Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA—Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related  A particularly useful application is to elucidate the functions of gene paralog families. As long as the family members are sufficiently related, a single siRNA can simultaneously knockdown all family members and thereby unveil the redundant function of the gene paralog family [RNA interference (RNAi) is a widely used approach to silence genes and thereby deduce their function. While primarily used for g family –3.reproductive homeobox (Rhox) gene cluster. This gene cluster harbors 33 homeobox genes that are selectively expressed in the male and female reproductive tract, suggesting that they encode transcription factors critical for reproduction [Rhox genes are expressed in reproductive tract, their spatial and temporal expression patterns are different. For example, RHOX10 is expressed specifically in spermatogonia and early spermatocytes [RHOX genes are expressed specifically in germ cells in the testes and are aberrantly methylated in infertile patients [We elected to take advantage of this approach to conditionally knockdown highly related gene paralogs in the oduction . Althougatocytes  and RHOXatocytes . RHOX5 iatocytes ,7,8. Thepatients ,10.Rhox3 gene paralogs, which comprise eight gene copies in mice that are highly homologous with each other—displaying 98.2–99.7% sequence identity [Rhox3 gene paralogs are expressed in the adult testes except Rhox3d , which is likely to be a pseudogene since it harbors a premature translation termination codon [Rhox3 gene paralogs appear to have expanded as a result of selection pressure exerted specifically in the mice lineage, as rats have only a single Rhox3 gene [Rhox3 paralogs have undergone purifying selection in the amino-terminal region and weak positive selection for changes in the homeodomain region [The subject of this report is the identity –14. Usinon codon . The Rhon region ,12,14. Tn region .Rhox3 paralogs are interspersed with other Rhox genes, a knockout strategy to determine their collective in vivo function is not feasible and thus we chose to use a RNAi approach instead. To provide specificity, we used a conditional RNAi approach in which the Rhox3 shRNA was selectively expressed in male germ cells, the cell type that we found normally express Rhox3. As described herein, this germ cell-specific shRNA approach successfully knocked down Rhox3 expression in the testis in vivo and it led to dramatic defects in spermatogenesis.Because these eight In vitro RNAi studies to determine gene function in cultured cells typically incorporate controls in their experiments to distinguish whether the phenotypic defects observed are the result of depletion of the target gene product or an off-target effect [in vivo rarely have such controls [in vivo studies, conditional/inducible shRNA expression methods have been developed that restrict the expression of the siRNA to specific cell types, developmental stages, and/or temporal windows [Rhox3 gene paralogs—reversed the phenotypic defects caused by the Rhox3 shRNA. An off-target effect seemed a distinct possibility given that we found that mice lacking the Rhox3 paralogs exhibited different defects from mice expressing Rhox3-shRNA. Indeed, we found that elimination of the intended target did not measurably reduce or change the defects caused by the Rhox3 shRNA, thereby demonstrating that the Rhox3 shRNA acts by a mechanism independent of its ability to knockdown Rhox3. We considered and tested different mechanisms by which the Rhox3 shRNA might cause off-target effects. This led to the discovery that Rhox3 shRNA mice have reduced production of endogenous (endo)-small interfering (si) RNAs. In contrast to microRNAs (miRNAs), which are expressed in most of mammalian cells, endo-siRNAs are only known to be expressed in germ cells and embryonic stem cells in mammals [t effect . In contcontrols . To redu windows ,19. Whil mammals –23. Our ad libitum. Euthanasia was performed by CO2 inhalation for 5 min followed by cervical dislocation. All efforts were made to minimize suffering.This study was carried out in strict accordance with the Guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego. The protocol was approved by the IACUC at the University of California, San Diego (Permit Number: S09160). All animals were housed under a 12 h light: 12 h darkness cycle and provided with food and water Rhox3 paralogs. U6 promoter driven-shRNA expression vectors (G-1256 and G-1257) targeting Rhox3 were generated by subcloning PCR products that contained both the target sequence and the mouse U6 promoter into the pGEM-T Easy vector, as previously described [Rhox3 target sequences used were 5’-GGAGCAGATTCCTGAGCAT-3’ (G-1256) and 5’-GCATGTTGAAGGAGAGAGT-3’ (G-1257). The construction of the shRNA expression vector to target firefly luciferase (G-472) is previously described [Rhox3 shRNA transgenic construct (G-1029), oligos MDA-4212 and-4213, which contained shRNA sequences, were annealed and ligated into the ApaI and EcoRI sites of the pBS/U6-ploxPneo plasmid , as described elsewhere [The siDESIGN algorithm  was used to design siRNA target sequences that have 100% complementarity with all 8 escribed . The Rholsewhere .Rhox3 shRNA plasmids. A U6-driven Luciferase shRNA plasmid was used as a control in transfection experiments. To delete the loxP-flanked neomycin cassette from the U6-ploxPneo Rhox3 shRNA transgenic construct, a Cre recombinase (pMC-Cre) expression plasmid was co-transfected. Total cellular protein extracts were prepared 48 h after transfection.HeLa cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and 50 mg/ml of penicillin and streptomycin and transfected with Lipofectamine-2000 following the manufacturer’s instructions. All cell culture reagents were purchased from Invitrogen Technologies . Cells were plated on 6-well culture plates and cotransfected with 500 ng of an expression vector encoding FLAG-tagged RHOX3 and various concentrations of U6-driven Rhox3 shRNA mice, a 2.7-kb DNA fragment containing the U6-ploxp-neo Rhox3 shRNA transgene in the G-1029 plasmid was excised using the restriction enzymes, AflIII and Asp718, and gel purified for microinjection. Positive transgenic mice were detected by PCR using tail DNA as a template and the primers MDA-4290 and-4291, which are specific for the U6 promoter and neomycin cassette. Four founder lines  containing the transgene were obtained. All lines were mated with Stra8-iCre mice to generate double transgenic lines (expressing the Rhox3 shRNA and CRE) to make germ cell-specific shRNA mice (Rhox3-shRNA mice) and the single transgenic littermates (either Rhox3-shRNA or Stra8-iCre) served as control (Control mice). Line 2 was subjected to detailed histological analysis and also bred with Rhox-c-KO mice.Transgenic mice were generated by pronuclear microinjection of FVB/NJ mouse embryos  at the M. D. Anderson Cancer Center transgenic core facility. To generate U6-ploxPneo Rhox-c-floxed mice, two targeting vectors were used to insert loxP sites at the beginning (upstream genomic region of Rhox1) and the end (the second intron of Rhox13) of the Rhox cluster. The targeting vectors were constructed from BAC clones . The two targeting vectors were electroporated sequentially into ES cells; in each case, cell clones that underwent homologous recombination were selected by Southern blot analysis. An ES cell clone containing both loxP sites was selected for blastocyst injection to obtain chimeric mice. Germline-transmitted Rhox-c-floxed mice were bred with EIIa-Cre mice to generate mice lacking the entire Rhox cluster (Rhox-c-KO or Rhox-c-heterozygote mice). The Rhox-c-heterozygote female mice were bred with Rhox3-shRNA transgenic mice to generate Rhox-c heterozygote; Rhox3-shRNA female mice and these mice were further bred with Stra8-iCre mice to obtain Rhox-c KO;Rhox3-shRNA;Stra8-iCre male mice (Rhox-c-KO/Rhox3-shRNA mice).To generate For quantitative RT-PCR (qRT-PCR) analysis, total cellular RNA was extracted from cell lines and tissues by the Trizol method  and converted to cDNA using iScript reverse transcription . Relative gene expression was determined by SYBR green incorporation in an Applied Biosystem StepOnePlus real-time PCR system, as described previously , with prCytoplasmic and nuclear protein extracts from adult testis were prepared as described elsewhere . WesternRhox3-shRNA mice  and control mice , using an Affymetrix GeneChip mouse exon 1.0 ST array. Raw data was normalised using robust multi-array averaging with correction for GC content of the probes (GCRMA) [Rhox3-shRNA testes were statistically tested using one-way ANOVA. All microarray data analysis was carried out using Partek genomics suite v6.3 . Microarray data is available from Gene Expression Omnibus under accession number GSE64324.Microarray analysis was performed at the University of California, San Diego (UCSD) GeneChip microarray core facility using total cellular RNA from P15 testis isolated from  (GCRMA) . Gene exin situ apoptosis detection kit , following the manufacturer’s instructions.For histology analysis, testes and epididymis were fixed in either 10% neutral buffered formalin or Bouin’s fixative and stained with hematoxylin-eosin or periodic acid Schiff-hematoxylin, as previously described . The sizRhox3 gene paralogs have the potential to function, we examined their developmental expression pattern during the first wave of spermatogenesis. Using qRT-PCR analysis with primers designed to amplify all eight Rhox3 paralogs, we found that Rhox3 mRNA is expressed at only trace levels during early stages of the first wave of spermatogenesis—postnatal day 5 (P5) to P18—when postnatal germ cells first proliferate and undergo meiosis. Rhox3 mRNA level is upregulated at P20  in testes nuclear lysates .. In addjsd mice testes, which lack virtually all germ cells except for undifferentiated spermatogonia [jsd mutant testes, implying that RHOX3 is expressed predominantly in germ cells (Rhox3 mRNA (at P20)  ., sugges(at P20) .in vivo, we elected to use a conditional RNAi approach developed by Shukla et al. [To determine the functional role of RHOX3 in male germ cells