineralization, as quantified by CO2 emissions is still unclear and needs further investigations. While some studies reported a decreased CO2 release following the application of NIs2352 emissions after their application during the crop growth phase there is no indication of a direct effect of the NIs on soil microbial activity. However, NIs appeared to indirectly influence CO2 emissions by affecting the availability of N to soil microbes and therefore residue decomposition.Similar to N4+, and NO3− as N sources, but often cannot successfully compete with soil microorganisms for organic N and NH4+ 4+ can be immobilised by adsorption onto the cation exchange sites of clay soils and is also used by soil microorganisms as an energy source via nitrification. For that reason, NO3− is the major N source for most plants4+ immobilisation and reduced NO3− supply which could potentially constrain plant N uptake, particularly in these heavy clay soils. However, the overall effect of the NIs on soil NH4+ and NO3− levels and the efficacy in reducing N2O over the cropping season in this study indicate that the use of NIs combined with modified fertiliser application rates could be a feasible strategy to reduce N2O emissions whilst sustaining high yields. More research is required to optimise N rates and timing for different NIs products and how these can be used to reduce N losses over the post-harvest fallow phase.There was no significant effect of N fertilizer addition on the green bean yield, which can most likely be attributed to the fact that green beans are a legume and therefore capable of fixing nitrogen under N limited conditions. There was also no significant effect of the NIs on crop yield compared to the conventional treatment. This is in contrast to other studies where it has been shown that N fertilizers combined with NIs, such as DCD or DMPP, may improve the yield and quality of vegetable crops15292O emissions from irrigated vegetable systems on a black Vertisol in sub-tropical Australia are comparably low and that the proportion of fertiliser N lost as N2O from such a system is likely to be considerably lower than the IPCC default value. This demonstrates the need to use the local emission factors to reliably estimate N2O emissions from vegetable cropping systems in Australia. It also demonstrates that the use of NIs can lead to elevated N2O emissions by storing N in the soil profile that is available to soil microbes during the decomposition of the vegetable residues over the post-harvest phase. Hence the use of NIs in vegetable systems has to be treated carefully and fertilizer rates need to be adjusted to avoid excess soil nitrogen during the postharvest phase. Further research is required to evaluate if the use of NIs in vegetable cropping systems allows for a reduction of the N fertiliser rate. Such a reduction would be necessary to compensate for the increased costs of NI fertiliser and could potentially avoid elevated N2O emissions post-harvest.This study highlights that NThe study was conducted from September 2013 to September 2014 an the Gatton Research Facility, located in the Lockyer Valley approximately 80 km west of Brisbane, Queensland, Australia . The Lockyer Valley is a major vegetable producing region in south-east Queensland, characterized by a humid subtropical climate with a humid summer and mild to cool winters. The mean daily minimum and maximum temperatures are 18.7 and 31.2 °C in the summer, and 6.8 and 21.4 °C in winter. Summer rainfall accounts for nearly 60% of the long term average annual precipitation of 770 mm . The soil was formed in an alluvial fan of basalt rock and is classified as a Black VertisolPhaseolus vulgaris), broccoli (Brassica oleracea) and lettuce (Lactuca sativa). Forage sorghum (Sorghum bicolor) was used as a catch crop to reduce N losses during the fallow period between the green bean and broccoli crop. A time line of the experiment is shown in A field experiment was conducted over a typical south-east Queensland vegetable crop rotation comprising a succession of three vegetable crops plus a catch crop; green beans  were planted from seed, with 2 rows and 267 plants per plot. Rows were 75 cm apart and plants 7.5 cm apart in the row; Broccoli (cv. Aurora) were transplanted using container grown seedlings, with 2 rows and 67 plants per plot. Rows were 55 cm apart and plants 30 cm apart in the row; Lettuce (cv. Icehouse 2) were transplanted from container grown seedlings, with 3 rows and 86 plants per plot. Rows were 35 cm apart and plants 35 cm apart in the row. A 1 m wide buffer zone was included between plots.The experiment was conducted using four fertiliser treatments with three replications arranged in a randomized complete block design. Each experimental plot was 1.5 m wide × 10 m in length. Fertilized plots received a total of 310 kg-N haThe plots were irrigated with bore water using a hand-shift sprinkler irrigation system. Irrigation was applied during times with low wind speeds to assure uniformity of application. Irrigation amounts were measured using a rain gauge installed at the centre of the experimental plot and were scheduled on a weekly basis, except when sufficient rain had fallen in the previous week.The fertilizer treatments were:(i) ZERO N – i.e. no added fertilizer.® and urea fertilizer application following standard grower practice.(ii) STANDARD GROWER PRACTICE (CONV) – i.e. Nitrophoska® to Nitrophoska and urea, basal and side dressing fertilizer, respectively.(iii) DMPP – addition of the nitrification inhibitor 3,4-dimethyl-pyrazole phosphate (DMPP), trade name ENTEC® to Nitrophoska and urea, basal and side dressing fertilizer, respectively.