BDNF mRNA levels were significantly decreased in layers IV and V of DLPFC in schizophrenia patients, in layer VI of ACC in schizophrenia and MDD and in layer VI of ITG in schizophrenia, BPD and MDD. BDNF mRNA levels were also significantly decreased in layer V and/or VI of STG in schizophrenia, BPD and MDD. TrkB−TK+ mRNA levels were only significantly decreased in the cortical layer VI of OFC in BPD. The shared and distinct patterns of neurotrophin transcript reductions, with some specific to each group, may compromise the function and plasticity of distinct cortical areas to various degrees in the different groups and contribute to the range and overlap of symptoms manifested across the diagnoses.Abnormalities in brain-derived neurotrophic factor (BDNF)/trkB signaling have been implicated in the etiology of schizophrenia and mood disorders. Patients with schizophrenia, bipolar disorder (BPD) and major depression (MDD) have reduced levels of neurotrophins in their brains when compared with normal unaffected individuals; however, only a few brain areas have been examined to date. Owing to the broad range of symptoms manifested in these disorders, we hypothesized that multiple associative areas of the neocortex may be implicated and that the degree of change in BDNF and trkB−TK+ mRNA expression and the cortical region or layers involved may vary according to Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnosis. We compared BDNF and trkB−TK+ mRNA levels across all layers of the prefrontal cortex , orbital frontal cortex (OFC), anterior cingulate cortex (ACC), inferior temporal gyrus (ITG) and superior temporal gyrus (STG) in four groups: schizophrenia, BPD, MDD and unaffected controls ( Abnormalities in BDNF and trkB−TK+ mRNA levels have been reported in various brain regions in those affected by psychiatric disorders and have been replicated in several cohorts. Specifically, BDNF mRNA and protein are decreased in DLPFC in schizophrenia14 and in DLPFC, ACC16 and hippocampus in mood disorders.18 TrkB−TK+ mRNA abnormalities have been described in the DLPFC,20 ACC21 and hippocampus17 in schizophrenia and in DLPFC, ACC16 and entorhinal cortex of mood disorders.17 Full-length TrkB protein levels are decreased in DLPFC in schizophrenia,20 and in the cerebellum in bipolar disorder (BPD).22 Several studies have also shown BDNF and TrkB mRNA and protein levels decreased in the DLPFC and hippocampus of depressed suicide victims.26 These studies to date, while primarily focusing on the DLPFC and hippocampal regions, nevertheless suggest that the abnormalities in BDNF and trkB−TK+ may be widespread across multiple brain areas; however, it is also possible that neurotrophin abnormalities may vary across cortical regions responsible for different aspects of cognition, emotion and behavior and may differentiate diagnostic categories as defined by Diagnostic and Statistical Manual of Mental Disorders (DSM). On the other hand, it is also apparent that many patients with mental illness do not always fit neatly into a DSM category but display features, both genetic, molecular and symptomatic, that overlap; thus, it is becoming increasing important to understand how the neuropathological changes in the brain of these patients may reflect both the common and different biological and symptomatic features of the disorders.Schizophrenia is a psychiatric disorder characterized by a variety of symptoms including hallucinations, social withdrawal, attention deficits and working memory impairments.in situ hybridization in the DLPFC, OFC, ACC, STG and inferior temporal gyrus (ITG) of 60 subjects in the Stanley Neuropathology Consortium comprising 15 subjects in each of the four groups: schizophrenia, BPD, major depression (MDD) and unaffected controls. None of the previous studies cited above examined neocortical areas in the Stanley Neuropathology Consortium cohort. By including the three diagnostic groups in the current study we will test for possible diagnostic differences in BDNF or trkB−TK+ expression levels across multiple association cortical areas and cortical layers of the brain. The regional, laminar and diagnostic specificities of BDNF/trkB−TK+ signaling deficits in schizophrenia and mood disorders may compromise the function and plasticity of the different areas to various degrees in the different groups and thereby contribute to the range of symptoms manifested across the diagnostic groups.Here we sought to replicate and extend the previous findings by quantifying BDNF and trkB−TK+ mRNA expression by 29 as described below. Details regarding the post-mortem diagnosis, collection and storage of this cohort have been reported previously30 and all the groups were matched for race, sex, age, brain pH, storage interval, post-mortem interval (PMI) and RNA integrity number  as a guide. Coronal 14-μm-thick slices were sectioned from frozen DLPFC, OFC, ACC, ITG and STG regions of interest (ROIs) cut on a cryostat at the Stanley Medical Research Institute. The cohort consists of 60 subjects divided into four groups: schizophrenia, BPD, MDD and unaffected controls . DLPFC sber RIN; .31The human BDNF complimentary DNA template, 511 bps corresponding to nucleotides 704–1214 (accession no. M61176), was inserted into the Apa1 site of the 2.96-kb Bluescript vector . Human TrkB−TK+ complimentary DNA, 216 bps corresponding to nucleotides 1753–1969 (accession no. U12140) was subcloned into the TA site of the 3.9 -kb pCRII vector  and this probe identifies the full-length tyrosine kinase containing version of the TrkB receptor. Probes were purified by DNase treatment, salt and ethanol precipitation followed by ethanol washes. The transcripts recognized with our BDNF and TrkB−TK+ riboprobes were previously confirmed by northern blotting.32 Slides were then incubated with a radiolabeled antisense probe  to a specific activity of ~2.2 × 109) in a hybridization cocktail (300 μl of 5 ng ml−1) and allowed to hybridize overnight at 55 °C in humidified chambers. Additional sections were used for the sense stand probes and were radiolabeled to similar specific activities and hybridized and washed along with the adjacent antisense strand sections. Post-hybridization RNAse digestions and stringent washes were performed as previously detailed.32 All slides were exposed to Biomax MR  film for 14 days (BDNF) and 5 days (TrkB−TK+).Two sections per subject from each of the four coronal levels containing the five anatomical ROIs were fixed, acetylated, delipidated and dehydrated according to standard protocols.−1, Amersham). Three lines (230 μM wide) were placed randomly within the BA of interest but traversing the cortical gray matter perpendicular to the pial surface and optical density measurements were recorded.Brain ROIs were analyzed blind to diagnosis using a calibrated densitometric image analysis (NIH Image 1.33) with a 14C standard , II (11–18%), III (19–45%), IV (46–56%), V (57–72%) and VI (73–100%). The cortical depth percentage of the OFC was obtained using the percentage of cortical widths according to the criteria of Rajkowska et al.28 The percentages of OFC cortical width were as follows: I (1–10%), II (11–20%), III (21–47%), IV (48–61%), V (62–78%) and VI (79–100%). Data for cortical lamina of the ACC were obtained using the percentage of cortical width according to the criteria of Vogt et al.29 The percentages of ACC cortical width were as follows: I (1–8%), II (9–16%), III (17–39%), Va (40–66%), Vb (67–78%) and VI (79–100%). The percent cortical depth occupied by individual cortical lamina of the ITG and the STG were calculated from Nissl-stained sections. The percentage of full cortical width corresponding to the individual lamina of ITG was as follows: I (1–10%), II (11–20%), III (21–48%), IV (49–58%), V (59–70%), VIa (71–84%) and VIb (85–100%) and for STG: I (1–10%), II (11–25%), III (26–52%), IV (53–62%), V (63–80%) and VI (81–100%). Sampling for individual statistical analysis of each lamina was taken from values within these boundaries.Cortical depth percentages of the DLPFC were obtained using the percentage of cortical widths according to the criteria of Rajkowska and Goldman-Rakicpost hoc test was performed in order to determine the diagnostic group responsible for the significant difference. In addition, outliers in the data set were identified after a close visual boxplot inspection, confirmed via the Grubb's test, that resulted in the removal of one to two individuals from each brain region examined.Statistica 6.0 software  was used to test whether differences existed in BDNF and trkB−TK+ mRNA expression in several anatomically defined cortical regions between the four diagnostic groups. Normal distribution was determined with the Kolmogorov–Smirnov test. Pearson's correlations were performed to examine the relationship between continuous variables  and BDNF and trkB−TK+ mRNA measures . If datat-tests for unequal sample size , those taking the medication at the time of death  and normal controls (n=15). ANOVAs were used to examine the effect of these medication categories on BDNF and trkB−TK+ mRNAs in all cortical areas. If an ANOVA reached significance, a post hoc least significance test (LSD) was performed to identify the group comparisons responsible for the significant finding.We determined whether BDNF or trkB−TK+ mRNA varied according to descriptive categorical characteristics  by using ple size . To evalBDNF and trkBTK+ mRNA hybridization signal was expressed throughout the gray matter of all cortical areas examined , whereasP=0.01; F=2.93, df=3, 49, P=0.04; F=4.03, df=3, 49, P=0.01, post hoc testing only showed significant reductions in individuals with schizophrenia compared with controls in layers IV and V  Effect of diagnosis: We found a significant main effect of diagnosis for BDNF mRNA levels in cortical layers I, IV and V of the DLPFC , pH , RIN . TrkB−TK+ mRNA levels correlated with PMI , pH , RIN  and MDD  compared with controls , pH  and RIN  compared with controls .Effect of diagnosis: ANOVAs of BDNF mRNA in the OFC revealed no significant effect of diagnosis  and RIN  compared with controls in layer V and a reduction in BPD , schizophrenia  and MDD  compared with controls in layer VI , pH , RIN , whereas trkB−TK+ mRNA was significantly correlated with PMI , pH , RIN , schizophrenia  and MDD  compared with controls , RIN . TrkB−TK+ mRNA levels correlated with PMI , pH  and RIN .BDNF and trkB−TK+ mRNA levels did not typically differ according to gender, hemisphere, suicide or smoking in any of the areas examined in the overall group . In the n=24), patients free of ADs at time of death (n=21), and unaffected controls (n=15). Whereas ANOVA revealed a significant effect of group in multiple areas, the results that distinguished between the groups on and off ADs were found for BDNF mRNA in DLPFC and trkB−TK+ mRNA in OFC. By LSD analysis, we found BDNF mRNA levels were reduced in layers II and