Macaca fuscata). Although reconciliation functions to repair bonds, such interactions sometimes entail risks of receiving further aggression. Vocalizations can be used at a distance from the former opponent; thus, we predict that vocalizations are used particularly by victims of a conflict, and are frequently used in situations of uncertainty when it is difficult for them to estimate whether the former opponent will resume aggression. In addition, we predict that vocalizations are effective in preventing further aggression. To test these hypotheses, we conducted observations of post-conflict and matched-control situations in female Japanese macaques living in a free-ranging group. We found that former opponents tended to be attracted to each other within the first minute following a conflict, thus demonstrating reconciliation behavior. Vocalizations were more frequently used by the victims in post-conflict interactions than under control situations; however, this tendency was not found in aggressors. When affiliation with the former opponent occurred, victims were more likely to use vocalizations towards less familiar opponents. These findings suggest that Japanese macaques used vocalizations more often when interacting with less predictable former opponents. Victims were more likely to receive aggression from former aggressors when engaged in affiliations with them than under no such affiliations. No significant differences were found in the probability of the victims receiving aggression, regardless of whether they used vocalizations; thus, whether the victim benefits from using vocalizations in these contexts remains unclear. Japanese macaques form despotic societies and therefore, further aggression was inevitable, to some degree, after a conflict. The use of vocalizations by a victim was found to depend on the nature of their relationship with the aggressor; however, the effectiveness of this behavior requires further investigation.We investigated the use of vocalizations called “grunts,” “girneys,” and “coos” accompanied by post-conflict affiliative interaction between former opponents (reconciliation) in Japanese macaques ( A post-conflict (PC) affiliative interaction between former opponents is generally labeled as reconciliation , 2. RecoMacaca arctoides) is categorized into two general forms: grooming/contact sitting and socio-sexual behaviors  [Papio hamadryas hamadryas) who are non-friends often engage in non-contact greetings, such as approaches or facial expressions  after conflicts [Mandrillus sphinx) direct facial expressions or postures toward former opponents who are likely to instigate renewed aggression [A previous research has indicated that forms of post-conflict interactions differ based on the nature of the relationship between opponents and the risk of renewed aggression. For instance, reconciliation among stump-tailed macaques (ounting) . The forounting) . Male haonflicts . Mandrilgression . These ngression . These sMacaca fuscata), use soft vocalizations known as grunts and girneys [Papio spp. [Macaques, including Japanese macaques , anpio spp. –23. A prpio spp. . This vopio spp. .Japanese macaques form despotic and nepotistic societies with steep dominance relationships and kin-biased social relationships , 26. In We aimed to clarify whether vocalizations are used on the basis of the level of uncertainty in post-conflict situations, and whether vocalizations play a specific role among Japanese macaques. We assume that uncertainty increases in post-conflict situations and during interactions with less-familiar or higher-ranking opponents. We first examined whether victims used vocalizations more frequently in post-conflict situations than in control situations. We then examined whether vocal behaviors occurred more frequently with less-familiar and higher-ranking individuals. Finally, we examined whether vocalizations function to prevent further aggression from the former opponent, that is, we examined whether the immediate risk of receiving aggression was lower following post-conflict interactions with vocalizations than those without vocalizations.We conducted this study in accordance with the Regulations on Animal Experimentation at Osaka University. The study was approved by the Animal Research Committee of the Graduate School of Human Sciences at Osaka University in Japan (No. 21-14-10). As the study area was privately owned, we conducted observations with the permission of the manager of Iwatayama Monkey Park, Kyoto, Japan.h' [The present study was conducted in a free-ranging group of Japanese macaques belonging to the Arashiyama group at the Iwatayama Monkey Park (35°00′N and 135°67′E), Kyoto, Japan. The Arashiyama group has been provisioned since 1954 , after wh'  and the Behavioral observations were conducted between 09:00 h and 17:30 h, when the monkeys remained near the provisioning area, based on the post-conflict-matched-control (PC-MC) method , 29. We The following behaviors were video-recorded (SONY HDR-CX560V) by the observer using the all-occurrence sampling method in PC and MC sessions: occurrence of aggression received by the subject, the first affiliative behavior(s) between the subject and the former opponent in PC sessions, and between the subjects and any other individuals (including the former opponent) in MC sessions. Affiliative behaviors were defined as non-aggressive physical contact, including grooming (giving and receiving); approaches within arm’s length; and giving vocalizations such as grunts, girneys, and coos, which are distinct vocalizations and therefore distinguishable by ear . If the In addition to PC-MC observations, we collected data on the baseline levels of affiliative interactions between adult females as an indicator of familiarity. We conducted line census observations, in which all visible adult females were recorded by an observer while walking along a predetermined route. We used this observation method instead of scan sampling because individuals were dispersed over a wide range and could not always be observed from a single location simultaneously. We recorded the activity of each adult female during: (1) physical contact including grooming, or within 3 m of another adult female, or (2) alone. Intervals between each census were at least 1 h in length, and a total of 172 census sessions were performed over 95 days.We calculated the index of familiarity between two adult females (A and B) by dividing the number of sessions that A had contact with or proximity to B by the number of sessions A was recorded in the line census. The familiarity index of A with B and that of B with A is not necessarily identical, given that the relative familiarity may not be equal for each individual (3). Because the level of familiarity between two individuals may change over time, we calculated the familiarity index separately into two evenly divided observation periods: (1) from March 2013 to March 2014 and (2) from April 2014 to March 2015. The former and latter periods included 87 and 85 census sessions, respectively. Each focal subject was recorded in 12–55 (median: 29) sessions in the former period and in 10–45 (median: 23) sessions in the latter period. The familiarity indices ranged from 0 to 0.73 (median: 0). The correlation between the familiarity indices of the former and latter period was significant, but relatively low . This is probably because presence or absence of an infant has an impact on social interaction between females in Old World monkeys , 32.Rank differences between subjects and former opponents were calculated by subtracting the absolute dominance rank of the former opponent from that of the subject. Positive values for rank differences indicated that the subject was subordinate to the former opponent, and negative values indicated that the subject was dominant. Values for the rank differences ranged from –89 to 89.We conducted all analyses in R version 3.2.0 . We firsP < 0.05.In the following analyses, we conducted generalized linear mixed models (GLMMs) with a binomial distribution (logit link) using the lme4 package in R . GLMMs aWe investigated the factors that influenced the occurrence of affiliative interactions between former opponents for all PC sessions Model 1; . The resWe investigated the probability of receiving aggression between all PC and MC sessions Model 2; . The resWe investigated whether vocal use increased after a conflict in PC and MC sessions in which affiliative interactions  occurred Model 4; . The resWe then examined factors affecting the vocal use of subjects in the PC session Model 5; . The resWe compared the risk of receiving aggression immediately after affiliations between the former opponents by whether affiliative behaviors included vocalizations. For PC sessions, in which affiliative interactions between former opponents occurred, we compared the probability of receiving aggression after affiliative behaviors between instances in which subjects used vocalizations and those in which they did not Model 6, . The resN = 76, mean ± SD: 12.5 ± 15.0%) exceeded those of dispersed pairs (8.3 ± 10.7%) for former opponents after the conflict . However, the proportions of dispersed pairs exceeded those of attracted pairs in the following timeframes . Thus, reconciliation occurred, but only in the first minute.Of the 300 pairs of PC-MC sessions in which aggressors were the focal subjects, affiliative behaviors occurred in 47 PC sessions with the former opponent and in 112 MC sessions with any individuals. Of the 305 sessions focusing on victims, affiliative behaviors occurred in 72 PC sessions and in 113 MC sessions. In the MC sessions, neither aggressors nor victims interacted with former opponents. The analysis revealed that in the first 1-min timeframe, the proportion of attracted PC-MC pairs  toward opponents and in 16 MC sessions (14.3%). Victims gave vocalizations in 33 PC sessions (45.8%) toward opponents and in 10 MC sessions (9.8%). The results of the GLMM for PC and MC sessions in which affiliative interactions occurred revealed that the effect of PC or MC was significant for victims  and those that did not Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


