The ability to fabricate nanoscale domains of uniform size in two-dimensional materials could potentially enable new applications in nanoelectronics and the development of innovative metamaterials. However, achieving even minimal control over the growth of two-dimensional lateral heterostructures at such extreme dimensions has proven exceptionally challenging. Here we show the spontaneous formation of ordered arrays of graphene nano-domains (dots), epitaxially embedded in a two-dimensional boron–carbon–nitrogen alloy. These dots exhibit a strikingly uniform size of 1.6 ± 0.2 nm and strong ordering, and the array periodicity can be tuned by adjusting the growth conditions. We explain this behaviour with a model incorporating dot-boundary energy, a moiré-modulated substrate interaction and a long-range repulsion between dots. This new two-dimensional material, which theory predicts to be an ordered composite of uniform-size semiconducting graphene quantum dots laterally integrated within a larger-bandgap matrix, holds promise for novel electronic and optoelectronic properties, with a variety of potential device applications. The nanoscale patterning of two-dimensional materials offers the possibility of novel optoelectronic properties; however, it remains challenging. Here, Camilli et al. show the self-assembly of large arrays of highly-uniform graphene dots imbedded in a BCN matrix, enabling novel devices. However, there has been only limited success in achieving uniform size or extremely small dimensions.Structures approaching the nanometre scale hold great interest, both for basic science and for potential applications in advanced technology. However, controlled fabrication at this scale represents an extreme challenge. Many applications such as quantum dots and metamaterials, moreover, require nanoscale heterostructures integrated within a solid matrix. Intriguing progress has been made using misfit stress to drive self-assembly of nanostructures on surfaces during heteroepitaxy3 opens up new opportunities and challenges. Most notably, confining lower dimensional domains within a 2D heterostructure could potentially lead to exciting and useful quantum effects. In particular, 2D lateral heterostructures formed by graphene and hexagonal boron nitride have recently been the subject of intense research10 owing to the similar lattice parameters but complementary electronic properties of their individual components. Heterostructures of different 2D semiconducting transition metal dichalcogenides are also of great technological interest12. However, a route towards fabrication of 2D lateral heterostructures at extreme nanoscale dimensions is still unavailable.The advent of two-dimensional (2D) materials14 between the dots. Furthermore, theory predicts graphene nanodots of the observed size to be semiconducting quantum dots16 and the BCN matrix to possess a larger bandgap17.Here we report the self-assembly and self-ordering of 0D graphene dots of highly regular size, 1.6 ± 0.2 nm in diameter, epitaxially embedded within a 2D boron-carbon-nitrogen (BCN) alloy. Using a combination of in situ microscopy, diffraction and spectroscopy techniques, we characterise the monolayer BCN grown on an Ir(111) substrate under different growth conditions. With an increasing C concentration, graphene dots appear in the BCN layer and self-organise into ordered hexagonal arrays. The arrays become denser when we increase the C to BN ratio during the growth process. We show that the observed uniform dot size can be explained by a model describing the competition between the graphene–BCN boundary energy and moiré-type interactions with the substrate, with the long-range organisation provided by the repulsion3N3H6) and 13.5 L of ethylene (C2H4) on a clean Ir(111) surface at 1250 K. The surface is covered by a patchwork consisting of interconnected triangular lines embedded in an apparently featureless background. These lines appear slightly raised relative to the background in the STM image. Sporadically, also small bright-contrast dots are found at the intersections of such lines.Figure 18, resulting from the exposure of a submonolayer of graphene on a Ru(0001) substrate at 900 K to a low dose of borazine. Notably, the disordered background was identified as a BCN alloy, while the lines were interpreted as segregated graphene ribbons18. A similar domain with disordered BCN alloy coexisting with bright-contrast faint lines was previously observed by Sutter et al.4, while synthesizing graphene-hBN planar heterostructures on Ru. As we increase the C fraction by co-dosing 22.5 L of borazine and 22.5 L of ethylene, a new arrangement arises within the BCN layer  or the dot is displaced (white arrow) from these intersection points. This suggests that the nanodot array is not a simple consequence of the brick-and-mortar pattern. Moreover, the graphene nanodots appear in the STM images with a different contrast than the mortar lines, suggesting a difference between the two structures. Indeed, in Fig. Figure 9. (B–C bonding motifs are discussed further below.)The non-uniform chemical composition of the mortar lines is also visible in Fig. 16.Figure white arrows. Additionally, Fig. white dashed lines indicating the termination of a row and inward relaxation of the neighbouring ones.Furthermore, the self-assembled arrays behave like a 2D superlattice, showing characteristic defects such as vacancies and dislocations. For instance, one or more missing dots  are highlighted in the STM images in Fig. 20. Figure Despite these defects, the graphene nanodot array has surprisingly strong hexagonal ordering, as seen in the sixfold-symmetric pattern in the Fourier Transform Fig.  of the S5.In situ low-energy electron microscopy (LEEM) experiments were performed in order to gain deeper insight into the growth dynamics of the system. Previous lines of work have already demonstrated the importance of such studies in investigating the growth of graphene–hBN heterostructures on Ru21. The lateral resolution of LEEM is not sufficient to distinguish between different nanoscale phases in the BCN area, but complementary information can be gained by micro-spot low-energy electron diffraction (µ-LEED) analysis, as shown in Fig. yellow arrow. Additional weaker diffraction features appear with a lattice constant slightly smaller than the Ir(111): six broad spots with a periodicity in real space of 2.57 Å (marked by the green arrow), and six faint, elongated spots at 2.44 Å (marked by the red arrow). The 2.44 Å features can be reasonably ascribed to the submicrometre graphene islands observed as bright-contrast areas in the LEEM image in Fig. 19. The fact that the diffraction spots exhibit an arc-like morphology implies that the islands have slightly different orientations22. We attribute the 2.57 Å spots to the BCN alloy, since they are larger than graphene and h-BN on Ir .Figure 19, and most likely originates from the bright-contrast graphene islands observed in the LEEM picture in Fig. Interestingly, close to the 00 diffraction spot six bright points with sixfold symmetry appear, followed by six elongated features. The distance of the six points from the 00 diffraction spot indicate a real space periodicity of 3.7 nm. This value is larger than both graphene/Ir and hBN/Ir moiré patterns, which are ~2.5 and 3 nm, respectively. We assign these spots to the periodicity of the graphene nanodot array. Indeed, this value lies within the range measured in the STM experiments for the sample with short periodicity (3.5 ± 0.7 nm), as displayed for instance in Fig. Figure s) and C(1s) core levels of four samples with varying ratios of partial pressures of borazine to ethylene of 70/30, 50/50, 30/70, plus either pure graphene (used as reference for C(1s)) or pure hBN (used as reference for B(1s)). The samples were grown with the same partial pressure ratios as the samples displayed in Fig. s) spectrum for hBN shows two peaks B0 (at 190.60 eV) and B1 (at 189.80 eV), which in agreement with previous studies can be both attributed to B–N bonds23, with the higher binding energy (BE) peak representing B atoms closer to the Ir substrate24. For all other samples the intensity shifts to lower binding energies, fit with peaks B2 (at 188.7 eV) and B3 (at 187.7 eV) in Fig. 25. On the basis of these previous reports and considering the electronegativity of the three elements at play , we attribute B2 to B atoms bound to at least one N atom , and B3 to B atoms bound to C or B, but not to N . The C1s spectra 27. The peak shifts to lower binding energies but keeps the same shape in all of the alloyed samples. We attribute this shift to the chemical heterogeneity of the layer and not to a change in the interaction of the graphene with the underlying Ir substrate . The C4 component we ascribe to carbon with two neighbouring B atoms28 and the C3 component to carbon with only one neighbouring B atom28. In all alloyed samples a higher BE shoulder, C2, appears at 284.7 eV. This peak may represent sp3 defects in graphene30 and/or C–N bonding motifs32, and it was not possible to distinguish between the two possibilities.In situ X-ray photoelectron spectroscopy (XPS) using synchrotron radiation was used to give insight into the average chemical composition of the BCN layer. Figure tra Fig.  are domi1 peak and the reduction and disappearance of the components associated with hBN (namely B0 and B1) in the 50/50 and 30/70 samples rule out the possibility that the nanodots can be hBN regions. In combination with the fact that, in the STM images, the dots appear remarkably different from the disordered alloy in the background, in terms of apparent height and lattice order, this confirms the hypothesis that the nanodots are indeed made of graphene.The survival of the intact C1 peak originates from C in an alloyed material, and that the most stable BCN systems have B:N ratio of 136, we propose a stoichiometry of approximately BC2.5N for the alloy matrix when the gas ratio during growth is 50/50. An alloy with such a low percentage of C is expected to be insulating, and thermodynamically stable structures show band gaps in excess of 1 eV in theoretical calculations36. A similar calculation for the alloy produced when a borazine-rich gas ratio was used during growth (70/30) gives a stoichiometry of BCN for the alloy. In all alloyed samples, we see some excess of B over N. This probably reflects primarily B atoms dissolved in Ir37. In addition, the detailed fitting shown in Fig. 9.The average element distribution across the sample can be estimated from XPS data  to borazine vapour. This exposure initially led to formation of the brick-and-mortar pattern, followed by a patchwork of graphene and hBN domains and eventually complete replacement of graphene by hBN.The behaviour observed here is strikingly different from that reported in previous studies, where a layer of one pure material (graphene or hBN) was exposed at high temperature to precursors of the other material. Such exposure leads to the formation of an alloy lattice via progressive