a et al. . In thisRhox3 genes in vivo, we first identified siRNAs exhibiting 100% complementary with all eight Rhox3 gene paralogs and then screened for those that efficiently depleted Rhox3 expression in vitro. This screen led to the identification of a siRNA sequence that reduced RHOX3 protein expression by ~90% in HeLa cells upon cotransfection with an expression vector encoding FLAG-tagged mouse RHOX3 (data not shown). We inserted this siRNA sequence (in a shRNA context) into the U6-ploxP neo construct  to create a Rhox3-shRNA transgene construct  [Stra8-iCre mice were crossed with mice from the four transgenic lines to generate double transgenic mice that carry both Stra8-iCre and the Rhox3-shRNA transgene. These Rhox3-shRNA;Stra8-iCre mice  were sacrificed at 6 weeks of age and analyzed for endogenous Rhox3 expression in the testis. The single transgenic mice that carry either the Stra8-iCre or Rhox3-shRNA transgene from same litter served as control mice. Testes from the 4 different Rhox3-shRNA mice lines displayed a ~50% to 85% reduction in Rhox3 mRNA level relative to control mice testes (Rhox3 mRNA level (Line 2) and observed a dramatic decrease in RHOX3 protein level (>95%) (The  and P7) . Stra8-ie testes . We examl (>95%) .Rhox3-shRNA mice lines 2, 12, and 13 decreased , compared to littermate control mice that had the Rhox3-shRNA transgene but lacked CRE  was decreased by ~50% . The majority of the tubules had vacuoles  increased numbers of apoptotic pachytene spermatocytes in seminiferous epithelial cycle stage IV, leading to missing generations of spermatocytes and/or spermatids in particular tubule areas in stages VI-VIII  fe VI-VIII ; (iii) m VI-VIII ; (iv) fe VI-VIII ; (v) a r VI-VIII . The redrol mice . We alsorol mice . Finallyrol mice .Rhox3-shRNA and control mice (Rhox3-shRNA adult testes relative to control adult testes. Some spermatocyte marker genes (Spo11 and Rec8) were also decreased significantly, but by less (~2- to 3-fold) than post-meiotic genes. Other spermatocyte marker genes (Sycp1 and Sycp3) did not exhibit a significant change in level. This confirmed that there was a defect in the progression of spermatocytes, probably at a mid or late phase. In contrast, spermatogonia markers (Stra8 and c-Kit) were increased in level by ~2- to 3-fold in Rhox3-shRNA testes, which is consistent with an enrichment of spermatogonia as a result of depletion of spermatocytes and spermatids. Similar increases were observed for Sertoli cell markers (Wt1 and Rhox5) and Leydig cell markers (Cyp450 and Lhgcr), suggesting that these cell populations were enriched too. Similar findings were observed in mid-postnatal (P20 and P25) testis samples . In brief, we introduced loxP sites at both ends of the Rhox cluster in ES cells (by homologous recombination), generated mice harboring these loxP sites, and mated them with EIIa-Cre mice (which express Cre from a ubiquitous promoter) to generate Rhox-cluster-KO (Rhox-c-KO) mice progeny. These Rhox-c-KO mice did not exhibit the defect in meiotic prophase we detected in Rhox3-shRNA mice, but instead had only a modest (~28%) reduction in testes size . If the Rhox3-shRNA mice phenotype is the result of depletion of Rhox3, Rhox-c-KO/Rhox3-shRNA mice would be predicted to show the same defect as Rhox-c-KO mice because Rhox3 is not there to be depleted by shRNA. Alternatively, if the defect from the shRNA is independent of the presence of Rhox3, Rhox-c-KO/Rhox3-shRNA mice would be predicted to show the same severe defect as Rhox3-shRNA mice. We found that the Rhox-c-KO/Rhox3-shRNA mice showed virtually the same phenotype as that of Rhox3-shRNA mice: 80% reduction in testis weight  that corresponds to mid-meiosis. The time point is ~10 days after Rhox3 shRNA would be predicted to be first expressed as a result of CRE-mediated transcriptional activation [Rhox3-shRNA mice at this developmental time point , we found that only one of the 8 down-regulated genes exhibit complementarity with the putative Rhox3 shRNA seed site—position 2–8. This gene, Cldn2, encodes a member of the claudin family of proteins, which are considered to be the most important components of tight junctions. While some claudins have been shown to function in spermatogenesis, Cldn2 does not appear to have such a role, as Cldn2-KO mice have only been reported to have kidney dysfunction; no fertility defects were observed [What is responsible for the severe spermatogenic defects in the mice expressing the tivation . We alsome point .. In conme point ., which me point . In totaentarity . Using Robserved .Rhox3 shRNA ties up a sufficient proportion of the small RNA processing machinery that it reduces the generation of small RNAs. We considered this to be an attractive possibility, as the histological defects we observed in Rhox3-shRNA testes were very similar to that of Dicer-conditional KO mice generated using the same Cre-driver mice as we used in our study—Stra8-iCre mice [Rhox3-shRNA mice  out of 78 known testicular endo-siRNAs [Rhox3-shRNA testes compared to littermate control . The line with no obvious defects (line 11) exhibited only modest Rhox3 depletion, suggesting a dose-response relationship rather than an insertional mutagenesis mechanism  reduced testes weight  res weight , (iii) fs weight , (iv) nus weight , (v) muls weight , (vi) ins weight ., and  RNA-binding proteins can change the substrate recognition and processing behavior of DICER in vitro [A surprising finding in our study is that siRNAs, but not miRNAs, were dysregulated by the x3 shRNA , as bothin vitro . For exain vitro . shRNA pin vitro , raisingRhox3-shRNA mice testes (T9 and T12) are not reduced in level in Dicer KO pachytene spermatocytes, suggesting that some siRNAs are processed in a DICER-independent manner [Rhox3 shRNA might sequester factors required for siRNA biogenesis other than DICER. Regardless of the precise mechanism, we suggest that the ability of shRNAs to inhibit the expression of endo-siRNAs in vivo can be used as a tool to study the biogenesis, stability, and/or function of these small RNAs.Another possibility is that shRNAs promote the destabilization of endo-siRNAs rather than inhibiting endo-siRNA biogenesis. This destabilization could be specific for endo-siRNAs, not miRNAs, explaining the selectivity we observed. For example, shRNAs might interfere with siRNAs forming RNA-induced silencing complexes (RISC), leading to their destabilization. In support of this, loss of AGO2, a key RISC component, results in decreased levels of siRNAs in mouse oocytes . Little t manner . This, iRhox3-shRNA mice may not be representative of all miRNAs; i.e., there may be a pool of shRNA-resistant miRNAs in male germ cells. We regard this is unlikely, as we tested endo-siRNAs with different expression characteristics  is consistent with them being subject to meiotic sex chromosome inactivation (MSCI), a transcriptional repression mechanism that transcriptionally silences the X and Y chromosomes during the meiotic phase of spermatogenesis [Rhox3 gene paralogs exhibit induced expression at a postnatal time point when round spermatids first appear   ., raisinia stage .in vivo highlights the need for controls to properly interpret shRNA-mediated knockdown studies. New strategies must be developed to specifically knockdown genes with reduced side effects.Other studies in addition to ours have identified unintended effects of shRNAs. For example, target-independent cellular toxicity and animal morbidity have been shown to result from the expression of some exogenous shRNAs ,62. SomeS1 FigRhox3 mRNA level  in total testicular RNA from mice (n = 3) of the age shown (Rhox3 mRNA level at P5 was arbitrarily set to 1). Values denote the mean fold change ±S.E. (B) Western blot analysis of adult testis lysate. The blot with lanes 1 and 2 was probed with purified RHOX3 IgG (α-RHOX3). The arrow points to the presumptive RHOX3 band (~18 kDa); the higher and lower migrating bands are proteins binding non-specifically to the RHOX3 IgG. As negative controls, blots were probed with IgG purified from the preimmune serum (Pre IgG) (Lane 3) or purified RHOX3 IgG that were preincubated with excess amounts of recombinant RHOX3 protein (α-RHOX3+recRHOX3) (Lane 4). Red boxes denote the lack of detectable RHOX3 in the negative controls. β-ACTIN is the loading control. N, nuclear protein extract; C, cytoplasmic protein extract.(A) qRT-PCR analysis of (TIF)Click here for additional data file.S2 FigRhox3-shRNA and control mice at 6 weeks of age. (B) Seminal vesicle weight of the Rhox3-shRNA and control mice lines at 6 weeks of age. Values denote the mean ± S.E. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.(A) Body weight of (TIF)Click here for additional data file.S3 FigRhox3-shRNA mice testes have elevated levels of spermatogonial and somatic cell markers and reduced levels of some meiotic and all post-meiotic germ cell markers. qRT-PCR analysis of testes from Rhox3-shRNA and control mice at 6 weeks of age. Values were normalized to the mRNA encoding the ribosomal protein RPL19 and denote the mean ± S.E. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.Adult (TIF)Click here for additional data file.S4 FigRhox3-shRNA and control mice from the indicated postnatal ages. Values were normalized to the mRNA encoding the ribosomal protein RPL19 and denote the mean ± S.E. Asterisk (*) indicates significant changes, p<0.05. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.(A–D) qRT-PCR of testes from (TIF)Click here for additional data file.S5 FigRhox3-shRNA testes (P25). (B) Quantification of the percentage of seminiferous tubules containing TUNEL-positive cells at the indicated developmental stages. Asterisk (*) indicates significant changes, p<0.05. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.