(iv) 3MP + TZ – addition of the nitrification inhibitor combination of 3-methylpyrazole (3-MP) and 1,2,4-triazole (TZ), trade name PIADINEach of the three vegetable crops was harvested when commercially mature. Multiple harvests were conducted for broccoli and lettuce and a once over harvest was conducted for green beans, as per commercial practice in the Lockyer Valley. Maturity was determined using colour, shape, size and defects descriptions listed in commercially available product specifications. The harvest date was determined by regular field inspections in the 10 days leading up to the expected harvest date. Actual harvest dates are listed in 2O fluxes were measured from every plot over one year using a mobile fully automated measuring system similar to the one described in Scheer, et al.Nin situ gas chromatograph (SRI GC 8610 C) equipped with a 63Ni electron capture detector (ECD) for N2O concentration analysis. Sample gas measurements were calibrated automatically by a single-point calibration using a certified gas standard of 0.5 ppm N2O. During the measuring season a multi-point calibration was performed using certified gas standards of 500, 980, 5030 ppb N2O  and the GC response over this range was determined to be linear. The detection limit of the system was ~1.0 μg N2O-N m–2 h–1 and sample dilution via leakage was considered negligible.Briefly, the system consisted of 12 acrylic static chambers (50 cm × 50 cm × 15 cm) equipped with pneumatically operated lids and fixed on stainless steel bases inserted 10 cm into the soil. The chambers were linked to a fully-automated system comprised of a computerised sampling unit and an 2O from the automated chambers were calculated from the estimated rate of increase in concentration over four time points measured during chamber closure as described in detail in Scheer, et al.r2) was used to quality check the flux measurements. Fluxes were discarded if the regression coefficient of determination (r2) was <0.80.Fluxes of NEmission factors of the N fertiliser applied to the soil were calculated using the following equation:2O-N); N2O-N is the total N2O over one year (kg N ha−1 year−1) for each treatment; total N applied is the amount of N fertiliser applied (kg N ha−1 year−1).where EF is the emission factor  was measured immediately before planting and weekly to fortnightly over the growing season and the fallow period. At each sampling date, eight samples were taken randomly from each replicate plot with a soil auger and combined to a bulk sample, resulting in three replicate samples per treatment. NO3− and NH4+ were extracted from the soil samples by adding 80 ml of 2 M KCl to 20 g of field-moist soil and shaking it for 1 h. Concentrations of NO3− and NH4+ in the extracts were measured calorimetrically using a discrete analyser .High resolution soil temperature (at a depth of 10 cm) and chamber air temperature was measured every minute in conjunction with the automatic sampling system using a PT100 probe . Soil moisture was measured from 0–10 cm continuously in one plot per treatment using a MP406 standing wave soil moisture probe  that was calibrated for the soil at the research site. Water-filled pore space (WFPS) was calculated using measured soil bulk density data (arithmetic means of four samples) and an assumed particle density of 2.65 g cmLinear mixed models were employed to estimate and compare trends in gas and soil nitrogen due to fertiliser regime. The models defined a linear response over time allowed to vary with treatment. The models included a cubic spline basis to allow for curvature about the linear response and to smooth through high frequency variation. A term allowing the cubic spline to depend on treatment was also included. Random effects defining a trend for each plot allowed for intra plot correlation between the repeated observations. Null hypothesis significance tests were assessed by Wald statistics. Estimated trends over time for each fertiliser regime were predicted and presented graphically with +/− twice the standard error of predictions used to approximate a 95% confidence interval.Cumulative gas emissions from each plot were approximated by integration of flux estimates over time using the trapezoidal rule. Effects of treatment on total emissions were assessed by two-way analysis of variance which estimated variability in total flux associated with experimental block and treatments. The null hypothesis significance test for treatment was conducted by F-ratio test.How to cite this article: Scheer, C. et al. Nitrification inhibitors can increase post-harvest nitrous oxide emissions in an intensive vegetable production system. Sci. Rep.7, 43677; doi: 10.1038/srep43677 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Yet, Ge and Sn are immiscible over about 98% of the composition range, which renders epilayers based on this material system inherently metastable. Here, we address the temperature stability of pseudomorphic Ge1−xSnx films grown by molecular beam epitaxy. Both the growth temperature dependence and the influence of post-growth annealing steps were investigated. In either case we observe that the decomposition of epilayers with Sn concentrations of around 10% sets in above ≈230 °C, the eutectic temperature of the Ge/Sn system. Time-resolved in-situ annealing experiments in a scanning electron microscope reveal the crucial role of liquid Sn precipitates in this phase separation process. Driven by a gradient of the chemical potential, the Sn droplets move on the surface along preferential crystallographic directions, thereby taking up Sn and Ge from the strained Ge1−xSnx layer. While Sn-uptake increases the volume of the melt, single-crystalline Ge becomes re-deposited by a liquid-phase epitaxial process at the trailing edge of the droplet. This process makes phase separation of metastable GeSn layers particularly efficient at rather