substitution of atoms, and eventually to complete conversion of graphene to hBN, or vice versa18 using Ir instead of Ru as the growth substrate. In this case, a mixing of C with B and N is not observed. Instead, on Ir we obtain interconnected pure phase graphene–hBN heterostructures with sharp interfaces . Although it was not reported in ref. 18, their images of the intermediate stages of transformation show features that might be graphene dots. On the other hand, the periodicity of those features could also be attributed to the moiré of graphene/Ru(0001). If further investigations should prove that dots can form on Ru, it would support the generality of the behaviour that we observe.It is expected that both the choice of metal and the growth protocol can affect the structure. To clarify the role of the metal, we have repeated the experiment of ref. Here, in contrast to previous work, which used sequential dosing, we simultaneously dose ethylene and borazine on an Ir(111) surface. As discussed above, only with such co-dosing do we observe graphene nanodots. While the mechanism is not clear, it seems reasonable that co-dosing could facilitate the self-organisation into discrete graphene dots within the BCN host phase. Sequential dosing requires nucleating graphene regions in pre-existing hBN, and, if the nucleation barrier is large, that could overwhelm any thermodynamic driving forces for self-organisation.14 (or electrostatic interactions between domains of different work functions14). In that case, if the fractional area of each phase is fixed, the equilibrium configuration can be a hexagonal lattice of compact regions (dots) of the minority phase. The size of the dots is then determined by a competition between interfacial line energy (favouring large dots) and stress-relaxation energy . However, we note that the graphene dots here have roughly the same size as the features in the moiré pattern of graphene on Ir(111). This suggests that the substrate interaction might play a dominant role in determining the size of the dots. Otherwise, the dot size is expected to vary exponentially with boundary energy14; therefore, it would be an improbable coincidence if that physics led to dots of similar size to the graphene moiré features.What is most remarkable is the degree of order observed, both in the dot-size uniformity and the spatial distribution. This surprising behaviour requires some discussion. At first glance, the dot formation and ordering seem strikingly similar to that predicted to arise driven by elastic relaxation in 2D domains of different stressR is thenλ is the energy per length of the graphene–BCN boundary, U is the graphene–substrate interaction, r is a 2D vector and the integral is taken over the dot. The interaction U varies laterally because of the varying alignment between graphene and substrate atoms, as reflected in the moiré pattern.  The simplest possible form for the interaction isU0 is the average interaction, that is the interaction energy per unit area for a large graphene domain; α is the amplitude of the variation; and the three g vectors of magnitude 2π/Lm, 120° apart, describe the hexagonal moiré pattern, which has period Lm = 2.5 nm for graphene on Ir. This interaction energy is minimised at a hexagonal lattice of points, and has approximate rotational symmetry up to distances of ~0.4 Lm  from each minimum.To gain qualitative insight into the dot formation, we consider what dot size will minimise the energy, for a fixed area of graphene embedded in BCN. For now we neglect elastic energy; the effect of this and other approximations is discussed below. The energy of a circular dot of radius Edot/(πR2). It is convenient to focus on the dot energy/area, relative to a large graphene domain, that is [Edot/(πR2)−U0], which is independent of U0. This is plotted in Fig. α, for different values of the boundary energy λ . We see that there are three distinct regimes. For sufficient small boundary energy, there is an energy minimum at dot sizes corresponding to a small fraction of the moiré period , with energy lower than a uniform graphene sheet. Thus, dots are the stable phase in this case. For larger boundary energy, there is still a minimum but at positive energy values; therefore, dots are metastable. They could easily form if the carbon mobility is limited. For still larger values of boundary energy, there is no minimum at any finite R, and dot formation cannot occur within our model. (It is worth noting that Eqs. (λ that dots would be unstable.)Assuming a fixed total area of graphene, we want to minimise the dot energy per area, hat Eqs.  and 2 coR = 0.8 nm = 0.32Lm, corresponds to the range where dots are robustly metastable; therefore, it is worth discussing this regime in more detail. Such dots are stable against coarsening—if they form with a range of diameters, any diffusion will actually drive the distribution to become more uniform. This is true as long as the distribution is confined to a limited range of R, as in our experiments. However, for a sufficiently large graphene domain, the energy/area drops towards zero. Therefore, within diffusion range of such large domains, the dots will shrink and disappear as material diffuses to the larger domains. Thus, metastable dot arrays are expected to form under conditions where, kinetically, there is no opportunity for the formation of very large domains nearby.The experimentally observed dot size, 14. These interactions also affect the dot self-energy. However, if they are not too strong, they can be thought of as mainly acting to renormalise λ, with only a weak dependence of the effective λ value on R14. The fact that the dot array is always oriented along the substrate lattice directions, as shown in the LEED images in Fig. α term may be interpreted as a net benefit from making the graphene optimally aligned. Also, high-resolution STM images suggest that the actual dot shape is not circular, but rather irregular with considerable hexagonal character. This does not change the qualitative picture above, but the coefficient of the edge energy will depend on the actual shape.Of course, this simple model neglects many factors that must also play a role. In particular, the organisation of the dots into a hexagonal array is consistent with the long-range repulsion expected to arise from elastic and/or electrostatic interactions16 epitaxially embedded in a larger-bandgap BCN matrix17. Nevertheless, the strong coupling with the underlying Ir substrate  to remove carbon contaminants. The clean Ir surface was exposed to a gas flux of ethylene and borazine, following standard background dosing technique, for 15 min at 1250 K. In all cases, aside from the LEEM experiments detailed below, the gases were not premixed prior to their introduction into the chamber and were therefore introduced one at a time. The ethylene/borazine ratio was controlled by adjusting the partial pressures of the individual gases to give a higher or lower ratio as required. We do not quote the absolute pressures used as variations between the chamber geometries and pressure gauge calibrations do not allow for precise determinations but typically the samples were exposed to a total background gas pressure of between 0.5 and 1 × 10−7 mbar.Data were collected in different ultrahigh vacuum chambers but following the same recipe for sample preparation in each. An Ir(111) crystal was cleaned by several sputter/anneal cycles using 2.5 keV Ne−10 mbar. Pt–Ir tips were used. STM images were processed using WSxM40 and Gwyddion41 software. The diameter of the graphene dots was estimated from the STM images, by assuming that the bright rim . The beamline is equipped with a SX-700 monochromator, allowing for light in the energy range of 20–700 eV. The end station consists of a single chamber, equipped with a Scienta electron energy analyser, and runs at a base pressure of <10−9 mbar during data acquisition. An Ir crystal was cleaned and the absence of any C, B, O and N contaminants was checked by XPS. Samples were prepared as described above, although during the growth process a higher total background gas pressure was used (0.5–1 × 10−6 mbar). The C(1s), and B(1s) core levels were collected in normal emission geometry (incidence/emission angles of 0°/45°) with photon energies of 375 and 270 eV, respectively. The BE was calibrated by measuring the Fermi edge for each photon energy. The C(1s) spectra for each sample were fit with 4 Doniach-Šunjić components convoluted with Gaussians. The Lorentzian full width at half maxima (FWHM) were fixed at 130 meV for each component and the Gaussian FWHMs were allowed to change between components. To account for doping of the graphene film, the BE position of the C1 component was allowed to change, while the relative positions of the C2, C3 and C4 components were fixed at +0.503, −1.413 and −0.992 eV, respectively. The B1s spectra were fit with 4 Voigt components using the same Lorentzian and Gaussian FWHM, 100 and 770 meV, respectively, for each component. Analogous to the C(1s) spectra, also in the case of the B1s core level, the B0 component was allowed to change in BE while the relative positions of the B1, B2 and B3 components were fixed at +0.680, −1.093 and −2.087 eV, respectively.The XPS experiments were performed at the The data sets generated during and/or analysed during the current study are available in the Zenodo repository (doi: 10.5281/zenodo.569453).Supplementary Information