V of the DLPFC in the patient group without recorded use of ADs as compared with the normal control group . TrkB−TK+ mRNA levels were significantly reduced in layers IV and VI of OFC in the patient group without recorded use of ADs as compared with normal controls .There was no correlation between the continuous variable of lifetime neuroleptic use  and BDNF or trk−BTK+ mRNA levels . Howevern=14), those not taking MSs (n=29) and normal controls (n=15). We found no effect of MS status on BDNF mRNA expression in any region . In contrast, by LSD analysis, we found that trkB−TK+ levels were significantly reduced in layer IV of OFC (25%) and layers III (17%), Va (24%) and Vb (21%) of ACC in the patient group not taking MSs as compared with controls . Similarly, trkB−TK+ mRNA levels were significantly lower in layers II (18%), III (17%), IV (19%) and V (19%) of ITG in the patient group not taking MSs as compared with normal controls . In each of these cases, the BDNF and trkB−TK+ mRNA levels in the subjects taking the medications are at levels not significantly different from the controls, whereas those not taking the medications are significantly lower than the controls, and thus the mRNA levels may be normalized by the medications.A similar ANOVA analysis was conducted to compare those taking MSs at the time of death  as compared with controls, they did not reach statistical significance . Specifically, BDNF mRNA levels were significantly decreased in layers IV and V of DLPFC in schizophrenia, in layer VI of ACC in schizophrenia and MDD and in layer VI of ITG in schizophrenia, BPD and MDD. BDNF mRNA levels were also significantly decreased in layer V of STG in schizophrenia and in layer VI of STG in schizophrenia, BPD and MDD. These results corroborate those of earlier studies that indicate low expression of BDNF mRNA in the DLPFC of individuals with schizophrenia to include identification of reductions in a 4th cohort.ificance . Taken t20 we did not find a decrease in trkB−TK+ mRNA in DLPFC, or in any other area, in schizophrenia. However, our data do show a 12–13% decrease in expression of trkB−TK+ in layers V and VIa in DLPFC in SCH as compared with controls that did not reach statistical significance (20 that reached a trend level of significance (P=0.059). Thus, it is not surprising that we were unable to detect a significant decrease with this smaller cohort (n=15 per group) with film-based analysis. Interestingly, the more significant change in trkB receptor levels was previously found in the truncated form of the trkB receptor. The trkB−TK− mRNA levels were significantly increased in the DLPFC in schizophrenia, thereby significantly altering the ratio of trkB−TK+ to trkB−TK− mRNA and protein levels.20 The truncated trkB form is capable of binding to BDNF; however, it is not able to mediate the classic neurotrophic response via tyrosine signaling. Thus, an excess of trkB−TK− could contribute to an overall reduction in BDNF/trkB signaling in the DLPFC in schizophrenia.In contrast to previous studiesificance . This isWhereas trkB−TK+ mRNA levels were not significantly reduced in any other cortical areas in schizophrenia either, we did find that levels tended to be lower in schizophrenia as compared with controls across layers II–VI (16–27%) in OFC, layers III−VI (14–22%) in ACC, layers II–VIa (13–17%) in ITG and layers II–VI (24.5–28%) in STG. These decreases in trkB–TK+ combined with the significant decreases described in various layers for BDNF would indicate that BDNF/trkB signaling is likely to be compromised across multiple cortical areas in schizophrenia.21 hippocampus33 and amygdala34 in subjects with mood disorder. We find that BDNF mRNA is significantly decreased in layer VI of ACC in MDD, and in layer VI of ITG and STG in both MDD and BPD. We also find that TrkB−TK+ mRNA is significantly decreased in layer VI of OFC in BPD. Thus, just as in schizophrenia, the abnormalities in BDNF/trkB signaling appear to be localized to the deep layers of the cortex and indicate that the neurons in subcortical areas that are supplied to the BDNF by the cortex, including thalamus and striatum, to which they project may have compromised neurotrophic support in these disorders. Many of these subcortical neurons, particularly the GABA neurons of the striatum require BDNF, which is anterogradely transported and released at the axon terminal, for their differentiation and survival.38Previous studies have also shown a decrease in levels of BDNF and/or trkB in cingulate cortex,39 or OFC40 in any diagnostic group. In the ACC, there is an increase in neuronal density in layer V in schizophrenia and BPD and in layer VI in schizophrenia and MDD,41 which would also indicate a downregulation of mRNA in existing neurons rather than a loss of neurons. There is evidence for a decrease in neuron soma size in deep layers of the cortex in this cohort43 that may reflect the decrease in BDNF particularly since BDNF was initially defined as a molecule necessary to promote cortical neuron somal size and dendritic differentiation during postnatal development and for the maintenance of viable neurons into adulthood.45 However, in the adult brain the primary functionof BDNF/trkB signaling maybe to potentiate glutamate synaptic transmission and plasticity46 and promote synaptic consolidation.47 Consequently, the decrease in BDNF that we find may contribute to the abnormalities that are found in markers of synaptic function and plasticity described in the cortex of these cases54 and which have also been described in other cohorts.58TrkB−TK+ is expressed homogenously throughout all layers of cortex with a modest increase in middle cortical layers, whereas BDNF synthesis appears to be enriched or exclusive to pyramidal neurons and is more highly expressed in layers V/VI than in more superficial layers I−IV. TrkB−TK+ is expressed almost exclusively in neurons  in the cortex and is necessary for activating the second messenger signaling and mediating the neurotrophic effects of BDNF. The decrease in TrkB−TK+ and BDNF mRNA that we find in the deep cortical layers is likely to be due to a downregulation of the mRNA in existing neurons and not due to a reduction in the number of neurons, as previous post-mortem studies in this cohort have revealed no clear change in neuron number or density in any layer of the DLPFC59 Likewise, it may be that the blood levels of BDNF in the periphery reflect the brain levels as has been shown in the development and aging of the rat.60 Thus, measuring peripheral BDNF levels may provide a biomarker for disease state or provide a marker for treatment response. However, meta-analysis of numerous studies shows that BDNF is generally decreased in the blood of patients with MDD,63 in both manic and depressive episodes of BPD64 and in schizophrenia.65 Thus, with the general decrease in peripheral levels across diagnostic groups in contrast to the specificity of brain deficits to particular areas, lamina and diagnosis, it is unclear as to how informative peripheral levels of BDNF will be.The current study is reporting on mRNA expression; however, it is likely that the deficits in mRNA levels are reflected in corresponding decreases in protein levels as has been described previously in several studies on human brain.19 Rodents treated with typical antipsychotics generally have reduced levels of BDNF expression, whereas those treated with atypicals tend to have increased expression.71 The patients in this study were prescribed both types of antipsychotics; therefore, it is not possible to extrapolate effects from the rodent studies. However, we can say that BDNF levels did not correlate with fluphenazine equivalent levels of antipsychotic in our study or in previous postmortem studies.19BDNF and trkB−TK mRNA levels were generally not affected by gender, hemisphere, suicide, history of substance abuse or by smoking. Lifetime intake of neuroleptics was also found to have no correlation with BDNF or trkB−TK+ mRNA levels. Previous human post-mortem studies also found no correlation between antipsychotic use and BDNF and trkB−TK+ mRNA levels.72 Several previous studies have also shown a decrease in levels of BDNF and/or trkB in various brain areas in mood disorders,73 and just as we show here, many find that patients free of ADs express significantly less BDNF and trkB−TK+ than the patients taking ADs at the time of death.73 Moreover, treatment with ADs will upregulate BDNF mRNA and protein in the rodent brain.79 Interestingly, the rapid-acting AD effects of ketamine appear to be mediated through an increase in BDNF protein synthesis.80 It is likely that these robust AD effects are a consequence of rapid changes in BDNF-mediated synaptic plasticity, which may provide a more direct target for future drug development.In contrast to the lack of antipsychotic affect, the use of ADs or MSs did appear to affect the level of BNDF and trkB−TK+ RNA levels in some layers and cortical regions. BDNF mRNA levels were significantly reduced in DLPFC in patients who had not taken AD at around the time of death as compared with patients who had taken them. This suggests that the reductions in BDNF that we found in all three major mental illnesses may have been greater if we had studied only those without a history of AD or MS use. Similarly, TrkB−TK+ mRNA levels were significantly reduced in the cortical regions in patients who had not taken ADs (OFC) or MSs  at around the time of death as compared with patients who had taken them. Thus, those individuals with major mental illness with a history of mood medication often had BDNF and trkB−TK+ mRNA levels comparable to the control group, thus suggesting that this medication may be normalizing an underlying deficiency in BDNF and trkB−TK+ mRNA levels. These studies support the hypothesis that one of the beneficial mechanisms of action for these compounds may include induction of neurotrophin gene expression in widespread areas of the brain.17 This anatomical separation of BDNF/trkB−TK+ signaling deficits may reflect the differences in affective symptoms that manifest between the different mental disorders. The shared patterns of reductions may contribute to the overlap of symptoms manifested across the diagnostic groups. Dissecting the various regional, laminar and cell-type-specific patterns of abnormality that are relevant to the different diagnostic categories may provide a useful guide when considering future therapeutic strategies.An understanding of the regional, laminar and diagnostic specificities of BDNF and trkB−TK+ deficits in schizophrenia and mood disorders may provide a guide for future therapeutic strategies aimed at this system. Our findings indicate that, whereas BDNF mRNA is decreased in all three diagnostic groups in cingulate and temporal cortices, the frontal cortex is more affected in schizophrenia and the hippocampus is more affected in mood disorders.