(A) Representative images of TUNEL assay of control and (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Accurate cervical intra-epithelial neoplasia (CIN) lesion grading is needed for effective patient management. We applied computer-assisted scanning and analytic approaches to immuno-stained CIN lesion sections to more accurately delineate disease states and decipher cell proliferation impacts from HPV and smoking within individual epithelial layers. A patient cohort undergoing cervical screening was identified (n = 196) and biopsies of varying disease grades and with intact basement membranes and epithelial layers were obtained (n = 261). Specimens were sectioned, stained (Mib1), and scanned using a high-resolution imaging system. We achieved semi-automated delineation of proliferation status and epithelial cell layers using Otsu segmentation, manual image review, Voronoi tessellation, and immuno-staining. Data were interrogated against known status for HPV infection, smoking, and disease grade. We observed increased cell proliferation and decreased epithelial thickness with increased disease grade . Analysis within individual cell layers showed a ≥50% increase in cell proliferation for CIN2 vs. CIN1 lesions in higher epithelial layers . Higher rates of proliferation for HPV-positive vs. -negative cases were seen in epithelial layers beyond the basal/parabasal layers in normal and CIN1 tissues. Comparing smokers vs. non-smokers, we observed increased cell proliferation in parabasal (low and high grade lesions) and basal layers (high grade only). In sum, we report CIN grade-specific differences in cell proliferation within individual epithelial layers. We also show HPV and smoking impacts on cell layer-specific proliferation. Our findings yield insight into CIN progression biology and demonstrate that rigorous, semi-automated imaging of histopathological specimens may be applied to improve disease grading accuracy. Predicting outcomes for cervical intra-epithelial neoplasia (CIN) lesions remains a complex challenge. Some lesions progress to later disease stages while others do not, meaning some patients experience risks and costs of treatment unnecessarily. Further, HPV infection status for normal and early CIN tissues may be insufficient for stratifying progression risk. New tests are needed to accurately stratify patients presenting with CIN and to reduce the number of women treated unnecessarily for high grade squamous intraepithelial lesions (HGSILs).Multiple biomarkers have been tested to identify CIN lesions with a high risk of progression. P53, p16, and Ki67/Mib1 are amongst the best accepted for patient management It is well-accepted that smoking is a cofactor for development of CIN Herein, we report our analysis on the effects of HPV infection and smoking on cell proliferation for normal and neoplastic cervical epithelia. We sought to apply a rigorous semi-automated approach to quantify these effects in CIN lesions. To achieve this, we analyzed individual epithelial layers in a well-annotated, thoroughly reviewed patient cohort. Through this, we have gained insights into the impact of these factors on cell behavior for different disease stages. This work provides a rationale for wider evaluation of a combined approach involving clinical features  and automated analysis of protein expression in epithelial layers as a biomarker for managing CIN.Samples were chosen amongst 1850 patients (3735 biopsies) aged ≥18 that were collected during a multi-center study to evaluate Mib1 and p16 staining as a means of improving diagnosis of HGSIL Non-pregnant women ≥18 years were enrolled from 1999–2006. The study protocol was approved by the University of British Columbia/BC Cancer Agency Research Ethics Board (protocol #C02-0476) and written consent was obtained from all enrolled patients. Patient demographic features  were obtHistopathological specimens were reviewed at BCCA by both WHO and Bethesda criteria. Initial reviews were performed by one of the gynecological pathologists on clinical duty (the “pool”). There were seven pathologists in the pool, which also included study pathologists. A second blinded review was performed by one of two study pathologists. If the first and second readings agreed exactly in the WHO system, third reviews were not performed. Discrepancy of two grades based on WHO criteria mandated a third blinded reading by both study pathologists. Kappa scores for pathology readings have been reported Endocervical samples were collected and tested for HPV using the Hybrid-Capture II system. This assay detects both low-risk  and high-risk  HPV types. All samples were processed using funds from public grants.2O2 in phosphate-buffered saline (PBS). Sections were immersed in sodium citrate buffer and microwave heated at power settings of 1000 W for 2 min, then 160 W for 15 min. Before immuno-staining, slides were soaked in PBS. Sections were incubated with biotinylated swine anti-rabbit antibody (DAKO) at 1∶100 for 30 min. Visualization of the complex was realized using diaminobenxidine/H2O2 for 10 min (room temperature). Two washes in PBS were performed before counterstaining with Mayer’s Hematoxylin. Sections were dehydrated using graded ethanol and mounted with a mixture of distyrene, plasticiser, and xylenes.Paraffin sections (4 µm) were mounted on slides, dried overnight (37°C), deparaffinised in xylenes, and rehydrated in alcohol. Next, endogenous peroxidase activity was blocked by 3% H2, and the effective pixel sampling area was 0.116 µm2. In each case, areas corresponding to diagnostic areas selected by study pathologists were carefully located by expert technicians. Images of that region were then captured. Regions of interest (ROI) were interactively and carefully delineated. Both basement membranes and superficial apical surfaces were separately defined.Getafics A simple, automated thresholding method followed by an Otsu segmentation algorithm was applied to extract centers of gravity of all cell nuclei Voronoi cell layers were defined thusly: all cells whose corresponding Voronoi polygons touched the basement membrane were assigned to layer 0 . Layer 1  consisted of those cells which were not in layer 0 and had a neighbor in layer 0. Layer 2 consisted of those cells which were not in layer 1 or layer 0 and had a neighbor in layer 1. Higher number layers were defined similarly. Generally, layers from layer 2 to the penultimate layer observed were interpreted as “intermediate” epithelial layers, while final layers were termed the “superficial” epithelial layer.The percentage of Mib1-positive cells in each layer was calculated as the number of positive cells divided by the number of cells belonging in the layer. Voronoi diagrams and layers-based analyses were undertaken as follows: given a set of points S in a plane, a Voronoi tessellation for S is the partition of the plane which associates a polygon V(p) with each point p of S in such a way that all locations in V(p) are closer to p than to any other point in S All analyses were done with STATISTICA10. All p-values were two-sided, with a two-sided p-value<0.05 considered significant. For comparisons between groups, ANOVA testing was done. When testing showed a statistical difference across groups, Least Significant Differences testing or planned comparisons were used to compare groups two-by-two.Two-hundred sixty-one biopsies obtained from this diagnostic population  were stained with Mib1 . Based oThe mean and standard deviation of epithelial thickness, number of layers, and percentage of proliferating cells for four pathological grades were determined . There wVoronoi layers were calculated as described above and in In normal epithelia, >60% of cell proliferation occurred in the parabasal layer and layer 2 . ProlifeWe next returned to assessing proliferation on a layer-by-layer basis for the four diagnostic groups . The perThis relative proliferation pattern changed in upper layers. The percentage of Mib1-positive nuclei for CIN1 cases decreased by ∼10% from layer 1 to 2 but increased by ∼6% from layer 1 to 2 in CIN2 lesions. The decrease in the percentage of Mib1-positive nuclei in CIN2 and CIN1 lesions was almost identical from layer 2 to layer 6, with an 18% decrease in CIN2 lesions and 14% decrease in CIN1 lesions, respectively.We noted several HPV impacts on proliferation patterns . This anNovel methods described herein allowed quantification of cell proliferation on a per-epithelial-layer basis . This yiOne key observation was that differences in proliferation rates between CIN1 and CIN2 lesions occurred in specific epithelial layers . While pWe also observed HPV-driven proliferation rate differences within a given disease stage. Moreover, these differences were detected in specific epithelial layers. Given high prevalence of HPV infection in advanced disease stages, we were only able to derive statistically meaningful results related to HPV status for normal and CIN1 tissues. For both these tissue types, we noted higher rates of proliferation for HPV-positive cases relative to HPV-negative cases in epithelial layers above basal and parabasal layers . These dFinally, we noticed marked smoking-mediated differences in the distribution of proliferating cells in CIN lesions. When epithelia were analyzed at full thickness, negligible separation in the percentage of proliferating cells was observed for normal and LGSIL tissues from smoking vs. non-smoking patients  . NonetheOur analyses of cell proliferation in normal cervical and CIN tissues have identified epithelial layer-specific features that delineate disease grades. Our results also show that proliferation within cell layers varies depending on HPV infection and smoking status. We have demonstrated the utility of computer-assisted histopathological slide analysis for identifying novel cell features associated with disease grade. More accurate clinical diagnoses based on this technique could improve patient management by facilitating more accurate estimations of disease progression risk. This work bolsters the argument that research into HPV and smoking impacts on cell proliferation must account for spatial differences in proliferation based on individual epithelial layers . Independent analyses using the approaches described here are needed to determine whether cell proliferation in epithelial layers beyond the basal and parabasal layers has utility for discriminating CIN1 and CIN2 lesions. Further analyses are also needed to determine the biological processes causing the HPV- and smoking-driven cell proliferation changes we observe within epithelial layers.