We investigated the PD-L1 expression in colorectal cancer (CRC) and in its microenvironment.PD-L1 was expressed in neoplastic cells (NCs) and tumor-infiltrating immune cells (IICs). All samples PD-L1+ on NCs were also on IICs. Three types of cancers could be grouped: group A(NCs-/ IICs-); group B (NCs-/ IICs+); group C (NCs+/IICs+). To group A belong tumors characterized by poorly immunogenic competence, poor immune response but massive granulocyte infiltrate, justifying the absence of PD-L1 as an immunoinhibitor receptor. To Group B probably belong more immunogenic CRCs, justifying the strong IICs-mediated immune response, and up-regulation of PD-L1 expression only on IICs. To group C belong CRCs probably characterized by a large amount of tumor neoantigens resulting in a marked infiltration of lymphocytes and PD-L1 upregulation also in NCs.Sixty-three colorectal cancer specimens from a cohort of 61 patients were retrospectively reviewed. Thirty-seven MSS and 26 MSI-H CRCs enrolled in this study. Immunohistochemical staining to PD-L1 was performed by using MAb E1L3N.Our study calls attention to the importance to assess PD-L1 expression in tumor microenvironment also evaluating type and density of infiltrating immune cells to better stratify CRCs with different immunological patterns. Colorectal carcinogenesis is driven by sequential genetic and epigenetic alteration of epithelial cells and is also influenced by tumor-host interaction –3. A welPD-1 is an immune-inhibitory receptor that is constitutively expressed by activated T lymphocytes and macrophages whereas other non-T lymphocytes, such as B cells and natural killer cells, express PD-1 only on induction , 10. HigPD-L1 is constitutively expressed by T and B cells, macrophages and dendritic cells and is up-regulated by many inflammatory mediators and cytokines –14. PD-LBecause immune checkpoints are one of the steps for immune escape and tumor development, inhibition of these molecules with monoclonal antibodies may restore host immune response and, consequently, stop tumor growth and even cause tumor regression. For this reason immunotherapy using immune checkpoint inhibitors is a rapidly growing modality for the treatment of some human cancer , 20–22. Although the majority of CRCs develops via a chromosomal instability pathway, approximately 12–15% have dMMR which is responsible for microsatellite instability (MSI) , 26. ThiSince it has been shown that not all MSI CRCs respond , 29, it The aim of this study is to investigate the immunohistochemical expression of PD-L1 in colorectal cancer cells and in its microenvironment and to correlate with microsatellite instability status and morphological and molecular characteristics.A total of 63 CRC specimens were included in this study. The clinicopathological characteristics of patients are listed in Table PD-L1 staining was observed with different rate both in neoplastic cells (NCs) and in infiltrating immune cells (IICs). NCs were positive in 25% of samples, while IICs in 78% (Table p = 0.04), right sided location (p = 0.004), medullary histology (p < 0.001), G3 tumor grade (p < 0.001), sporadic tumor associated neutrophils (TANs) (p = 0.003), BRAF mutation (p = 0.001), and MSI (p = 0.003). On the contrary, the other features such as sex, pattern of advancing border, tumor budding, IICs density, pN status, and RAS status were not associated with PD-L1 expression in tumor cells. By multivariate analysis, after adjusting for sex and age, right sided location , medullary histology , sporadic TANs , N1 status , BRAF mutation  and MSI status  were significantly associated with PD-L1 expression.The association between PD-L1 expression in NCs and clinicopathological and molecular parameters is shown in Table p = 0.005), moderate/marked IICs density (p < 0.001), and MSI (p = 0.03). Instead the other features  were not associated with PD-L1 expression in IICs.The association between PD-L1 expression in IICs and clinicopathological and molecular parameters is shown in Table p = 0.01) and MSI status  were significantly associated with PD-L1 expression.By multivariate analysis, after adjusting for sex and age, right sided location . Samples of group C compared with those of group A are prevalently localized on the right colon (p < 0.0001), are associated with medullary histological type (p < 0.0001), G3 tumor grade (p < 0.0001), BRAF mutation (p = 0.001) and MSI status (p < 0.0001). Samples of group B compared with those of group C are prevalently localized on the left colon (p = 0.02), are associated with adenocarcinoma histological type (p < 0.0001), G1/G2 tumor grade (p < 0.0001), massive TANs (p = 0.0005), N0 status (p = 0.05), BRAF wild-type (p = 0.007) and microsatellite stability status (MSS) (p = 0.008).All specimens PD-L1 positive in NCs showed PD-L1 expression also in IICs. There were no specimens positive only in NCs. Following these findings three groups can be assembled: group A (14 samples negative both in NCs and in IICs); group B (33 samples positive only in IICs); group C (16 samples positive both in NCs and IICs). The comparison among groups is shown in Table PD-1/PD-L1 pathway is a significant mechanism of immune suppression within tumor microenvironment. MAbs to PD-1 and PD-L1 could block this pathway and reverse T cell exhaustion inducing tumor regression. In the treatment of malignancies including melanoma, NSCLC, renal cell carcinoma, bladder cancer these MAbs have an overall response rates ranging from 16% to 100%, 31. AltLe et al.  in a phaDespite these results being encouraging, it is clear that most MSI CRCs do not respond to immunotherapy and this represents the rational for testing other markers able to predict cancer population who be eligible for personalized immune approaches.There is an apparent paradox: both MSI CRC and medullary carcinoma which have many overlapping clinical and morphological features, are generally characterized by better prognosis . It is bPD-L1 immunohistochemical expression in NSCLC and melanoma is used as marker to select patients who may be successful in the treatment with Pembrolizumab and Nivolumab , 31.Assessing PD-L1 expression is complicated by many challenges: different immunohistochemical assays (i.e. different primary antibodies and assays conditions) and different PD-L1 evaluation methods (i.e. different scoring methods and PD-L1 positivity cut-offs) complicate the comparative evaluation between clinical studies , 39–44.We used MAb E1L3N to PD-L1 immunohistochemical detection and cut-off of 5% positive cells. Most studies on CRC evaluated the expression of PD-L1 using a 1% or 5% cut-off value –44. We uWe found 25% of cases with PD-L1 positive in NCs and 78% in IICs. Our positivity rate is higher than that described in other studies using the same MAb E1L3N and the same 5% cut-off , 42, 43.BRAF mutation, and MSI status , moderate/marked IICs (p < 0.001), and MSI (p = 0.03)  Table .Our results have clearly shown that there is no positivity of PD-L1 in NCs without positivity in IICs. These data allows to outline three types of tumors: tumor PD-L1 negative both in NCs and in IICs (group A), tumors PD-L1 positive only in IICs (group B), tumors PD-L1 positive both in NCs and IICs (group C). The association between the three groups and the variables in observation are shown in Table Many cells with immune functions populate the tumor microenvironment: T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells. This heterogeneous population, together with stromal cells such as fibroblasts and endothelial cells, contribute to forming an activated immune system against the cancer. The crosstalk between these different cellular populations exploits cytokines and other molecules that interact with other cells in the microenvironment. Unfortunately the complex of mechanisms that regulate the antitumor response of the immune system is yet to be completely elucidated.In CRC, especially MSI ones, it is believed that the neo-epitopes burden produced by a large number of mutation induces a T-cell mediated immune-response with three possible sequential scenarios. In a first phase (elimination phase), the stimulated immune system produced many costimulating cytokines  and less inhibitors  resulting in efficient tumor cell killing. Subsequently, an equilibrium phase takes over, in which cancer cells activate biochemical pathways inducing a dynamic state of immune tolerance. The third phase (escape phase) begins when cancer cells become capable of binding and activating the co-inhibitory molecules on the T lymphocytes , and secreting soluble immunosuppressive or inducing the secretion of mediators such as IDO, TGFβ, IL10. The final result is the blockade of the immune system and the cancer survival [In addition to T-mediated mechanism, another line of defense against cancers are neutrophil granulocytes, the most abundant leukocytes. Recent studies have shown the independent unfavorable prognostic role in several humans tumor –50 incluShaul et al. , in a muTherefore, a delicate and still not fully known crosstalk mechanism seem to play a fundamental role in determining the density and function of immune population involved in the tumor microenvironment.The data of this small retrospective study on a CRC cohort with and without microsatellite instability clearly demonstrate that the type and density of inflammatory cells populating the tumor microenvironment together with PD-L1 expression identify three different tumor groups .Group A, maybe due to neo-antigen scarcity, shows a poor IICs-mediated immune response and a strong granulocytes-mediated immune response. It would then be formed by very little immunogenic CRCs, so that the lymphocyte response is absent/mild and on the contrary it is massive the granulocytic one. In fact to the group A belongs prevalent MSS CRCs and not PD-L1 expressing (both in NCs and IICs) tumors.Group B, maybe due to the presence of more neo-antigens, has a strong IICs-mediated immune response, with or without granulocyte participating and the positivity of IICs to PD-L1 is probably linked to the perfect and vigorous functioning of the immune response that elicits self-control mechanisms. To this group belong a prevalence of MSS CRCs. In addition, Housseau et al.  suggest Group C, often shows a moderated/marked component of IICs with sporadic TANs. In this group there is probably a remarkable neo-antigens production with an equally remarkable T-cell immuno-mediated response counterbalanced by checkpoint inhibitor signaling, e.g. PD-L1, on both IICs and NCs, that would guarantee tumor tolerance and favor tumor progression. Indeed in group C there is an high percentage of MSI and medullary carcinomas, probably the subset of these tumors with unfavorable prognosis.If this theoretical setting is true, you might suggest that therapy with immune checkpoint inhibitors is useless in group A because another type of response works, while it might be strongly recommended in group C where the immunological response could be exhausted.Group B is less clearly outlined. It is generally conspicuous both the IICs and the TANs. Although the biologic significance of this distinct pattern of PD-L1 expression is currently not clear, it likely reflects the combined effect of innate and adaptive cellular and soluble factors that shape the tumor microenvironment . In addiIn conclusion our study shows that: it is important the comprehensive assessment of PD-L1 expression both in NCs and IICs; PD-L1 on NCs may have heterogeneous and focal expression; in agreement with other published data in CRCs the PD-L1 expression on NCs associates with right sided tumors and high grade, medullary histological type, MSI status, marked IICs; the granulocyte infiltration also contributes to separating CRCs with different immune microenvironments; the combination of density and type of inflammatory cells together with the expression of PD-L1 on NCs and IICs defines three tumor groups variously associated to MSI status, medullary histology and other clinicopathological features; theoretically the group A  would not benefit from anti-PD-L1 therapy otherwise the third group  would be the ideal candidate for this type of therapy; further studies are needed to validate these hypotheses and to check within the group B  other predictive features able to select candidate patients for the blockade immunotherapy.This study encompasses 63 formalin-fixed paraffin-embedded colorectal carcinoma specimens from 61 patients  underwent surgical resection (between the years of 2010 and 2016) at our National Institute of Gastroenterology.For selection, we have reviewed written pathology reports searching for keywords “adeno”, and “medullary” and in addition, within these histological types, we have selected those carcinomas with known MSS or MSI status. All the tumors had been surgically resected in a single surgery unit of our Institute and underwent pathological and molecular testing in our molecular pathology laboratory.Therefore, we collected 37 MSS (33 adenocarcinomas and 4 medullary), 26 MSI-H (19 adenocarcinomas and 7 medullary) CRCs. All MSI were sporadic CRCs.The same paraffin block on which molecular assays had been performed was chosen. Hematoxylin and eosin-stained sections of all formalin-fixed paraffin-embedded blocks were reviewed by a pathologist (MP) to confirm the presence of adequate tumor tissue and to evaluate tumor grade, pN status, pattern of advancing border, tumor budding, TANs, and IICs encompassing intra and peritumoral lymphocytes, macrophages, plasma cells. The IICs were evaluated as absent/mild when few cells were highlighted within the tumor and/or at the tumor-stroma interface, moderate/marked when the infiltrate was of greater density with the tendency to flow into plagues and infiltrate neoplastic epithelium. We evaluated TANs at the tumor-stroma interface and graduated as sporadic  when onlImmunohistochemistry (IHC) analysis was carried out in sections (4 micron thick) mounted on positively charged slides and dewaxed in an oven for 20’ at 60°C followed by two serial xylene dippings. Sections were then rehydrated in grades alcohols and incubated in 3% hydrogen peroxide for 10’ to block endogenous peroxidase activity. Sections were retrieved in EDTA buffer (pH 8) at 98°C for 30′. For PD-L1 IHC, has been used a monoclonal antibody against cytoplasmic domain of PD-L1 , on an automated autostainer . The dilution factor of this antibody and target retrieval buffer were optimized by using tonsil and placental tissues as positive control.Dako Real ENVISION  was used as visualization reagent and the 3,3’-DAB as chromogen, according to the manufacturer´s instructions.Human placenta was included as positive control on each staining run. Negative rabbit IgG controls were used and did not show any staining.PD-L1 immunoreactivity was evaluated separately for NCs and IICs. PD-L1 positivity was defined as PD-L1 expression on ≥ 5% of membranous positive cell staining of any intensity. Cytoplasmic staining was not considered in this study. The immunostains were independently evaluated by two pathologists . For discordant cases, the results were confirmed by discussion of the two pathologists.CD3  and CD68  immunostains were performed on each specimen by using the same protocol of PD-L1, except antigen retrieval that was carried out in 10mM Citrate buffer (pH 6). The CD3 and the CD68 stains were used to better interpret the topographical distribution and the composition of PD-L1 expressing immune infiltrate .KRAS codons 12, 13 of all specimens enrolled in this study. Later, on 12/13 wild type samples, it was performed RAS extension  and BRAF (V600E) analysis [The protocols were performed according to the manufacturer’s instructions. Two sections (10 micron thick) of FFPE tissue were used for DNA extraction using the QIAamp DNA FFPE Tissue Kit . As a first step we evaluated, by pyrosequencing , the status of analysis . BrieflySamples with a reported potential low-level mutation were considered positive for the mutation, if confirmed by re-running in duplicate with wild type DNA control.MSI testing was performed on paraffin-embedded tumor tissue using a PCR-based assay with five National Cancer Institute recommended markers  , 57.2 and 200 ng DNA. PCR cycling conditions were: denaturation at 95° for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final elongation at 72°C for 5 minutes. The PCR products were controlled on 1.5% agarose gel and were separated on a vertical denaturing 6M Urea polyacrilamide gel (acrylamide/bis-acrylamide 19:1). Polyacrilamide gel was stained with Silver Nitrate. On the basis of the MSI status, CRCs can be classified into three groups: MSI-Low if only one of the five microsatellite markers shows instability, MSI-High if two or more of the five microsatellite markers show instability and MSS if none of the markers shows instability [MSI testing required DNA from both normal and tumor tissue for each patient. PCR amplification was performed in a total reaction volume of 20 µl, containing 100 µM dNTPs, 0.5 µM each primer, 0.25 U of Taq Polymerase Platinum, 1.5 mM MgCltability , 57.Means and standard deviations (M ± SD) for continuous variables, and relative frequency (%) for categorical variables, were used as indices of centrality and dispersion of the distribution.Chi-square test or Fisher exact test, depending on the number of observations, for categorical variables, Kruskal-Wallis rank test and Wilcoxon rank-sum test (Mann-Whitney test) for continuous variables were used to test differences among the groups. In the logistic regression models the absence of PD-L1 expression was considered as a reference category.p-value was < 0.05, two tailed for all analysis. All statistical computations used STATA 10.0 Statistical Software, (StataCorp), College Station, TX, USA.All models were adjusted for gender and age. Results were presented as Odds-Ratio (OR) and with 95% Confidence Intervals (C.I.). The Odds-Ratio represents the risk for one-unit variation of the predictor variable. When testing the hypothesis of significant association,


The first type is composed of arrays of aligned equidistantly separated parallel wrinkles propagating over large distances, and originates from line interfaces in the graphene, such as thin, long wrinkles and graphene grain boundaries. The second type of wrinkle arrangement is composed of non-aligned short wrinkles, formed in areas without line interfaces. Besides the presented hybrid graphene topography with distinct wrinkle geometries induced by the pre-patterned substrate, the graphene layers are suspended and self-supporting, exhibiting large surface area and negligible doping effects from the substrate. All these properties make this wrinkled graphene a promising candidate for a material with enhanced chemical reactivity useful in nanoelectronic applications.We present an approach that allows for the preparation of well-defined large arrays of graphene wrinkles with predictable geometry. Chemical vapor deposition grown graphene transferred onto hexagonal pillar arrays of SiO Corrugating graphene via creating folds, crumples, ripples, and wrinkles produces a material that combines the intrinsic  and enhanced properties of graphene.In recent years, graphene-based research has moved from investigating flat ideal two dimensional (2D) graphene to exploring structures that are topologically distorted8, corrugated graphene exists even if it is not exposed to artificial manipulation with the topography. In fact, real graphene is never completely flat; it always exhibits surface corrugations, which prevents the monolayer from collapsing2. In general, surface corrugations manifesting as a local topological disorder significantly modify the electrical, optical, and chemical properties of graphene in comparison to the intrinsic properties of non-corrugated graphene10 by varying the electrostatic potential of the layer and by further modifying the band structure. For example, a change in the relative position of the Dirac point (described by energy ED) with respect to the Fermi level (with energy EF)11, the creation of the mid-gap states12, and trigonal warping13 have been observed. Moreover, the creation of surface corrugations in graphene results in a change in the charge transport5, the suppression of weak localization11, the appearance of electron–hole puddles15, band-gap opening16, and carrier scattering9, or enhancement of the spin-orbit interaction allowing for spin polarized transport at low magnetic fields17.Because wavy-like structures are observed everywhere in nature3, such as graphene ripples, wrinkles, folds, or crumples. Graphene ripples18 have a length/width aspect ratio close to 1 and a height <1 nm; they act as out-of-plane deformations stabilizing the 2D graphene layer. Graphene wrinkles have an aspect ratio larger than 1, reaching a length of up to a few micrometers, and they are formed when graphene experiences a uniaxial exterior force19. Wrinkles are also formed in the area surrounding the defects in the graphene26.Depending on the physical dimensions, different types of corrugations can be defined5. Graphene crumples with dimensions that are more or less comparable to the dimensions of the wrinkles are dense isotropic corrugations in two or three directions. Crumples are formed when multidirectional forces are applied to graphene28, and crumpled graphene exhibits tunable wettability and transparency28. Based on experimental observations, crumpled and wrinkled graphene are both promising candidates for energy-related research (such as fuel cells and supercapacitors), which requires high conductivity, high surface area, thermal stability, and mechanical and chemical robustness30.Graphene folds can be described as collapsed wrinkles. When the wrinkle exceeds the critical height, it is not standing perpendicular to the substrate; rather, it bends and collapses upon the fold formation31 and crumpling so the corrugated graphene will be beneficial for targeted graphene-based device fabrication. The wet transfer of graphene onto any substrate (flat or patterned) often leads to the creation of wrinkles and crumples33. Crumple formation is typically connected to the fast evaporation of the liquid used for the wet transfer. Formation of the specific graphene wrinkle networks can be done via the transfer of graphene onto patterned substrates36. Many approaches have been presented in the literature37, such as graphene transferred onto nanoparticle-decorated substrates, when parts of graphene are still in contact with the substrate41, or graphene transferred over arrays of pillars43. It has been shown that transfer of graphene over substrates decorated with homogeneously distributed nanoparticles leads to the formation of wrinkles with specific density and surface/projected area40. Those systems demonstrated the tunability of the wrinkle network morphology and the graphene’s intrinsic properties by changing the distribution of the nanoparticles on the substrate40.Considering the application of corrugated graphene, it is essential to fully control the wrinkling43, forming either a smooth or wrinkled graphene layer depending on the pillar density and geometry46. After reaching the critical pillar distance (minimizing the inter-pillar distances), all the large wrinkles that are formed are localized near/on the pillars, oriented in the direction of the symmetry axis of the pillar arrays. Finally, line defects in graphene has been found to introduce defined wrinkle and ripple networks47. As demonstrated in previous research, graphene corrugation is a highly complex issue with many degrees of freedom. Therefore, a rigorous analysis of the wrinkle patterns and the substrate topography