It was found that continuously distributed nano-scale patterns formed on NSiCLs which were about submicrons in-plane and about 100 nanometers out-of-plane in size. After HTTs under vacuum, pattern sizes reduced, and the sizes of the remains were inversely proportional to the treatment time. Referring to Raman spectra, the establishment of multi-layer graphene (MLG) on NSiCL surfaces was observed. MLG with  First, thermal decomposition is a relatively simple process, since it can be performed at a wide temperature range, e.g., 1400~2000 K under high or medium vacuum, even at Ar ambient conditions . Second,bstrates  which pebstrates ,18 and cbstrates ,20, wereIn addition to growth on flat, rigid substrates to facilitate the planar device fabrication process, methods have been found to combine graphene with nano-materials, forming nanostructures which are expected for use in potential applications in nanoelectronics and optoelectronics . AlthougDue to high density nano-terraces on the epitaxial surfaces, which guide epitaxial growth in the so-called “step-control growth mechanism”, 4H–SiC homoepitaxial layers with smooth and uniform morphologies are usually grown on 4H(0001) substrates by low pressure chemical vapor deposition (LPCVD) ,27. Acco−3 Pa for one hour and two hours, respectively . Nano-features still appear on the surfaces of the mentioned samples; however, sizes of the features are miniscule compared to those of NSiCLs. Although binary compound SiC is chemically inert and it is difficult to break the Si–C bonds, when subjected to high temperature treatment, silicon and carbon bonds break from surface level to a few monolayers beneath, and silicon atoms detach from the SiC surface while the remaining carbon atoms rebond with each other and rearrange on the surface. Nano-texture surfaces will be more active for decomposition than flat surfaces due to their larger surface area. They are also especially reactive on the edges since there are more unsaturated bonds and fewer bonds that have to break to detach an atom from an edge. At high temperature, those Si–C bonds at lower-energy barriers will break more easily, while unbound SiC molecules or clusters will detach from surfaces until all lower-energy barrier bonds are broken. In the end, nano-texture surfaces will lose many SiC fractures, becoming smoother with smaller features, as shown in Nano-textures evolve after high temperature treatments. −3 square microns, as it is below), as shown in f(x), expressed as: a0, ia, it, and x0 are fitting constants, and the variable parameter x is the in-plane lateral position.Feature sizes of those samples and their evolution are illustrated in Apart from feature sizes, the morphologies of graphene G-1h-1700K and G-2h-1700K are similar to each other and to that of NSiCL . We chosTo further investigate the in-plane and out-of-plane feature sizes of NSiCL and graphene, we performed line-sectional analyses on AFM images. Using a horizontal line across the middle of the images, sectional data were extracted from AFM images in −1–2800 cm−1. Usually named D mode, G mode, and 2D mode, respectively, peaks located at 1350 cm−1, 1580 cm−1, and 2700 cm−1 relate to graphene signals [We used Raman spectra to judge whether NSiCL is a 4H polytype, as well as find layer and defect information of the obtained graphene.  signals , which i signals .I2D/IG) is used to identify the number of graphene layers [I2D/IG ~ 2 indicate monolayer graphene, while those with 1 < I2D/IG < 2 indicate bilayer graphene [I2D/IG for our samples are all less than 1, indicating that the obtained graphene are multi-layer graphene (MLG)  c.−1, plus the 2D peak can be fitted by a one-peak Lorentz fitting curve  of the 2D peak is another indicator of graphene quality . G-2h-17ng curve c. For a urve , it often forms an interfacial buffer layer of carbon between the Si face and graphene . This ise C-face  and the e C-face . FurtherC wafers . In thisaminated . This reaminated ,7,43, in15 cm−3, respectively, were grown on commercially available, on-axis 4H–SiC(0001) substrates, using low pressure chemical vapor deposition. In order to obtain surfaces with nano-textured morphology, an unconventional growth technique was adopted. Before epitaxial growth, 4H–SiC(0001) substrates were etched under H2 atmosphere at 1600 K for one hour to prepare clean and smooth surfaces. After etching, the temperature was raised to 1850 K rapidly, while SiHCl3 and C2H4 as precursors, diluted in highly pure H2 (99.999%), were fed into the chemical vapor deposition (CVD) chamber for epitaxial growth. Precursors were turned off after a 30 s growth period, and the 4H–SiC samples cooled down to room temperature naturally under H2 atmosphere. Pressures were kept at 104 Pa during the whole process.Nano-textured 4H–SiC homoepitaxial layers (NSiCLs) with nominal thickness and doping concentration of 620 nm and 5.8 × 10−3 Pa for one and two hours, respectively, while some samples were treated at 1900 K under Ar atmosphere of 1.013 × 105 Pa (1 atm) for one hour. The corresponding samples are denoted as G-1h-1700K, G-2h-1700K, and G-1h-1900K, respectively.NSiCLs were subjected to high temperature treatments in an induction furnace. Samples were treated at 1700 K under medium vacuum of 10Morphologies of both NSiCL and graphene were characterized by atomic force microscopy , while morphology features were characterized by statistical histogram and sectional analyses. Raman scattering spectra  were used to evaluate graphene quality, and were performed at room temperature using an He-Ne laser operating at the 632.8 nm line mode with a confocal microscope at about 1 μm spatial resolution.−3 Pa for one hour, while multi-layer graphene (MLG) grew in two hours. MLG without sp2 disorders was also grown under argon once NSiCL was treated at the higher temperature of 1900 K. This investigation produces an avenue of refined technique for graphene production, expanding the pathway for future nano, SiC-derived graphene growth.In summary, nano-textured 4H–SiC homoepitaxial layers (NSiCLs) were grown on 4H–SiC(0001) by an unconventional epitaxial method using low pressure chemical vapor deposition, producing as-grown NSiCLs with feature sizes of about 0.01–0.29 square microns. After subjection to different high temperature treatments, feature sizes of the NSiCLs were reduced, while remaining features were at least two orders of magnitude smaller than on as-grown NSiCLs. Graphene was grown on these 4H–SiC homoepitaxial layers. The initial growth of graphene took place after a high temperature treatment at 1700 K under medium vacuum of 10


Graphene as one type of well-known solid lubricants possesses different nanotribological properties, due to the varied surface and structural characteristics caused by different preparation methods or post-processes. Graphene nanosheets with controllable surface wettability and structural defects were achieved by plasma treatment and thermal reduction. The nanotribological properties of graphene nanosheets were investigated using the calibrated atomic force microscopy. The friction force increases faster and faster with plasma treatment time, which results from the increase of surface wettability and the introduction of structural defects. Short-time plasma treatment increasing friction force is due to the enhancement of surface hydrophilicity. Longer-time plasma treatment increasing friction force can attribute to the combined effects of the enhanced surface hydrophilicity and the generated structural defects. The structural defects as a single factor also increase the friction force when the surface properties are unified by thermal reduction. The surface wettability and the nanotribological properties of plasma-treated graphene nanosheets can recover to its initial level over time. An improved spring model was proposed to elaborate the effects of surface wettability and structural defects on nanotribological properties at the atomic-scale. Graphene derived from graphite is a highly attractive two-dimensional lubricating material, as well as a wear-resistant coating12479et al. prepared a hydrophobic trilayer film of APTES-GO-OTS and investigated its tribological behaviors. The low adhesion and the greatly reduced friction force were found on the film, which was attributed to the OTS-introduced surface hydrophobicityet al. studied the effect of structure on the nanotribology of ultrathin graphene oxide films with varying C/O ratio through friction force microscopy, and found higher C/O ratio GO exhibited much improved tribological properties and wear resistance which approached that of the graphene sampleset al. discovered the friction coefficient of structural disordered graphene treated by oxygen plasma was larger than pristine graphene based on the micro-scale scratch testset al. investigated the friction behaviors between a diamond tip and three-layer graphene with single defect or stacked defects by using MD simulations, and showed that the friction force on defective graphite was significantly influenced by the defect location and type8131415Junfei Ou 3 hybridization defects, vacancy-like defects and substitution-like defects, can be introduced into graphene by oxygen plasma treatment21The roles of surface properties and structural characteristics in nanotribological properties of graphene need to be further enriched, and the mechanism is also acquired to be understood more deeply. Moreover, their roles in nanotribological properties are always confused in previous studies. It is meaningful to control the nanotribological properties of graphene with controllable surface properties and structural defects. Plasma treatment is an effective way for the modification of surface properties and etching of graphene171819The graphene nanosheets with controllable surface wettability and structural defects were achieved by plasma treatment and thermal reduction. The roles of surface wettability and structural defects in nanotribological properties were investigated separately using calibrated atomic force microscopy (AFM). The theoretical calculation and atomic model were adopted to uncover the underlying mechanism. The time-dependent nanotribological properties of plasma-treated graphene nanosheets were also explored.−1) consists of six components. On the basis of the height acquired from AFM topographic images and the number of sub-2D peaks fitted from Raman spectra, it can be concluded that the number of layers of graphene nanosheets is about three-layer23A series of characterizations of graphene nanosheets with same thickness after plasma treatment are conducted by AFM, Raman spectroscopy and WCA measurements. The measured WCA values on the surface of graphene nanosheets before and after plasma treatment are shown in −1) appeared after plasma treatment for 0 s, 1 s and 2 s, and the intensity ratio ID/IG is also nearly zero. It can be concluded that plasma treatment for 1 s and 2 s do not destroy the structures of graphene nanosheets. When the treatment time reaches 3 s, the structural defects start to produce as D peak emerges, and since then the intensity of D peak and the intensity ratio ID/IG increase continuously. In addition, the approximate linear relationship between ID/IG and plasma treatment time is unexpected, which means that the degree of structural defects is proportional to the plasma treatment time.Raman spectra of the plasma-treated and thermal-reduced graphene nanosheets are presented in 2 networks of graphene, but the existing defects can also be extended and some new defects can be producedD/IG of thermal-reduced graphene nanosheets is larger than that before thermal reduction. While for long-time plasma-treated graphene nanosheets, the intensity of D peaks and the intensity ratio ID/IG get smaller after thermal reduction. These results suggest that the controllable surface properties can be obtained on short-time plasma-treated graphene nanosheets, while the controllable structural defects can be obtained on thermal-reduced graphene nanosheets.Thermal reduction is widely used for the change of the surface properties via removing the hydrophilic functional groups, but also affects the structures inevitably. Structural disorders can be reduced by restoring SP3 hybridization defects because of the existence of oxygen-containing functional groups etc.1718192 hybridization zones after carbon deoxidization and induce vacancy-like defects, since C atoms are also taken away during the removal of oxygen-containing functional groups21D/IG increases after thermal reduction.In addition, the shapes of D peaks are different in both cases . The D pSince no structural defects are induced by plasma treatment for 1 s and 2 s, only the surface properties of