The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results. Preparing biological material for electron microscopy (EM) involves harsh processing steps that can poorly preserve cellular ultrastructure. Here the authors apply a single layer of graphene onto wet cells to enable direct EM using low voltage, and correlate actin filaments and mitochondria using super-resolution microscopy. A major challenge in the application of electron microscopy to biological samples has been faithful preservation of cellular ultrastructure during the laborious dehydration and embedding/coating procedures required for sample preparation124568910111213Here we utilize graphene, a single-atom-thick honeycomb lattice of carbon atoms1617182023252619We report that monolayer graphene can hermetically seal and protect large areas of mammalian cells, cultured on conventional coverglass, from external environments, including the high vacuum typically encountered in an electron microscope. This protection, combined with the high electrical and thermal conductivity of graphene and its ultimate thinness, enables facile electron microscopy of wet and untreated cells with excellent contrast and resolution, as well as correlated super-resolution microscopy directly on the culturing substrate. In particular, individual actin filaments are resolved in wet cells through electron microscopy and well correlated with super-resolution results.2) areas of cells conventionally cultured on coverglass  growth on copper foil and wet-transferred to cover large  was readily reached during pump down.Having verified that graphene can provide a hermetic seal for cells, we moved forward to examine its applicability to electron microscopy of wet cells under high-vacuum conditions. Graphene sheets were deposited onto wet cells cultured on coverglass such that most of the coverglass surface was overlaid with graphene. Silver paint was used to contact a corner of the deposited graphene sheet to the sample holder for dissipation of electric charge during electron microscopy , ‘Ag'. TWe first examined fixed cell samples that were briefly stained with a 0.5% uranyl acetate solution but otherwise remained fully hydrated. Under SEM, the non-covered, non-conductive parts of the sample rapidly accumulated electric charge, leading to excessively bright and unstable signals . Zoomed-12V0) of 3 kV, substantial contrast was obtained for the internal structure of the graphene-covered, untreated cells (V0 (2 kV) led to less transparent images, but was helpful in outlining the overall cell morphology (V0 (5 kV), the untreated cells became overly transparent with only the nuclei providing contrast (V0 (>10 kV) to penetrate the relatively thick (>∼50 nm) membranes, thus providing limited contrast on unstained animal cells1123V0. The fact that graphene is an excellent thermal and electrical conductor further reduces damage by the electron beam so that the same unfixed cells can be imaged multiple times and under different conditions without noticeable structural changes , and then post-fixed for 20 min in 2% (v/v) glutaraldehyde in CB12−1) were used to label Tom20, respectively. For fluorescent labelling of actin filaments, samples were incubatedMammalian cells  were cultured on common glass coverslips  following standard tissue culture protocols. For live cell experiments , cells wFor data presented in CVD graphene on copper foil−7–2 × 10−5 torr) was readily reached during pump down. Calibration of magnification was verified with a replica of a 2,160 lines per mm waffle-pattern diffraction grating (Ted Pella 604-A).The graphene-covered coverslip was mounted on a standard metallic sample mount with carbon tape, and a small amount of silver colloid paint (Ted Pella 16031) was used to create a conductive bridge between graphene and the sample mount. SEM imaging was performed under standard secondary electron mode on a FEI Quanta 3D FEG system or a JEOL JSM-6340F system. Normal operational vacuum (5 × 102 triangular) at the centre of the coverslip, which was readily identifiable both under optical microscope and in SEM under the coverage of graphene. 3D-STORM imaging32−2; for STORM of AF647) or 560-nm laser  was used to excite fluorescence from labelled dye molecules and switch them into the dark state. Concurrent illumination of the 405-nm laser was used to reactivate the fluorophores to the emitting state. The power of the 405-nm laser  was adjusted during image acquisition so that at any given instant, only a small, optically resolvable fraction of the fluorophores in the sample were in the emitting state. For 3D-STORM imaging, a cylindrical lens was inserted into the imaging path so that images of single molecules were elongated in x and y for molecules on the proximal and distal sides of the focal plane (relative to the objective), respectively−1 glucose oxidase and 40 μg ml−1 catalase. After STORM imaging, the coverslip was stored in PBS before processing for graphene-based SEM imaging (as described above). To align the obtained STORM and SEM images, the STORM image was mapped to the coordinate system of the SEM image through a two-dimensional affine spatial transformation (MATLAB) on the basis of corresponding features (control points). About 20 control points were selected in each data set for inferring an averaged, global, affine transformation matrix.To facilitate location of the same cells under STORM and SEM, a diamond scribe was used to make a scratch mark .Supplementary Figures 1-12.


The demand for nanocomposites of graphene and carbonaceous materials decorated with metallic nanoparticles is increasing on account of their applications in science and technology. Traditionally, the production of graphene-metal assemblies is achieved by the non-environmentally friendly reduction of metallic salts in carbonaceous suspensions. However, precursor residues during nanoparticle growth may reduce their surface activity and promote cross-chemical undesired effects. In this work we present a laser-based alternative to synthesize ligand-free gold nanoparticles that are anchored onto the graphene surface in a single reaction step. Laser radiation is used to generate highly pure nanoparticles from a gold disk surrounded by a graphene oxide suspension. The produced gold nanoparticles are directly immobilized onto the graphene surface. Moreover, the presence of graphene oxide influences the size of the nanoparticles and its interaction with the laser, causes only a slight reduction of the material. This work constitutes a green alternative synthesis of graphene-metal assemblies and a practical methodology that may inspire future developments. Graphene sheets decorated with metal nanoparticles (MNPs) are excellent materials that can be used in several fields such as energy storage, optoelectronics, biosensors or catalysis, due to their physical and chemical properties13489101113The vast majority of graphene-MNPs are synthesized by the simultaneous reduction of metal salts and highly oxidized graphene derivatives15161820Ligand-free MNPs have been successfully prepared by laser irradiation using an alternative and easy top-down technique that fulfils the principles of green chemistry2224252628Decoration of ligand-free gold NPs created separately by PLAL and subsequently mixed with graphene derivatives has been reported3335In this work, we have focused on producing ligand-free gold NPs anchored to graphene oxide sheets in a single reaction step. The impact of the femtosecond radiation-based technique on the control of the production of ligand-free gold NPs in an environment filled with graphene oxide sheets has been studied. With this aim, AuNPs were produced in two different liquid environments for comparison purposes: (i) deionized water and (ii) a graphene-oxide homogeneous suspension. Our results demonstrate that it is possible to create ligand-free MNPs directly anchored to graphene oxide sheets in a single reaction process. This methodology has been proved to provide an effective way to obtain graphene metal assemblies that could potentially be used in several applications where undesirable cross chemical effects need to be avoided.Gold nanoparticles were obtained from the ablation of a gold solid target immersed in deionized water using an amplified Ti:Sapphire femtosecond laser source working at a maximum energy per pulse of 0.8 mJ, with a pulse duration of 30 fs full width half maximum and 1 kHz of repetition rate. The schematic drawing of the set-up is presented in 2. This is the characteristic MNP shape obtained through the PLAL process. During laser radiation, fragmented material is released from the bulk material and suspended in the liquid medium with a certain density. The average size of the obtained gold particles was 26 nm with a size distribution of 18 nm  files for cubic Au crystals (JCPDS: 4-784).The EDX spectrum shown in 2. The immobilization of ligand-free AuNPs happens in a natural way after particle formation. The particles are formed in an environment filled with the carbonaceous material, which becomes a support for the newly formed nanoparticles.The decoration of graphene oxide sheets with AuNPs was achieved through the PLAL process by radiation a gold disk in a graphene oxide (GO) suspension in deionized water (1 mg/mL). The experimental set-up and conditions were exactly the same as those previously described using water as the medium. As such, the fluence was set to ca. 1 J/cmThe size of the particles, the morphology and the purity of the AuNP/GO material were characterized by TEM, EDX, Fast Mapping with SDDs (FS Mapping) and Dark-Field Scanning Transmission Electronic Microscopy (DF-STEM). The micrographs depicted in A statistical analysis showed that the average size of the AuNPs in the final material is 3 ± 1.1 nm. The majority of the particles are in the average size, but a few particles of size >20 nm were also found in the sample. The average size distribution achieved is lower than that using only water as the solvent. When water was used as the medium, the average particle size was 26 . Under tThe FS Mapping measurements, obtained by means of the EDX system and the special HAADF detector, show that this laser-assisted fabrication route produces highly pure AuNPs anchored on GO with an heterogeneous distribution . The DF-The EDX spectrum shows that, when the irradiated area by the electron beam is large, some extra elements  can be detected, similarly to what was observed in the samples produced in water . The mosd inter-planar spacing for hexagonal graphite are included in the Joint Committee on Powder Diffraction Standards (JCPDS): 41-1487).The interaction between a high power electromagnetic field, such as the one associated to femtosecond laser radiation, and almost any kind of material promotes a considerable interchange of energy that can result in structural changes in the material. In order to study how femtosecond laser radiation centered at 800 nm modifies the graphene oxide sheets, the AuNP/GO material was further analyzed by powder X-ray diffraction. The XRD pattern of rGO is completely different, the [001] signal disappears; however, the peak corresponding to the [002] plane appears at 24.85°, and the peak for [101] is observed at 43.1°. The reduction of GO using hydrazine to obtain rGO removes many oxygen functionalities and restores part of the π-network of the materiali.e. deionized water and a graphene oxide suspension in water.Using PLAL, we have decorated