Graphene has been actively investigated as an electronic material owing to many excellent physical properties, such as high charge mobility and quantum Hall effect, due to the characteristics of a linear band structure and an ideal two-dimensional electron system. However, the correlations between the transport characteristics and the spin states of charge carriers or atomic vacancies in graphene have not yet been fully elucidated. Here, we show the spin states of single-layer graphene to clarify the correlations using electron spin resonance (ESR) spectroscopy as a function of accumulated charge density using transistor structures. Two different electrically induced ESR signals were observed. One is originated from a Fermi-degenerate two-dimensional electron system, demonstrating the first observation of electrically induced Pauli paramagnetism from a microscopic viewpoint, showing a clear contrast to no ESR observation of Pauli paramagnetism in carbon nanotubes (CNTs) due to a one-dimensional electron system. The other is originated from the electrically induced ambipolar spin vanishments due to atomic vacancies in graphene, showing a universal phenomenon for carbon materials including CNTs. The degenerate electron system with the ambipolar spin vanishments would contribute to high charge mobility due to the decrease in spin scatterings in graphene. Graphene has attracted a great deal of attention because graphene shows excellent physical properties, such as quantum anomalous Hall effect24589To further understand the high mobility and to apply graphene to the high-speed transistors, the elucidation of the spin and charge-impurity states that cause charge carrier scatterings11121315161718Electron spin resonance (ESR) spectroscopy using device structures is the most effective method to study the spin states in electronic materials under device operations222324252223242527Here, we report the ESR study of electrically induced spin states in single-layer graphene that are carried out using driven transistors fabricated with large-area high-quality graphene. To carry out the ESR measurements sensitively, we use an ion gel which is capable of forming high charge-density states in graphene2930To attain high signal-to-noise (S/N) ratio of the ESR signal by increasing the active area of the device, we utilized a rectangular graphene-transistor structure 3 mm × 30 mm) in an ESR sample tube with an inner diameter of 3.5 mm. 29 mm in anVG) region to present the microscopic investigations of the spin states due to the Fermi-degenerate two-dimensional electron system and those due to the atomic vacancies in graphene. VG. In the negative VG region, the ESR signal intensity increased with increasing the absolute value of the VG. The parameters of the ESR signal at VG = −1.5 V were obtained as the g factor of g = 2.0036 and the peak-to-peak ESR linewidth (ΔHpp) of 1.45 mT. In contrast, the ESR spectrum and the VG dependence dramatically changed under the positive VG region, as shown in VG increased and showed a maximum at VG = 0.8 V, and then decreased when the VG increased from VG = 0.8 V. The ESR parameters at VG = 0.8 V were obtained as g = 2.0033 and ΔHpp = 0.74 mT. As discussed later in detail, the signals at VG = −1.5 and 0.8 V are ascribed to the charge carriers and the atomic vacancies in graphene, respectively.The spin states of single-layer graphene are clearly reflected in the ESR signals. We show the ESR signals of graphene transistors under a wide gate-voltage (VG dependence of the ESR intensity and the sheet conductivity (σ2D) of the graphene transistor. To present the ESR intensity, we evaluated the spin susceptibility (χ) from double integral value of the ESR spectrum, considering the active area of the graphene transistor. The σ2D value showed a minimum at VG = 0.8 V or at VG = 0.6 V when the source and drain electrodes of Ni/Au or Au were used, respectively. The observation of the σ2D minimum indicates an existence of the charge neutral point, namely, the Dirac point in the graphene transistors. Such behavior is consistent with that reported for single-layer graphene transistors32VG at the charge neutral point as VCNP. χ and the σ2D on VG − VCNP. The χ value shows a maximum at VG = VCNP where the σ2D shows the minimum. The increase in the χ has been observed for VG − VCNP < −1 V. It is worth noting that the maximum of the χ at VG = VCNP with the drain-current minimum is fully consistent with that observed for the CNT transistorsThe correlation between the transport characteristics and the observed ESR signals were examined by measuring the σ2D of the graphene transistor. We extracted the Lorentzian component from the ESR spectrum of the graphene transistor because mobile charge carries show an ESR signal with the Lorentzian lineshape. We define the spin susceptibility due to the charge carriers as χL, which was evaluated from double integral value of the ESR spectrum with the Lorentzian lineshape. The χL at different