Repression of retrotransposons is essential for genome integrity and the development of germ cells. Among retrotransposons, the establishment of CpG DNA methylation and epigenetic silencing of LINE1 (L1) elements and the intracisternal A particle (IAP) endogenous retrovirus (ERV) is dependent upon the piRNA pathway during embryonic germ cell reprogramming. Furthermore, the Piwi protein Mili, guided by piRNAs, cleaves expressed L1 transcripts to post-transcriptionally enforce L1 silencing in meiotic cells. The loss of both DNA methylation and the Mili piRNA pathway does not affect L1 silencing in the mitotic spermatogonia where histone H3 lysine 9 dimethylation (H3K9me2) is postulated to co-repress these elements.Here we show that the histone H3 lysine 9 dimethyltransferase G9a co-suppresses L1 elements in spermatogonia. In the absence of both a functional piRNA pathway and L1 DNA methylation, G9a is both essential and sufficient to silence L1 elements. In contrast, H3K9me2 alone is insufficient to maintain IAP silencing in spermatogonia. The loss of all three repressive mechanisms has a major impact on spermatogonial populations inclusive of spermatogonial stem cells, with the loss of all germ cells observed in a high portion of seminiferous tubules.Our study identifies G9a-mediated H3K9me2 as a novel and important L1 repressive mechanism in the germ line. We also demonstrate fundamental differences in the requirements for the maintenance of L1 and IAP silencing during adult spermatogenesis, where H3K9me2 is sufficient to maintain L1 but not IAP silencing. Finally, we demonstrate that repression of retrotransposon activation in spermatogonia is important for the survival of this population and testicular homeostasis. This Ln Figure E. Given a Figure F. We nexa Figure G-J; howea Figure G. The abcapacity . EnrichmCKO; MiliKO mice with either the complete loss of all germ cells, or with spermatogonia being the only germ cells present within the seminiferous tubules. Thus, not only are meiotic cells acutely sensitive to L1 reactivation and the ensuing DNA damage but spermatogonia are as well. The observed genomic damage in G9aCKO; MiliKO spermatogonia could be a result of L1 derepression alone or in conjunction with the observed IAP deregulation. The fact that IAP reactivation is observed in MiliKO spermatogonia without DNA damage could indicate that other post-transcriptional mechanism (s) are in place to inhibit IAP translocation such as the APOBEC RNA editing pathway that has been shown to restrict ERVs inclusive of IAP elements [KO spermatogonia and does not elicit a robust DNA damage response. It is very interesting that both L1 and IAP retrotransposons that depend upon the piRNA pathway for the establishment of epigenetic silencing [KO spermatogonia indicates that DNA methylation and or the piRNA pathway are predominantly required for the maintenance of IAP silencing therein. In contrast to L1, the conditional loss of Mili or specifically its endonuclease activity in meiotic cells that does not affect CpG DNA methylation patterns, has no impact on IAP silencing [CKO spermatocytes that are devoid of the piRNA post-transcriptional silencing pathway would indicate that CpG DNA methylation is sufficient and the key mechanism for the maintenance of IAP silencing. In summary, here we show that G9a-mediated H3K9me2 is sufficient to maintain L1 silencing in the absence of L1 CpG DNA methylation and the piRNA pathway within spermatogonia. Finally, as G9a is broadly expressed our findings may indicate a role for G9a and H3K9me2 in L1 silencing in other cell types.Here we demonstrate a role for G9a and H3K9me2 in the repression of L1 elements during adult spermatogenesis. This function of G9a is redundant with CpG DNA methylation and the Mili-piRNA pathway; it is only when all three mechanisms are ablated that L1 becomes derepressed within spermatogonia. In the absence of both DNA methylation and the Mili piRNA pathway, G9a is both necessary and sufficient to maintain L1 silencing. Thus, with respect to other adult germ cell populations, spermatogonia are unique in employing three distinct mechanisms to repress L1. The SSC that maintains spermatogenesis throughout adult life resides within the population of undifferentiated spermatogonia. Therefore having three distinct L1 defensive mechanisms in place within SSCs has major protective advantages for the long-term genomic quality of the gametes. The consequence of L1 activation in spermatogonia are dire as evidenced in the G9aelements -24. Alteilencing ,7-9 haveMili-, G9aFl and Rosa26ERT2Cre alleles were described previously [o116) under license from the Italian health ministry. Requests for G9aFl mice should be addressed to Alexander Tarakhovsky (The Rockefeller University). The Mili- allele is available from European Mouse Mutant Archive  on a non-collaborative basis.The eviously ,15,25. Thttp://fiji.sc/Fiji).Rabbit polyclonal L1 ORF1 antisera were made through immunization of rabbits with recombinant ORF1 protein. The following antibodies were used at the indicated dilutions for IF: anti-G9a (1:50) , anti-ORF1 L1 (1:500), mouse monoclonal anti-GLP  (1:100), anti-IAP Gag (1:500) , mouse monoclonal anti-γH2AX  (1:500), mouse monoclonal anti-H3K9me2  (1:100) and rabbit polyclonal anti-Plzf  (1:100). Immunofluorescence and histology were performed as described [Northern blotting detection of L1, IAP and MuERV-L transcripts from total testicular RNA was performed as described  using anQuantitative PCR from total testis was performed as previously described . H2Afz win vitro cultured SSC cells [Chromatin immunoprecipitation with both H3K9me2 and isotype control antibodies were performed as previously described [SC cells  were FACCKO: conditional knock out; ERV: endogenous retrovirus; HMTase: histone methyltransferase; H3K9me2: histone H3 lysine 9 dimethylation; IAP: intracisternal A particle; i.p.: intraperitoneally; KO: knock-out; LINE1 or L1: long interspersed elements 1; MuERV-L: murine endogenous retrovirus-like; ORF1: open reading frame 1; piRNA: Piwi-interacting RNA; qRT-PCR: quantitative reverse-transcriptase PCR; SSC: spermatogonial stem cell; TMX: tamoxifen.The authors declare that they have no competing interests.Fl allele. Srinath CS generated the G9a antibody. DO’C conceived and supervised the experiments and wrote the manuscript. All authors read and approved the final manuscript.MDG designed and performed the majority of the experiments. SC performed the northern blotting, ChIP and qRT-PCR experiments. Srihari CS provided the G9a


We here report on the influence that the CVD parameters  have on the morphology, transmittance, and electrical conductivity of the graphene films grown with pyridine. A temperature range between 930 and 1070 °C was explored and the results were compared to those of pristine graphene grown by ethanol-CVD under the same process conditions. The films were characterized by atomic force microscopy, Raman and X-ray photoemission spectroscopy. The optical transmittance and electrical conductivity of the films were measured to evaluate their performance as transparent conductive electrodes. Graphene films grown by pyridine reached an electrical conductivity of 14.3 × 105 S/m. Such a high conductivity seems to be associated with the electronic doping induced by substitutional nitrogen atoms. In particular, at 930 °C the nitrogen/carbon ratio of pyridine-grown graphene reaches 3%, and its electrical conductivity is 40% higher than that of pristine graphene grown from ethanol-CVD.Graphene films were produced by chemical vapor deposition (CVD) of pyridine on copper substrates. Pyridine-CVD is expected to lead to doped graphene by the insertion of nitrogen atoms in the growing sp Transparent conductive electrodes (TCEs) are an indispensable component of many kinds of electronic devices, such as displays, touch-screens, light emitting diodes, and solar cells –8. SinceThe impact that graphene will have on many fields of electronics ultimately depends on the real properties it will be able to provide. Thus, doping of graphene is currently considered a promising way of enhancing its carrier density and improving the electrical conductivity to satisfy the requirements of various electronic applications . NeverthGraphene can be doped through surface proximity by layering it with other materials  that can fit within the graphene lattice . When bo5H5N) is a liquid precursor akin to ethanol (C2H5OH). The main difference between the two is that ethanol is generally used to make pristine graphene while pyridine can be used to form nitrogen-doped graphene. Our group recently showed that highly-crystalline, pristine graphene can be produced by ethanol-CVD on copper above 1000 °C [2H2) and hydrogen cyanide (HCN) [2 flow in the vapor mixture) to assess their effect on the graphene properties. In order to better evaluate such effect and assess the occurrence of nitrogen doping, we further run a systematic comparison with the graphene films made by ethanol-CVD under analogous conditions [In this paper, we grew graphene films by CVD of pyridine on copper surfaces, evaluating the occurrence of doping by substitutional nitrogen atoms in the films. Several groups recently started to produce graphene using liquid carbon precursors such as ethanol  because of their effectiveness, safety and low-cost. Pyridine (C 1000 °C –31. Howe 1000 °C . Ethanol 1000 °C . Likewisde (HCN) , which ide (HCN) . To datenditions .2 (300 nm) and analyzed by micro-Raman spectroscopy. At the optical microscope, the graphene films showed the typical features of those grown by other carbon precursors (such as methane and ethanol) on commercial copper foils. They are overall uniform and have some darker areas running along parallel stripes, which are graphene grains developed at sites of secondary nucleation induced by the lamination process undergone by the copper foil [The graphene films grown by pyridine for 10 min at different temperatures  with two different hydrogen flows (1 and 100 sccm) were transferred on Si/SiOper foil –37.ID/G, I2D/G, ID/D’) are listed in −1 and ≈2700 cm−1, respectively) are present. The D and D’ peaks  are also visible and usually indicate the presence of defects in the graphitic lattice such as disordered carbons, edges and wrinkles [In all the Raman spectra, the typical G and 2D peaks of graphene  are reported in Table S1 , whereas at 930 °C the films seem to be composed of 1 to 3 layers (I2D/IG between 1.29 and 1.55). This situation is in line with graphene grown through ethanol-CVD  λ4(ID/IG)−1 (with λ being the Raman exicitation wavelength and ID/IG integrated intensity ratio) [La values obtained at 930 °C indicate a low average inter-defect distance; however the narrow full width half maximum (FWHM) of ≈28 cm−1 seems to point out that these highly dense defects should belong to a single type [ID/G ratios of the samples grown by pyridine (0.92 at 1 sccm H2 and 1.16 at 100 sccm H2) are consistently higher than those of the ethanol samples (0.68 at 1 sccm H2 and 0.25 at 100 sccm H2). This ID/IG increment for pyridine-CVD might be thus ascribed to the insertion of nitrogen in the graphene film.Concerning the D and D’ peaks, the highest intensities are observed at the temperature of 930 °C (y ratio) . The lowgle type . CompariID/ID’ intensity ratio can be used experimentally to get information about the nature of defects in graphene [ID/ID’ ratio is found to be maximal (about 13) for defects associated with sp3 hybridization. It decreases for vacancy-like defects (about 7), and reaches a minimum for boundary-like defects (about 3.5). In the spectra of the pyridine-CVD films grown at 930 °C, the values of ID/ID’  [2 hybridized) for nitrogen within a carbon lattice  [Samples grown at 930 °C were further analyzed in detail by XPS . The widaphitic)  and oxidaphitic) –48. Pyriridized) . Specifiridized) –51: in t3-hybridized component in XPS is found to be in agreement with the measured Raman ID/ID’ ratio, which