graphene oxide sheets with ligand-free AuNPs in a practical and single step reaction. The laser-based method causes a slight reduction of graphene oxide but avoids the use of extra chemicals that can alter the properties of the gold nanoparticles. The irradiation of a gold disk containing a GO suspension in water leads to the immediate immobilization of the generated ligand-free AuNPs on the graphene surface. According to the experimental set-up , the lasComparison of the results of both experiments suggests that graphene oxide has an influence on the AuNP formation and in graphene oxide itself by i) decreasing the average particle size, ii) immobilizing the particles on the graphene surface, iii) heterogeneously distributing the anchored AuNPs at the graphene wrinkles, and iv) slightly reducing the graphene oxide.2 onto the surface of the gold target disk. The effect of graphene oxide as a density filter was evaluated experimentally by performing an additional experiment: AuNPs were produced using only water as the medium at a fluence of ~0.25 J/cm2 which is the fluence calculated to reach the gold disk through the GO dispersion  was then added to the resulting solution and the mixture was stirred for 12 h. TEM analysis revealed that all the AuNPs previously formed in water had been immobilized onto the graphene oxide surface , but also the peak corresponding to the [002] plane . These results are further confirmed by XPS. After laser radiation, we have observed an increase of C/O ratio indicating that there is a decrease of oxygen functional groups. In this work, we have shown the convenience of using a laser-based method for the production of ligand-free AuNPs that are directly immobilized on the surface of graphene oxide. The modulation of laser properties and the presence of the graphene material have an influence in the size of the AuNPs. The properties of graphene oxide are not altered considerably after laser irradiation and we have only observed a slight increase in the reduction degree of the material. Considering previously reported results, we can make a comparison of gold nanoparticles anchored onto graphene obtained by chemical methods and laser ablation. We have observed that chemical methods allow a better control of the size dispersion of nanoparticles and produce a complete reduction of GO44in-situ femtosecond laser radiation strategy known as PLAL. The particles obtained using this procedure are in agreement with previous reported literatureIn order to produce the nanomaterials and understand the influence of graphene sheets in the synthesis of AuNPs, two different experiments were performed. For the first experiment, deionized water (18 MΩ) was used as the liquid medium to fabricate gold NPs by the 2O and H2O + GO respectively) and the ejected material that comes from the ablation process was captured in the surrounding medium. The gold disk was placed at the bottom of a glass chamber filled with H2O for the first experiment and H2O + GO for the second. The thickness of the fluid layer above the gold target in all cases was set to be ~2 mm. Then the target was irradiated from the air-liquid interface as shown in In all cases, a gold disk of thickness 1 mm and diameter 6.5 mm (99.99% purity) was used as a target to produce ablation. The target was surrounded by the liquid medium  emitting pulses of 30 fs, with a central wavelength of 800 nm, a maximum energy per pulse of 0.8 mJ, and 1 kHz repetition rate. The delivered energy was monitored by means of an analogical power meter  and controlled using a set of calibrated neutral density filters. The diameter of the incoming laser beam was controlled using an iris of 6 mm of diameter placed before the focusing lens.2 in order to avoid the plasma breakdown of water, while having enough energy to promote ablation. For the complimentary experiment where GO was considered a neutral optical density filter, we replaced the absorption contribution of H2O + GO by a neutral optical density filter which promotes the same power decrement, achieving this way a fluence of ~0.25 J/cm2 in the target. The dispersion introduced by either of the liquids, and the optical elements in general, was partially compensated in a post-compression stage by changing the relative position of two fused silica Brewster prisms, which guaranteed the minimum pulse duration at the sample.The optimized fluence of the laser beam at the sample plane was set to ~1 J/cmThe samples were analyzed by X-ray diffraction and electronic microscopy. High Resolution Transmission Electron Microscopy (HRTEM) images and High-Angle Annular Dark-Field (HAADF)-STEM images of the samples were obtained using a Jem-2100 LaB6 (JEOL) transmission electron microscope coupled with an INCA Energy TEM 200 (Oxford) energy dispersive X-ray spectrometer (EDX) operating at 200 kV. The preparation of the samples for TEM measurements was carried out by deposition of a droplet of the colloidal suspension onto a carbon-coated copper-based TEM grid. The liquid content was removed with the help of absorbent paper and, then, the sample was air-dried for several hours. The solid material collected on the grid surface was used for analysis. This technique allows the preparation of dry and highly dispersed particles. The crystallographic structure of the samples was analyzed by X-ray powder diffraction (XRD) using a Bruker D4-Endeavor diffractometer with Cu-Kα radiation at a wavelength of 0.1542 nm. X-ray photoelectron spectroscopy (XPS) spectra were acquired on a Kratos AXIS ultra DLD spectrometer with a monochromatic Al Kα X-ray source (1486.6 eV) using a pass energy of 20 eV. The photoelectron take off angle was 90° with respect to the sample plane. To provide a precise energy calibration, the XPS binding energies were referenced to the C1s peak at 284.6 eV.How to cite this article: Torres-Mendieta, R. et al. In situ decoration of graphene sheets with gold nanoparticles synthetized by pulsed laser ablation in liquids. Sci. Rep.6, 30478; doi: 10.1038/srep30478 (2016).


Graphene-based carbon materials such as fullerenes, carbon nanotubes, and graphenes have distinct and unique electronic properties that depend on their dimensionality and geometric structures. Graphene wrinkles with pseudo one-dimensional structures have been observed in a graphene sheet. However, their one-dimensional electronic properties have never been observed because of their large widths. Here we report the unique electronic structure of graphene nanowrinkles in a graphene sheet grown on Ni(111), the width of which was small enough to cause one-dimensional electron confinement. Use of spatially resolved, scanning tunnelling spectroscopy revealed bandgap opening and a one-dimensional van Hove singularity in the graphene nanowrinkles, as well as the chemical potential distribution across the graphene nanowrinkles. This observation allows us to realize a metallic-semiconducting-metallic junction in a single graphene sheet. Our demonstration of one-dimensional electron confinement in graphene provides the novel possibility of controlling its electronic properties not by chemical modification but by ‘mechanical structuring'. Wrinkles in graphene can restrict the motion of electrons to one dimension. Here, the authors observe a one-dimensional van Hove singularity and bandgap opening in a five nanometre wide wrinkle, which enables them to create a metallic-semiconducting-metallic junction in a single graphene sheet. Graphene wrinkles, which are one-dimensional 1D) folded graphene structures, have generally been observed in graphene produced by chemical vapour deposition. These structures have been thought to be the result of the difference in the thermal expansion coefficient between graphene and its substrateD folded 345In the following, we demonstrate the 1D electron confinement in graphene nanowrinkle (GNW) by scanning tunnelling microscopy/spectroscopy (STM/STS), whose width is <5 nm. Moreover, spatially resolved electronic structures have been investigated, and the manipulation of graphene geometry by STM tip has been demonstrated. Our results imply that a semiconducting property can be realized by the mechanical deformation of the graphene geometry not by chemical modification, which is analogous to the case of a strain-induced pseudo magnetic field7Epitaxial graphene with GNWs was synthesized by dissociating acetylene on a clean Ni(111) surface. A rapid cooling process is necessary, which is the most critical step to synthesize GNWs. Most of the GNWs were observed in the region where the terrace width of the underlying Ni surface was as small as several tens of nanometres . These GVs) of 1 V and a feedback current (If) of 1 nA, whereas the centre region was scanned with a smaller tip–sample distance (Vs=0.05 V and If=1 nA), so that individual carbon atoms could be clearly imaged. If up to 6 nA on the same area as h) and width (w) of the GNW were precisely measured. The translation vector (T), chiral vector (Ch) and chiral angle (θ: angle between T and the armchair direction) were defined as shown in w=2.14 nm, h=0.42 nm and θ≈10°, we could estimate the indices of the Ch to be  for the GNW ; this behaviour is similar to the ‘band-gap opening' feature observed in a single-wall carbon nanotube (SWCNT)910121314I/dV images in Vs, including the voltages at peak positions (Vs=–0.6 V (v1) and 0.5 V (c1)) and between v1 and c1  as well At the peak positions, the measured dI/dV intensities, which are indicative of the LDOS, were higher on the GNW area than on the pEG area. In contrast, lower LDOSs were observed on the GNW at Vs values in the energy gap between v1 and c1 (ΔEg) (I/dV images at the second set of peaks (Vs=−1.6 V (v2) and 1.5 V (c2)), and Vs between the first and second peaks (Vs=−1.0 V and 1.0 V)). The dI/dV images clearly show the metallic-semiconducting-metallic junction across a GNW, which has never been observed in the dI/dV spectra previously measured on other graphene wrinkles6The electronic structures of GNWs located on the terraces of the underlying Ni111) were investigated by STS and d1 were inI/dV spectra in the present work were compared with Landau levels observed in monolayer graphene under magnetic fields18n=0) should be observed near Fermi level (EF), since electrons behave like a Dirac fermion. In addition, the position of the nth Landau level (En) follows the relationship v1 or c1) are too far from the EF to assign one of them as the zeroth Landau level, even when considering the doping effect. Also, the energy gaps between the peaks do not match the relationship, We considered two plausible origins of these peaks, a strain-induced magnetic field and a 1D electron confinement. An induced magnetic field due to the high curvature in graphene can result in Landau levels in the graphene, as observed in graphene nanobubbles291011ip  ref. . In our ΔEg, which should show a relationship to w (ΔEg∝1/w), if ΔEg originates from 1D electron confinement. I/dV spectra obtained from the other GNWs within the width range 1.67 nm (a) to 3.65 nm  is used rather than width of the GNW or the diameter of the SWCNT, because L is more directly related to ΔEg induced by 1D electron confinement. Hence, ΔEgs measured in GNWs, and calculated ΔEgs for both SWNCTs20L, that is, arc length of GNW, circumference of SWCNT and width of AGNR, respectively. The arc length of the GNW was estimated on the assumption that the GNW arc shape of its cross section is a part of a circle 131415π