graphene nanosheets are changed. The effects of the controllable surface properties as a single factor on nanotribological properties can be performed on these plasma-treated graphene nanosheets. The dependences of adhesion force on plasma treatment time and friction force on normal force are summarized in The significance of water contact angle (WCA) is also reflected that it contains information about the surface energy of solid surfaces through the Young equationSV, γLV, γSL represent the solid surface free energy, liquid surface free energy, and solid-liquid interfacial energy respectively. θ is the contact angle between the solid surface and liquid. Referring to the value of γLV = 72.7 mJ/m2, γSL = 29 mJ/m2 29where γOnce the surface energy is acquired, the work of adhesion of graphene nanosheets can be obtained based on the equationa represents the work of adhesion. The work of adhesion can be used for approximatively characterizing the adhesion strength between the AFM tip and the graphene nanosheets. Larger work of adhesion corresponds to greater adhesion force. The calculated surface energy and corresponding water contact angle as a function of plasma treatment time are presented in where WThe reasons for the increase of the surface energy after plasma treatment are concluded as follows. The nitrogen-containing and oxygen-containing functional groups are induced into the surface of graphene nanosheets by plasma171833po) is generally a combination of the capillary force (Fcap), the van der Waals force (FvdW), the electrostatic force (Fel) and the forces due to chemical bonds (Fchem)As for the increased adhesion force in po) and the work of adhesion (Wa) per unit area measured at the pull-off point isTheoretical calculation is used to quantitatively estimate the adhesion force between the AFM tip and the plasma-treated graphene nanosheets. The Johnson-Kendall-Roberts (JKR) theory for mechanical contacts between elastic solids is adopted to account for adhesion forceswhere R is the radius of curvature of the tip apex. While at the instability point where the tip is pulled from the surface, the contact radius is given byt) and sample (Es) materials:where K is the reduced modulus of the tip-sample contact. The reduced modulus is related to the Young’s modulus of the tip (Et and νs represent the respective Poisson ratios. The contact area can be written as where νpo) values of 3.83 nN, 5.59 nN and 6.13 nN for the graphene nanosheets after plasma treatment for 0 s, 1 s and 2 s respectively. These values are comparable to the adhesion forces acquired through experiments . So the total friction force can be expressed as Ff = τA + Fp. Since the shear strength scales with the adhesion forceAs for the variation of the friction force after plasma treatment in Surface roughness is always considered as a factor increasing the friction forceWhen long-time plasma treatments are performed on the graphene nanosheets, the variation of the adhesion force and friction force are displayed in D/IG in Thermal reduction can recover the surface properties and restore part of the structural defects, but it will also create new defects2543442544As summarized in When thermal reduction is performed on the long-time plasma-treated graphene nanosheets, structural defects decreases, but the total amounts of the residual and the new generated structural defects increase with the increase of plasma treatment time before thermal reduction . The varD/IG of thermal-reduced graphene nanosheets increases continuously with the increase of the plasma treatment time  refers to the interaction potential energy between the tip and the surface, which usually approximates by a sinusoidal form: 0 is the corrugation of the interaction potential  and a is the substrate (graphene) lattice constant.where UAccording to the model mentioned above, the atomic-scale mechanism of the increase of friction force can be explained. Before the tip starts to move, its atoms are assumed to reside at the location of the minimum potential. Later, the deformation of the spring will increase continuously as the tip moves from right to left , becauseint) is weak, the variation of the potential barriers (ΔE) in total potential and the tip atomic trajectory correspond to int) corresponds to int) caused by structural defects is shown in When the tip-surface interaction potential (Uint) and the deformation energy of spring (Uspring). The greater the adhesion force, the stronger the tip-surface interaction potential, which more energy consumption is needed to overcome the larger potential barriers  locally52The structural defects can strengthen the tip-surface interaction potential  theory are comparable to the experimental values. An improved spring model was proposed to explain the increase of the friction force caused by the enhanced surface hydrophilicity and the generated structural defects. The enhanced adhesion between the tip and the surface increases the tip-surface interaction potential and the deformation energy of the spring, thus increases the friction force. The structural defects lead to the local improvement of the tip-surface interaction potential, further increase the friction force.Our findings could provide insights in the reasonable design and use of graphene with controllable nanotribological properties as solid lubricants in nanomechanical applications. In addition, this work will propel the fundamental study of the nanotribology of grahene and help understand the underlying mechanism of the roles of surface properties and structural characteristics in nanotribological properties.The graphene nanosheets detached from the highly oriented pyrolytic graphite (HOPG) were deposited on a Si substrate with 300 nm thick oxide layer via the mechanical exfoliation method171819The thickness of graphene nanosheets on the substrate was identified by optical microscopy (Motic/PSM 1000), AFM  and Raman spectroscopy. The silicon probes with a nominal normal spring constant of 3 N/m and tip radius less than 10 nm  were used for imaging and friction tests. The adhesion force, friction force and stick slip phenomena of atomically thin graphene nanosheets were measured in ambient conditions (20–25 °C and 30–40% R.H). For quantitative force measurement, normal and lateral force calibrations of AFM tips were performed via noncontact methodHow to cite this article: Zeng, X. et al. Controllable Nanotribological Properties of Graphene Nanosheets. Sci. Rep.7, 41891; doi: 10.1038/srep41891 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


An evaporative colloidal assembly enabled the fabrication of highly crystalline opal template, and a subsequent photopolymerization of PHEMA followed by solvent-etching and final soaking in IL produced a humidity-responsive IOPG showing highly reflective structural color by Bragg diffraction. Three IOPG sensors with different crosslinking density were fabricated on a single chip, where a lightly crosslinked IOPG exhibited the color change response over entire visible spectrum with respect to the humidity changes from 0 to 80% RH. As the water content increased in IL, thermodynamic interactions between PHEMA and [BMIM+][BF4−] became more favorable, to show a red-shifted structural color owing to a longitudinal swelling of IOPG. Highly porous IO structure enabled fast humidity-sensing kinetics with the response times of ~1 min for both swelling and deswelling. Temperature-dependent swelling of PHEMA in [BMIM+][BF4−] revealed that the current system follows an upper critical solution temperature (UCST) behavior with the diffraction wavelength change as small as 1% at the temperature changes from 10 °C to 30 °C.We demonstrate a fast response colorimetric humidity sensor using a crosslinked poly(2-hydroxyethyl methacrylate) (PHEMA) in the form of inverse opal photonic gel (IOPG) soaked in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM As a d or the effective index of refraction (neff) can be varied by certain stimulus, the structural color will be changed as well, and such a PC will act as a stimuli-responsive colorimetric sensor. A light diffraction can occur either longitudinally or laterally. A longitudinal light diffraction is utilized in one-dimensional (1D) PC  to which Bragg equation can be applied, while a lateral diffraction occurs in two-dimensional (2D) PC such as diffraction grating or colloidal monolayer. To be utilized as a colorimetric sensor, likewise, d should be changed longitudinally in 1D-PC, while a lateral variation of d should occur in 2D-PC. Comprehensive studies have been conducted towards the fabrications of stimuli-responsive sensors using 1D- or 2D-PC structures via opal templating techniques. From the mechanistic point of view, PC sensors can be either field-responsive  [+][BF4−]), a non-volatile hydrophilic room temperature ionic liquid (IL) to demonstrate a fast-response and high-sensitivity humidity sensor.If ic field ,6,7,8, mic field , pressuric field , temperaic field ,13) or mic field ,15,16. Iield [BF4−] was supplied by Future Chem, . Chloroform and acetonitrile (ACN) was respectively purchased from Duksan Chemical  and Daejung Inc. . Deionized (DI) water was produced from the water purifying system (human technology).Materials: Styrene (98%), potassium persulfate, sodium dodecylsulfate (SDS), activated alumina, trichlorooctadecyl silane, ethylene glycol dimethacrylate (EGDM) (98%), and iso-octane were purchased from Sigma-Aldrich, . 2-hydroxyethyl methacrylate (HEMA) (96%) was supplied from Junsei,  and Irgacure-651 was obtained from Ciba specialty chemicals, . Ionic liquid, [BF4−] IL, and finally vacuum-dried at 40 °C overnight in order to completely remove ACN leaving the dried IL behind. An as-prepared IOPG in IL was placed in a custom-made desiccator (LK Lab Korea) in which the relative humidity had been kept very low (<1.0% RH) by using CaSO4 drierite. The relative humidity was monitored by using a digital humidimeter . The humidity was increased by placing wet cotton balls in the desiccator. The color changes of IOPG were characterized by a digital camera or a custom-made reflectance measurement system equipped with a reflective microscope  and a UV–vis spectrometer . Refractive index of IL with varying water content was measured using a digital refractometer .Methods: The polystyrene (PS) μ-sphere with narrow size distribution was synthesized by emulsion polymerization . In an NThe sensor material used in this study is a cross-linked PHEMA which is well known for hydrophilicity, biocompatibility, and rubber elasticity . Opal-te+][BF4−], while the thermodynamic interaction between PVA and IL is controlled by inclusion of water in IL [+][BF4−], and an IOPG film of PHEMA in [BMIM+][BF4−] was found to exhibit a humidity-dependent color changing response. At a low humidity, IL molecules are equilibrated with PHEMA IOPG. Upon the increase of humidity, water is dissolved in IL since BF4−, a kosmotropic ion strongly interacts with water molecule by hydrogen bonding [BF4−] under various humidities at 25 °C are shown. nD of a dried [BMIM+][BF4−] IL was measured to be 1.4230, and that of DI water was 1.3330. Even though neff,D showed a decreasing tendency with an increased humidity due to inclusion of water with low index of refraction, the values were maintained close to that of pure IL regardless of relative humidity, implying that apparent water content in IL is kept low. Using Equation (2), water content (fwater) was calculated for the measured neff,D values  with respect to HEMA were respectively prepared, and the IOPGs with different crosslinking densities were fabricated on a single glass substrate as shown in The calculated +][BF4−], open to air. The IOPG/IL humidity sensors were subject to the controlled humidity, to reveal the color changes as shown in Entire IOPGs were fully soaked with [BF4−], a non-volatile hydrophilic IL at room temperature to demonstrate a colorimetric humidity sensor. Under varying humidity from 0% to 80% RH, IOPGs with different crosslinker contents were exposed, to exhibit structural color changes over entire visible ranges especially for a lightly crosslinked IOPG. Fast color change responses with exponential time constants of 0.6~2.8 min were obtained due to highly porous IO structure of the sensor. From a temperature-dependent color change test, temperature increase resulted in a swelling of IOPG, implying that the thermodynamic interaction between PHEMA IOPG and [BMIM+][BF4−] is supposedly UCST behavior. However, the dimensional change was less than 1% at the temperature variations from 10 °C to 30 °C.In summary, we fabricated a crosslinked IOPG via DEECA followed by photopolymerization of HEMA and crosslinker which was subsequently soaked in [BMIM