should always precede the interpretation of the resulting graphene arrangement.Studies examining graphene-nanopillar systems have revealed that tuning of the pillar geometry  should lead to the transformation of collapsed graphene into fully suspended graphene43, our graphene layers are self-supporting and do not form tent-like structures, which touch the substrate beneath the pillars. A thorough analysis of the wrinkling networks, primarily using Atomic Force Microscopy (AFM) and Raman Spectroscopy (RS), shows an unexpectedly large number of different wrinkles in terms of the preferred propagation direction and propagation length, despite the regularly-ordered hexagonal pillar arrays that locally support the graphene monolayer. These wrinkles can be categorized into two main types, and their origin explained by the presence or absence of distinct long features that are characteristic of chemical vapor deposition (CVD)-grown graphene: the grain boundaries and/or thin wrinkles originating in the graphene transfer.In this paper, we present an approach to prepare well-defined large arrays of graphene wrinkles with predictable geometry, and we report the existence of fully-suspended graphene on nanopillar arrays with inter-pillar distances up to 1 μm. In contrast to a previous study2 substrates. Two pillar-array geometries with graphene suspended from the substrate were made, each with a different nanopillar density, and, consequently, a different geometry and morphology of the transferred graphene layer. The used nanopillar arrays form a hexagonal (hcp) lattice  and the GN@pillars_B (1 nanopillar/ μm2) samples.We prepared model structures of suspended topographically-corrugated graphene by transferring the CVD-grown graphene monolayers onto the top of the nanopillar arrays that were fabricated using electron beam lithography on Si/SiOice Fig. , their hInspection of both samples by RS revealed no apparent D mode within the graphene layers, indicating the high quality of the graphene layer see Figs  and S3, −2 and 2.53 μm−2 for the GN@pillars_A and the GN@pillars_B samples, respectively , and the major wrinkles extend over multiple inter-nanopillar lengths  aligned, (ii) mixed and (iii) random areas, depending on the wrinkle type, the direction of the wrinkle propagation, and the symmetry of the wrinkle network. Specification of the individual wrinkle area morphology is summarized in Table -p Table . The so--p Table .Table 2PRtm, , and it is two-times smaller for the mixed area (Rtm ~ 5 nm).The median peak-to-peak amplitude of the wrinkled graphene layer, The aligned and mixed areas are formed in the proximity of specific line boundaries, which are visible as thin dark lines on the AFM phase images see Figs , S7–S10.lp-p away from the graphene edge in the perpendicular direction; hence, the central parts of the sheets that are larger than 6 × 6 (lp-p)2 are completely wrinkle-free. For details, please see Table The morphology of the wrinkle networks is completely changed when the nanopillar array dimensions are increased four-times (the GN@pillar_B sample). The graphene layer is then almost smooth, with scarce wrinkles created near the edges of the graphene sheet. The formed wrinkles do not propagate farther than 3 48. In analogy with the system of wrinkled graphene on particle-decorated Si/SiO2 substrate38, an elaborate fitting of the G and 2D modes was conducted to analyze separately the graphene on the pillars (G2 and 2D2 components) and between the pillars . In principle, it is necessary to distinguish between the aligned and non-aligned wrinkle networks of the graphene in the GN@pillars_A sample. We observed that the aligned areas containing long wrinkles are clearly recognized by the RS, forming the lines with enhanced intensity on the Raman maps . Note that the strain determined for the GN@pillars_B sample is higher in several regions of the graphene layer (see the Supplementary information file) in comparison to the GN@pillars_A sample.As can be deduced from the Raman maps Fig. , the Ram49 using AFM, which is capable of obtaining higher resolution than RS  should be lower for the strained areas in comparison to the mechanical deformation of the unstrained graphene layer, as is known from the standard mechanics of materials49. Hence, the relative strain can be estimated measuring the mechanical deformation of the graphene layer  than for the arrays with low-pillar density (GN@pillars_B). This suggests that the graphene in the GN@sample_B sample is stiffer and better attached to the pillars.The strained graphene areas were also examined by mapping the mechanical properties Figures  and S11. Figures  and S11.43 however, this was observed even for the 1 μm pillar separation length (the GN@pillars_B sample). Nevertheless, this observation is also in contrast to the findings reported by Reserbat-Plantey et al.43, which demonstrated a collapse of the graphene layer and the formation of a tent-like structure for the pillar arrays with an lp-p of approximately 1 μm. A possible explanation for the self-supporting nature of the graphene layer in the case of the GN@pillar_B sample can be found in the mechanism used to prepare the sample50. We assume that the key factors for preventing the graphene from gliding down the pillars and, subsequently, adhering to the substrate, are the solvents that are used, the polymer that is used for the wet transfer55, and the strength of adhesion of graphene to the nanopillars.Generally, one would expect full delamination of the graphene from the flat part of the substrate decorated with pillar arrays with interpillar distances reaching 240 nm (the GN@pillars_A sample);lp-p, whereas the mixed areas are found near the interface edges. On the other hand, the interface-free graphene is characterized by short-range propagation of the wrinkles (the random wrinkle area). However, the formation of the wrinkle networks with predictable hierarchy on the line defects is not surprising.It is important to discuss the origin of the most interesting issue addressed in this paper, which is the formation of the equidistantly separated parallel aligned wrinkles. We observed that the aligned and mixed areas of the wrinkles are formed on the graphene line interfaces, which are visible on the phase images as the dark thin lines Figs , 4 and 558 and demonstrated experimentally for the grain boundaries47, presence of any constraint in a thin sheet (constraint means 2D layer boundary such as a line defect represented by the graphene grain boundary or edge of the membrane) leads to the formation of the wrinkle networks with a predictable hierarchy. In general, specific mechanical stimulation57 and any confinement of the 2D membrane allows artificial control over wrinkle formation, resulting in a universal self-similar hierarchy of wrinkles59. This was proven for strained graphene32, hanged fabric56, and graphene grain boundaries/line defects formed by pentagons and distorted hexagons58. Origin of folding and wrinkling on the constraint is based on the competition of the stretching and bending of the constrained membrane56, because the bending rigidity of thin membrane is smaller than the stretching rigidity and membrane tends to reduce the elastic energy via formation of folds or wrinkles60. The periodic wrinkling of thin membrane in the direction perpendicular to the constraint represents the equilibrium state of the constrained membrane, with the wrinkle wavelength increasing as λ~xm, where x is the distance from the constraint (boundary) and m = 1/2 for graphene sheet56.As was suggested by several theoretical papersThis implies that the existence of graphene line interfaces is critical for the formation of aligned wrinkle areas on our pre-patterned nanopillar substrates. The explanation of the origin of these graphene line interfaces is based on the analysis of the AFM phase images, correlated with the topography images.61 that are different from the rest of the graphene layer.Phase imaging and direct measurement of the phase shift, which, in general, is related to the local energy dissipation of the surface, enables visualization of the regions with different material properties. Moreover, the phase images of the graphene layers examined in this present study simultaneously visualize the topography corrugations and the objects hidden beneath the graphene layer Figs , S7–S9, 62  and (ii) the graphene grain boundaries (marked with green arrows in the selected images)47. The transfer-wrinkles (i) are visible both on the topography and phase images, whereas the graphene grain boundaries (ii) are observable only on the phase images. To prove this presumption directly, we examined the graphene transferred onto the Si/SiO2 substrate using the same transfer method , the initial wavelength of the wrinkles emerging at the boundary corresponds to λboundary. In our case, λboundary is equal to the interpillar distance of our pillar array. The difference in the wrinkle propagation in a free thin membrane and our system is that the wrinkles are not propagating as λ~xm over their whole propagation length in our aligned wrinkle arrays, but they follow the symmetry of the pre-patterned substrate, hence the inter-wrinkle distance is kept equal to interpillar distance lp-p. Moreover, if we assume that the wavelength of a long wrinkle follows the λ~xm dependence at least between two nearest pillars, the estimated increase of the λ along the distance to the nearest pillar  is smaller than pillar diameter (27.5 nm). When the next pillar is reached, the wavelength is ‘reset’. This implies that the first order wrinkle will always propagate to the nearest-neighbor (NN) pillar in the wrinkle propagation direction and does not pass between the pillars. Therefore, it is not surprising that the long parallel equidistantly separated wrinkles are formed on the grain boundaries and that they take on the dimensions of our nanopillar array. Furthermore, the particular orientation of the long wrinkle with respect to the grain boundary is given only by the mutual orientation of the nanopillar array and the grain boundary.The wrinkles, the pillar tops, and the graphene line interfaces are manifested in the phase images as dark areas with low-phase values see Figs , and S1bxm is not expected. The longest formed wrinkles exceed in maximum to the 1st NN pillar, if their length corresponds at least to the lp-p. If shorter wrinkles are created on the individual pillars (lwr < lp-p = 240 nm), the two individual wrinkles emerging from two individual NN pillars form the so called ‘avoiding pairs’ of wrinkles, preventing their joining. The observation of ‘avoiding pairs’ of wrinkles in the random areas corresponds well with the simulations of Budrikis et al.63, who simulated the conditions under which two wrinkles formed on two separated objects can join or form the ‘avoiding pair’. Comparing the geometry of our system with the simulations, the length of the wrinkle created on individual pillars with no external anisotropic effects should reach almost 140 nm, and prevents joining