low temperatures.The revival of interest in Ge Sn-containing group-IV alloys are attractive because their band gap can be engineered all the way from a semiconductor to a semimetal10, and, moreover, they are the only known group-IV semiconductors that can assume a direct band gap without additional strain- or defect engineering14. In this respect, a seminal breakthrough was achieved with the recent demonstration of lasing in strain-relaxed Ge1−xSnx epilayers with Sn concentrations around 10%16.In the last few years, interest in direct-gap group-IV heterostructures1−xSnx films with such high Sn concentrations has always been a concern. Sn has a lattice mismatch of 14.6% with respect to Ge concomitant with a miscibility gap over almost the entire composition range17. Moreover, at 13.2 °C pure Sn undergoes an allotropic phase transition from the diamond lattice of semi-metallic α-Sn to the tetragonal lattice of metallic β-Sn. Thus, a precondition for utilizing Ge1−xSnx alloys in meaningful device applications is the feasibility of metastable epitaxial growth with Sn concentrations far above the solid solubility limit of xsl ≈ 1%17.Despite this major accomplishment, the thermal stability of Ge1-ySny or Ge1−xSnx alloys. It has been demonstrated that far from thermal equilibrium metastable Ge1−xSnx epilayers can be grown with x ≫ xsl24. Still, for CMOS-compatible device integration the low eutectic temperature TEC = 231 °C of the binary Ge-Sn alloy, which is less than 1 °C lower than the melting point of Sn17, remains a problem.Over the last 30 years a wide range of growth parameters has been investigated aiming at the implementation of metastable epitaxial films and layer sequences containing Si29. In particular, Sn precipitation at the growth front of Ge1−xSnx films on Ge(001) was observed already at growth temperatures TG > 150 °C30. Also, the authors of ref.30 were the first to report trails behind the Sn precipitates as evidence for their movement over the surface. Moving Sn precipitates have meanwhile been confirmed by several groups on material either grown by molecular beam epitaxy (MBE)33 or by the CVD  process34 that led to the first demonstration of lasing in this material system15.The temperature stability of substitutionally incorporated Sn in diamond-type host lattices has been investigated by several groupsEC. Therefore, a comprehensive understanding of the underlying physics is required to devise measures that can suppress this type of phase separation at comparably low temperatures in device applications.Still, little is known about the mechanisms that propel Sn precipitates while simultaneously, as we will demonstrate in this contribution, inducing the phase separation of the underlying GeSn films into its constituents Ge and Sn. Such a mechanism can jeopardize any application of the GeSn heterosystem that requires processing and/or operation temperatures above T1−xSnx films grown by MBE on Ge(001). Commercial Ge(001) substrates were employed to rule out any influence of the high threading dislocation densities and local strain variations35 typically associated with Ge-buffered Si substrates, alias virtual substrates36. Details of layer growth and the subsequent characterization by X-ray diffraction (XRD), Rutherford back-scattering (RBS), scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDXS) are described in the section on experimental methods. The growth parameters of the four sample series A–D used in this study are listed in Table For this purpose, we conducted systematic growth and annealing experiments on uncapped Ge1−xSnx films were grown with increasing compositions x either at TG = 120 °C with a deposition rate of r = 0.015 nm/s, or at TG = 200 °C and r = 0.1 nm/s  side of the substrate peak 1−xSnx film at its leading edge into liquid Sn and a corrugated trail region. The volume of the Sn precipitates increases gradually as a function of time until the droplets come to a halt as they run into trail regions of other droplets. This can be seen in the sequence of still images in Fig. A = 350 °C, demonstrates the remarkable efficiency of the underlying transformation mechanism at a temperature that is still moderate in comparison with typical processing temperatures in standard Si technology.The video sequences clearly show that each Sn droplet defines the very location of a transformation process that converts the intact GeFrom the video sequences, we estimated the velocity of the Sn droplets, and thus the speed of the transformation front. We found that the velocity increases with droplet size  through a large Sn droplet along its trail and the projected trajectory. A pair of electron transparent windows was then prepared in regions ahead and behind the Sn droplet , a technique in which a precursor species is dissolved in a supersaturated melt from where it precipitates epitaxially when brought in contact with a crystalline substrate47.46.In analogy to conventional LPE, epitaxial Ge growth at the trailing edge of each droplet is described by a negative difference of the Gibbs free energies of melt and crystalline Ge, 48:48. Also, The feeding part of the LPE process occurs at the leading edge of each Sn droplet, where it is in contact with the strained GeSn film. This contact region is described by 1−xSnx layer. However, producing a Sn melt supersaturated with Ge costs energy. This we express by Feeding and growth are coupled in the droplet by entclass1pt{minimaA more detailed discussion of Equation  can be f51, but not for our case of far-from-equilibrium MBE-growth. It is, however, clear from the experimental observations that the system gains free energy when Ge and Sn from the metastable GeSn film become dissolved in the Sn melt, and simultaneously almost pure (x ≤ 1%)17 Ge is deposited