points only to sp2, boundary- or point-like defects.It should be noted that the curve deconvolution of the N1s spectra does not seem to show trace of pyrrolic N, i.e., N atoms contributing two p electrons to the π system . When present, the peak from pyrrolic N is expected to be located in between the pyridinic and quaternary peaks, at a distance of about 1.1–1.2 eV in binding energy from each of them . In our The samples grown at 1000 and 1070 °C were also investigated by XPS. The 1000 °C sample showed evidence of nitrogen, but the signal-to-noise was not high enough to reproducibly assess the N/C content and/or ascertain the binding components. In the 1070 °C samples it was not possible to univocally determine the presence of nitrogen. Overall, the XPS spectra at each temperature were observed to be not affected by the hydrogen flow, as the Raman analysis.2 surface. Occasionally, some folded regions can be found, as well as some wrinkles, as expected. The thickness was measured by taking profiles of the films grown at different temperatures. The measured values are in the range of about 1–2 nm, and a monotonic increase in thickness (t) with the temperature is observed. All these results are consistent with the analysis of the Raman I2D/IG ratios. These values can be used to estimate the number of layers of the graphene films: 1–2 at 930 °C, 3–4 at 1000 °C, and 6 at 1070 °C.The graphene films were analyzed by atomic force microscopy (AFM) to investigate their morphology and evaluate their thickness . The filIn T = (1 + Z0/2 σOp·t)−2, where Z0 is the impedance of free space  [t is the thickness. The value of t at each temperature was set taking in consideration the maximum number of layers as estimated by AFM and the optical transmittance of the film. This is a conservative approach, as the graphene films can occasionally appear thicker when analyzed by AFM in tapping mode [t930 °C = 0.9 nm, t1000 °C = 1.3 nm, t1070 °C = 2.1 nm [The optical conductivity was calculated as o 377 Ω) , and t iing mode . Conside= 2.1 nm .We plotted the optical transmittance vs the CVD temperature for ethanol- and pyridine-grown films  to gain Rs = (σDC·t)−1. We have also calculated a figure of merit (FoM) that can be used to compare the electrical/optical properties of thin transparent conductors made from various materials. Such FoM is defined as the conductivity ratio, σDC/σOp [The electrical properties of the graphene films grown by pyridine-CVD are reported in  σDC/σOp .The temperature is the main parameter dictating the characteristics of the pyridine-CVD graphene films, as also observed for the optical properties and the thickness of the films. At each temperature, the sheet resistance is observed to sweep over a narrow range of values when adding the two different hydrogen flows. Hydrogen seems to lead to a slight decrease in sheet resistance at 1000 and 1070 °C, while this trend is reversed at 930 °C. The electrical characteristics of the pyridine films can be compared to those of ethanol-CVD graphene in DC up to 11.9·105 S/m for the pyridine sample). This points to a negligible effect of doping in the pyridine samples at 1070 °C, as confirmed by Raman and XPS, and as expected at such a high temperature [To account for the intrinsic electrical properties of the graphene films, without reference to the thickness, a study of their electrical conductivity is in order. Overall, the electrical conductivity of graphene from both pyridine and ethanol is observed to increase with the CVD temperature, as expected (and as demonstrated by the progressively lower D peak in the Raman spectra) ,57. At 1perature .DC up to 10.4·105 S/m). Instead, in the case of pyridine, the film grown at 1070 °C has a lower conductivity (11.9·105·S/m) than the film grown at 1000 °C (14.3·105 S/m). Upon decreasing the temperature to 930 °C, the electrical conductivity of the samples from both precursors decreases due to the evident defectiveness of the growth at such low temperature . This situation strongly supports the occurrence of nitrogen doping during pyridine-CVD below 1000 °C, and in particular at 930 °C, as evidenced previously by the Raman and XPS analysis. Such values of electrical conductivity also demonstrate that the ID/G Raman ratios observed in pyridine-derived graphene, which are higher than those of ethanol-derived graphene, are due to quaternary nitrogen doping and not to a more defective graphene lattice. In fact, a defective lattice would rather impair the electrical conductivity making it lower than that of ethanol-CVD graphene [Upon lowering the temperature to 1000 °C, some differences in the two kinds of graphene films emerge and a weak effect of doping can be detected in the pyridine-derived films . With the CVD of ethanol, at 1070 and at 1000 °C pristine graphene films were grown with the same conductivity  The film thickness is directly proportional to the CVD temperature. ii) The electrical conductivity generally improves when increasing the CVD temperature and hydrogen flow, due to a higher graphitization level and to a lower defect density. iii) The electrical conductivity is also linked to the doping level, which is however less pronounced above 1000 °C.5 S/m, i.e., consistently higher values than the pristine samples grown by ethanol-CVD.Graphene films were grown by chemical vapour deposition (CVD) using pyridine as liquid carbon precursor, exploring a range of temperatures between 930 and 1070 °C, and assessing the effect of hydrogen in the CVD gas mixture. A comprehensive range of characterizations  was run to ascertain the properties of the graphene samples and the occurrence of doping. These results were compared to the case of pristine graphene films grown by CVD of ethanol, a liquid precursor akin to pyridine. The graphene films grown at high temperatures (1070 °C) are in both cases multilayered with high crystalline quality. Upon lowering the temperature below 1000 °C, the graphene films are on average thinner (down to one or two layers) and with a higher defect density. In particular, at 930 °C significant differences emerge in the Raman spectra of the graphene films grown by the two precursors: the pyridine samples show G, 2D and D band shift which are usually ascribed to n-type doping, while the ethanol samples do not show such trends. XPS spectra confirmed the presence of nitrogen in pyridine-films grown at temperatures below 1000 °C. Graphene grown from pyridine at 930 °C (with 1 sccm of hydrogen flow) had a nitrogen/carbon ratio of 3%, and from the N1s peak analysis the nitrogen atoms appear in quaternary form . We conclude that when the CVD temperature is lowered below 1000 °C, non-carbon atoms or groups can insert in the C lattice forming doped graphene. As a result, below 1000 °C the effect of nitrogen doping emerges among these effects and contributes to bring the electrical conductivity of the pyridine samples up to 14.3·10Graphene films were grown onto 25 μm thick copper foil  cut to the desired size and placed over a quartz boat, after cleaning with acetone and ethanol.2. The quartz boat supporting the samples was then inserted into the hot zone and annealed for 1200 s at the growth temperature. Liquid pyridine was contained in a steel “bubbler” vessel pressurized in Ar at 3 bar, which was kept at 20 °C (about 15 mbar equilibrium pressure). After the annealing, the H2 gas flow was set to the desired value and the Ar flow was switched from the bubbler vessel at a flow rate of 20 sccm. The amount of vapor entering the chamber was estimated to be 0.5% (15 mbar/3 bar) of the carrier flow, thus 0.1 sccm. After the desired growth time, the samples were extracted from the hot zone, let to cool to near room temperature, and then extracted from the vacuum vessel and further processed for extracting the graphene. When performing the growth with ethanol, the procedure was the same, with the only difference being that the steel “bubbler” vessel in this case contained liquid ethanol (pressurized in Ar at 3 bar) and was kept at 0 °C. It can be noted that the CVD of pyridine requires the same experimental setup as the CVD of ethanol, being both precursors liquid under standard conditions and having similar vapor pressures (about 15 mbar at 20 °C for pyridine and at 0 °C for ethanol).Graphene was grown in a low-pressure CVD reactor and the graphene films were transferred onto the substrates of use by a wet transfer method. The reactor consists of a 2 m long, 38 mm inner diameter quartz tube, coaxial to a high temperature furnace. The tube is connected to a rotary vane vacuum pump, a mass flow controlled gas feed system and absolute pressure gauges. The long reactor tube allows the samples to be inserted in and extracted from the heated section without breaking the vacuum or perturbing the gaseous atmosphere. The system design implements a fast cooling scheme allowing several samples to be grown within a single furnace heating cycle, without exposing them to the atmosphere when hot. After the initial ramping of the furnace temperature the pressure was stabilised at 4 mbar by flowing 20 sccm Ar and 20 sccm HGraphene was transferred onto 300 nm thermal oxide-coated Si wafers for Raman, XPS, AFM and sheet resistance measurements, and onto glass substrates for optical transmittance. The graphene transfer was performed using a cyclododecane protective layer, a novel method recently developed by our group. Cyclododecane is a waxy solid which sublimates at ambient temperature in a few hours without leaving residues and it was recently demonstrated not to alter Raman nor XPS spectrum of graphene when used, with respects to the free floating method . To furt2 crystallinity and mechanical strain were investigated using Raman spectroscopy, with a HORIBA Scientific LabRAM HR Evolution Raman spectrometer with an integrated Olympus BX41 microscope. Laser excitation wavelength of 532 nm (2.33 eV) was focused on the sample surface using a 100× objective with a spot size of approximately 1 μm. Low laser power (below 1 mW) was used to minimize sample heating and possible damages. Because of possible inhomogeneity in the films, the spectra were recorded at ten different spots and averaged.Doping level, degree of sp2 were probed by X-ray photoemission spectroscopy (XPS) with Mg Kα X-ray radiation at 1253.6 eV . The samples were treated in air at 200 °C for a few minutes before XPS to remove possible organic contaminants.The electronic and structural properties of the graphene films on Si/SiOGraphene-films transferred on silicon have been characterized by tapping-mode atomic force microscopy (using a Bruker-Veeco Dimension Icon AFM). The images were acquired in tapping mode at 0.5 Hz, using Sb-doped Si cantilevers (Bruker) with resonant frequency around 300 kHz. At least five areas for each sample were measured in order to take into account thickness inhomogeneity which might derive from the film transfer (the AFM images shown in 2 was measured in a controlled environment under constant temperature (23 ± 0.5 °C) and humidity (35 ± 5%). The test system consisted of a probe with four collinear WC tips , fixed on a stand (Signatone S301) and connected to a current source and a low-voltage meter. The measurements were repeated multiple times over an area of at least 1 × 1 cm2 on each sample and then averaged.The electrical sheet resistance of the films transferred to Si/SiOTransmittance spectra at normal incidence have been recorded in the 400–1100 nm wavelength range by a fiber optics spectrophotometer (HR4000CG-UV-NIR model by Ocean Optics), equipped with a tungsten halogen source and connected to the sample stage by quartz optical fibers  with quartz collimating lens mounted on their ends (spot size on the sample about 1 mm). For each sample, the measurement was repeated in three sites to average over film inhomogeneity.In the Supporting Information the results of the Raman spectral fitting for pyridine- and ethanol-CVD are reported.File 1Results of the Raman spectral fittings.