VG was evaluated by performing the ESR spectrum simulation using Lorentzian and Gaussian functions. VG = −1.5 V (or VG − VCNP = −2.3 V), where most of the signal component is found to be described by the Lorentzian function. In contrast, the fitting analysis for the ESR spectrum at VG = 0.8 V (or VG − VCNP = 0 V) exhibits a different result, where the signal component is found to be described practically by the Gaussian function, as shown in VG = 0.8 V because the χL is proportional to the square of the ΔHpp and the ΔHpp of the Lorentzian component is considerably broader than that of the Gaussian component. σ2D and the χL of the graphene transistor on VG − VCNP, respectively. It is worth noting that the χL value shows a minimum at VG = VCNP, which is consistent with the minimum of the σ2D. The increases in the χL for VG < VCNP and VG > VCNP have been observed, which are ascribed to the accumulation of holes and electrons, respectively. Therefore, the clear correlation between the χL and the σ2D demonstrates that the ESR signal with the Lorentzian lineshape is originated from the charge carriers in graphene. Schematics of the energy diagram and the spin states in the graphene transistor are shown in χL intensity reflects the density of states at the Fermi surface in graphene shown in The ESR signal due to the charge carriers should have a correlation with the transport characteristics. To demonstrate the correlation, we discuss the relationship between the spin susceptibility due to the charge carriers and the χ, which is called as Pauli paramagnetism. Thus, it is interesting to discuss the temperature dependence of the χ. The previous studies have reported the temperature dependence of the ESR intensity of the graphene samples27χ of the graphene samples followed the Curie law or the Curie-Weiss law27χ using the graphene transistor to observe the Pauli paramagnetism in graphene. χ of the graphene transistor. The χ was evaluated from the ESR signal at VG = −1.5 V (or VG − VCNP = −2.3 V) for the hole accumulation state by double integrating the ESR spectrum. As shown in χ hardly depends on the temperature, which means the Pauli paramagnetism, not the Curie paramagnetism shown by dashed line in The Fermi-degenerate electron system is known to show no temperature dependence for the spin susceptibility ΔH1/2), which was obtained by integrating the ESR spectrum. As shown in ΔH1/2 increases with increasing temperature. This result can be explained by the Elliott mechanism observed for Fermi-degenerate electron systems, where the spin-lattice relaxation time (T1) is shortened with increasing temperature and the ESR linewidth is proportional to 1/T1, that is, ΔH1/2 ∝ 1/T1  due to the atomic vacancies shows a maximum at a charge neutral point (VG = VCNP) with a drain-current minimumNspin due to the atomic vacancies in the graphene transistors with that of the CNT transistors to present the universality of the electrically induced ambipolar spin vanishment in carbon materials. We evaluated the Nspin in the graphene transistors from the double integral value of the ESR spectrum with the Gaussian lineshape. The Nspin is related to the χG as μB is the Bohr magneton, S is a spin quantum number due to an atomic vacancy (S = 1/2), kB is the Boltzmann constant, and T is the temperature. Nspin of the graphene transistor and the CNT transistors on VG − VCNP, respectively. The data for the CNT transistor are taken from the literatureNspin similarly has the maximum at VG = VCNP and decreases for VG < VCNP and VG > VCNP in the ideal two-dimensional material single-layer graphene and in the one-dimensional material CNTs, respectively. Therefore, the electrically induced ambipolar spin vanishment seems a universal phenomenon for carbon materials with honeycomb structures formed with carbon atoms regardless of the dimensionality of the electron systems.The electrically induced ambipolar spin vanishments have been reported in the ESR study of the CNT transistors, where the number of spins (VG = VCNP where the Nspin (or the χG) due to non-bonding orbitals (NBOs) at the atomic vacancies in graphene shows the maximum value  in the CNT thin film due to a thick film thickness of CNT (approximately 300 nm)VG, resulting in the almost linear change in the Nspin . In the case of graphene, such charge-density distribution is not basically expected due to a monolayer film thickness.The electrically induced ambipolar spin vanishments are schematically explained in alue see . In the spin see . This ree VG see . That isIn contrast to the similar observation of the electrically induced ambipolar spin vanishments in the graphene and CNT transistors, the ESR signal due to charge carries was unable to be observed in the CNT transistors26352635Our result demonstrates the observation of the ESR signal due to the charge carriers and the electrically induced Pauli paramagnetism in the Fermi-degenerate two-dimensional electron system in graphene. The result is