orbitals remain at the GNW owing to the freestanding feature of GNW from the Ni surface  can have the 1D electronic structure. The strong interaction between pEG and the Ni(111) substrate may play a critical role in making a 1D electron channel along GNWs. Owing to the strong interaction, the  surface . These det al.EF in the dI/dV spectra. This implies that the strong interaction at the pEG is essential for the 1D electron confinement. Because the graphene nanoribbons were physically deposited by a spin-coating method on a Au(111) substrateTao I/dV were measured at different positions across the GNW , the STM image became very unstable (scratched lines appeared), and the width of the GNW became broader  corresponding the threshold width showing vHS. Another possibility that cannot be excluded is possible modification of the tip apex during the manipulation that can affect the spectral shape to some extent.Finally, we attempted to manipulate the GNW by the STM tip . An STM  broader . Subsequ broader . To expl broader . The mec broader , the pEG broader . The preOur results constitute the demonstration of electron confinement in GNWs with dimensions <5 nm and the demonstration of an open bandgap, which implies that GNWs have semiconducting properties. Many efforts have been made to manipulate the electronic properties of graphene by chemical modification25+ sputtering-and-annealing cycles. EGs consisting of GNWs were synthesized in a ultrahigh-vacuum chamber (base pressure: 2 × 10–7 Pa) by dissociating C2H2 (–5 Pa) on Ni(111) at 650 °C for 5 min. For rapidly cooling, the sample was immediately removed from the heating stage after the reaction. All STM images were obtained in a constant-current mode. We measured the differential conductance (dI/dV) with a lock-in amplifier by applying an a.c. bias of 50 mV (797 Hz) to the tunnelling bias. We focused only on the GNWs on the terrace of the Ni(111) surface. A total of 27 individual GNWs were observed with atomically resolved STM images.The experiments were performed in an ultrahigh-vacuum, low-temperature STM (Omicron LT-STM) at 4.7 K. The Ni(111) single crystal was cleaned by repetitive ArHow to cite this article: Lim, H. et al. Structurally driven one-dimensional electron confinement in sub-5-nm graphene nanowrinkles. Nat. Commun. 6:8601 doi: 10.1038/ncomms9601 (2015).Supplementary Figures 1-12, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary References


Dielectric-graphene-dielectric (DGD) structure has been widely used to construct optical devices at infrared region with features of small footprint and low-energy dissipation. The optical properties of graphene can be manipulated by changing its chemical potential by applying a biased voltage onto graphene. However, the excitation efficiency of surface wave on graphene by end-fire method is very low because of large wavevector mismatch between infrared light and surface wave. In this paper, a dielectric-semiconductor-dielectric (DSD) tapered waveguide with magnetic tunability for efficient excitation of surface waves on DGD at infrared region is proposed and analyzed. Efficient excitation of surface waves on DGD with various chemical potentials in graphene layer and incident frequencies can be attained by merely changing the external magnetic field applied onto the DSD tapered waveguide. The electromagnetic simulations verify the design of the proposed structure. More importantly, the constituent materials used in the proposed structure are available in nature. This work opens the door toward various applications in the field of using surface waves. Surface plasmon polaritons (SPPs) on metal surface have attracted a lot of attention from all over the world for its promising optical property, such as supporting subwavelength surface wave234810111214The wavelength and extension length of surface waves can be reduced to dozens of nanometer under infrared illumination by replacing metal with two-dimensional material, graphene. Therefore, the use of graphene provides us a path to make an ultra-compact device2021There are many methods for launching the surface wave on graphene, such as prism coupling excitationIn this work, a semiconductor tapered waveguide with magnetic tunability for efficient end-fire excitation of gaphene surface waves at infrared region for the demand of real application is proposed and analyzed. High coupling efficiency of excitation of graphene surface waves with different chemical potentials in graphene layer and different incident frequencies can be attained by merely changing the external magnetic field applied onto the tapered waveguide. Compared to previous investigations, this work focuses on adding another degree of freedom for manipulating the coupling efficiency between semiconductor surface waves and graphene surface waves by applying an external magnetic field.. x = 0) . The disWhen the external magnetic field is applied onto InSb, its dielectric constant becomes a dielectric tensor whose components depend on the magnitude of magnetic field. Therefore, the optical property of InSb can be manipulated by the applied magnetic field. This is the physical mechanism of magnetic tunability for the proposed structure. The simulated dielectric tensor of InSb under Voigt configuration is expressed ase, m* and N represent the electron charge, the effective mass and the electron density of free electron, respectively, and B is the magnitude of applied external magnetic field; and m* = 0.014 m0 (m0 is the electron mass in vacuo), where For modeling the monolayer graphene, the numerical formula for conductivity (from Kubo formula) and relative permittivity are described as below, respectively30313233e, 2 V−1s−1, which is calculated from where shown in  is almosshown in . The carThe physical mechanism of efficient excitation of graphene surface wave in the proposed semiconductor tapered waveguide is described as follows. First, the surface waves on the first section of DSD are effectively excited by a plane wave normally incident upon the end face of tapered waveguide (i.e. the end-fire method). . This effective excitation of surface wave is ascribed to a much smaller wavevector mismatch between incident light and surface wave. Second, the surface waves propagate in the tapered DSD with wavelength compression due to the gradual increase of their wavevectors. Since InSb is a lossy material (the loss is modeled by the collision frequency in the simulation), the decay of surface waves on the tapered DGD is inevitable. Finally, the graphene surface waves on DGD are efficiently excited at the interface between the fifth section of DSD and DGD because of their wavevectors close to each other. The magnetic tunability of the tapered DSD stems from the fact that the applied magnetic field can manipulate DSD’s dispersion curve.12 rad/s, which is excited directly by the end-fired method. In the simulation, the TM-polarized plane wave  is launched at the beginning of DGD structure (i.e. x = 0). However, the coupling efficiency is only 5.53%. (The method to determine coupling efficiency is listed in Methods).d). In d, as shown in insets (i)~(v) of d in front of graphene can effectively reduce the wavevector mismatch for exciting surface wave on graphene. Furthermore, since the thickness of InSb layer is larger than that of monolayer graphene, the extinction cross section of the DSD-graphene composite structure also increases. After the surface wave is excited at position of x = 0 (shown in cm−1) vs. momentum (k/k0). (See k0 is the wavevector in free space). The propagation lengths of surface waves (defined as the lengths after which the intensities decrease to 1/e) on DGD and DSD at incident frequency of 26.8 × 1012 rad/s are also calculated. Based on d = 300 nm  are 25.9 shown in , its wavd and magnitude of applied external magnetic field B) on the dispersion curves of DGD and DSD structures are also investigated. d and d = 80 nm intersect each other  as For improving the feasibility and dynamical reconfigurability of the proposed tapered waveguide, the effects of changing key parameters . The dispersion relation of DSD with an applied magnetic field under Voigt configuration is also derived in Methods. B. Insets (i), (ii), and (iii) of B = 0, 1, and 1.67 Tesla, respectively, at incident frequency of 28.6 × 1012 rad/s. They confirm that the surface waves can propagate on DSD structure and the wavelength decreases with the increase of B’s magnitude. Comparing B = 0 is suitable as a waveguide connector to efficiently excite the surface wave on graphene with its 12 rad/s. When B between 1 and 1.67 Tesla. B’s values equal to 0, 1.33 and 1.67 Tesla with incident frequency of B = 1.33 Tesla. This result is ascribed to that the dispersion curves of surface waves on DGD with B = 1.33 Tesla are very close to each other at this incident frequency . Here the DSD structure is the same as the fifth section of the proposed tapered waveguide  in 12 rad/s, respectively. They show that, except for 30 × 1012 rad/s, the surface waves with all other frequencies are efficiently excited on DGD. The surface waves of 30 × 1012 rad/s are only excited in the DSD region. All the results agree with the prediction of dispersion curves in 12 rad/s is very small (5.53%) due to momentum mismatch between surface wave on DGD and light line. However, when the tapered waveguide is adopted, the coupling efficiency at the same frequency increases to 68.7% (insets (ii) of B = 0). For 23 and 17.2 × 1012 rad/s with B = 1 and 1.67 Tesla, respectively, the coupling efficiencies of surface waves on DGD are also substantially improved (insets (iii) and (iv) of d of DSD structureFinally, the frequency tunability of surface wave on DGD that is excited by using the proposed tapered DSD waveguide is examined. The dispersion curves of surface wave on DGD with ases see . Here thIn this work, parylene thin film can be used as the substrate in our proposed design. Its relative permittivity is close to 3.0 at the operating frequenciesases see .In summary, the DSD tapered waveguide with magnetic tunability for efficient excitation of surface waves on DGD at infrared region is proposed and analyzed. Excitation of surface waves on DGD with various chemical potentials in graphene layer and incident frequencies can be attained by merely changing the values of external magnetic field applied onto the DSD tapered waveguide. The FEM electromagnetic simulations verify the design of the proposed structure. More importantly, the constituent materials used in the proposed structure are available in nature. Hence, our methodology can be applied in the field of use of surface waves, such as multi-functional material (device), real-time subwavelength imaging, and high-density optoelectronic components. It will provide us a path to overcome the major issue of energy dissipation of surface waves in conventional metallic material .All the simulated results shown in The dispersion relation of surface wave on each three-layered DSD structure without applying the external magnetic field is described as,where When the external magnetic field is applied onto the DSD structure, the dispersion relation is transformed into the following form:where The coupling efficiency of excitation of surface wave on DGD is defined as How to cite this article: Cheng, B. H. et al. Tunable tapered waveguide for efficient compression of light to graphene surface plasmons. Sci. Rep.6, 28799; doi: 10.1038/srep28799 (2016).