This article presents four different plasmonic structures using Graphene which yielded an efficient plasmonic mode with low loss for Supercontinuum(SC) generation. At an operating wavelength of 1550 nm in these structures, we generated a multi-octave broadband SC spectrum ranging from 1.5 um–25 um at a low input peak power of 1 W. Due to pumping in the anomalous dispersion region with two Zero Dispersion Wavelengths (ZDWs) and the process of cross phase modulation with soliton fission, red-shifted dispersive waves were generated which led to large broadening from 1.5 um–25 um. Two other Supercontinua ranging from 1–10 um and 0.85–2.2 um also at low input peak powers of 2 W and 0.1 W respectively were generated. These three supercontinua are useful for applications in the fields of biomedical sensors, spectroscopy, fluorescence lifetime imaging and in the design of many other new optical devices. Furthermore, we have also discussed our results on behaviour of Graphene as a metal, even without the negative real value of dielectric constant. However, they require very high input peak powers ranging from 1 kW to 1000 kW or more9. From the literature in the area13, low input peak powers (of the order of Watts) has limited the spectral broadening and thereby their usability for wide range of applications14. Dispersion engineered waveguides and/or fibres using different materials have been developed but their main limitation is the control of peak input power with low loss for broadband SCG.Supercontinuum Generation (SCG) has been widely studied over the past 40 years. Detailed analysis of non-linear dynamics in solids, organic and inorganic liquids, gaseset al. generated a SC spanning from 1.8 to 10 μm by pumping a Ge-As-Se/Ge-As-S chalcogenide fibre with 330 fs pulses at 4 μm using ~3000 W input3. In 2016, a three-layer index guided lead silicate (SF57) photonic crystal fibre4 was used to generate three octaves spanning SC from 900 to 7200 nm using 50 fs sech optical pulses of 5 kW peak input power with a large nonlinear coefficient of ~1078 W−1 km−1. Recently, Cheng et al. experimentally demonstrated mid-infrared (MIR) SCG spanning ~2.0 to 15.1 μm in a 3 cm-long chalcogenide step-index fiber with a pulse width of ~170 fs at 9.8 um with input peak power of 2.89 MW5. This is the highest average power SC covering 2–15.1 μm reported to date. Also, the broadening beyond 15 um for this design is not possible due to material absorption of the chalcogenide. Our results thus emphasize on solutions to these problems – the peak input power and loss by using Graphene as one of the core materials for the waveguide designs.For example, in 2015, Yu 15 is a two-dimensional (2D) Carbon material with a honey-comb lattice. Recently Graphene nano-ribbon and other waveguides have emerged showing possible outstanding applications in the fields of on-chip interconnects, bright visible light emission, flexible electronics and more17. However, in all these designs, Graphene has been used as the outer core or the cladding material. Thus, from our research, we have shown that with Graphene as the inner core in the designed waveguides, we can exploit its remarkable non-linear properties including a very high Kerr coefficient of –1.1 × 10−13 m2/W18 for broadband SCG at a low input peak power, while exhibiting low material losses.Graphene2) substrate with the inner and outer core surrounded by a low-index medium(air), thereby providing strong optical confinement.In our design of Graphene waveguides for SCG: the structure of a ridge waveguide rests on a Silicon dioxide (SiO3N4) with hexagonal crystalline structure capable of controlling the overall waveguide loss, with tunable band-gap properties for electro-optic applications is used as the outer core. This design thereby produces an efficient mode to guide the light leading to broader SC. The low refractive index contrast of the waveguide materials was kept in mind to avoid large non-linear effects which can narrow the Supercontinuum broadening with losses.The inner core of the designed ridge waveguides consists of a single or bi layer of Graphene, of thickness 0.335 nm and 0.67 nm respectively. A material  curve and the respective higher order dispersion coefficients . Supercontinuum for these designs were further calculated by solving the Generalized Non-Linear Schrödinger Equation (GNLSE) using the Split-step Fourier method.Four Graphene waveguide designs  were simulated using Finite Element Method(FEM)c = 450 meV and 500 meV, which can be achieved by applying a gate voltage to the 2D material. This range of chemical potentials were specifically used to tune Graphene as a metal in the operating wavelength region (1550 nm) with low waveguide loss, thereby generating a plasmonic mode at the dielectric-metal-dielectric interface.In all the simulations performed, Graphene was tuned to two chemical potentials, µg) and Permittivity (ɛg) of single and bi layer of Graphene were calculated from the Kubo formula23 using a FORTRAN code developed by us. The wavelength variation of the real and imaginary parts of the permittivity is shown in Fig. The Conductivity (σ3N4(Silicon Nitride) is used with permittivity, εSi3N4 = 6.0945364 on top of the Graphene layer with Silica (SiO2), εSiO2 = 2.0852 as the substrate, and Air, εAir = 1 for the cladding operating at 1550 nm. Figure x, Ey and Ez field components generated in this structure with the plasmonic mode shown in Fig. z and the plasmonic mode plots from Fig. The waveguide with a single layer of Graphene Fig.  was used25 in Graphene layer of the designed waveguide geometries is formed from coupling between Surface Plasmon Polaritons (SPPs) generated on the top and bottom interfaces of the 2D material with Silicon Nitride and Silica layers respectively. Thickness of Graphene26 also plays a vital role in tight electric field localization at the metal interface, thereby guiding the EM energy with sub-wavelength confinement to micron/sub-micron propagation lengths. Similar physics but with slightly different values of GVD, Aeff and loss was observed in these waveguide designs at 450 meV, 371 K, with plasmonic mode-shifting from z to y - axis at 500 meV–300 K and 371 K. This property makes these designs sensitive for sensor applications26 that will be investigated in the future.The plasmonic mode2), effective mode area (Aeff) and loss plots calculated at a step size of 0.025 µm from 1.5–2 µm wavelength range for all the four designed waveguide structures at 450 meV, 371 K and 500 meV, 300 K respectively. The GVD Curves27 show the presence of increasing and decreasing slopes after reaching a peak dispersion value which results in two ZDWs.Figure 2 and 0.9593657 µm2 at 300 K, and 0.9613992 µm2 and 0.9578994 µm2 at 371 K, for 500 meV chemical potential. These structures also showed an average loss of only ~15 dB/km. The overall dispersion in bi and tri layer structures (with buffer) was moderate with excellent Aeff and loss, thereby making them highly favourable for SCG. Similar properties of this GVD curve in Fig. At 1.55 um, bi and tri layer graphene waveguides induced a total effective area of 0.962449 µmSupercontinuum in the designed Graphene waveguides was generated operating at 1550 nm, at a pulse width of 10, 2.5 and 1fs for 1 mm, 10 um and 1 mm long waveguides as shown in Fig. −13 m2/W. This Kerr coefficient parameter28 combined with the tailored dispersion of the waveguide generated fundamental optical solitons thereby stimulating dispersive wave radiation in the anomalous dispersion region. The spectral expansion is mainly due to those successively ejected fundamental solitons from the input pulse30 during soliton fission.The broad expansion of the SC in Fig. 32 in the following material.The largest spectral bandwidth is observed in the Graphene Single layer structure shown in Fig. 31 LNL = 1/(γP0), where γ is the nonlinear coefficient and P0 is the peak input power. From calculations, we get LNL at the 1550 nm pump wavelength as 21.584 μm. The dispersion length is LD = T02/|β2|, where T0 is the pulse width and β2 = −1.068446552230430 ps2∕m at 1.55 μm is the dispersion parameter calculated from Fig. NL is 21.584 × 10−6 m, and LD is 9.3594 × 10−5 m. Also, the characteristic propagation distance (≈5LD) where the ejected soliton separation begins to become apparent in the temporal and spectral characteristic is calculated as ≈ 4.6797 × 10−4m. Since the waveguide length L = 1 mm is slightly larger than 5LD and is greater than both LNL and LD, the spectrum leads to a number of ejected solitons appearing with a significant decrease of spectral energy in the vicinity of the pump thereby limiting the spectral bandwidth30. The soliton order(N) of the waveguide for these input parameters is calculated as ~2.0824.For the 1 mm-long Graphene waveguide at a pulse width of 10fs, shown in Fig. NL and LD are calculated as 1.0792 um and 5.8496 um respectively with the characteristic propagation distance (≈5LD) as 29.248 um. In this case, the waveguide length L = 10 um is much smaller compared to 5LD resulting in only one clearly separated soliton peak in the temporal intensity despite the calculated soliton order(N) being ~2.3281.From Fig. 31 from Fig. NL and LD are calculated as 2.1584 um and 0.93594 um with the characteristic propagation distance calculated as ≈5LD = 4.6797 um. The waveguide length (L) of 1 mm for this design is much larger compared to the 5LD resulting in a greater number of ejected solitons with distinct spectral peaks in the anomalous GVD regime that can be clearly identified as due to soliton fission33. The extension of the spectral broadening to the long wavelengths is however due to the generation of red shifted waves formed by coupling of negative dispersion slope  with the anomalous GVD.For the broadest Supercontinuum generated2, and its temporal width and peak power adjust themselves to conserve a unit soliton number during propagation. This effect is clearly seen in the broader Supercontinuum generated in Fig. 5c1,c2. The flatness of the spectrum is however achieved due to the low third order dispersion (β3 = 2.251048248652541 × 10−3 ps3/m) for this design at 1550 nm.As the pulse width is less than 12fs