with the wrinkle formed on the NN pillar .It remains to answer the origin of the wrinkle shapes found in the random areas, where the short-range wrinkles are present. We have not observed any boundary or constraint in the random areas, consequently formation of the long wrinkles with λ~Even though there is a non-zero probability that the two wrinkles join, the probability of creating an ordered array in such a way is negligible, as can be seen, e.g. in Figs Stop) is more than ten-times larger for the pillars in the GN@pillars_B sample than for the GN@pillars_A sample (2.53 vs. 0.29 × 10−2 μm2), the graphene/pillar contact region, hence its adhesion, is much larger for the GN@pillars_B sample. Consequently, the graphene is prevented from slight gliding down from pillars and forming of wrinkles, as the graphene membrane can be stretched more efficiently between the large pillars. This wasindirectly confirmed by larger strain for the GN@pillars_B sample than for the GN@pillars_A sample reaching 0.1%; and smaller median deformation for the GN@pillars_B sample (8 nm) than for the GN@pillars_A sample (12 nm), both together proving better adhesion of graphene to the large pillars. Therefore, the GN@pillar_B sample is proposed to be a candidate for strain-induced band-gap engineering64  and the creation of pseudomagnetic gauge fields67.In addition, the strain determined for both samples corresponds qualitatively with the different sample morphologies. The GN@pillars_B sample exhibits larger strain than the dense system of pillars (the GN@pillars_A sample) when comparing the free-standing regions, which is confirmed both by the RS and the lower measured mechanical deformation of the more strained layer Figures  and S11.In conclusion, we prepared a model system of fully-suspended, self-supporting graphene on nanopillar arrays with inter-pillar distances up to 1 μm. We showed that, for the dense nanopillar array with inter-pillar distances ~240 nm, topographically-corrugated graphene formed three well-defined types of wrinkle networks with a predictable wrinkle hierarchy. After observing equidistantly separated parallel aligned graphene wrinkles on the graphene line interfaces (thin wrinkles and graphene grain boundaries), we discussed how the geometry of the wrinkle networks can be tuned up. If the position of the graphene line interfaces is controlled, one can prepare large regions of aligned wrinkles propagating over large distances with the inter-wrinkle spacing corresponding to the inter-pillar distances. By demonstrating that it was possible to manipulate the wrinkle network morphology and that the present line defects allowed 3D patterning of 2D material, we were able to prove existence of the self-supporting nature of the graphene layer with periodic wrinkle patterns.2 nanopillar arrays with different geometries were prepared. The nanopillar arrays were made by electron beam lithography, evaporation and lift-off-technique to create the hard mask pattern and subsequent Inductively Coupled Plasma (ICP)-etching. The wafer layout consists of 60 numbered chips with a size of 5 × 10 mm². Each chip contains a 1 × 1 mm²-field of nanopillar gratings. Hexagonal nanopillar gratings with different periods (240 and 1000 nm) were fabricated.Two types of samples of the graphene transferred onto the top of Si/SiO2-thickness for 240 nm gratings, 365 nm oxide for 1000 nm pitch gratings). The pre-cleaned Si wafers were coated with a two-layer lift-off -resist  and additionally covered with a 10 nm thick gold conduction layer. The e-beam exposure was performed using the shaped beam writer SB350 OS  at an electron dose of 750 to 2000 µC/cm² to vary the structure sizes. The resist was developed for 60 s in AR600–546 and 30 s in MIBK:IPA = 1:3, always rinsed in IPA. Afterwards 40 nm of aluminium were deposited on top of the wafer by thermal evaporation under normal incidence. The lift-off process was carried out in a 10:1- mixture of 2-methoxyethanol and acetone. The lifted aluminium patterns serve as hard mask for etching into the oxide film. By using an ICP etching process with CHF3/SF6 and with some over-etching into the Si-wafer, needle-shaped nanopillar patterns are formed  H2. The Cu foil was exposed to 30 sccm CH4 for 20 min, after which the Cu foil was cooled to room temperature. The as-grown graphene was subsequently transferred to the substrate with the pillars using nitro-cellulose , according to procedures reported previously69. The nitro-cellulose was removed from the graphene by annealing the sample at 180 °C for 30 min in air.A graphene was prepared using chemical vapor deposition (CVD) growth on the Copper (Cu) foil, as described previously inThe prepared samples were characterized using complementary methods, including Field-Emission Scanning Electron Microscopy (FESEM), Atomic Force Microscopy (AFM), and Raman Spectroscopy (RS). FESEM S-4800 (Hitachi) was used for direct visualization of the prepared nanopillar arrays as well as the final samples of the nanopillars covered with graphene, using acceleration voltage of 1–5 kV.1 and 2D1 components account for the wrinkled suspended graphene, the other pseudo-Voigt functions  account for the graphene that is in contact with the Si/SiO2 pillars38. In the aligned wrinkle areas, an extra peak function (G3) accounts for the long wrinkles. Finally, the fourth pseudo-Voigt function of the G mode accounts for the D’ mode  laser excitation, a laser power of approximately 1 mW, and a grating of 600 lines/mm to obtain the entire spectrum from the Si TO mode to the 2D’ mode at once and lateral steps of 50 nm in both directions. The laser was focused on the sample with a 100× objective to a spot with a diameter of approximately 500 nm. All the spectra were fitted with pseudo-Voigt functions to obtain the Raman shift ω, full width at half-maximum (FWHM), intensity (I), and the fraction of the peak that is Lorentzian. To account for the wrinkles in graphene and the graphene that is in contact/not in contact with a pillar, the G and 2D modes were fitted with 4 and 2 pseudo-Voigt functions, respectively; Gk = 3 N/m, f0 = 75 kHz, nominal tip radius = 8 nm; Bruker, Inc.) were used for imaging the individual samples, preserving the comparable wear of the tip. The scan rate (0.6 Hz) and the amplitude setpoint were chosen in order to the minimize forces acting on the graphene layer. The images were captured with a maximum image size of 10 × 10 um2. High quality images were processed in the standard way using Gwyddion software70, applying line-by-line 1st order levelling and scar removal. The images were further analyzed using image analysis, 2D Fast Fourier Transform (2D FFT) analysis and filtering, image arithmetic and grain analysis in Gwyddion software, and analysis of profile height distribution implemented using custom-made software. This study used 2D FFT analysis to determine the periodicity of the nanopillar pattern, the inter-pillar distances, and the preferential propagation of the graphene wrinkles. We used 2D FFT filtering to separate the long-range and short-range topography components and to separately visualize the long wrinkles and fine wrinkle structure. Image arithmetic was applied to the topography and phase images to obtain a combined visualization and evaluation of correlated morphological and phase features. Grain analysis was used to evaluate the surface coverage by the nanopillars and to determine the cross-section areas in the defined height and contact areas between the graphene and the nanopillars. Profile height distribution was analyzed to determine the median peak-to-valley distance, Rtm, of the graphene layer topography profile that was attributed to the hanging of the graphene layer. The topography AFM image was cut into individual lines using home-built procedures, in order to identify all the minima and maxima attributed to the highest parts of the wrinkles and the lowest parts in the inter-wrinkle valleys. Then, the median difference of all maxima and minima was calculated as:pμ is the median wrinkle height and vμ is the median valley depth, calculated over all the lines in the image. For details on the procedure, see the Supplementary Information File  equipped with a JV scanner, with a resolution of 1024 lines. Fresh RFESP probes . The captured topography, adhesion, and deformation images were processed using Gwyddion software, as described in the previous paragraph. The adhesion and deformation images of the graphene were qualitatively compared with the phase images captured in tapping mode.Peak Force quantitative nano-mechanical mapping (PeakForceQNM) was done using the Dimension Icon Atomic Force Microscope ; this complementary method was used to reveal the origin of the phase contrast. The images were captured using a SCANASYST-AIR probe (Supplementary Information File


Circular questions are based on social constructivism. They include a variety of systemic interviewing methods. The goals of circular questions are to explore the mutual dependency of team members’ behavior and recurrent behavior patterns, to generate information, to foster perspective taking, to “fluidize” problems, and to put actions into relational contexts. We describe the nature of circular questions, the benefits they offer, and ways of applying them during debriefings.We submit that interaction patterns within healthcare teams should be more comprehensively explored during debriefings in simulation-based training because of their importance for clinical performance. We describe how  In the OR, do you feel that your colleagues share their points of view more frequently before or after the attending enters the OR?Who can typically speak up the most to the attending when she seems stuck in unsuccessfully performing an unexpected difficult intubation?circular questions [These questions differ from the questions that we usually ask during debriefings, and they certainly differ from questions we use in our daily conversations. They are called uestions –3. Origiuestions –6.In this manuscript, we describe the nature of circular questions, the benefits they offer, and ways of applying them during debriefings. Debriefings—the instructor-guided conversations among learners aiming to reflect on the relationships among events, actions, thought and feeling processes, and performance outcomes of the simulation—play a crucial role during simulation-based training –10. NotaTeamwork and its components  are typical content of learning objectives in simulation-based training , 11–20. individual behaviors and frames about their individual actions [First, discussions during debriefings tend to be focused on team members’  actions . This fo actions , 26. Yet actions –32. For  actions  Also, te actions , 34. So fundamental attribution error: we overestimate the individual learner’s disposition and personality and underestimate their environment and situational dynamics. With respect to teamwork, this means that instructors might overestimate the linear causality of teamwork behaviors and underestimate the circular causality of