In this paper we detail a novel semi-automated method for the production of graphene by sonochemical exfoliation of graphite in the presence of ionic surfactants, e.g., sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). The formation of individual graphene flakes was confirmed by Raman spectroscopy, while the interaction of graphene with surfactants was proven by NMR spectroscopy. The resulting graphene–surfactant composite material formed a stable suspension in water and some organic solvents, such as chloroform. Graphene thin films were then produced using Langmuir–Blodgett (LB) or electrostatic layer-by-layer (LbL) deposition techniques. The composition and morphology of the films produced was studied with SEM/EDX and AFM. The best results in terms of adhesion and surface coverage were achieved using LbL deposition of graphene(−)SDS alternated with polyethyleneimine (PEI). The optical study of graphene thin films deposited on different substrates was carried out using UV–vis absorption spectroscopy and spectroscopic ellipsometry. A particular focus was on studying graphene layers deposited on gold-coated glass using a method of total internal reflection ellipsometry (TIRE) which revealed the enhancement of the surface plasmon resonance in thin gold films by depositing graphene layers. The double-bonded structure of graphene is responsible for the electrical properties of the material as the movement of π-bonds between adjacent carbon atoms can be used to transmit an electrical current. Because of this electrical activity in particular, graphene is being examined as a base material in a number of different applications including sensor applications, for use in flexible electronics [Since its initial discovery and development by Novoselov et al.  graphenectronics .Graphene has reportedly been produced in a number of different ways. The method chosen for this research is sonochemical exfoliation in water in the presence of a surfactant, as first reported by Notley et al. . This meIn previous work  we manufIn this work a semi-automated technology of graphene production by sonochemical exfoliation of graphite in the presence of ionic surfactants is described in detail. The composite graphene-surfactant materials produced were characterised with NMR and Raman spectroscopy to confirm the formation of graphene. Thin films of graphene composites were deposited using the techniques of Langmuir-Blodgett (LB) and electrostatic layer-by-layer (LbL) deposition. Films composed of these new graphene composite materials were then characterised using SEM, AFM, and spectroscopic ellipsometry. The study of SPR in gold films coated with graphene using total internal reflection ellipsometry was carried out for the first time.−1 while the CTAB solution concentration was made up to 49.7 mg·mL−1. These solutions were prepared and then placed into a water bath heated to 50 °C to aid dissolution.Two different surfactants were used to synthesise graphene–surfactant complexes: sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Firstly the surfactant solutions were prepared by dissolving in water. The SDS solution was made to a concentration of 462.9 mg·mL−1 giving an addition rate of 16.2 mg·min−1 for SDS and 1.74 mg·min−1 for CTAB. The addition rate was crucial to maintain a surface tension of 41 mJ·m−2, which is both the optimum surface tension for graphene production and also the surface free energy of graphene. The graphite suspension (10–50%) was then placed into the reactor, and sonicated continuously for 120 min at a power of 50 W. A total of 3.15 mL of surfactant solution was added in each case over the course of the synthesis. The sonication step was carried out in a fume cabinet, as an aerosol containing potentially harmful graphene nanoparticles is produced at this stage. A schematic diagram of this apparatus is shown below  was re-suspended in 40 mL of deionised water. This was then centrifuged at 3500 rpm for 20 min. This process was repeated until no more graphene was contained in the supernatant. If a dry sample was required, then the graphene suspension was frozen and then placed into a freeze dryer (Labogene) for 3–4 days (depending on the sample size) and then subsequently into a vacuum oven at 70 °C overnight to remove all traces of water.−1·cm−1 [2O and placed into an NMR tube. Another tube containing the surfactant alone was also prepared. Both were scanned individually and then the spectra were compared to show shifts in peak position corresponding to interaction between the surfactant and the graphene. NMR experiments were performed on a Bruker 400 MHz spectrometer running topspin analysis software. This analysis works according to the nuclear Overhauser effect (NOE). The NOE, which is present in nuclear magnetic resonance (NMR) spectroscopy, can be used to determine the amount of contact between the surfactant and the graphene sheet. This is achieved by observing peak shifts that occur when two nuclei are within 5 Å of each other. The dried graphene sample was deposited onto a glass microscope slide and then scanned using a Renishaw In-Via Raman microscope at an excitation wavelength of 532 nm. Data were recorded between 100 and 3200 cm−1.Graphene–surfactant complexes were characterised by using a range of different techniques. The presence of graphene was determined by Raman microscopy. The concentration of graphene in solution was obtained by spectrophotometry. The absorbance of a graphene solution at a wavelength of 650 nm was determined and the Beer–Lambert law applied, using an extinction coefficient of 13.9 mg·mL−1·cm−1 . The int3+ and SO3−, respectively. Yet, the presence of alkyl chains and π-systems facilitated their solubility in chloroform, hence the use of the Langmuir–Blodgett (LB) technique (Nima 610 trough) was an obvious choice for the deposition of thin films. The standard LB procedure was applied [−1 solution of graphene(+)CTAB in chloroform was spread onto the surface of deionised water (Millipore). Surface pressure was then recorded using a Wilhelmy plate-based sensor. Because of the unknown ratio of graphene/surfactant within the complex the area per molecule (or repeated unit) was difficult to calculate, so the area was presented in actual units of cm2. Another method known as Langmuir–Schaefer (LS) deposition [Both composite materials obtained, e.g., graphene(+)CTAB and graphene(−)SDS, appeared to be soluble in water due to the presence of ionic groups, NMe applied : a 1 mg·position  was also−1 solutions of the above materials in deionised water. The mutlilayered films obtained were then characterised with scanning SEM combined with EDX (energy dispersing X-ray) elemental analysis (SEM NOVA) and AFM.Much better results (in terms of adhesion and surface coverage) were obtained by using a simple technique of electrostatic layer-by-layer (LbL) deposition, a well-established technique developed first for polyelectrolytes  and lateUV–vis spectra of graphene–surfactant samples are featureless showing almost constant absorbance over the spectral range of 350–800 nm, while the main absorption band of graphene lies in the UV region at about 280 nm. Therefore, optical properties of novel graphene–surfactant composites were studied via spectroscopic ellipsometry using a J. A. Woollam M2000 instrument operating in the spectral range of 370–1000 nm. The measurements were performed on graphene–surfactant films deposited on different substrates, i.e., glass, silicon, and gold-coated glass. Experimental parameters were found by fitting data, the procedure for which is explained in the Results and Discussion section below.−6 Torr using an Edwards 360 unit. Prior to LbL deposition, gold-coated slides were treated overnight in cystamine hydrochloride solution in order to make the gold surface positively charged. Then LbL deposition started with the layer of graphene(−)SDS (negatively charged) followed by deposition of graphene(+)CTAB (positively charged). This procedure was repeated four times, so that four graphene bilayers were deposited. Layer by layer deposition onto other substrates was performed in a similar manner.For the LbL deposition of alternating layers of graphene(−)SDS and graphene(+)CTAB on gold-coated glass slides, a gold film of approximately 25 nm thickness was thermally evaporated onto a 3 nm under layer of chromium (which was used to improve adhesion between the gold and the glass). Metal evaporation was carried out in a vacuum of 10The surface plasmon resonance (SPR) phenomenon in graphene films deposited on thin films of gold was studied in more detail using the method of total internal reflection ellipsometry (TIRE), which was developed in the last decade –14. TIREGraphene was synthesised using graphite suspensions of 10–50% using either SDS or CTAB as the surfactants. The concentration of the final graphene solution obtained from each synthesis was determined by spectrophotometry .1H NMR measurements of the graphene–surfactant complex, when compared to 1H NMR measurements of the surfactant alone, shows shifting of peaks representing hydrogens involved in the complexation interaction  is shifted downfield by complexation with the graphene. This means that the protons are deshielded by the presence of graphene. Since the carbon–sulfur bond is polar the electron density around the carbon atom is already lower than it would normally be in a carbon–carbon bond. Repulsion between the graphene and the sulfate group could cause lengthening of the carbon–sulfur bond. This could in turn lead to a lower electron density around the nuclei responsible for this peak .