epitaxially at the opposite side of the melt.The contributions entclass1pt{minimaquations  are only17  Growth by CVD under kinetic conditions very far from equilibrium has been shown to suppress Sn precipitation during growth up to temperatures of at least 390 °C15. This extension of the metastable range to temperatures substantially above TEC is most likely based on two beneficial properties of CVD. For one, hydrogen termination of the surface during CVD is expected to efficiently suppress surface segregation of Sn, and thus the initial source of molten Sn during growth. Secondly, suppressed Sn segregation and far-from-equilibrium growth conditions allow for higher growth temperatures, thus reducing the density of point defects that may play a role for solid-state Sn diffusion in low-temperature MBE material. Still, the results in ref.34 gained from such CVD materials suggest that Sn precipitation can be shifted to higher processing temperatures, but not totally suppressed.Efficient phase separation at temperatures as low as 230 °C imposes severe limitation to potential applications based on metastable GeSn films. It is therefore a necessary precondition for any conceivable application to suppress Sn precipitation or at least to shift it to higher processing temperatures. Several such measures appear feasible: (i) Capping of the GeSn epilayers affects Sn diffusion in the solid phase and thus delays the formation of sufficiently large Sn precipitates that are required to initiate the transformation process. (ii) Rapid thermal annealing, which is routinely applied in high-temperature CMOS processes, limits the amount of segregated Sn0.9Sn0.1 films grown by MBE on Ge(001) substrates. Above the eutectic temperature of 231 °C, we find an efficient phase separation mechanism based on molten Sn precipitates that move in a self-propelled manner over the surface. The free-running Sn droplets induce phase separation by taking up Sn and Ge from the pristine GeSn film at their leading edge, and precipitating crystalline Ge at their trailing edge. This behavior is attributed to a liquid-phase epitaxial process that is driven by the free-energy difference between the GeSn and the Ge regions which are both in contact with the molten Sn droplet during movement.In summary, we investigated the thermal stability of uncapped Ge52. The samples are heated radiatively with temperature control calibrated to within ±25 °C. Polished Ge (001) substrates with a diameter of 100 mm and a specified resistivity of 8 Ωcm were purchased from Umicore and subsequently diced into 9.5 × 9.5 mm² pieces to fit into solder-free adapters milled from high-purity Si ingots53. Commercial Ge(001) substrates were employed to rule out any influence of the high threading dislocation densities and local strain variations typically associated with virtual  substrates35. The Ge substrate pieces were chemically pre-cleaned54 immediately before being introduced into the load-lock chamber of the MBE system. Before growth, the Ge-substrates were degassed for 30 min at 300 °C and then heated for 15 min to 750 °C for oxide desorption. Growth always commenced with a 50 nm thick Ge buffer layer deposited at 400 °C, which results in smooth surfaces with double-atomic height steps only53. The substrate was then ramped down to the respective growth temperature TG of the Gex1−Snx epilayer  of GeSn superlattices in an analogous way as described in ref.hed Ge 00 substratx1−Snx epilayers under systematic and well-controlled experimental conditions we grew four series (A–D) of un-capped Gex1−Snx films −7 mbar range that is attached to an AN-700 van de Graaf accelerator. The chamber is equipped with two semiconductor surface barrier (SSB) detectors, namely a LN2-cooled high-resolution detector55 featuring ≈3 keV full-width-at-half-maximum (FWHM) for protons and ≈7 keV for helium ions , and a standard SSB detector of larger solid angle . Energy spectra of the samples were recorded using 550 keV He+ ions. To avoid channeling effects and to optimize depth resolution, two angles of incidence of the ion beam (α = 0° and α = 60°) were chosen. The respective Sn contents were deduced from simulations of the experimental spectra employing the SIMNRA simulation software56.Since XRD can only provide the lattice constants of strained epitaxial films, we also performed RBS experiments on samples A1–A4 and A9 to assess the composition of the respective films. By comparison with the XRD experiments we then determined the relation between composition and lattice constant  detection of so-called SE1 secondary electrons which are predominantly generated by the incident electron beam. In addition, a conventional Everhart-Thornley (SE2) detector is available, which provides a higher degree of material contrast due to a higher sensitivity to SE2 and SE3 electrons that are generated by back-scattered electrons on the sample surface and at structural components of the SEM instrument, respectively57 were extracted from the AFM raw data with the free Gwyddion analysis software58.A Digital Instruments Veeco Dimension 3100 AFM was used in a non-contact tapping mode to assess both the surface roughness and height profiles of the trails left behind by Sn precipitates moving on the surface. Either MICRON or Olympus TESP cantilevers were employed for this purpose. Height images and surface angle plots3 80–300 at 300 kV, or in Linz with a JEOL JEM-2200 FS at 200 kV. Specimens were either prepared by conventional dimple grinding and subsequent argon sputtering in Karlsruhe, or in Linz with the focused ion beam (FIB) technique using a ZEISS 1540XB Cross-Beam system. The FEI TITAN3 is equipped with an image aberration corrector, which was used for high-resolution (HR)TEM investigations. Also, scanning TEM (STEM) experiments were performed with the FEI TITAN3 in combination with energy dispersive X-ray spectroscopy (EDXS) for composition mappings. The annealed samples were investigated with the JEM-2200 FS using both HRTEM imaging and STEM-EDXS.TEM was performed either at the Karlsruhe Institute of Technology , with a FEI TITANSupplementary MaterialsSupplementary Video Sequence V1Supplementary Video Sequence V2Supplementary Video Sequence V3Supplementary Video Sequence V4