interesting due to the first direct ESR observation of the electrically induced Pauli paramagnetism for organic, inorganic, and carbon materials. The observation also demonstrates the existence of the Fermi surface of the two-dimensional material graphene from a microscopic viewpoint. Moreover, the electrically induced ambipolar spin vanishments due to the atomic vacancies in graphene are observed, which indicates that the spin vanishment is a universal phenomenon in carbon materials including carbon nanotubes regardless of the dimensionality of the electron systems. The spins due to the atomic vacancies vanish owing to the antiferromagnetic interactions with electrically induced electrons or the discharges of the vacancies’ spins due to hole doping, which would decrease the carriers’ spin scatterings due to the atomic vacancies. Therefore, the Fermi-degenerate electron system with the electrically induced ambipolar spin vanishments would contribute to high charge mobility in graphene. Our approach using the electrically induced ESR technique is the most appropriate method for investigating the spin states and their correlations with the transport characteristics in electronic materials from a microscopic viewpoint because the method can directly observe the correlations between the spin states and the transport characteristic. Applying the method to other various electronic materials would give new understanding of the microscopic properties which are also useful for improving the device performance.c-plane sapphire [α-Al2O3(0001)] by magnetron sputtering at high temperature. The Cu/sapphire substrate was placed inside a quartz tube (inner diameter 26 mm) and then annealed at 1000°C in the flow of H2 (2.5% in Ar) gases for 40 min. After elevating the temperature to 1075 °C, CH4 gas (10 ppm) was introduced for 90 min to grow graphene. High purity gases were used for this experiment . Confocal Raman (Tokyo Instruments Nanofinder30 with 532 nm excitation) measurements were performed for the graphene transferred on a quartz substrate by a poly(methyl methacrylate) (PMMA) mediated transfer technique. The obtained Raman spectra are consistent with that previously reported for single-layer grapheneWe synthesized large-area single-layer graphene with a chemical vapor deposition (CVD) method37b-methylmethacylate-b-styrene) (PS-PMMA-PS) (3 wt%) , and a solvent ethyl acetate (61 wt%) ; the mixture was stirred for over one and half day, drop-casted on the gate electrode and then thermally annealed at 70 °C under vacuum for over one and half day. The ion-gel insulator shows large electric double layer (EDL) capacitance and high ionic conductivity. The EDL capacitance is generally very large (~10–100 μF cm−2), leading to significant charge accumulation with low voltage and high on/off current ratios. The source and drain electrodes of Ni (1 nm)/Au (30 nm) or Au (30 nm) which had a channel length of 1.0 mm and a channel width of approximately 25 mm were fabricated with a vapour-deposition method on the quartz substrate. Finally, the PET substrate was placed on the quartz substrate, completing the transistor fabrication. The fabricated transistor was sealed into an ESR sample tube under a nitrogen glove-box atmosphere  after the transistor was connected to wires by Ag paste.The transistors were fabricated using two types of nonmagnetic substrates; one was a polyethylene terephthalate (PET) film with dimensions of 30 mm × 3 mm × 100 μm , and the other was a quartz glass with dimensions of 30 mm × 3 mm × 1 mm . Gate electrodes of Ni (1 nm) and Au (30 nm) were vapour-deposited on the PET substrate using an ULVAC VPC-260F vacuum evaporation system. Ion-gel solutions consisted of an ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([EMIM][TFSI]) (36 wt%) , a gelator ABA-type triblock copolymer poly of the first integrated ESR spectrum. The spin susceptibility  was evaluated by integrating the ESR spectrum twice and comparing it with that of the Mn2+ marker sample. The number of spins, Nspin, was evaluated by integrating the ESR spectrum twice and by comparing it with that of the Mn2+ marker sample. The absolute value of the number of spins of the Mn2+ marker sample was calculated using a solution (220 μL) of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) as a standard. The calibration of the g factor was performed using a software program from the JEOL RESONANCE ESR system considering high second-order correction to the effective resonance field. Its correctness was also confirmed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as another standard sample.The ESR measurements were performed with a JEOL RESONANCE JES-FA200 X-band ESR spectrometer and a Keithley 2612A source meter. The ESR signals were measured as a function of How to cite this article: Fujita, N. et al. Direct observation of electrically induced Pauli paramagnetism in single-layer graphene using ESR spectroscopy. Sci. Rep.6, 34966; doi: 10.1038/srep34966 (2016).