A systematic series of experiments were carried out to outline the relation between inserted defect creation and the fluorination process. Based on a subsequent X-ray photoelectron spectroscopy (XPS) analysis, a 6-fold increase of the fluorine concentration on graphene under simultaneous irradiation was observed when compared to fluorination under normal conditions. The fluorine atoms are predominately localized at the defects as indicated from scanning tunneling microscopy (STM). The experimental findings are confirmed by density functional theory which predicts a strong increase of the binding energy of fluorine atoms when bound to the defect sites. The developed technique allows for local fluorination of graphene without using resists and has potential to be a general enabler of site-selective functionalization of graphene using a wide range of gases.The functionalization of graphene remains an important challenge for numerous applications expected by this fascinating material. To keep advantageous properties of graphene after modification or functionalization of its structure, local approaches are a promising road. A novel technique is reported here that allows precise site-selective fluorination of graphene. The basic idea of this approach consists in the local radicalization of graphene by focused ion beam (FIB) irradiation and simultaneous introduction of XeF Light and ion beams have been used intensively to enhance the surface functionalization, both for removing and adding atoms12456Graphene functionalization is a key enabler to open graphene to applications and has attracted significant interest, because it can alter the chemical, electronic and structural properties of this fascinating material89101112131617182 atmosphere in high vacuum. The main idea of this approach is to radicalize the graphene surface locally by utilizing high-energy ion irradiation and, simultaneously, provide a fluorine containing precursor molecule to the carbon atom while it is in the radicalized state. In this method, the fluorine atoms are predominately localized in the nanometer regime surrounding the inserted defects. X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, scanning tunneling microscopy (STM) and density functional theory (DFT) calculations were performed to verify the formation of fluorinated graphene and explain the mechanism of focused ion irradiation induced fluorination.In this work, we carried out the site-selective fluorination of graphene by using focused ion beam irradiation in XeF2 exposure time of 1013 ions/cm2 and 167 s respectively. A selected irradiation area of 100 × 100 μm2 (shown in The graphene was fluorinated by using ion dosage and XeFshown in  SEM imag+) irradiation on graphene samples in FEI Strata DB235 (FIB/SEM) under high vacuum conditions (5.5 × 10−5 mbar), while simultaneously, XeF2 was supplied during the irradiation by the gas injection system (GIS) inside FIB/SEM chamber with a partial pressure at the nozzle exit of 600 PaThe fluorination was performed by a low dosage focused ion beam , which is nearly negligible in the pristine graphene sample, increases clearly after irradiation. In contrast, the intensity of the 2D band (at 2700 cm−1) decreases sharply, which indicates the translational symmetry of the sp2 carbon bonds in graphene is broken in both irradiated samples. Compared with DG, the intensity ratio of D and G band (ID/IG) in FG is significantly lower, which could correspond to a lower degree of structural disorder. From the ID/IG ratio that we obtained, an effective crystalline size that is bigger in FG (7 nm) than in DG (5.7 nm)24When high-energy Ga spectra  display 2 in fluorinated graphene and the surface is covered by defects, corrugations and bright features. The corrugations correspond to the standing waves with different structure particularly around the defects. The Fast Fourier Transform of 2 bonds in To understand the atomic structure further, more STM experiments were performed on FG sample shown in F2) is higher than that of F1 (IF1), and IF1 is around five times larger than IF4. To explain the result, a simple model could be descripted taking sample F1 as an example. In sample F1, when the gas nozzle is open, XeF2 will be injected into the vacuum chamber and form a thin film on graphene surface+ ions remove carbon atoms from the graphene lattice and then propagate into the substrate deeply, resulting in the local radicalization of graphene surface. The nature of this radicalization is the formation of carbon dangling bonds as well as the activation of carbon atoms surrounding defects. These carbon atoms can efficiently react with XeF2. When the ion irradiation and gas exposure are closed simultaneously, the gas film on graphene surface evaporates and the fluorinated graphene remains. Accordingly the study of Utke et al.2 will not be decomposed under ion irradiation, which confirms our explanation that the fluorination occurs between defects and XeF2. The difference between F1 and F2 is that, before gas exposure, F2 has an extra ion irradiation, implying more defects are formed. The higher fluorine intensity of F2 versus F1 could be explained by the defects created in the first step of F2 being at least partially saturated by fluorine. In contrast to F1, F3 shows lower fluorine intensity which can be explained as the partial saturation of defects by residual gas molecules present in the vacuum chamber within the time gap between ion irradiation and gas exposure. The intensity increase in F3 as compared to F4 equally can be understood by this mechanism. F4 is the control experiment, and the fluorine is mainly from the direct reaction between graphene and XeF22 exposure of graphene17In order to better understand the mechanism of the fluorination in graphene, more comparison experiments were designed and the schematic details are shown in 13 ions/cm2. In these two models, there are four typical positions marked as site A-D. The calculated energetics shows that the adsorption energy 32272, epitaxial graphene on SiC has better surface quality and thus is suitable for STM imaging. However, SiC can react with injected XeF2 gas, making the analysis of graphene using XPS more ambiguous, while SiO2 substrate is more inert to fluorination17Fluorination experiments were performed on large scale graphene synthesized by chemical vapor deposition (CVD) and transferred to SiOBefore and after irradiation, Raman spectroscopy  and X-ray photoelectron spectroscopy (Quantum 2000 Scanning ESCA) spectra were taken to verify the formation of fluorinated graphene. Scanning tunneling microscopy  was utilized to observe the atomic structure of irradiated graphene.−5 eV and 10−2 eV/Å, respectively. For geometry optimization, a 5 × 5 × 1 Gamma centered k grid was used. For total energies and electronic structures, we have used an 11 × 11 × 1 Gamma centered k grid. The adsorption energy (Eads) of F atom adsorbed in graphene sheet is calculated using the following formula,Ab initio density functional theory calculations are performed by using a plane wave based pseudo potential method (VASP) to study the interactions between fluorine and carbon atoms3638E(Sheet + F) is the total energy of the F atom adsorbed in on the sheet, E(Sheet) is the total energy of the sheet without F atom and E(F) is the total energy of an isolated F atom kept in a box.where How to cite this article: Li, H. et al. Site-selective local fluorination of graphene induced by focused ion beam irradiation. Sci. Rep.6, 19719; doi: 10.1038/srep19719 (2016).