behaviors, that is the mutual dependency of behaviors; a person’s behavior at one time is considered both effect and cause of the interactional partner’s behavior [the more internally willing individual team members were to speak up, the more effort they would put into actually speaking up would be based on linearity; the assumption that if team member A speaks up and team member B reacts with verbal appreciation, team member A might feel encouraged to speak up in future teamwork would be based on circularity. So far, circularity is rarely systematically explored during debriefings, missing an essential element of teamwork.Second, by focusing on individual thought and feeling processes rather than on team interactions, instructors might overestimate the individual’s capacity and underestimate the influence of the context on the individual. Thus, we make, as Ross  describebehavior . For exasystem. For example, by askingWhy didn’t you speak up? – (Learner: “I don’t know.”)Third, using linear rather than circular assumptions, we tend to underestimate the meaning and messages of feelings, thoughts, and actions within the team as not speaking up for the system such as preserving hierarchy within the team, maintaining responsibilities or respecting sub-team territory [not much is yet explored about the potential function of erritory , 18. Theerritory . As a fuerritory . Instrucerritory .In sum, we submit that circular interactional patterns within healthcare teams and their importance for clinical performance should be explored more comprehensively during debriefings. We propose that by applying systemic thinking and asking circular questions, instructors can actively use debriefings to walk the talk of teamwork.behavior rather than personality traits, ranking and classification, change in the relationship before and after an event, and differences in respect to hypothetical conditions.Circular questions are based on social constructivism and on circular assumptions about an issue , 39. Theintent dimension refers to intended locus of change, that is, whether to orient oneself or change the others [assumptions dimension refers to whether the instructor considers predominantly linear or circular assumptions about an issue [circular questions and we follow that tradition [To provide an overview, classify, and illustrate different types of questions, we used Tomm’s  framewore others . The assan issue . Accordian issue . Linear an issue , 43. Notradition , 44–47. radition . For exaradition .Fig. 1FoDebriefing with Good Judgment Approach can be used to identify frames that drive actions [Notably, we do not consider circular questions a stand-alone debriefing method. Recent literature has highlighted the benefits of applying blended debriefing approaches , 25, 48. actions . Circulasystemic mindset. One essential part of that mindset is based on what Rudolph and colleagues have also described in their Debriefing with Good Judgment Approach as holding the learner in high regard and combining honesty with curiosity [A trauma patient arrived in the emergency department. While handing the patient off, there seems to be confusion, many voices are heard, each team member seems to be engaged in action. There is no structured information exchanged or verbal planning. The paramedic repeats himself frequently, getting louder each time.Approaching this episode systemically, the instructor might consider the paramedic as competent and experienced in performing patient handovers (respect), be curious about how team members mutually influenced each other and the paramedic’s behavior  and the differences and similarities in each team members’ perception of the handover .In order to use circular questions during debriefings, the instructor needs to adopt a uriosity , 49, 50,uriosity . Anotheruriosity , 3, 47. uriosity , 40. As Circular questions differ in their objectives: understanding the learners  vs. explicitly helping learners to change . Asking a on Table  to multicontext-oriented question above and difference-oriented questions  by asking linear questions, instructors may tend to underestimate the meaning and messages of feelings, thoughts, and actions within a system, missing new information and team phenomena.In this manuscript, we have introduced circular questions as a way of debriefing team interactions. We have proposed that the interdependency of team members’ actions, and their importance for clinical performance should be more comprehensively explored during debriefings because (1) recent research has shown that rather than individual actions of single team members, it is the interaction pattern among team members that discriminate higher- from lower-performing teams –32, (2) We have described the nature of circular questions and how they allow for exploring team behavior patterns, generating new information, and fostering perspective taking and observation skills in the debriefing. We have offered ways of applying circular questions and have recommended using them especially when the instructor (1) feels s/he would like to learn something new or explore something deeper, (2) feels that s/he is taking sides, (3) feels that s/he is moving into an oppositional stance to the learners, (4) wants to highlight different points of view among team members, and (5) wants to explore circularity among team members. We have highlighted that requirements such as creating a respectful and engaging learning environment and adopting a systemic mindset are ideally in place before using circular questions.Debriefing with Good Judgment Approach [Notably, we do not consider circular questions a replacement for other debriefing methods. Within the framework of blended debriefing approaches , 25, 48,Approach . This emApproach . LearnerApproach , 50. InsSo far, empirical research on circular questions is sparse and almost non-existent in the context of healthcare simulation debriefings . ResearcWe hope this introduction of circular questions in simulation-based training will stimulate interesting debriefings, more research on debriefings, and help to walk the talk of teamwork during debriefings.


We study spin transport in a fully hBN encapsulated monolayer-graphene van der Waals heterostructure at room temperature. A top-layer of bilayer-hBN is used as a tunnel barrier for spin-injection and detection in graphene with ferromagnetic cobalt electrodes. We report surprisingly large and bias-induced  spin-injection (detection) polarizations up to 50% (135%) at a positive voltage bias of + 0.6 V, as well as sign inverted polarizations up to −70% (−60%) at a reverse bias of −0.4 V. This demonstrates the potential of bilayer-hBN tunnel barriers for practical graphene spintronics applications. With such enhanced spin-injection and detection polarizations, we report a record two-terminal (inverted) spin-valve signals up to 800 Ω with a magnetoresistance ratio of 2.7%, and achieve spin accumulations up to 4.1 meV. We propose how these numbers can be increased further, for future technologically relevant graphene based spintronic devices. In 2D spin-valve devices, effective spin injection and detection can be potentially realised combining graphene with an ideal hBN tunnel barrier. Here, the authors report that a bilayer hBN tunnel barrier allows up to 100% spin-injection and detection in a fully hBN-encapsulated graphene heterostructure. The promising nature of crystalline hexagonal boron nitride (hBN) layers as pin-hole free tunnel barriers3 for spin injection into graphene8 has been recently demonstrated. However, due to the relatively low interface resistance-area product of monolayer-hBN barriers, there is a need to use a higher number of hBN layers for non-invasive spin injection and detection9. Theoretically, large spin-injection polarizations have been predicted in FM/hBN/graphene systems as a function of bias with increasing number of hBN layers10.Recent progress in the exploration of various two-dimensional materials has led to special attention for van der Waals (vdW) heterostructures for advanced graphene spintronics devices. For graphene spin-valve devices, an effective injection and detection of spin-polarized currents with a ferromagnetic (FM) metal via efficient tunnel barriers is crucial11 reported an inversion of the spin-injection polarization for different thicknesses of chemical vapour deposited (CVD)-hBN tunnel barriers, as well as an asymmetric bias dependence of the polarization using multilayer CVD-hBN/FM tunnel contacts. The observed behavior was attributed to spin-filtering processes across the graphene/multilayer-hBN/FM tunnel contacts.Kamalakar et al.13.In order to explore the potential of hBN tunnel barriers for graphene spin valve devices, one can study the role of current/voltage bias for spin-injection and detection with FM electrodes. Application of a bias across the FM/hBN/graphene tunneling contacts (a) allows to widen the energy window up to ~1 eV for additional spin polarized states in the FM and graphene to participate in the tunneling spin-injection and detection processes, (b) induces a large electric-field between the FM and graphene, which can modify the tunneling processes, (c) provides electrostatic gating for the graphene, which could change the carrier density between electrons and holes, and (d) is predicted to induce magnetic proximity exchange splitting in graphene of up to 20 meVHere we show that bilayer(2L)-hBN tunnel barriers are unique for spin-injection and detection in graphene, with  polarizations unexpectedly reaching values close to ±100% as a function of the applied DC bias at room temperature. Furthermore, we demonstrate a two-terminal (inverted) spin-valve with a record magnitude of the spin signal reaching 800 Ω with magnetoresistance ratio of 2.7%.14 to obtain clean and polymer free graphene-hBN interfaces4 (see “Methods” section for device fabrication details). We use a four-terminal non-local measurement geometry to separate the spin current path from the charge current path  is applied between two Co/2L-hBN/graphene contacts to inject a spin-polarized current in graphene. The injected spin accumulation in graphene diffuses and is detected non-locally (v) between the detector contacts using a low-frequency (f = 10–20 Hz) lock-in technique. For the spin-valve measurements, the magnetization of all the contacts is first aligned by applying a magnetic field By along their easy axes. Then By is swept in the opposite direction. The magnetization reversal of each electrode at their respective coercive fields appears as an abrupt change in the non-local differential resistance Rnl (=v/i). Along with a fixed amplitude i of 1–3 μA, we source a DC current (Iin) to vary the bias applied across the injector contacts. In this way, we can obtain the differential spin-injection polarization of a contact, defined as Is (is) are the DC (AC) spin currents, and study in detail how pin of the contacts depends on the applied bias. We observe with bilayer-hBN tunnel barrier that the magnitude of the differential spin signal ΔRnl at a fixed AC