for the generated SCs above, dispersive perturbation induces soliton fission and this can be modified in two ways. Primarily, as an ejected fundamental soliton shifts to longer wavelengths because of the Raman effect which is small in our case, the soliton encounters a varying value of β13. This is clearly seen in Fig. 5a1,b1 although the effect is quite small in SC generated in Fig. 36.The second effect is due to the presence of higher-order dispersion which also leads to the transfer of energy from soliton to a narrow-band resonance in the normal GVD regime38. The selected Graphene bilayer with buffer structure can be tuned40 to 500 meV and practically fabricated by exfoliating graphene and defining its contacts around a particular flake using standard photolithography methods43. An alternative method for this fabrication is to define the contacts first, transfer large area CVD Graphene on top, then add another layer of photoresist and use O2 plasma to isolate devices45.These generated Supercontinua have many practical applications such as in DNA sensing, speed/pressure sensor monitoring and medical therapeutics as they cover the full functional band of lipids, proteins, genes, bacteria, viruses, chemicals and spectral region of most biomolecules47 which tells us that at a metal-dielectric interface, SPPs are formed when ε1 and From our results, an important observation is the behaviour of Graphene as a metal, and formation of SPPs on interaction with a dielectric at optical frequencies even without the negative real permittivity value of Graphene at 450 meV (300 K and 371 K temperature). This is different from the theory on Surface Plasmons3N4 of thickness - 0.6 um and 0.8 um for the outer core. This fundamentally tailored the plasmonic mode in the dielectric-metal-dielectric interface. With typical waveguide parameters, we achieved a large negative non-linear coefficient of γ = −4.6330088494 × 105 1/m-W due to negative non-linear Kerr coefficient of Graphene of the order, n2 = −1.1 × 10−13 m2/W that played a major role in stimulating a small Raman effect and thereby leading to broader Supercontinuum by the influence of solitons and dispersive waves.We have designed four waveguide structures using Graphene as the inner core, with SiWe have shown that the Graphene Bilayer with Buffer waveguide  exhibits a SC extending from 1.5–25 um at a very low input peak power of 1 W, with overall waveguide loss of only 16.5 dB/km. This is the broadest SC to the best of our knowledge.The modal solutions in the designed Graphene waveguides were obtained using Full Vectorial H-field formulation with penalty term to eliminate the spurious solution. It is one of the most accurate and numerically efficient approaches to obtain the modal field profiles of a waveguide. Various quasi-TE and quasi-TM modes was calculated from equation ,1\documeentclass1pt{minimaeff by equation (3) was calculated using equation (As chromatic dispersion of the waveguide manifests through the wavelength dependence of the refractive index n(λ) , TOD (Third order Dispersion) and subsequently other higher-order dispersion coefficients were calculated from NFor SCG, the Generalized Non-Linear Schrödinger Equation (GNLSE) was solved using the Split-step Fourier method using equation ,5\documeet al.48, moderated and tested for accuracy and efficiency with experimental studies. The non-linear coefficient (γ) in equation .The results of FEM have been benchmarked


The current cancer research strongly focuses on immune therapies, where the PD-1, with its ligands plays an important role. It is known that PD-L1 is frequently up-regulated in a number of different cancers and the relevance of this pathway has been extensively studied and therapeutic approaches targeting PD-1 and PD-L1 have been developed. We used a non-invasive, real-time biopsy for determining PD-L1 and PD-L2 expression in CETCs of solid cancer patients.® method.CETCs were determined from blood of 128 patients suffering from breast (72), prostate (27), colorectal (18) and lung (11) cancer. The number of vital CETCs and the expression of PD-L1 and PD-L2 were investigated using the maintracPD-L1 expressing CETCs were detected in 94.5% of breast, 100% of prostate, 95.4% of colorectal and 82% of lung cancer patients whereas only 75% of breast cancer patients had PD-L2 positive CETCs. In the PD-L1 and PD-L2 expressing patients the cell fraction of PD-L1 positive CETCs is significantly higher than the fraction of PD-L2 positive CETCs . Breast cancer patients with metastatic disease had significantly more PD-L1 positive CETCs as compared to patients without metastasis .PD-L1 seems to be a major factor in immune evasion and is highly expressed on CETCs regardless of the type of cancer. Monitoring the frequency of PD-L1 positive CETCs could reflect individual patient's response for an anti-PD-1/PD-L1 therapy and may be a promising target of anticancer treatment. Metastatic disease is responsible for over 90% of cancer-related deaths. To date the characteristics of the primary tumor are used to predict the probability of tumor progression and metastatic relapse. However, the clonal landscape of the overall tumor burden is very heterogeneous and a single biopsy may fail to represent the whole cancer cell population. Furthermore, the biopsy is invasive and repeating the procedure is not always feasible due to safety concerns , 2.Most of metastases are due to hematogenous dissemination of tumor cells from the primary tumor which starts at an early stage of cancer growth. Single tumor cells or cell clusters shed from the primary tumor travel to distant organ sites and can grow into metastatic lesions . AggressTherefore, using circulating tumor cells as a “liquid biopsy” holds great potential to better represent the actual composition of tumor cells with minimal risk for patients. It can be repeated frequently for real-time monitoring of cancer treatment and can give important information on therapeutic targets and drug resistance mechanisms , 7, 8, 9In order to successfully evade immune surveillance, tumor cells use a variety of different strategies. One of them is the upregulation of surface programmed cell death ligand 1  expression . PD-L1 iTo date, however, there is no reliable predictive biomarker for determining the response rate for a targeted PD-1/PD-L1 therapy. It has been shown that PD-L1 expression by tumor and/or infiltrating immune cells correlates with a therapeutic response . The intFor the development of an approach to detect PD-L1 or PD-L2 on CETCs the specificity of antibodies was determined. Therefore we analysed different cancer cell lines H820, Sk-Br-3, MCF-7 and SW-620 by using fluorescence scanning microscope. H820 was strongly positive for PD-L1 only with clones 29E.2A3 and MIH1 and completely negative with clone 130021  patients received adjuvant radiotherapy, of which 20 patients were irradiated with standard regime (50 Gy in 25 fractions + 16 Gy boost). 10 patients older than 60 years obtained hypofractionated regime with 42.56 Gy in 16 fractions. Patients after radiotherapy (n=30) had a higher fraction of PD-L1 positive CETCs as compared to patients without radiotherapy (n=31) , regardless of radiation regime  breast cancer tissue sections varies widely in the literature. Ghebeh et al.  and MuenIn contrast, circulating tumor cells, which are the precursors of metastatic disease, are accessible and can be detected in a comparable way as other blood cells .Tumor cells that are invasive and able to effectively evade cytotoxic T cells would have a greater selective advantage and likely contribute more to the progression of cancer disease. Very little data is available for PD-L1 expression on these cells and its significance in circulating tumor cells. For this reason we investigated PD-L1 expression on circulating epithelial tumor cells in breast, prostate, colorectal and lung cancer patients.For the establishment of the PD-L1 assay on CETCs we tested different cancer cell lines  and different clones of PD-L1 antibodies . Clone 29E.2A3 reacted positively only with H820 cells. In contrast to Mazel et al, we found no specific PD-L1 staining with clone 130021 neither with the H820 nor with the Sk-Br-3 cell line even after IFNγ treatment. However, with clone 29E.2A3 the most specific and unambiguous staining was observed in the H820 but not in the Sk-Br-3 cell line. The latter showed minimal binding in two other studies , 27. TheWe found neither an association between the number of CETCs and different cancer types nor between the fraction of PD-L1 positive CETCs and different cancer types. In our approach 94.6% of breast cancer patients had PD-L1 positive CETCs although in different proportions. Mazel et al. using the CellSearch system found PD-L1 positive circulating tumor cells in 11/16 metastatic breast cancer patients (68.8%). The fraction of PD-L1 positive circulating tumor cells varied from 0.2-100% in individual patients , which i+ T cells mediating up-regulation of PD-L1 expression on tumor cells. Additionally, there is a strong correlation between PD-L1 expression on tumor cells and lymphocytic infiltration not only among tumors but also within regional sites in a tumor [In the adjuvant situation chemo- and radiotherapy are the major components of cancer treatment but many patients get local recurrence or metastasis. The association of PD-L1 expression and obtaining radiotherapy found in our study correlates well with the known inflammatory effect of radiotherapy. Dovedi et al. showed that fractionated radiotherapy is responsible for an increased IFNγ production by CD8 a tumor , 34, 35.We were surprised to observe that 100% of prostate cancer patients had PD-L1 positive CETCs. Also in prostate tumor tissue PD-L1 expression seems to be elevated in comparison to other tumor types. Gevensleben et al. detected PD-L1 expression in 61.7% of primary prostate cancers . MassariThe results reporting PD-L1 expression in colorectal cancer are highly diverse. Whereas Masugi et al.  found thAlso in NSCLC reports on PD-L1 expression varies highly ranging from 7.4% to 72.7% . With reDetection of copy number variants which, differently to the difficulties in antibody-dependent approaches, is independent of most fixation procedures can be an alternative method to IHC. The PD-L1 gene is located on chromosome 9p24.1 and the amplification of this gene locus has been reported in lymphomas , triple The role of PD-L2 in evading the immune system is not fully understood. Comparing the expression level of both PD-1 ligands on CETCs in breast cancer patients we observed that frequency of cells with PD-L2 expression was significantly lower compared to the frequency of PD-L1 expression. Therefore PD-L2 may play only a marginal role in immunotherapy. This is in agreement to previous studies which found that in comparison to PD-L1, PD-L2 expression was observed less frequently in tissue samples. PD-L2 expression may be relatively restricted to macrophages, dendritic cells, and fibroblasts , 44. To Breakthrough therapy with checkpoint inhibitors in the treatment of cancer may gain even more importance in the near future. Drugs inhibiting PD-L1 and PD-1 exhibit a favorable toxicity profile, but so far treatment is applied only to a subset of patients. There is a need to identify reliable biomarkers to predict response to these therapies and to facilitate patient selection . Taken tPeripheral blood (7.5 ml) from altogether 128 patients with breast (72/56%), prostate (27/21%), colorectal (18/14%) and lung (11/9%) cancer in different stages of disease was drawn into normal blood count tubes with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and processed within 48 hours of collection. In parallel, healthy control blood samples were collected from 25 female and male donors aged from 20-40 years. In patients with primary breast cancer (n=55) the sampling of peripheral blood was carried-out 6-12 weeks after end of standard therapy . In patients with local or distant recurrence the blood was collected prior to treatment of recurrent disease.