Plasmons in the visible/UV energy regime have attracted great attention, especially in nano-materials, with regards to applications in opto-electronics and light harvesting; tailored enhancement of such plasmons is of particular interest for prospects in nano-plasmonics. This work demonstrates that it is possible, by adequate doping, to create excitations in the visible/UV regime in nano-carbon materials, i.e., carbon nanotubes and graphene, with choice of suitable ad-atoms and dopants, which are introduced directly into the lattice by low energy ion implantation or added via deposition by evaporation. Investigations as to whether these excitations are of collective nature, i.e., have plasmonic character, are carried out via DFT calculations and experiment-based extraction of the dielectric function. They give evidence of collective excitation behaviour for a number of the introduced impurity species, including K, Ag, B, N, and Pd. It is furthermore demonstrated that such excitations can be concentrated at nano-features, e.g., along nano-holes in graphene through metal atoms adhering to the edges of these holes. Research in plasmonics has dramatically increased in recent years, with a number of suggested, possible applications, including biosensors, solar cells and sub-wavelength optics1234624Since graphene has been ‘put on the map’, apart from the attention it has received due to its unusual electronic properties, such as high carrier mobility81012141618Here we present electron energy loss studies in the 2–4 eV range of dosed and doped CNTs and graphene. We investigate spectral features, spanning the range from visible (green) to far UV frequencies, introduced by doping of CNTs with alkali and earth alkali metals Ag and B, and doping or dosing of graphene with N and transition metals. To achieve doping we carry out low-energy implantation, which enables direct impurity incorporation and modification of the electronic structure.As mentioned above, few-layer and single wall nanotubes (SWNTs) and graphene have a pronounced absorption feature at ~4.6 eV; in the case of SWNTs this is deemed to be a collective interband excitation, the π-plasmon. As also mentioned above, the existence of plasmons in graphene at these energies has been disputed. Here we show via electron energy loss spectroscopy (EELS) studies that there is a new, unambiguous absorption component at ~3 eV, whose origin is still under discussion: it could be a collective mode component or a single particle excitation or both. Assessing energy losses in the low loss/optical regime via EELS in a transmission electron microscope (TEM) is usually restricted by the energy spread of the electron beam, although monochromation and post-acquisition deconvolution methods can push the limits to which spectral features can be resolved down to 1 eV, enabling observations of variations in the extent and structure of the 3-eV feature. These techniques reveal that the peak at 3 eV varies in intensity and structure when dosing/doping has been applied; there is usually a significant increase in intensity with increasing doping level. We provide evidence that this excitation has indeed plasmonic character by the revelation of a zero-crossing of the extracted dielectric function. Furthermore, a highly localised enhancement of this feature has been observed in graphene, where strong spatial inhomogeneities of impurity atom distributions, e.g., after dosing with metals, occurred. Since metal atoms move preferentially to edges in graphene sheets, this might be a promising way to ‘nano-sculpt’ light emission.4 and H2 precursor gasses on a copper foil substrateCNTs and graphene were produced via chemical vapour deposition (CVD). Graphene was produced via CVD of CHResults presented in this paper are obtained on CNTs and graphene treated/modified by adding foreign atom species as listed in CNTs were doped directly by introducing alkali and earth alkali metals  as well as Ag, B and N. Results of implantation with B and N have been presented in14 and 1015 cm−2. The exact retained dose is hard to determine via HRTEM methods due to the large number of images needed to gather adequate statistics. In support of the ultra-low energy ion-implantation experiments simulations of the implantation process in graphene were previously performed in Göttingen using the Monte Carlo program SDTrimSP2524Substitutional doping of graphene with N and B was achieved using ultra-low-energy ion implantation as demonstrated previously25Metals (Pd and Ti) were deposited onto the graphene using electron-beam or thermal evaporation. An evaporation rate of 0.1 nm/s was used and the amount of metal deposited varied between 0.1 nm and 0.3 nm.Measurements on the Ag-implanted CNTs , were cand and 3rd order and partially 4th order corrected prisms. EFTEM images were acquired with a slit width of between 0.4 and 1 eV and with energy step sizes of between 0.1 eV and 0.5 eV. Low loss EFTEM data cubes were then collected, consisting of a stack of EFTEM images (with the x- and y-directions defining the image coordinates and the z-direction acting as energy axis) spanning an energy range from typically −2 eV to 18 eV. EEL spectra were (i) extracted from such EFTEM data cubes or (ii) directly acquired in scanning (STEM) mode. Monochromation of the electron beam enabled the FWHM of the zero-loss peak to be reduced to around 0.1 eV.All energy filtered TEM (EFTEM) imaging  and enerIn order to access data below 5 eV it is necessary to remove the zero-loss peak (ZLP). This was done in Gatan Digital Micrograph (DM) by fitting a power-law background to the ZLP using a vacuum spectrum (obtained far from the sample) as a guide. An alternative approach, using the reflected-tail function in DM, was used for the EFTEM images recorded on the monochromated Titan Pico. This method assumes that the ZLP is symmetric. This is not strictly true for FEG sources, where the ZLP has an extended tail towards higher energies; however, monochromation has been shown to reduce the effect of this tailCalculations and modelling of low loss EEL spectra within the frame of the dielectric theory in order to support the measured EEL spectra are referred to in the εm, extracted by Kramers-Kronig analysis in εm crosses zero at the energy of the maximum of the absorption peak. B-implants, although their presence in CNTs cannot easily be revealed in HREM or HAADF images due to B’s low atomic number, also introduce a distinct absorption feature at a slightly lower energy than K- and Ag-implants (orange curve), again with a zero crossing of Reεm at that energy. It should be noted that a non-zero Reεm does not necessarily imply non-collective nature, but can indicate a heavily damped plasmon313233In the following we will give evidence of new excitations with plasmonic character, induced by impurities that are incorporated into or between lattice planes of sheet-like nano-carbons. In order to investigate/consolidate dopant-introduced optical/near-UV plasmon behaviour in sheet-like carbon nanostructures, K, Na, Ba, Ag, N and B were introduced via low-energy ion implantation into few-walled carbon CNTs, primarily SWNTs and DWNTs, succeeded by EELS investigations. A spectrum of N-implanted graphene is included in the results shown in the following, together with one of Pd-dosed graphene, to demonstrate the similarity of doping effects in sheet-like carbon nanostructures. εmextracted via Kraemers-Kroenig analysis from the experimental spectrum for N-doped graphene (green curve in λ). There is a zero crossing of Reεm at around 3 eV, at the energy of the new absorption feature. As in the case of the CNTs this feature is very likely to represent a new plasmon. N-doping of single- and few-wall CNTs (not shown here) introduces a similar feature at ~3 eV, so it can be assumed that N-doping in CNTs induces similar electronic structure modifications as in graphene. Detailed measurements of graphene doped at various levels with N and also B, supported by calculations of electron energy loss spectra, are presented in a separate publication. Pd-dosing of graphene induces features similar to that of N-doped graphene  represent Pd nanoparticles, whilst the darkest regions are holes in the graphene film. Example spectra from different locations, extracted from EFTEM cubes after subtraction of the zero loss peak are shown in th and 5th image in th image in We now focus on EFTEM studies of metal-dosed graphene, which demonstrate an enhancement of the ~3 eV absorption feature along the edges of holes in graphene, especially in the presence of Pd-atoms. Dosing with metal always results in non-uniform distributions. This is shown in the HREM image in The origin of the enhancement is not known. It could be due to interband transitions affected by electronic structure changes of graphene due to the Pd atoms, or highly localised edge plasmons. There could also be occurrence of diffraction effects or edge folding/scrolling. Interestingly an energy-filtered image in the region of the π-plasmon energy, that is between 4.5 and 5 eV , shows aIn a study combining high resolution electron microscopy imaging and energy loss spectroscopy we show that excitations in the 2–4 eV region are produced in nano-carbons, e.g., carbon nanotubes and graphene, upon doping and dosing with alkali and transition metals as well as group V and III elements. A new, distinct absorption peak at ~3 eV is introduced, which is most prominent in samples doped/dosed with K-, B-, N-, Ag- and Pd- atoms. These foreign atoms either adhere to the nanocarbons (on the surface or between atomic sheets) or are incorporated in the carbon lattice; they produce absorption features which comply with criteria for collective character, i.e, those for charge carrier plasmons arising by dopant induced Fermi level shifts in the nano-carbons, and not due to hybridization between the nano-carbons and the dopants. This is confirmed by observations and calculations of the real part of the dielectric function, which shows a zero crossing for the above dopants. However, more experiments as to the exact origin of this feature, and whether it is coupled to single particle excitations, are required. We furthermore demonstrate a localisation of this feature around the holes in edges of graphene dosed with metals (here Pd), which are preferred sites for metal atoms. This bears promise for the creation of plasmonic structures via nano-sculpting. A most noteworthy point to make is that the occurrence of this UV/vis feature at graphene edges conforms with k-vector requirements for coupling of plasmons to light, which would then be emitted from such edges, and vice versa. Hence graphene with nano-cavities, due to preferred metal-atom bonding to edges, is promising for incorporation as light emitters and detectors in nano-scale integrated photonic devices.How to cite this article: Bangert, U. et al. Collective electronic excitations in the ultra violet regime in 2-D and 1-D carbon nanostructures achieved by the addition of foreign atoms. Sci. Rep.6, 27090; doi: 10.1038/srep27090 (2016).