Apart from the conventional negative magnetoresistance, we observe an unusual nonlocal response with negative field dependence up to room temperature, originating from the diffusion of valley polarization. Furthermore, a large magneto-optic Kerr effect generated by parallel electric and magnetic fields is detected. These new experimental approaches provide a quantitative analysis of the chiral anomaly phenomenon which was inaccessible previously. The ability to manipulate the valley polarization in topological semimetal at room temperature opens up a route towards understanding its fundamental properties and utilizing the chiral fermions.Chiral anomaly, a non-conservation of chiral charge pumped by the topological nontrivial gauge fields, has been predicted to exist in Weyl semimetals. However, until now, the experimental signature of this effect exclusively relies on the observation of negative longitudinal magnetoresistance at low temperatures. Here, we report the field-modulated chiral charge pumping process and valley diffusion in Cd et al. report magnetic field-modulated chiral charge pumping and valley diffusion in Cd3As2 up to room temperature.Magnetotransport signature of topological semimetal states has been observed but restricted at very low temperature. Here, Zhang  Conservation laws arising from preserved symmetries are the base of modern physics22Despite its crucial role in the description of elementary particles, the study of Weyl fermions in condensed matter physics has only been paid intensive attentions in recent years56429108In Weyl semimetals, electrons can be pumped from one node to the other due to the chiral anomaly. This chirality-dependent charge pumping rate can be characterized byχ is the pumped charge, e is the electron charge, E and B are the external electric and magnetic fields, respectively. Such a pumping process will be compensated by the depletion of valley charge, characterized by the inter-valley scattering time τv. Eventually, the Weyl nodes with opposite chirality χ will acquire a different chemical potential as schematically illustrated in k-space16where ρE and B14x1−Sbx system at the critical state between topological insulator and trivial band insulator3Bi and Cd3As2 have been theoretically predicted and experimentally confirmed as Dirac semimetals91020212224x1−Sbx, has been frequently observed in these materials at low temperatures2627282930One important signature of the chiral anomaly is the large negative MR with unusual anisotropy with respect to the angle between 3As2 based on three independent experimental evidences: the negative longitudinal MR, the valley transport, and the E·B-generated magneto-optical Kerr effect (MOKE). Surprisingly, in stark contrast to the strong temperature dependence of negative MR, the chiral pumping process is robust against thermal perturbation and persists up to room temperature as revealed by the following valley transport and MOKE experiments. It is noted that such chirality-polarized states are directly coupled with spin, orbit and valley degree of freedom6Here, we demonstrate the existence of the chiral anomaly in Cd3As2 nanoplates. These nanostructures were grown by chemical vapour deposition method  to ensure their semimetallic nature. 3As2 nanoplates at 2.5 K. As illustrated in the inset, B was rotated from in-plane to out-of-plane with θ defined as the angle between B and the applied current I. A large positive and linear MR along with a strong anisotropy respective to the field direction was obtained, similar to those observed in Cd3As2 bulk crystalsB approaches I, we find a clear dip emerging in the MR curve, which vanishes rapidly even if θ changes only a few degrees  and nonlocal resistance RNL (R34 and R56). As shown in R34 and R56 acquire a negative field dependence from 0 to 9 T, as shown in R34 and R56) is partially originated from valley diffusion, we plot the resistance ratio between two different channels (R56/R34 and R56–NL/R34–NL) as a function of B. The ratio of R56/R34 is close to 0.25 around zero field at different temperatures . Meanwhile, the observation of negative MR in both sides also excludes the influence of the current jetting effect  adopts a complete opposite dependence on B in comparison with the local resistance R12. After extracting the Ohmic contribution , as demonstrated in −2. The ROT–MOKE curves adopt a cosine-function dependence on the angle θ between the electric and magnetic fields. The absolute value of Kerr rotation reaches a maximum when the electric and magnetic fields are parallel (θ=0°), and in turn it becomes zero when they are perpendicular (θ=90o). In addition, we performed the ROT–MOKE experiments with the magnetic field in the x-z plane , the Weyl nodes will be in non-equilibrium configurations where Hall conductivity of doped Weyl nodes becomes complex due to the finite frequency electromagnetic response  deduced from the Drude model as an estimation of the intra-valley momentum relaxation rate (τintra). So we can in turn verify the initial assumption on the occurrence of Kerr effect (finite frequency electromagnetic response from the charge pumping process). Note that these two relaxation rates are calculated based on the data of the Cd3As2 nanoplates. In single crystals, the corresponding values are expected at least one order of