® approach was used, as reported previously [7 cells/100 μl cell suspension were added and incubated for 15 min in cold. The corresponding isotypic control for EpCAM  was used at the same final concentration. The samples were subsequently diluted with 430 μl PBS-EDTA. A defined volume of the cell suspension and propidium iodide (PI)  was transferred to wells of ELISA plates . Analysis of red and green fluorescence of the cells was performed using a Fluorescence Scanning Microscope, ScanR, , enabling detection and relocation of cells for visual examination of EpCAM positive cells. For data analysis we used the ScanR Analysis software . Vital CETCs were defined as EpCAM-positive cells, lacking in CD45-/ PI-staining and with intact morphology, and only these cells were counted. We used fluorospheres  for daily verification of optical components and detectors of the microscope, which are required to ensure the consistent analysis of samples.For CETC enumeration and further characterization the maintraceviously . Briefly2. MCF-7 cells were grown in Minimum Essential Eagle ready-to-use medium . For immunofluorescence analysis cells were detached from cell culture flasks using StemPro® Accutase® Cell Dissociation Reagent  washed and stained for PD-L1 with the same protocol like a patient sample.H820 lung cancer cells which were used as a positive control for PD-L1 analysis were obtained from the American Type Culture Condition . Three different clones of anti-PD-L1 antibodies were tested: (1) clone 29E.2A3 , (2) clone 130021 , (3) clone MIH1  -conjugated antibody  at a final concentration of 0.2 μg/ml and for PD-L2 we used an anti-human PD-L2 Alexa Fluor® 350 conjugated antibody  at a final concentration of 2 μg/ml. The corresponding isotypic controls for PD-L1  and PD-L2  were used at the same final concentration. Finally, cells were visually inspected looking for a green, red and blue surface staining, but also a well-preserved nucleus /CEN9q FISH Probe, abnova, Taiwan) containing the CD274 (PD-L1) gene  and CEN9q . Patient cells were transferred onto Poly-L-Lysin coated slides. Before hybridization slides were fixed with 4% paraformaldehyde for 10 min and treated for 10 min with proteinase K at room temperature. In a next step, cells were denatured for 5 min at 72°C in 70% formamide - 2 x standard saline citrate solution, air dried and dehydrated in 70%, 85% and 96% ethanol. After overnight hybridization at 37°C in a humidified chamber, slides were washed, air dried and counterstained with 0.2 μM DAPI in an anti-fade solution. At least 20 nuclei per sample were counted. CETCs were positive for PD-L1 amplification when more than 3 PD-L1 signals in one cell were counted. The final results for PD-L1 amplification were calculated as percentage of 20-30 visually expected EpCAM positive cells.For FISH analyses the


ABCB1 (ATP-Binding Cassette Subfamily B Member 1) gene which encodes a P-glycoprotein and underlies dwarfing traits in maize (br2), sorghum (dw3) and pearl millet (d2) displayed considerable variation in intron composition.The presence of non-coding introns is a characteristic feature of most eukaryotic genes. While the size of the introns, number of introns per gene and the number of intron-containing genes can vary greatly between sequenced eukaryotic genomes, the structure of a gene with reference to intron presence and positions is typically conserved in closely related species. Unexpectedly, the ABCB1 gene structure in 80 angiosperms revealed that the number of introns ranged from one to nine. All introns in ABCB1 underwent either a one-time loss (single loss in one lineage/species) or multiple independent losses  with the majority of losses occurring within the grass family. In contrast, the structure of the closest homolog to ABCB1, ABCB19, remained constant in the majority of angiosperms analyzed. Using known phylogenetic relationships within the grasses, we determined the ancestral branch-points where the losses occurred. Intron 7, the longest intron, was lost in only a single species, Mimulus guttatus, following duplication of ABCB1. Semiquantitative PCR showed that the M. guttatus ABCB1 gene copy without intron 7 had significantly lower transcript levels than the gene copy with intron 7. We further demonstrated that intron 7 carried two motifs that were highly conserved across the monocot-dicot divide.An analysis of the ABCB1 gene structure is highly dynamic, while the structure of ABCB19 remained largely conserved through evolution. Precise removal of introns, preferential removal of smaller introns and presence of at least 2 bp of microhomology flanking most introns indicated that intron loss may have predominantly occurred through non-homologous end-joining (NHEJ) repair of double strand breaks. Lack of microhomology in the exon upstream of lost phase I introns was likely due to release of the selective constraint on the penultimate base (3rd base in codon) of the terminal codon by the splicing machinery. In addition to size, the presence of regulatory motifs will make introns recalcitrant to loss.The The online version of this article (10.1186/s12862-017-1077-x) contains supplementary material, which is available to authorized users. Introns are a characteristic and common feature in eukaryotic genomes. They likely accumulated very early in eukaryotic evolution and some introns have remained in conserved positions across kingdoms for a period close to two billion years –3. Altho−10 per intron per year by Roy and Gilbert [−13 by Coulombe-Huntington and Majewski [Arabidopsis thaliana -mediated intron loss and intron deletion triggered by repair of double strand breaks (DSBs) via non-homologous end joining (NHEJ). RT-mediated loss occurs when a cDNA recombines with its genomic copy. Characteristics of RT-mediated loss include precise removal of introns, preferential removal of small introns, deletion of adjacent introns and a 3′ bias of intron removal –13. A siosophila , 15, 16.osophila .Drosophila uncovered a total of 1754 intron loss events [A. thaliana and A. lyrata revealed a combined loss of 105 introns [Brachypodium) using Arabidopsis and banana as outgroups revealed a total of 745 intron loss events, including 93 cases of parallel intron loss whereby the same intron was lost independently in multiple lineages [GAPDH) gene which has 10 introns showed parallel loss of intron 9 in opossum, dog and the primate/rodent lineages [white gene in the distantly related dipteran (true fly) lineages contains 14 introns, three of which  underwent parallel loss 3, 4 and 5 times, respectively [Drosophila and mosquito species, convergent loss of one intron, intron Z, was observed in the MRP1 gene [white and MRP1, are ABC transporters. In Angiosperms, the isochorismate synthase gene, which comprises 15 introns, underwent parallel loss of intron 2 in the grass lineage and the Medicago truncatula/Glycine max lineage [Intron loss has been documented in whole genome studies of multiple species. Comparison of 10 fully sequenced genomes within the genus s events . A compa introns , and a slineages . The whiectively . In the RP1 gene . Inciden lineage . The oveABCB1 gene across Angiosperms. ABCB1 encodes a P-glycoprotein that modulates basipetal auxin transport in stems [ABCB1 in grasses significantly reduce plant height and these mutants have been exploited extensively in agriculture to reduce lodging under high inputs and hence to increase yields [ABCB1 mutants of global economic importance are sorghum dw3 and pearl millet d2 [ABCB1 was first observed when pearl millet ABCB1 was isolated as a candidate for the d2 dwarfing trait [ABCB1. We subsequently expanded the structural analysis of the ABCB1 gene to other sequenced and non-sequenced members of the grasses and beyond, including selected non-grass monocots, dicots and the basal species Amborella trichopoda.In the current study, we conducted a detailed analysis of intron loss in the in stems . The proin stems . Mutatioe yields –25. Examillet d2 , 24. Intng trait  and compArabidopsis ABCB1 (locus AT2G36910) and ABCB19 (locus AT3G28860) proteins as queries against plant proteins in Phytozome 11.0 (phytozome.jgi.doe.gov/), Gramene (www.gramene.org) and the non-redundant protein section of GenBank (www.ncbi.nlm.nih.gov). In first instance, proteins with >70% similarity to Arabidopsis ABCB1 and >75% similarity to Arabidopsis ABCB19 were considered ‘true’ orthologs of ABCB1 and ABCB19, respectively, and were retrieved for analysis together with their corresponding genomic and cDNA sequences  had 67% similarity to Arabidopsis ABCB19 and was also included in the analysis. Because no ABCB1 protein homolog was identified in Carica papaya, a BLASTN analysis using Arabidopsis ABCB1 cDNA as query was performed against the genome sequence of C. papaya. We found that the ABCB1 gene in C. papaya (evm.TU.supercontig_102.36) had been misannotated due to gaps in the sequence but was likely full-length. This ABCB1 homolog was included in the gene structure analysis. ABCB1 gene sequences from additional monocot species for which whole or partial genome sequence was obtained prepublication  was included as outgroup. We used either the proteins retrieved from the various databases or, when the protein structure appeared to be incorrect, in silico translations from cDNAs generated from genomic sequence alignments in the protein alignment. Phyllostachys edulis and Chasmanthium laxum were not translated in silico because of gaps in the ABCB1 coding sequence. A maximum likelihood tree was constructed from the MUSCLE alignment using RAxML v.8.2.4 [A BLASTP analysis was performed using settings . ArabidoABCB1 and ABCB19 genomic sequences were aligned at the nucleotide level using MUSCLE v3.8.31 with default settings [ABCB1, dicot ABCB1, monocot ABCB19 and dicot ABCB19. Where necessary, sequence alignments were edited manually using Jalview [ABCB1 and ABCB19 of Arabidopsis and rice identified the introns in each of the genes and their correspondence across the two genes, allowing conclusions to be drawn from the separate alignments on the differential presence of introns across species and genes.The retrieved settings . Because Jalview . A sequeDanthoniopsis dinteri, Sacciolepis myosuroides and Arundinella hirta, and leaf material from Brachiaria spp., Andropogon gerardii, Bambusa spp. and Acroceras macrum were obtained from Melanie L. Harrison, Plant Genetic Resources Conservation Unit (PGRCU), Griffin, GA. The seeds were planted in 3 in. pots and grown in a greenhouse under natural light and day/night temperatures of 32 °C/28 °C. Paspalum vaginatum cultivar ‘SeaDwarf’ was provided as a clonal propagule by Paul Raymer, University of Georgia (UGA). Leaves from the Zoysia grass cultivar ‘Emerald’ (Zoysia japonica X Zoysia pacifica) were collected from the lawn outside the UGA Miller Plant Sciences building. Leaves