This study examined the effects of individual differences in temperamental reactivity (fear) and self-regulation  on attentional biases toward threat in a sample of school-aged children (age range was between 9 years 1 month and 13 years 10 months). Attentional biases were assessed with pictorial Dot-probe task, comparing attention allocation toward angry (threat-related) vs. neutral and happy faces. Children also completed self-report temperamental measures of fear and attentional control. We compared attentional bias scores in 4 groups of children: high/low fear and high/low attentional control. Results indicated that, in the case of children with high fear and low attentional control, attention was significantly biased toward angry faces compared with children who had low fear and low attentional control. Findings are discussed in terms of the moderating role of individual differences in attentional control in the context of threat, anxiety-related attentional biases in children. Cognitive theories have proposed that anxious individuals tend to direct their attention toward threatening information during early stages of processing  or non-verbal stimuli   and another 10 items that measure the ability to shift attention . Children are answering the items by reporting how frequently certain things happen to them and they respond on a 4-point Likert scale. A good capacity of attentional control is reflected by higher scores obtained on this scale. Different studies conducted with different samples report good internal consistency of the ACS-C Attentional biases were measured with a pictorial version of the Dot-probe task, adapted from Bradley et al.  and SusaData from both the questionnaires and the Dot-probe task were collected from children in two schools, in the presence of a research assistant. Children who voluntarily consented to participate were asked to have their parents sign the informed consent form. In order to prevent children's fatigue, questionnaire data were collected first and then, approximately 2 weeks after, children completed the Dot-probe task.For the Dot-probe task, children were seated in front of the computer at a distance of approximately 40 cm from the screen. At this distance, they were able to comfortably reach the laptop keyboard throughout the task. The task was presented to the children as a computer game and they were asked to read the instructions displayed. Participants were instructed to press key A when the probe replaced the picture on the left side of the screen and key L when the probe replaced the picture on the right side of the screen (on a QWERTY keyboard). Before starting the task, the research assistant summarized for each child what he or she was asked to do. For each child, the program presented the picture pairs in random order. At the end, each child received positive feedback and a small reward.During the Dot-probe task all children included in the analysis completed 160 experimental trials and 8 practice trials. There were a total of 80 pairs of stimuli, 32 of them showing angry-neutral facial expressions, 32 showing happy-neutral facial expressions and 16 pairs showing neutral- neutral facial expressions. By including neutral-neutral pairs, we could analyze the two mechanisms of attentional biases discussed in the literature: attentional faster engagement by angry faces or difficulty of disengagement from angry faces . Trials with reaction times greater than 1500 ms are considered to represent outliers and are likely attributable to error, therefore not excluding these reaction times would have influenced individually trimmed means . This is similar to the mean reported by Muris and Meesters .The total mean fear score on the fear subscale of the EATQ, for the whole sample of children in this study was 2.85 . This is somewhat different from the mean reported by Muris et al. .The total mean attentional control score on the ACS-C for the whole sample of children in this study was 26.74 . The Romanian version of the SCAS is currently under validation but preliminary data .Mean anxiety score on the SCAS, in the whole sample, was 29.28  = 0.87, ns or anxiety scores, F = 1.10, ns. Also, the four groups did not significantly differ in overall reaction times in the Dot-probe task, F = 0.49, ns, or in accuracy, F = 0.27, ns.By using the median split of the ratings of children's fear level  and attentional control level  we formed four groups  = 0.23, ns, or when face stimuli were neutral vs. happy, F = 1.99, ns.In order to control for behavioral interference effects in the Dot-probe task, we conducted a preliminary 2 × 2 × 2 ANCOVA  analysis with Age as a covariate, for comparing reaction times in the four groups between all conditions, with neutral faces collapsed and all conditions with angry faces collapsed. We ran separately a 2 × 2 × 2 ANCOVA analysis between all conditions with neutral faces and all conditions with happy faces. Results indicated no significant differences in overall reaction times when face stimuli were neutral vs. angry, The theoretical focus of our study was on estimating the impact of temperamental variables and of their interaction on attentional biases, while controlling for the effect of other variables that may influence both the measured independent and the dependent variables. Therefore, because anxiety may influence attention to threat and individual differences in temperamental traits are associated with anxiety, we included anxiety as a covariate. Also, the quasi-experimental design of the present study, in which participants were not randomly assigned to groups, requires the inclusion of this covariate  = 7.72, p < 0.01, partial η2 = 0.05 (Bonferroni correction applied). In order to understand the three-way interaction we completed two separate ANCOVAs, one for the angry bias scores and another for the happy bias scores.As our hypotheses were concerned with differences between groups, we conducted a mixed ANCOVA with Emotion valence (angry or happy) as a within-subjects factor, Fear and Attentional Control levels as between-subjects factors, and Age (in months) and Anxiety as covariates. This analysis indicated no significant main effect, but a significant three-way interaction effect of Emotion valence by Fear level by Attentional Control level, F = 5.58, p = 0.01, partial η2 = 0.03 (Bonferroni correction applied). No main effects of Fear, F = 1.22, ns, Attentional Control, F = 0.05, ns, Age, F = 0.002, ns, or Anxiety, F = 0.20, ns, reached significanceThe 2 × 2 ANCOVA (Age and Anxiety as covariates) for angry bias scores indicated a significant interaction effect of Fear and Attentional Control levels on bias scores, t-tests. We looked at the main effect of fear on attentional biases toward angry faces as a function of attentional control. When comparing the low fear low attentional control group with the high fear, low attentional control group, for threat bias scores we observed a significant difference, t(80) = −3.03, p = 0.003, d = 0.68 two-tailed. Inspecting the means from Table t(74) = 0.59, ns.Further we decomposed the interaction effect with follow-up t-tests in order to compare bias scores for each group to 0. When bias scores are significantly different from 0, they indicate a clear attentional bias. For the low fear, low attentional control group, the mean bias score was significantly different from 0, t(38) = −2.26, p = 0.02, d = 0.51. The same was true for the high fear, low attentional control group t(42) = 2.06, p = 0.04, d = 0.45. In the low fear high attentional control group, the mean bias score was not significantly different from 0, t(52) = 0.09, ns. Also, the mean bias score did not significantly differ from 0 in the high fear, high attentional control group, t(22) = −0.58, ns. Consequently, attentional biases appear to be present in the two groups of children that have low attentional control, at both high and low levels of fear. Specifically, children with high fear and low attentional control are significantly vigilant toward angry faces, whereas children with low fear and low attentional control present a significant attentional avoidance of angry faces. Children high in attentional control, with either low or high levels of fear, are not significantly biased in their attentional responses when confronted with an angry face  = 0.004, ns., Attentional Control, F = 2.99, ns., and no interaction effect, F = 0.23, ns. Also, the effects of Age F = 0.19, ns., and Anxiety F = 0.24, ns., did not reach significance. Therefore, it seems that the relation between fear, attentional control, and attentional biases is not a significant one in the case of happy faces.We conducted a second ANCOVA for the happy-neutral trials. We looked for possible effects of Fear and Attentional Control on bias scores for the happy-neutral stimuli, also controlling for the effects of Age and Anxiety. Results indicated no main effect of Fear, r = −0.30 in this sample), which may lead to inflated ANCOVA interaction if dichotomous groups are formed through median splits . However, steps 1–4 were not significant . We examined the particular form of this interaction by plotting the regression of threat bias scores on temperamental fear at high (one standard deviation above the mean), medium, and low (one standard deviation below the mean) levels of fear and attentional control. As shown in Figure t(154) = 2.73, p < 0.01. More specifically, there was a significant positive association between fear and attentional biases toward angry faces only for children with low attentional control. At high or medium values the slopes were not significantly different from zero, t(154) = −55, p = 0.57 and t(154) = 1.63, p = 0.10. These results indicate that there is no significant relation between temperamental fear and attentional vigilance toward threatening stimuli for children with good abilities for attentional control.Consistent with the results from ANCOVA, this analysis , attentional control , or interaction term  in explaining attentional biases toward happy faces.Consistent with the results from ANCOVA, no significant results were found for fear  − Disengagement score =  − We also conducted two regression analyses for the engagement score, respectively disengagement score.b = −1.37, p = 0.01, f2 = 0.06). Also, steps 1–4 were not significant . In the regression analysis for the disengagement score no significant results were found for fear , attentional control , or interaction term  in explaining difficulty to disengage from angry faces. Therefore, it seems that only the faster engagement to threat is implicated in the variations of attentional biases as a factor of temperamental fear and temperamental attentional control in the current study.The regression analysis for the engagement score yielded a significant Fear × Attentional control interaction on the fourth step  (Reinholdt-Dunne et al., In our study we also examined attentional biases for happy faces. However, we did not formulate any specific predictions regarding the direction of attentional processes for happy faces, given that some studies conducted with children (Waters et al., per se, especially at longer stimulus durations (e.g., Wolters et al., There are several limitations to be considered when interpreting our current findings. First, temperamental traits and attentional biases were assessed concurrently. Therefore, no conclusion can be inferred regarding the directionality of the observed effects. From a developmental perspective, it is important to shed light on the specific ways these variables influence each other, so that longitudinal studies assessing these factors will be needed. Second, our study investigated only one part of the model formulated by Lonigan and his collaborators. In order to adequately test the full model, data should be collected longitudinally. For example, future studies should analyze the impact of attentional biases on anxiety symptoms in children with certain temperamental characteristics. Third, as this study included only children without anxiety disorders, the observed effects cannot be generalized to clinically anxious children, for whom the nature of attentional processes and their relations with temperamental traits may be different (Vervoort et al., In conclusion, despite the inherent limitations, the present results point to the importance of studying threat-related attentional biases in relation to temperamental traits. Results indicate that heightened vigilance toward angry faces is characteristic only of children with high fear and low attentional control.The present study indicates that temperamentally-based attentional control plays a regulative role, modulating reactivity that characterizes temperamental fearfulness. Therefore, based on our data, we advance the hypothesis that attentional control can be seen as a possible early protective factor for the development of attentional biases toward threat, and further for the manifestation of anxiety problems. Future work, using a longitudinal design with both clinical and non-clinical samples, is required to examine this hypothesis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BMC Medicine contributes to the ultimate goal of early identification and better management of patients with ILD, and IPF in particular, worldwide.Idiopathic pulmonary fibrosis (IPF) is the prototype of a large and heterogeneous group of pulmonary disorders, mainly chronic and progressive, usually known as interstitial lung disease (ILD). Over the last few decades, IPF has been increasingly recognized as a major unmet medical need in respiratory medicine and has become the focus of intense research activity. This is due to the fact that IPF incidence is increasing worldwide, with rates (and unfortunately prognosis) which are very similar to those of many forms of cancer. Basic and clinical research on IPF has been enormously advancing over the last few decades, culminating in the recent discovery of two safe and effective drugs, now finally made available to patients. For all these reasons, missing a diagnosis of IPF is not acceptable anymore and there is a need for spreading the knowledge about IPF across various specialties of medicine globally. In this context, this article collection in  BMC Medicine, the flagship medical journal of the BMC series, has invited a number of IPF experts worldwide to contribute commissioned pieces and original research articles for this special collection titled, Idiopathic pulmonary fibrosis: diagnosis, management and new therapies. Some particularly hot topics have been covered in this collection, which is being launched concomitantly with the opening of the 2015 International Congress of the European Respiratory Society. Other articles will also be featured in the collection and will be published in the next few weeks.The landscape of respiratory medicine has been transformed over the last few decades by the emergence of a group of variegated diseases, commonly referred to as interstitial lung diseases (ILDs). Once considered rare, difficult to diagnose and substantially untreatable, today ILD still represents a medical challenge. We know now that IPF incidence is increasing worldwide and rates are coming together across countries. Recent evidence suggests that incidence of IPF is increasing worldwide and it is very similar to that of conditions such as liver cancer . HoweverFew fields of modern respiratory medicine have been studied in their genetic component as IPF. In their Opinion article, Susan Mathai and her colleagues address the increasingly relevant topic of incorporating genetics in the diagnosis and the treatment of patients with IPF . In addiIn addition to genetic factors, a number of other, largely unknown, determinants are causing IPF. As this disease is idiopathic by definition, answering the patient’s question of ‘why did I get IPF?’ is a major challenge for any physician. Reading the Commentary by Pierre-Simon Bellaye and Martin Kolb will help readers understand the complexity of the field, but also the recent and promising advancements achieved by the expanding community of basic and translational IPF researchers . We haveSome patients, when diagnosed with IPF, have a sense of relief because they have not been diagnosed with lung cancer. Unfortunately, this sensation of good luck disappears when they discover that the two diseases, in their eyes so different, have a very similar prognosis. Carlo Vancheri in his Opinion article discusses the many (often hidden) links between IPF and cancer, thus highlighting the potential positive pitfalls of considering these two entities as closely inter-related . In realIf it is a challenge discussing with IPF patients the causes of their disease, it is maybe even more difficult to address their expectations and anxieties with regard to their future. Kerri Johannson and colleagues have been exploring this topic over the last few years and they summarize the current status of knowledge in their Commentary . AlthougThis is another reason why patients have to be considered the centre of IPF research. In their Opinion, Anne-Marie Russell and her colleagues clearly point to the need for putting patients before the condition . PatientUltimately, safe and effective drugs reach patients through regulatory agencies. Laura Fregonese and Irmgard Eichler, both working with the European Medicines Agency, describe in their Commentary the failures and the successes in the pathway to approved treatments in IPF . They hiFinally, in the first of the Research Articles in this collection, Simon Walsh and his colleagues explore the complex relationship between pathology and imaging in lung fibrosis . This arBMC Medicine will remain committed to advancing and spreading knowledge about IPF. In particular, a set of linked articles involving world IPF leaders will address the importance and the limitations of using an evidence-based approach in the management of IPF patients. This issue has enormous potential repercussions, in a global context of increasing attention to the possible areas of clash between guidance and guidelines. Finally, an upcoming Forum article will explore the status of IPF care in a part of the world which is home to the majority of human beings. Four experts, representing the so-called BRIC countries  will provide, for the first time, an authoritative assessment of the potentials and the pitfalls of managing IPF in these areas of the globe.After the launch of this collection, We are confident that this article collection will contribute to moving forward towards better care for IPF patients worldwide.