magnitude larger owing to the much higher mobility11ω is 2.8 × 1015 rad s−1. ωτv≫1 ensures the independence of charge pumping in each Weyl node. And ωτintra≫1 indicates that the Weyl nodes are in fact not able to reach equilibrium within the light frequency. Thus a finite frequency electromagnetic response is anticipated with a complex Hall conductivity of gyrotropic tensor and the effective time-reversal symmetry breaking, leading to the emergence of the Kerr effectOne possible origin is the variable optical gyrotropic coefficient that is modulated by E·B fields. The chiral anomaly leads to the polarization of Weyl nodes. The charge transfer between a pair of Weyl nodes is accomplished through the surface Fermi arcs. As demonstrated by recent photoemission studies, the Fermi arcs are highly spin polarized46E·B field suggests its intimate relationship with the chiral anomaly. So far, this novel Kerr effect is still not fully understood and thus deserves further theoretical and experimental investigations.Another possible origin for the Kerr effect is the spin-polarization induced by the 3As2 and further demonstrates the field-modulated valley transport. We find that the nonlocal response can serve as an accurate way to measure the inter-valley relaxation rate. The ability to manipulate the chiral charge at room temperature is a significant step towards quantum electronics. The robustness of the chiral anomaly not only reflects the topological character of the Weyl nodes, but it also reveals the prospects of utilizing chiral fermions in the future low-dissipation valleytronic applications.In conclusion, our work presents several evidences for the chiral anomaly in Dirac semimetal Cd3As2 nanoplates were grown using Cd3As2 powders as the precursor in a horizontal tube furnace, in which argon was a carrier gas. Before the growth, the furnace was pumped and flushed with argon several times to remove water and oxygen. The temperature was ramped to the growth temperature within 15 min, held constantly for 20 min, and then was cooled down naturally over ∼2 h in a constant flow of argon before the substrates were removed at room-temperature. The precursor boat was placed in the hot center of the furnace (held at 720 °C), while the smooth quartz substrates were placed in the down-stream within a very small temperature range from 150 to 200 °C. The smooth quartz substrates then appeared shining to the naked eyes.The CdThe crystal structures of the synthesized products were characterized by X-ray diffraction, recorded on an X-ray diffractometer equipped with graphite monochromatized, Cu Kα radiation (λ=1.5418 Å). The morphological, structural, and chemical characteristics of the synthesized products were investigated by scanning electron microscopy  and transmission electron microscopy  equipped with energy-dispersive X-ray spectroscopy.3As2 Hall bar devices were fabricated by electron beam lithography technique and plasma-etched by argon for 2 min at the electrode regime before the deposition of Cr/Au (5 nm/150 nm) electrodes. The FIB process is achieved using a low current of 50 pA, 30 kV to avoid strong damage to the sample. The magnetotransport measurements of the devices were carried out in a Physical Property Measurement System (Quantum design) with constant DC current (μA range) through the devices.The Cd3As2 single crystals were synthesized by self-flux growth method in a tube furnace with stoichiometric amounts of high-purity Cd powder (4N) and As powder (5N). Mixed elements were sealed in an alumina crucible inside an iron crucible under argon atmosphere. The iron crucible was heated to 800–900° and kept for 24 h, then slowly cooled down to 450 °C at 6 °C per hour. Then the crucible was kept at 450 °C for more than one day before cooled naturally to room temperature. The superfluous Cd flux was removed by centrifuging in a vacuum quartz tube at 450 °C. To avoid possible surface contaminations and oxidation, the bulk crystal was measured within three days after exposed to the atmosphere. High-quality Cdy-z plane, which is parallel to the sample surface as illustrated in p-polarized with a wavelength of 670 nm. In the MOKE measurements, the Kerr rotation angle from the p-polarized light can be quantified as a function of field strength and orientations3As2 bulk flake with thickness around 100 μm. A constant DC electric bias was applied across the sample. The electrical bias applied on the sample was in the range of 0–500 mV, mainly dropping on the contact regime. The actual voltage drops on the sample was <5 mV with 115 mA current applied on the Cd3As2 bulk crystal. The Kerr signal was re-checked by switching the direction of the applied current.A two-dimensional vector rotate magnet was used to generate a rotating magnetic field in the The data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Zhang, C. et al. Room-temperature chiral charge pumping in Dirac semimetals. Nat. Commun.8, 13741 doi: 10.1038/ncomms13741 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-18, Supplementary Tables 1-2, Supplementary Notes 1-11 and Supplementary References