from a Phyllostachys spp. were collected from a bamboo stand on the UGA North Campus. Leaves from Typha latifolia were collected from the UGA Plant Biology greenhouse teaching collection. Leaves from Pharus mezhii and Streptochaeta spp., and DNA from Anomochloa spp., Phragmites australis and Micraira subulifolia were provided by Elizabeth Kellogg, Donald Danforth Plant Science Center, St. Louis, Missouri. DNA was extracted from leaf samples using the Qiagen DNA extraction kit. DNA of Mayaca spp. and Ecdieocolea spp. was provided by Jim Leebens-Mack (UGA), of Zea mays by Kelly Dawe (UGA) and of Oryza sativa spp. japonica (Nipponbare) by Jeff Bennetzen (UGA).Seeds of the grass species ABCB1 gene sequences, primer sets were designed against conserved exon regions to amplify the introns in a range of monocot species. The primer pairs were developed to amplify across introns 1 + 2, 3 + 4, 5, 6, 7, 8 and 9 . Amplification conditions consisted of initial denaturation at 95 °C for 5 min followed by 34 cycles of 95 °C for 30 s, the appropriate Tm , in the first and last exons of ABCB1  version 4.10.1 (http://meme-suite.org/tools/meme) [Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Zea mays, Setaria italica; one non-grass monocot: Musa acuminata; four dicots: A. thaliana, Glycine max, Populus trichocarpa and Phaseolus vulgaris) and extracted 1080 bp upstream of the start codon. A MEME analysis was conducted on this set of 10 sequences. This was repeated 100 times using different genes for each analysis. A similar analysis was conducted with 1080 bp of randomly selected genic sequence. In addition, we conducted MEME analyses on intron 9 (median length: 1266 bp) and on concatenated introns 1, 2, 4, 5, 6 and 7 (median length: 1073 bp) across 59 ABCB19 genes as well as on ABCB1 intron 7 across the same 59 species (median intron length: 1084 bp). The first five and last five bp of each intron were removed before concatenation to remove the conserved splice sites.The intron 7 sequences of 81 ls/meme) . DefaultMimulus guttatus inbred lines IM62 and IM767 were provided by Andrea Sweigart (UGA). Primers were designed targeting the two ABCB1 homologs Migut.J00652  and Migut.L01707  to amplify a 212 bp fragment from cDNA, and 308 bp (Migut.J00652) and 298 bp (Migut.L0107) fragments from genomic DNA. Amplification with actin primers MgACTIN.F (5′-ATGGTAACATTGTGCTCAGTGGT-3′) and MgACTIN.R (5′-GATAGAACCTCCAATCCAGACACTGTA-3′) [® Flexi PCR buffer (Promega), 1.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 μM forward and reverse primers, 1 U GoTaq DNA polymerase (Promega) and 1 μl of a 1:10 cDNA dilution. PCR conditions were as follows: initial denaturation at 95 °C for 2 min followed by 31 cycles (Migut.J00652 and Migut.L01707) or 26 cycles (Actin) of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. PCR products were separated on a 1.5% agarose gel. The same reaction and PCR conditions were used for amplification from genomic DNA using 20 ng of DNA as input.cDNA from leaves, buds and flowers  and genomic DNA from TGTA-3′)  was usedABCB1 and ABCB19 genes fell into two distinct clades  with ABCB1 in the panicoid species sorghum , maize  and foxtail millet  had revealed that ABCB1 in pearl millet and foxtail millet carried two introns while ABCB1 in maize and sorghum carried four introns  and Cucumis sativus (Cusca.306720), and one of the two ABCB1 copies in Mimulus guttatus (Migut.L01707) had eight introns and lacked introns 8, 9 and 7, respectively  carried two genomic deletions, one that spanned the region from the 3′ end of exon 2 to the 5′ end of exon 4, and one that spanned part of exon 7 and part of intron 7. Lus10014427.g is likely a pseudogene and was removed from the analysis. The presumed functional ABCB1 copy in L. usitatissimum (Lus10023929.g) lacked intron 2. Zostera marina, a seagrass which belongs to the order Alismatales, lacked introns 2, 8 and 9. The number of introns in ABCB1 in grasses varied from 1 to 4. All sequenced grass species carried intron 7, lacked introns 1, 3, 4, 8 and 9, and had varying combinations of introns 2, 5 and 6  lacked intron 7 in B. rapa and one copy (Bo9g008680) lacked intron 7 in B. oleracea  in Z. marina due to the presence of a stop codon in exon 2. The other non-grass monocots analyzed contained one or two copies of ABCB19 with nine introns each  and rice  [ABCB homologs LOC_Os08g05690 and LOC_Os08g05710, and ABCB1 and ABCB19 in the basal angiosperm Amborella also carried an intron in that region. Intron8-ABCB19 was present in all ABCB genes analyzed, except ABCB1 and the rice ABCB ortholog (LOC_Os02g46680) most closely related to ABCB2/10 [ABCB1 in Amborella also lacked intron8-ABCB19. The presence of both intron8-ABCB1 and intron8-ABCB19 in ABCB19 in Amborella suggests that presence is the ancestral state for both introns in ABCB genes. Arabidopsis ABCB2 and ABCB10 carried three additional introns after the 9th intron . Introns 11 and 12 were also present in rice ABCB2/10, but intron 12 was absent from rice ABCB2/10. Amborella ABCB19 carried an additional intron, designated Amborella-intron10, which differed in its position from intron 10 in ABCB2/10. This intron was absent in all dicot and monocot ABCB genes analyzed and may represent an intron gain in Amborella  . The ricCenchrus americanus, O. sativa and Z. mays, three species for which the ABCB1 gene structure was known, were used as controls. For most introns, a single amplification product was obtained in the analyzed species, allowing unambiguous determination of the presence/absence of that intron. Some primer pairs, however, generated two amplicons of different sizes with one fragment corresponding in size to an intron-less amplicon and the other to an amplicon containing an intron. This was observed especially, although not exclusively, in species outside the Poaceae. Multiple fragments might have been due to allopolyploidy of the analyzed species (the ploidy level of many non-sequenced species was unknown) whereby different subgenomes carried ABCB1 genes with a different intron composition. However, especially for the non-grass species, multiple amplicons might have been due to non-specific amplification of other ABC genes. As the identity of these amplicons was not confirmed by sequencing, we conservatively scored intron presence/absence in species that displayed two PCR fragments as ‘not determined’ (ND)  Table . Exon 2 ABCB1 introns was achieved for Typha, Ecdieocolea, Streptochaeta, Mayaca, Paspalum vaginatum, Zoysia and Phyllostachys and the amplification products sequenced  had a highly conserved core motif of 8 bp (GTAACATG) that was present without mismatches in 44 angiosperms (55 ABCB1 genes), and with 1 and 2 bp mismatches in an additional 13 angiosperms (15 genes) and six angiosperms (seven genes), respectively  contained four mismatches in the 8 bp motif suggesting that the motif may have been eliminated in this species. The size of intron 7 in Z. marina is 68 bp, the smallest size for intron 7 across the analyzed species. A second motif  was highly conserved  in 76 ABCB1 genes (57 angiosperms) ; Plant Cistrome Database ; Plant DHS (www.plantdhs.org); miRBase (www.mirbase.org)).With a width setting of 12 letters, four motifs were identified by MEME in intron 7 with e-values <1.0eABCB1 intron 7, we conducted MEME analyses on six concatenated ABCB19 introns (median length 1073 bp), on ABCB19 intron 9 (median length 1266 bp), and on ABCB1 intron 7 (median length 1084 bp) across 59 species. Motif ‘GTAACATG’ was the top motif identified in intron 7 with an e-value of 5.7e−64 followed by ‘TTTKGTCARSAA’ with an e-value of 1.6e−55. The most significant motif identified in intron 9 in ABCB19 was ‘GCAYGTGCTTYC’ (6.9e−61) which comprises a G-box motif (‘CACGTG’) [ABCB19 introns, the top motif was TYAGATCYMA’ (2.2e−47). This motif was largely found in intron 1 in dicots (83% of dicots) and in intron 4 in monocots (75% of monocots). ‘AGATCCAA’ (AG-motif) has been identified as a cis-regulatory motif in the promoter region of NtMyb2 involved in the response to wounding and elicitor treatment [ABCB1 intron 7 for the presence of motifs. For both datasets, all motifs identified in 100 MEME analyses in six monocots and four dicots had e-values higher than the e-value of the ‘GTAACATG’ intron 7 motif across the same 10 species   and zero out of 100 for random genic sequence  had e-values equal to or lower than the target motif. We can thus reject the null hypothesis at alpha level of 0.05 that these motifs were found by chance.To determine the probability of finding motifs in sequences similar in length to CACGTG’) . In the reatment . We alsoTranscript levels of Migut.J00652 were significantly higher than those of Migut.L01707 in all three organs in both inbred lines tested Fig. .Fig. 2AgABCB1 gene consisted of 10 exons and nine introns. In the discussion below, we will refer to introns by their ancestral intron number. The ancestral gene structure has been maintained in the majority of dicot species and about half of the non-grass monocots analyzed. In most grass species, however, the maximum number of introns in ABCB1 was four. Although our analysis focused on only a single gene, ABCB1, a number of observations could be made. Firstly, the overall frequency of intron loss events in ABCB1 was less than 1% in dicots and 6% in monocots when we assume conservatively that neighboring introns were lost together and that losses in related species occurred in a common ancestor. The total number of ABCB1 introns lost in the monocots was close to 50%. Hence, intron loss was not random across the phylogenetic tree. Secondly, intron removal was precise in 100% of the cases for which exon-intron boundary sequence was available. Thirdly, in both dicots and monocots, all ABCB1 introns except intron 5 were flanked by two to four bases of microhomology . Introns 2 and 6 were phase I introns, and hence the second to last base of the upstream exon was the third base in a codon. Following intron loss, these bases no longer played a role in splicing, and hence were free to mutate provided amino acid identity was maintained [ABCB1 involved non-adjacent introns. Irrespective of whether intron removal occurred through recombination with a cDNA or NHEJ, most genes underwent multiple independent loss events. Finally, intron loss, including parallel intron loss, occurred more frequently than previously thought. An initial analysis of ABCB1 in Arabidopsis, Musa, rice, Brachypodium, pearl millet, foxtail millet, sorghum and maize had suggested that introns 1, 3, 4, 8 and 9 had been lost before the radiation of the grasses, intron 5 had been lost in the Paniceae lineage, intron 6 in the Pooideae lineage and intron 2 had been lost independently in both the Paniceae and Pooideae. However, as more genomes were added to the analysis, many additional intron loss events were observed. Intron 2 was lost independently at least eight times in angiosperms with seven of the events being in monocots  after it split from the Panicinae  sometime during the past 13.1 MYA [Ecdeiocolea lineage. Considering the highly dynamic nature of the genomic structure of ABCB1 in the sample of analyzed species, further expansion of the species set will likely identify additional events of intron loss. Interestingly, the genomic structure of ABCB19, which also has nine introns and, in Arabidopsis, is functionally redundant to ABCB1 [Structural data from a range of sequenced dicot and monocot