Growth of graphene by chemical vapor deposition on metal supports has become a promising approach for the large-scale synthesis of high quality graphene. Decoupling of the graphene from the metal has been achieved by either mechanical transfer or intercalation of elements/molecules in between the metal and graphene. Here we show that metal stabilized two-dimensional (2D)-oxide monolayers can be grown in between graphene and the metal substrate thus forming 2D-heterostructures that enable tuning of the materials properties of graphene. Specifically, we demonstrate the intercalation-growth of a 2D-FeO layer in between graphene and Pt(111), which can decouple the graphene from the metal substrate. It is known that the 2D-FeO/Pt(111) system exhibits a moiré-structure with locally strongly varying surface potential. This variation in the substrate surface potential modifies the interface charge doping to graphene locally, causing nanometer-scale variation in its work function and Fermi-level shifts relative to its Dirac point. Here we describe the fabrication of a 2D-FeO layer in between vacuum-CVD grown graphene and a Pt(111) single crystal surface. The FeO monolayer is a truly 2D material with strongly varied properties compared to bulk FeOHigh-quality graphene can be grown on many late transition metal surfaces by chemical vapor deposition (CVD) even in ultrahigh vacuum (UHV)12344791071213141516171417not due to variation in the chemical interaction with the substrate but due to physical charge transfer as a consequence of the periodic surface potential of the FeO/Pt(111) substrate. Generally, interface charge doping of weakly physisorbed graphene is determined by the substrate work function36Weak graphene-support interaction is a promising approach for modifying the materials properties of graphene while maintaining many of the desired properties of pristine graphene, like its high charge carrier mobility, which is not possible by e.g. chemical functionalization. The effect of a weak periodic potential induced in graphene by a substrate has been recently highlighted by the observation of Hofstadter’s butterfly for graphene supported on hex-BN2428292331323323Pt111 = 2.77 Å) results in a moiré superstructure. In the case of FeO on Pt(111) a moiré periodicity of ~26 Å is measured31323334The 2D-FeO layer on Pt(111) has been studied extensively and thus many details of this system are well-known23313233343538394038−6 Torr show that even at sample temperatures up to 250 °C the iron may not be oxidized. To achieve oxidation, the sample was transferred into a ‘high-pressure’ cell. In the high pressure cell the sample could be exposed at a much higher oxygen chemical potential by increasing the O2 pressure to 40 Torr and heat the sample to 235 °C. It is known that graphene remains stable at these temperatures and oxygen partial pressure, e.g. annealing of transferred graphene in air to 350 °C is frequently used for ‘burning-off’ PMMA-residueGraphene grown on Pt(111) exhibits different orientational domains that give rise to various moiré-unit cells as shown on 9To confirm that the 2D-FeO, schematically shown in 4749In addition to STM we conducted photoemission studies. XPS shows that the iron is in a 2+ charge state. Comparison of the Fe-2p spectra for a 2D-FeO/Pt(111) film with that of the graphene covered surface are very similar, further supporting the formation of the 2D-FeO monolayer structure underneath of graphene despite the different preparation conditions that need to be employed to oxidize the iron underneath a graphene layer. A comparison of the Fe-2p spectra is shown in Pt = 5.4 eV for Pt(111), slightly less than some literature valuesFeO/Pt = 5.69–5.88 eV due to the surface dipole of the FeO-layer. Importantly, rather than one sharp cut-off two cut-off energies can be discerned indicating locally varying work function of the sample. This is in agreement with the expectation that different regions within the 2D-FeO-moiré structure exhibit different surface potentials. However, we only measure a difference of ~0.2 eV between the two cut-offs, which is smaller than what has been calculatedgraphene/FeO/Pt = 4.85 eV and 4.64 eV.To learn more about the interface charge transfer doping of graphene supported on FeO/Pt(111) we conducted work function measurements by evaluating the secondary electron-cut off in ultra violet photoemission spectroscopy (UPS) as well as detailed analysis of the C-1s peak position. Interface charge doping of graphene as a consequence of work function difference between graphene and the substrate are well documented and well understood especially for metals365According to the above arguments a variations in the charge doping should also cause a variation in the C-1s core level. The C-1s peak position for HOPG, graphene on Pt(111), and graphene/FeO/Pt(111) is shown in In a broader view, this study demonstrates the potential of interface engineering of graphene by direct growth processes without any mechanical graphene transfer. A similar 2D FeO oxide layer to that observed on Pt(111) has also been grown on other late transition metals, namely Ru(0001)60616263646566676869−10 Torr. The first UHV system was equipped with an Omicron VT-scanning tunneling microscope (STM) operated at room temperature. The second system was a UHV chamber for x-ray photoemission spectroscopy (XPS) and ultraviolet photoemission spectroscopy (UPS). An Mg/Al dual anode x-ray source and He-VUV lamp were used for XPS and UPS, respectively. An Omicron-Sphera II hemispherical energy analyzer was used for measuring the photoelectron spectra. Both chambers were also equipped with sample preparation equipment, including sample heating, argon-ion sputter gun, precision-leak valves for oxygen and ethylene dosing, and a mini electron-beam evaporation source (tectra GmbH) for iron deposition.Graphene/FeO/Pt(111) interfaces were studied in two different UHV systems with base pressures of 2 × 10−7 Torr ethylene at 750 °C for 10 min. This procedure resulted in a full monolayer coverage of the Pt(111) sample with graphene. Several graphene domains with different rotational angles were observed, as reported previously. Iron was evaporated by electron-beam bombardment from a 4 mm-diameter iron rod inside a water-cooled copper shroud. The iron was deposited with the graphene/Pt(111) sample at room temperature. Subsequently, the Fe/graphene/Pt(111) sample was annealed in vacuum at 300 °C to intercalate Fe. The inert nature of graphene enables us to transport the graphene covered surface through air. The graphene/Fe/Pt(111) sample was transferred to an UHV-XPS system equipped with an attached high-pressure cell. The high-pressure cell contained a sample holder fitted with a ceramic button heater allowing the samples to heat in reactive gas environments. Static volumes of O2 were introduced in the high-pressure cell. The O2 pressure was monitored with an Inficon capacitance gauge. The graphene/Fe/Pt(111) sample was at room temperature when first exposed to O2 at 40 Torr pressure, ramped to 235 °C, and allowed to cool to below 50 °C in the oxygen atmosphere. Compositional analysis of the sample prior and after high-pressure oxygen treatment was performed with XPS without air exposure to ensure oxidation of the iron and maintenance of the graphene C-1s peak intensity. Then the sample was transferred through air for characterization by UHV-STM and detailed measurements by XPS/UPS. Prior to these measurements, the sample was annealed in UHV at 500 °C. Evaluation of the C-1s core level was done by fitting it with a Doniach–Sunjic peak shape to accommodate for the typical asymmetric line shape of sp2 carbon.The Pt(111) sample was cleaned by repeated ion-sputtering annealing cycles. Graphene was grown on the clean Pt(111) sample by exposure to 1 × 102 atmosphere at 10−6 Torr pressure.For comparison of the XPS/UPS spectra with a non-graphene covered surface, FeO films were prepared in-situ following standard preparation conditions for FeO/Pt(111)(i)Fe-2p XPS data for Fe on Pt(111), FeO/Pt(111), and graphene/FeO/Pt(111): (ii) calculated relationship between work function difference between substrate and graphene and the corresponding Fermi-level shift in graphene, adapted from ref. .How to cite this article: Dahal, A. and Batzill, M. Growth from behind: Intercalation-growth of two-dimensional FeO moiré structure underneath of metal-supported graphene. Sci. Rep. 5, 11378; doi: 10.1038/srep11378 (2015).