−1 (D) and 1620 cm−1 (G & D’). Additionally the peak labelled 2D is slightly broader, between 2650–2700 cm−1. This is indicative of graphene flakes with a high number of edge defects [The sample was also analysed using Raman spectroscopy  which, w defects .2-hybridised carbon materials, and is caused by stretching of the C–C bond. The reduction in intensity in the graphene spectrum compared with the graphite spectrum is caused by the reduced number of layers. The D and D’ bands are caused by disorder in the graphene flakes. The D’ band is present when there are surface defects, such as charging or other impurities adsorbed onto the surface. The D band is caused by edge defects such as a “zig-zag” or “chair” shape on the edge. Edge defects provide an enhancement to electrochemical systems by increasing the total capacitance of the electrode surface. Both the D and the D’ band are not present in pristine graphene with straight edges [The G band is present in all spht edges .2-hybridized systems and can be used to estimate the number of layers [The 2D band is also present in many spf layers . Howeverf layers .Typical surface pressure vs area diagrams of graphene(+)CTAB in - groups, positively charged graphene(+)CTAB was chosen; as this provided good adhesion between the substrate and monolayer.However, for LB deposition onto hydrophilic substrates such as glass or oxidised silicon, which are slightly negatively charged due the presence of OHAttempts at depositing thin films of graphene(+)CTAB using a traditional LB process, e.g., vertical dipping and withdrawing the substrate through the monolayer, were not successful since the transfer ratio was poor. The first withdrawal yielded about 60%. After that the layer was shown to peel off during subsequent dipping cycles. The overall transfer ratio by area (when substrate surface area was compared with graphene LB isotherm) was 10–20%. The most significant cause of this was poor adhesion of the first graphene layer to hydrophilic substrates. This could be improved in future work through the use of substrates with surface modifications that either enhance the surface charge or make the surface more hydrophobic. Much better results were obtained using the horizontal lifting method known as Langmuir–Shaefer (LS) deposition , in whicOrganised monolayer films obtained in this fashion were then characterised by using AFM (Nanoscope III) operating in tapping mode using Veeco cantilevers with silicon nitride tips having a radius of less than 10 nm. A typical AFM image of graphene(+)CTAB flakes deposited onto a piece of silicon wafer using LS method is shown in Sectional analysis of AFM image along the lines shown, allows for an estimation of the graphene(+)CTAB flake thickness at 2 nm, which is significantly higher than the reported thickness of pristine graphene of 0.355 nm . The obtIt is clear from these images that the surface coverage is not optimal. Additionally the graphene flakes were seen to change position and shape during scanning, suggesting poor adhesion to the silicon surface. This could potentially be overcome in future work by using surface-modified silicon wafers for sample deposition.The multi layered films obtained from layer-by-layer deposition method were characterised with scanning SEM combined with EDX (energy dispersing X-ray) elemental analysis (SEM NOVA) and AFM.Not all alternating combinations worked well, however. For example, the most promising combination of graphene(+)CTAB with graphene(−)SDS was not successful, while the alternation of graphene(+)CTAB with PAH (or with PEI) proved to be optimal. Deposition on glass or silicon samples was performed by electrostatic adsorption of PAH (or PEI) for 20–30 min followed by dipping into a solution of graphene(−)SDS for 10–15 min. This sequence was repeated several times with a typical incubation time of 10–15 min.Adhesion between graphene and substrate was greatly improved when using a branched polycation such as PEI. The AFM image of graphene(−)SDS deposited onto a layer of PEI in Sectional analysis performed on the sample shows the double and triple layers, and shows the thickness of a single flake at approximately 2.5 nm. This is a somewhat higher than that for LS films in The analysis of graphene films by spectroscopic ellipsometry has already been carried out by other groups, and so is fairly well described –21. A tyd) and dispersions, e.g., spectra of refractive index (n) and extinction coefficient (k) of graphene films can be found by fitting the above spectra to the model using the dedicated software by J. A. Woollam. In this particular case, the model of the reflecting system consists of the following three layers: (1) Si substrate; (2) the layer of native oxide (SiO2) which is typically present on the surface of Si; (3) the deposited layer of PAH/graphene(−)SDS. The ambient was air. Optical parameters for Si and SiO2 were taken from the J. A. Woollam database. The fitting was first performed for ellipsometric spectra of the Si/SiO2 substrate. For the set of data in 2 layer was performed first using the data for the bare Si substrate. The thickness of SiO2 layer obtained (d = 3.2 nm) was then fixed for consecutive fittings. The PAH/graphene(−)SDS film was considered as one layer in the following fittings which is justified by the fact that aliphatic chains of SDS interlock with PAH and form a mixed composite layer of PAH/graphene(−)SDS. Several possible models were tried for fitting PAH/graphene(−)SDS layers, and the best results were achieved using a Lorentz oscillator model from the J. A. Woollam data analysis software, which is given below as a dispersion function of a complex dielectric permittivity, ε(hν):The thickness values 2, Bk = 3.86 eV. The presence of the Lorentz peak in the IR region gives a featureless dispersion of k for graphene–surfactant composite films in the visible spectral range similar to that reported in [d value of the first bilayer appeared to be too high, the average thickness increment Δd = 3.63 ± 2 nm is reasonable and close to that observed with AFM.where εorted in –23. The orted in –23 at abd, n(λ) and k(λ) of the chromium/gold layer were found by fitting the data for uncoated samples, and then used as fixed parameters for subsequent fittings. The graphene layers were modeled through a Lorentz oscillator as before, the values for thickness obtained are plotted against the graphene layers deposited in Spectroscopic ellipsometry measurements were carried out on samples after each layer was deposited and similarly to the previous experiments, Ψ spectra did not change much while the Δ spectra exhibited downward shifts upon deposition of the layers. The ellipsometry data fitting was performed in a similar way as described above using a three layer model containing the substrate glass, the chromium/gold layer, and the graphene layer. The ambient was air. The parameters As mentioned above, the use of PEI as a binding layer alternating with graphene(−)SDS improving the deposition of graphene. The ellipsometry spectra recorded on samples of PEI/graphene(−)SDS deposited on gold-coated glass are shown in It is quite remarkable to observe spectral features around the plasmon-resonance frequency of the gold substrate, which are not related to graphene itself –23 but rThe samples were constructed by electrostatic LbL deposition of PEI and graphene(−)SDS on chromium/gold-coated glass slides, as described above. The results obtained are shown in Deposition of graphene layers made the SPR minimum even lower on the Ψ spectra while causing an additional phase shift on Δ spectra. The enhancement of the SPR peak due to the deposition of graphene layers is observed for the first time. It is interesting to note that graphene itself does not show plasmonic behavior in the visible spectral range as was proven by ellipsometry measurements shown before . At the A simple semi-automated technique for graphene production by aqueous sonochemical exfoliation of graphite in the presence of ionic surfactants, e.g., CTAB or SDS, was developed. Full automation could be potentially achieved by adding surface tension sensors to control the amount of surfactant being added to the reactor, thus maintaining a constant and optimum surface tension. The formation of individual graphene flakes and the interaction of alkyl chains of the surfactants with graphene were, respectively, confirmed with Raman spectroscopy and NMR measurements.The two different graphene-surfactant complexes produced (graphene(+)CTAB and graphene(−)SDS) appeared to be soluble in water and thus suitable for electrostatic LbL deposition. Both compounds were also found to be amphiphilic and soluble in chloroform, hence it was possible to form stable monolayers on the water surface. Thin films of the above graphene composites were deposited onto different solid substrates, i.e., silicon, glass and gold-coated glass, using either electrostatic LbL or LB (LS) deposition techniques. SEM and AFM study showed that LB (or LS) films of graphene(+)CTAB had poor surface coverage and adhesion to the substrate. Electrostatic LbL deposition of graphene by alternation of graphene with oppositely charged polyelectrolytes was much more promising in these aspects. Several combinations of materials were tried including the alternation of graphene(+)CTAB and graphene(−)SDS. The best results were achieved by alternation of graphene(−)SDS with PEI. AFM study allowed the estimation the thickness of an individual graphene–surfactant flakes of about 2.0–2.5 nm.The spectroscopic ellipsometry study of graphene thin films gave similar values for the thickness of the graphene–surfactant composite layer. While the dispersions of refractive index and extinction coefficient were modelled by a single Lorentzian peak lying in the IR region, the absorption peak of graphene at approximately 260 nm was outside the spectral range (370–1000 nm) of the ellipsometric instrument. Interesting results were obtained when studying the SPR effect in gold films coated with a few layers of graphene using both external and internal (TIRE) reflection ellipsometry. Even though graphene itself axhibits no spectral features associated with plasmon oscillations in the above spectral range, the deposition of graphene layers on gold progressively enhances the plasmon resonance in TIRE Ψ spectra and caused an extra phase shift in TIRE Δ spectra. This phenomenon can be explored in the future for enhancing the performance of SPR-based biosensors.