injection current increases with the DC bias applied across the injector  with the stationary solutions to the steady state Bloch equation in the diffusion regime; Ds∇2μs − μs/τs + γBz × μs = 0. Here, the net spin accumulation μs is the splitting of spin chemical potentials spin-up μ↑ and spin-down μ↓, i.e., (μ↑ − μ↓)/2, and γ is the gyromagnetic ratio. In order to obtain reliable fitting parameters, we probe the Hanle signals for a long spin transport channel of length L = 6.5 μm. We measure the Hanle signals for different DC current bias and obtain the fitting parameters τs ~ 0.9 ns, Ds ~ 0.04 m2 s−1, and λs ~ 5.8 μm. We estimate the carrier density −2 from the Einstein relation and the carrier mobility μ ~ 3000 cm2 V−1 s−1 form the Drude’s formula, by assuming Ds = Dc15, where Dc is the charge-diffusion constant. Both the mobility and spin relaxation time are relatively low, which could be due to the ineffective screening of the very thin (~0.7 nm) top-layer of bilayer-hBN from the contamination on the top surface. Due to non-functioning backgate of the device, we could not measure the carrier density dependence of these parameters. For the calculation of mobility, see Supplementary Note In Hanle spin-precession measurements, where the magnetic field λs does not change due to the bias applied between the injector contacts, the bias dependence of the non-local differential spin signal ΔRnl in Figs. Rnl in Fig. 16.L8−9 = 1 μm, the square resistance Rsq ~ 400 Ω, and the width W = 3 μm of graphene. The non-local spin signal as a function of bias due to the spin injection through 8 is obtained from Iin when the magnetization of contacts 7, 8, and 9 are aligned in ↑, ↑, and ↑ configuration, respectively. We find that p cannot be obtained from the spin transport measurements and we define it to be positive for the majority of the unbiased contacts  polarization is dramatically different from what has been observed so far for spin-injection in graphene, or in any other non-magnetic material. For spin-injection/detection with conventional FM tunnel contacts, the polarization does not change its sign close to zero bias. It can be modified at high biaspin for four contacts plotted as a function of the voltage bias obtained from the respective ΔRnl(Iin). All contacts show similar behaviour, where the magnitude of pin increases with bias and changes sign close to zero bias. For the same range of the applied voltage bias, contacts with either 1L-hBN or TiO2 tunnel barriers do not show a significant change in the spin polarization  measured at the detector due to a change in the spin accumulation underneath (Δμs) . However, we find that pd of contact 9 can reach more than 100% above +0.4 V (corresponding electric field is +0.06 V Å−1). We note that the presence of a non-zero DC current in the graphene spin transport channel between injector and detector could modify λs due to carrier drift, and consequently the calculated polarizations have a typical uncertainty of about 10% (Supplementary Note pd cannot exceed 100% (Supplementary Note pd(I) as compared to pin(I)  spin detection polarization (I) Fig. . Due to Concluding, we have obtained a dramatic bias-induced increase in both the differential spin-injection and detection polarizations, reaching values close to ±100% as a function of applied bias across the cobalt/bilayer-hBN/graphene contacts.i + I) is sourced between contacts 8 and 9, and a spin signal  is measured across the same pair of contacts as a function of By  and the two-terminal DC voltage Vt2, respectively, measured as a function of By. As a result of the two-terminal circuit, both the contacts are biased with same I but with opposite polarity, resulting in opposite sign for the injection and detection polarizations. Therefore, we measure an inverted two-terminal differential spin-valve signal R2t with minimum resistance in anti-parallel configuration. We observe a maximum magnitude of change in the two-terminal differential (DC) signal ΔR2t (ΔVt2) of about 800 Ω (7 mV) at I = +20 μA, where R2t and ΔV2t can be observed as a function of DC current bias  spin-independent background. Since we have found that the injection and detection polarizations of the contacts can be enhanced with DC bias, the two-terminal spin signal can now be large enough to be of practical use. For the two-terminal spin-valve measurements, a current bias  ratio of the two-terminal differential spin signal is a measure of the local spin-valve effect, and is defined as R2t(I) is plotted in Fig. V2t(I)  geometries. This confirms the accurate determination of the individual spin-injection and detection polarizations of the contacts.Since we have already obtained the differential spin-injection and detection polarizations of both the contacts 8 and 9 as a function of bias Fig. , we can entclass1pt{minimaI = +20 μA. It is noteworthy that such a large magnitude of spin accumulation in graphene at room temperature has not been reported before.Furthermore, we can now calculate the total spin accumulation in graphene, underneath each contact in the two-terminal biased scheme, due to spin-valve effect. The results are summarized in Table 12 have predicted that an applied electric field in Co/hBN/graphene system can reverse the sign of the proximity-effect-induced equilibrium spin polarization in graphene . Although this study is related to our experimental geometry, the exchange interactions are not relevant for the current discussion because we do not observe any signature of (bias-induced) exchange coupling on the shape of the Hanle signals  except for the magnitude of the spin signals. Another study by Lazić et al.13 on the tunable proximity effects in Co/hBN/graphene has predicted that the system can be effectively gated, and both the magnitude and the sign of the equilibrium spin polarization of the density of states at the Fermi level can be changed due to transverse electric field. However, the spin polarization of the injected current is not calculated. Although these results are relevant to our study, further research is required to understand the results.Recent first-principles calculations of the proximity exchange coupling induced in graphene by Zollner et al.10, the bias-dependent spin-current injection efficiency from Ni into graphene increases up to 100% with the number of hBN tunnel barrier layers. However, these calculations do not show any sign inversion of the spin injection efficiency and do not predict any special role of bilayer-hBN. Therefore, we will not speculate further here on possible explanations of our fully unconventional observations. We note, however, that further research will require the detailed study of the injection/detection processes as a function of graphene carrier density, in particular, the interaction between contact bias induced and backgate induced carrier density. Via these measurements, one could also search for possible signatures of the recently proposed magnetic proximity exchange splitting in graphene with an insulator spacer, hBN13.According to the first-principles transport calculationsIn conclusion, by employing bilayer-hBN as a tunnel barrier in a fully hBN-encapsulated graphene vdW heterostructure, we observe a unique sign inversion and bias induced spin-injection (detection) polarizations between 50% (135%) at +0.6 V and −70% (−60%) at −0.4 V at room temperature. This resulted in a large change in the magnitude of the non-local differential spin signal with the applied DC bias across the Co/2L-hBN/graphene contacts and the inversion of its sign around zero bias. Such a large injection and detection polarizations of the contacts at high bias made it possible to observe the two-terminal differential and DC spin signals reaching up to 800 Ω, and magnetoresistance ratio up to 2.7% even at room temperature. Moreover, we obtain a very large spin accumulation of about 4.1 meV underneath the contacts in a two-terminal spin-valve measurement.20, we expect that we can increase the polarizations even further. Also, one can increase the width of the contacts by a factor of 5 to about 1 μm (yet far below λs), which will reduce the background resistance of two-terminal spin-valve signal by the same factor, and allow to apply a maximum current bias up to 100 μA21. This could result in two-terminal spin signal above 50 mV and MR ratio beyond 20%. The corresponding change in spin accumulation could reach up to 40 meV underneath the contacts, exceeding the room temperature thermal energy (kBT ~ 25 meV). Such high values of spin accumulation will open up an entirely new regime for studying spin transport in graphene and for applications of graphene based spintronic devices2.Note that we have been conservative in biasing the contacts to prevent breakdown of the 2L-hBN barriers. By increasing the bias to the maximum theoretical limit of ~±0.8 V14. The graphene flake is exfoliated from a bulk HOPG (highly oriented pyrolytic graphite) ZYA grade crystal (SPI) onto a pre-cleaned SiO2/Si substrate (2/Si substrate (2/Si is brought in contact with a viscoelastic PDMS (polydimethylsiloxane) stamp which has a polycarbonate (PC) film attached to it in a transfer stage arrangement. When the sticky PC film comes in a contact with a 2L-hBN flake, the flake is picked up by the PC film. A single layer graphene (Gr) flake, exfoliated onto a different SiO2/Si substrate is aligned with respect to the already picked up 2L-hBN flake in the transfer stage. When the graphene flake is brought in contact with the 2L-hBN flake on the PC film, it is picked up by the 2L-hBN flake due to vdW force between the flakes. In the last step, the 2L-hBN/Gr assembly is aligned on top of a 10 nm thick-hBN flake on another SiO2/Si substrate and brought in contact with the flake. The whole assembly is heated at an elevated temperature ~150 °C and the PC film with the 2L-hBN/Gr is released onto the thick-hBN flake. The PC film is dissolved by putting the stack in a chloroform solution for 3 h at room temperature. Then the stack is annealed at 350 °C for 5 h in an Ar-H2 environment for removing the polymer residues.A fully encapsulated hBN/graphene/hBN heterostructure is prepared via a dry pickup transfer method developed in our group−7 mbar during the deposition. To prevent the oxidation of the cobalt, the ferromagnetic electrodes are covered with a 3 nm thick aluminum layer. The material on top of the unexposed polymer is removed via the lift-off process in hot acetone solution at 50 °C, leaving only the contacts in the desired area.The electrodes are patterned via the electron beam lithography on the PMMA (poly(methyl methacrylate))-coated 2L-hBN/Gr/hBN stack. Following the development procedure, which selectively removes the PMMA exposed to the electron beam, 65 nm thick FM cobalt electrodes are deposited on top of the 2L-hBN tunnel barrier for the spin polarized electrodes via electron-beam evaporation. Vacuum pressure is maintained at 1 × 10The data that support the findings of this study are available from the corresponding authors upon request.Supplementary Information