They induce ROS-regulated transcripts including for genes implicated in pathogen defense, biotic and abiotic stress. Mutant cryptochrome alleles that are non-functional in photomorphogenesis retain the capacity to induce ROS-responsive phenotypes. We conclude that nuclear biosynthesis of ROS by cryptochromes represents a new signaling paradigm that complements currently known mechanisms. This may lead to novel applications using blue light induced oxidative bursts to prime crop plants against the deleterious effects of environmental stresses and toxins.Cryptochromes are evolutionarily conserved blue light receptors with many roles throughout plant growth and development. They undergo conformational changes in response to light enabling interaction with multiple downstream signaling partners. Recently, it has been shown that cryptochromes also synthesize reactive oxygen species (ROS) in response to light, suggesting the possibility of an alternate signaling mechanism. Here we show by fluorescence imaging and microscopy that H They are localized in the nucleus and function both directly and indirectly as core regulators of more than 20% of total cellular transcripts6. Structurally, cryptochromes are flavoproteins consisting of a conserved N-terminal light sensing domain (designated the PHR domain) and a less well-conserved C-terminal domain (designated CCE) of variable length that is important for nuclear localization and signaling7. Upon illumination, Arabidopsis cryptochromes interact with protein partners including phytochromes (PhyA and PhyB), the WD-repeat protein SPA1, the E3 ubiquitin ligase COP1, and bHLH transcription factors CIB2, PIF3 and PIF49 which have a signaling role in photomorphogenesis or flowering initiation. Most of these partners attach to the PHR domain and release the CCE domain from the protein surface11. The ensuing suppression of COP1/SPA1-regulated degradation of transcription factors, such as HFR1 and HY5/HYH, results in photomorphogenesis and interaction with a large network of phytochrome, hormone, and stress signaling pathways15.Cryptochromes are blue light sensing receptors that regulate multiple processes of plant growth and development, including photomorphogenesis, de-etiolation, flowering initiation, stress response, and hormone signaling2O2) and superoxide (O2•−) subsequent to illumination17. This follows from reoxidation of the bound flavin cofactor from the light activated radical (FADH°) or reduced (FADH-) redox state back to the oxidized (FADox) resting dark-adapted state18. ROS are damaging byproducts of metabolism originating in the chloroplasts, peroxisomes or mitochondria. They are also induced by several environmental stresses and function as core regulators of cellular processes including stress tolerance, root growth, senescence, hormonal responses, pathogen defense, defense mechanisms against ROS and induction of ROS scavenging pathways22.Recently, it has been reported that plant cryptochromes release reactive oxygen species (ROS) including hydrogen peroxide  and cryptochrome overexpressing (Ws01) lines of the Wassilewskija (Ws) ecotype described previously28 were illuminated in the presence of the fluorescent probe Singlet Oxygen Sensor Green (SOSG) to detect singlet oxygen26, a byproduct of flavin reoxidation after illumination of cryptochromes  and overexpressing (WsO1) Arabidopsis seedlings showed approximately similar levels of ROS, although levels were somewhat reduced in cry1cry2 mutants  can be observed within minutes of blue light illumination in the nuclei of Sf21 insect cell cultures expressing recombinant mes Fig. 18. The a27; reviewed also in refs20. These indicate considerable overlap between light-regulated and ROS regulated gene expression. To verify the role of cryptochromes in a variety of ROS signaling pathways, we chose to analyse at random a number of genes reported to respond to ROS across several distinct functional categories implicated in economically important traits such as defense against pathogen attack, and other forms of biotic and abiotic stress. These include ROS induced ethylene responsive transcription factors 29 and WRKY33 the ROS regulated Zat zinc finger protein ZAT1030, Sigma Factor Binding Protein 1 (SIB1)29, Heat shock protein (HSPRO2)27, ROS detoxifying enzyme (APX2)30, oxidative stress responsive (ANACO47)31 and pathogen resistance gene FMO132. The expression of these genes was analysed in Arabidopsis wild type (Wt) and mutant  seedlings after a short (3 hour) illumination with saturating blue light to induce the cryptochrome response33  domain. Several such mutants had been previously described as inactive in hypocotyl growth inhibition and anthocyanin accumulation35, and as such can be concluded to be unable to undergo conformational change and partner protein interactions necessary for photomorphogenesis. To confirm that mutation of the C-terminal domain does not also affect light sensitivity or the formation of reactive oxygen (ROS) by the PHR domain of cryptochrome, we analysed protein from a mutant allele E531K34 which has a point mutation in the CCE domain outside of the region required for nuclear localization36. The recombinant E531K mutant protein was expressed and purified from a baculovirus expression system by established methods33, and compared to wild type AtCry1 for in vitro light sensitivity and flavin photoreduction. The mutant protein was comparable to the wild type AtCry1 both in the ability to undergo flavin photoreduction and the time course of reoxidation  from the radical (FADH°) state back to the oxidized (FAD) inactive redox state , which also shows no detectable protein expression28. We first analysed these mutant alleles for hypocotyl growth inhibition at a range of blue light intensities  which were located outside of any known functional region for signaling allele hy-3, which38. However, anthocyanin accumulation also occurs more specifically in response to multiple forms of stress, and we therefore investigated whether C-terminal mutants of Cry1 which continue to synthesize ROS may also retain some capacity to regulate this response , consistent with previous reports35. However, at higher blue light intensity (90 μmol m−2 s−1), anthocyanin accumulation in the C-terminal mutant alleles R611K, E623K and E652K was significantly enhanced as compared to both of the null mutant alleles 39. Since cryptochromes bind to COP1 independently of light, they would be in permanent