genomes Table  suggestsintained . Fourthl13.1 MYA , 55. Intto ABCB1  has largABCB1. Some studies have shown intron loss to be correlated with gene duplications [ABCB1, however, is single copy in many of the species that underwent intron loss. It has also been shown that smaller introns have a higher tendency to undergo evolutionary loss than larger introns [ABCB1 genes, where we found the majority of intron loss events, introns 1 to 5, 8-ABCB1 and 9 have mean and median lengths of <100 bp , intron 4  and intron 9  were larger Additional file 2:Maximum likelihood protein tree of analyzed ABCB1 (in blue) and ABCB19 (in red) sequences. The maximum likelihood tree was generated by RAxML from alignments of analyzed ABCB1 and ABCB19 proteins. Protein names are as listed in Additional file Additional file 3:ABCB1 genes. A multiple sequence alignment was generated for genomic and, for a few species, cDNA sequences of monocot ABCB1 genes obtained from available whole genome sequence data or sequenced ABCB1 genes. Differentially colored regions indicate exons and introns. (PDF 962 kb)Multiple sequence alignment of monocot Additional file 4:ABCB1 genes. A multiple sequence alignment was generated for genomic and, for a few species, cDNA sequences of dicot ABCB1 genes obtained from available whole genome sequence data or sequenced ABCB1 genes. Differentially colored regions indicate exons and introns. (PDF 1331 kb)Multiple sequence alignment of dicot Additional file 5:ABCB19 genes. A multiple sequence alignment was generated for genomic and, for a few species, cDNA sequences of dicot ABCB19 genes obtained from available whole genome sequence data or sequenced ABCB19 genes. Differentially colored regions indicate exons and introns. (PDF 1643 kb)Multiple sequence alignment of dicot Additional file 6:ABCB19 genes. A multiple sequence alignment was generated for genomic and, for a few species, cDNA sequences of monocot ABCB19 genes obtained from available whole genome sequence data or sequenced ABCB19 genes. Differentially colored regions indicate exons and introns. (PDF 1568 kb)Multiple sequence alignment of monocot Additional file 7:ABCB1 and ABCB19 from Arabidopsis and rice. The multiple sequence alignment of genomic and cDNA sequences of ABCB1 and ABCB19 from Arabidopsis and rice provides information on the comparative location of introns in ABCB1 and ABCB19. Differentially colored regions indicate exons and introns. (PDF 193 kb)Multiple sequence alignment of genomic and cDNA sequences of Additional file 8:Arabidopsis and rice, and of ABCB1 and ABCB19 from Amborella. A multiple sequence alignment was generated of protein sequences of selected ABCB family members from Arabidopsis and rice, and of ABCB1 and ABCB19 from Amborella. The positions of intron 8-ABCB1 in the variable linker region (~amino acids 681–762) in Arabidopsis and Amborella ABCB1, Amborella ABCB19, and rice ABCB homologs LOC_Os08g05690 and LOC_Os08g05710 are indicated by a black box around the relevant amino acid. (PDF 119 kb)Multiple sequence alignment of selected ABCB proteins from Additional file 9:ABCB1 obtained by long-range PCR across all nine introns or amplification across one or a small number of introns. (PDF 337 kb)Multiple sequence alignment of genomic sequences from regions of Additional file 10:ABCB1 orthologs from 27 monocots, 32 dicots and the basal angiosperm Amborella trichopoda. Four sequence motifs were identified by MEME in intron 7 at an e-value threshold of 1.0e-10. (PPTX 70 kb)MEME output of the motifs discovered in intron 7 in 81 Additional file 11Mimulus guttatus accessions IM62 and IM767 using primers designed against ABCB1 gene copies Migut.L01707 and Migut.J00652. Similar amplicon levels were obtained from genomic DNA for Migut.L01707 and Migut.J00652 in both accessions, indicating that both primers/gene regions have similar amplification efficiencies. (PPTX 2838 kb)Agarose gel showing amplification products obtained in Additional file 12:ABCB1 intron variance in the Poales explained by both intron loss and intron gain events. Intron loss events are shown by red stars and a minus sign (−) followed by the intron that was lost. Intron gain events are shown by green stars and a plus sign (+) followed by the intron that was gained. Members of the grass family are represented in black while non-grass monocots are in blue. The vertical bars indicate different grass subfamilies. (PDF 922 kb)Mixed model for


The deposition results are studied by Raman micro-spectroscopy and hyperspectral analysis using large fields of view to allow for the characterization of the whole deposition area. Results confirm that laser pre-patterning of the HOPG block prior to cleaving generates anchor points favoring a more homogeneous and defect-free HOPG surface, yielding larger and more uniform graphene depositions. We also demonstrate that a second patterning of the HOPG block just before exfoliation can yield features with precisely controlled geometries.This work describes a method for electrostatic deposition of graphene over a large area using controlled electrostatic exfoliation from a Highly Ordered Pyrolytic Graphite (HOPG) block. Deposition over 130 × 130 µm Sgrapheme ,36. Elecgrapheme ,36. Neve2 square islands with 30 μm wide and 50 μm deep etched spacing between them using a UV laser marking system, the HOPG block with the etched features is shown in Shown in −1 and 2710 cm−1) for the same region. The 2D peak center is taken as the center of a single-fitted Gaussian obtained from the Raman data between 2550–2750 cm−1. In the optical images, single-layer graphene cannot be resolved due to its low light absorption [Then, graphene is directly transferred onto a silicon wafer with a 1 µm-thick thermal oxide layer by using an exfoliation voltage of 3 kV. The complete details of the sample preparation and electrostatic deposition setup are provided in the methods section. The deposition results are shown in sorption , while ssorption ,38,39. Asorption ,38,39. F−1 and 34 cm−1, with the exception of the single-layer Lorentzian curve measured at 55 cm−1 and the lowest Lorentzian used to fit the bi-layer curve at 50 cm−1. This deviation from the 24 cm−1 FWHM can be attributed to unintentional doping of charge carriers from the deposition method which is known to widen the 2D peak in single-layer graphene [−1 to avoid any effect from the D’ shoulder (1620 cm−1). The variation of the G peak position is found to be fairly large, especially for mono- and two-layer graphene, this also suggests edge effects and excess charge carriers from the deposition method [Meanwhile, graphene ,41,42. Tn method ,44.Since the micro-Raman stage’s travel is restricting the scan size, hyperspectral analysis with a much larger field-of-view (FOV) can be used over the same deposition to fully appreciate the deposition scale. Our study has clearly shown that the cleaving process is critical to achieving such large-scale depositions. Micro-Raman analysis of the cleaved HOPG surface with and without laser pre-patterning offers a direct assessment of the improved cleaving method and highlights the importance of the cleaving process. Indeed, the Raman spectra averaged over a 100 µm × 100 µm area of the HOPG block after regular scotch tape cleave with and without laser pre-patterning are compared in If features with a precisely defined geometries are desired, a second  patterning of the HOPG block can be performed after the cleaving and before the exfoliation step. In ® with mosaic angles of 0.4° ± 0.1° and a typical lateral grain size of 3 mm. The substrate used for deposition is a silicon wafer with a 1 µm-thick thermal-oxide layer. In the experimental setup shown in ®, Dusseldorf, Germany) UV cured PVC bonding adhesive to create an insulating layer between the bottom electrode and the deposition substrate. The bottom electrode is attached to a gimbal levelling stage to ensure that the HOPG and the deposition substrate are parallel to each other so they come into complete contact. A Stanford Research Systems model PS375 +20 kV 10 W power supply is used as the high voltage power supply to connect the top and bottom electrodes with a high voltage cable. The set-up is placed in a Precise Basic Glove Box  with a controlled-atmosphere and purged with nitrogen to prevent arcing between the two electrodes.The electrostatic deposition of graphene starts from a bulk graphitic material. In this case, we use HOPG grade SPI-1 from SPI Supplies®  Professional. This process was done originally to limit the size of the deposited graphene; however we will demonstrate it significantly improves the homogeneity of the cleaving process between depositions. Cleaving of the HOPG surface is performed between each deposition using Scotch-tape . On some occasions, the cleaving process completely removes the etched features from the HOPG and leaves a flat surface. Prior to deposition, the HOPG block is pre-patterned to create an array of square islands using a Samurai UV laser marking system . operating at 355 nm with a spot size of ~25 μm. An etching depth between 0–300 μm can be achieved using this system on HOPG and is precisely controlled via the number of etching laser passes. The etching pattern is drawn using WinLaseIndeed, we find that large-scale deposition of graphene occurs after the complete cleaving of the etched features while using an exfoliation voltage of 3kV with a contact time of 3 s. 2 using a 10× objective.The large-scale depositions of graphene are probed using Raman micro-spectroscopy  to precisely identify the number of graphene layers within these depositions. A Witec Alpha 300R Raman system is used for the imaging and characterization of the deposited graphene. Raman measurements are performed with a 532 nm excitation laser at a power density of 0.57 mW/μm2 is used. A 50× objective is used to show the deposition in its entirety. Further Raman analysis is performed on the HOPG cleaved surface with and without the etched island anchoring process, to emphasize the role of the cleaving method.Hyperspectral analysis is also performed, both to confirm the Raman results and to analyze the size of the deposition with a larger field of view. For this hyperspectral analysis, a Raman imaging system, RIMA™ NANO  with an excitation wavelength of 532 nm at a power density of 0.12 mW/μmSEM micrographs were also taken using a SU 8230 FE-SEM  with a cold field emission gun to characterize the deposition and confirm the Raman and Hyperspectral layer identification methods. In this report, we demonstrate large-scale graphene deposition, up to 130 μm × 130 μm with 96% coverage (including 92% coverage with single-or double-layer graphene) using electrostatic exfoliation. This is achieved by improving the cleaving process used in preparation for the electrostatic deposition process. Indeed, an array of island features etched into the HOPG substrate seemingly act as anchors to the underlying graphene sheet when scotch tape cleaving is performed, thus significantly improving the homogeneity of the cleaving process as a whole. This critical preparation step is responsible for the superior results reported in this work. We also demonstrate that a second patterning of the HOPG block between the cleave and the exfoliation can be used to achieve features with precisely controlled geometries. We believe this work can have a significant impact as a promising approach to integrating graphene in production of next-generation devices.