close proximity to the G-box binding family of transcription factors, which bind to COP1 through the intermediary of HY5/HLH transcription factors40. Therefore, a pulse of blue light illumination would provide a localized burst of H202 sufficient to induce a transcriptional response from GBF-regulated promoters. Along these lines, biosynthesis of ROS by cryptochromes may  contribute to its signaling role simply by increasing the local concentration of ROS near transcription factors to which cryptochromes have not been reported to bind directly, but which are assembled into the same complexes. Recent studies indicating a multiplicity of signaling molecules that are regulated directly by oxidation/reduction at cysteine residues greatly adds to the potential signaling substrates of cryptochromes41.One likely substrate, would be the family of G-Box binding transcription factors including GBF1 and MYC2, which are known to bind promoter elements related to high light stress signaling30. Given this process of redox homeostasis, induction of ROS through cryptochrome activation would not be expected to change intracellular levels of ROS under steady state, long-term growth conditions. Furthermore, under subsaturating light conditions, the efficacy of ROS formation as a signaling mechanism is doubtful as the quantum yield of the receptor is relatively low33, so that a mechanism of conformational change, wherein the activated form of cryptochrome is stable over a period of at least several minutes38 is the optimal signaling mechanism. As a result direct induction of ROS signaling pathways through cryptochrome is likely to occur primarily under short-term illumination at high light intensities, both to ensure saturating light intensities for cryptochrome-dependent ROS biosynthesis and to evade eventual cellular ROS scavenging and downregulation mechanisms. As a validation of this prediction, we observe that under the short term, high intensity illumination conditions used in this study we have observed robust cryptochrome-dependent induction of the ROS regulated FMO1 gene  and cry2 mutant reference alleles28 lacked detectable protein. Other mutant alleles were either as previously described35 or screened and sequenced from genetic screens for blue light insensitive mutants as in ref.35. All seedling growth was on agar petri plates at 1/2xMS salts, 2%sucrose, pH 6.0, 0.8% agar. Seeds were sterilized as in ref.28, plated, retained at 4 °C for two days, then allowed to germinate for 24 hours in red light before being returned to darkness or the test light condition. Hypocotyl growth and anthocyanin content was measured as previously described35.All Arabidopsis seedlings were of ecotype WS. Cry1 (−2 s−1) for 10 minutes and observed immediately with a Zeiss AxioImager.Z1/ApoTome microscope using a 20X objective, excitation/emission 350 nm/460–490 for HOECHST and 372/395–416 for SOSG reagent. Z- series were performed with a z-step of 1.5 µm. For quantification, the images were captured using a CCD-camera. Single z section were analysed for colocalization on ImageJ software . Briefly, colocalized pixels were obtained by using colocalization color map plugging. Red structures on Hoechst images have been segmented to define each nucleus as a region of interest (ROI), then by using ROI manager tool, integrated density of cyan and red signal have been quantified for each ROI and exported to LibreOffice calculator.10 plantlets (8–10 days old etiolated seedlings) were used per experiment and incubated in 2 ml of 200 mM potassium phosphate buffer (pH 7.4) comprising 2 µL HOECHST reagent , 2 µL DMSO  and 4 µL of SOSG  for 30 minutes in the dark at room temperature. Plantlets were then rinsed 4 times in potassium phosphate buffer (200 mM), then exposed either to dark or to saturating blue light , CRY1 null mutant, CRY2 mutant, CRY1-CRY2 double mutant and C-terminal mutants of Arg611Lys, Glu623Lys and Glu652Lys were grown and germinated as described above. Four-day-old dark-grown seedlings were transferred to blue light (60 μmol m−2 s−1) for 3 h or kept in dark. Seedlings were then collected and ground with liquid nitrogen. Total RNA was isolated using RNeasy Plant kit (Qiagen), and the first-strand cDNA synthesis was done using a Reverse Transcription Kit (Qiagen) following the manufacturer’s instructions. qRT-PCR was performed using a SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions. All qRT-PCR reactions were amplified as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, 57 °C for 30 s, and 72 °C for 30 s. The expression levels were determined and normalized to the UBIQUITIN (UBQ1) expression using the comparative 2−∆∆Ct method. Primers of the 12 ROS genes used for gene expression analysis are listed in Supplementary Table The seedlings of 16. Briefly, the mutant allele E531K of cry1 was cloned into the pBakPak9 baculovirus expression vector including the presence of a 6 amino acid HIS tag at the N-terminus. The vector was transfected into Sf21 insect cell cultures and protein expression carried out according to the manufacturer’s instructions. Recombinant protein was isolated over a nickel affinity column as described48. Concentration of protein used in the experiments was between 30–50 micromolar. Photoreduction experiments were peformed in a buffer of 50 mM NaPO4 pH 7.5, 5 mM DTT at18 °C. Subsequent to illumination, spectra were taken in a Cary Scan 300 UV/Vis spectrophotometer. For detection of ROS, samples of wild type Cry1 and E531K mutant proteins at the identical concentration were illuminated in a buffer of 50 mM NaPO4 pH 7.5 maintained on ice. Ilumination was at 1000 μmol m−2 s−1 in the absence of reducing agents. Aliqots were taken at the indicated times .All methods were as previously describedSupplementary Information