The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene at optical frequencies. When the resonance wavelengths of the magnetic dipole modes are tuned to approach one another by changing the diameters of the Ag nanodisks, a broadband absorption enhancement can be achieved. The position of the absorption band in monolayer graphene can be also controlled by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks. Our designed graphene light absorber may find some potential applications in optoelectronic devices, such as photodetectors.It is well known that a suspended monolayer graphene has a weak light absorption efficiency of about 2.3% at normal incidence, which is disadvantageous to some applications in optoelectronic devices. In this work, we will numerically study multiband and broadband absorption enhancement of monolayer graphene over the whole visible spectrum, due to multiple magnetic dipole resonances in metamaterials. The unit cell of the metamaterials is composed of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO Graphene, a monolayer of carbon atoms tightly arranged in two-dimensional 2D) honeycomb lattice, was first separated from graphite experimentally in 2004 . Since tD honeycoTo overcome this problem, various physical mechanisms –43 to en2 spacer on an Ag substrate. The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into four-band absorption enhancement of monolayer graphene. When the magnetic dipole modes are tuned to be overlapped spectrally by changing the diameters of Ag nanodisks, a broadband absorption enhancement is achieved. The position of the absorption band in monolayer graphene can be also controlled by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks.In this work, by employing similar multi-resonator approach, we will numerically demonstrate multiband and broadband absorption enhancement of monolayer graphene in the whole visible wavelength range, which arise from a set of magnetic dipole resonances in metamaterials. The unit cell of metamaterials consists of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO2 spacer on an Ag substrate. We calculate the reflection and absorption spectra, and the distributions of electromagnetic fields by the commercial software package “EastFDTD, version 5.0,” which is based on finite difference time domain (FDTD) method (www.eastfdtd.com). In our numerical calculations, the refractive index of SiO2 is 1.45, and the frequency-dependent relative permittivity of Ag is taken from experimental data [σ of graphene is the sum of the intraband term σintra and the interband term σinter [The designed metamaterials for multiband and broadband absorption enhancement of graphene at optical frequencies are schematically shown in Fig. tal data . Under tm σinter , 68, whiω is the frequency of incident light, e is electron charge, ħ is reduced Planck constant, Ef is Fermi energy , τ is the relaxation time of electron-phonon, kB is Boltzmann constant, T is temperature in K, and i is the imaginary unit. Graphene has an anisotropic relative permittivity tensor of εg expressed aswhere ε0 is the permittivity of the vacuum, and tg is the thickness of graphene sheet.where λ1 = 722.9 nm, λ2 = 655.7 nm, λ3 = 545.5 nm, and λ4 = 468.8 nm. At four absorption peaks, the light absorption in graphene can reach as high as 65.7, 61.2, 68.4, and 64.5%, respectively. Compared with a suspended monolayer graphene whose absorption efficiency is only 2.3% at optical frequencies [Figure quencies , 17, thequencies –75, whicquencies –81.Fig. λ1, λ2, λ3, and λ4. At the resonance wavelength of λ1, the electric fields are mainly concentrated near the left and right edges of the first Ag nanodisk with a diameter of d1  absorption efficiency more than 50% (73%). For the diameters of the Ag nanodisks to be increased gradually, this broad high-absorption band is red-shifted, as shown in Fig. In our designed metamaterials, the near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene in the visible wavelength range from 450 to 800 nm, with an average absorption efficiency exceeding 50% , so the center distance l between the nearest-neighbor Ag nanodisks is 200 nm. By varying l, we can also tune the position of the absorption band in monolayer graphene. Figure l to be decreased from 220 to 160 nm. With the decreasing l, the absorption band in monolayer graphene is slightly blue-shifted, owing to the plasmon interactions among the Ag nanodisks.In the above calculations, the coordinate points of four Ag nanodisks are (±2 spacer on an Ag substrate. The near-field plasmon hybridizations between individual Ag nanodisks and the Ag substrate form four independent magnetic dipole modes, which result into multiband absorption enhancement of monolayer graphene in the visible wavelength range. When the magnetic dipole modes are tuned to be overlapped spectrally by changing the diameters of Ag nanodisks, a broadband absorption enhancement is achieved. The position of the absorption band in monolayer graphene can be also controlled, by varying the thickness of the SiO2 spacer or the distance between the Ag nanodisks. The numerical results may have some potential applications in optoelectronic devices, such as photodetectors.In this work, we have numerically investigated multiband and broadband absorption enhancement of monolayer graphene at optical frequencies from multiple magnetic dipole resonances in metamaterials. The unit cell of the metamaterials consists of a graphene monolayer sandwiched between four Ag nanodisks with different diameters and a SiO


K+) ions on air-cleaved mica is important in many interfacial phenomena such as crystal growth, self-assembly and charge transfer on mica. However, due to experimental limitations to nondestructively probe single ions and ionic domains, their exact lateral organization is yet unknown. We show, by the use of graphene as an ultra-thin protective coating and scanning probe microscopies, that single potassium ions form ordered structures that are covered by an ice layer. The K+ ions prefer to minimize the number of nearest neighbour K+ ions by forming row-like structures as well as small domains. This trend is a result of repulsive ionic forces between adjacent ions, weakened due to screening by the surrounding water molecules. Using high resolution conductive atomic force microscopy maps, the local conductance of the graphene is measured, revealing a direct correlation between the K+ distribution and the structure of the ice layer. Our results shed light on the local distribution of ions on the air-cleaved mica, solving a long-standing enigma. They also provide a detailed understanding of charge transfer from the ionic domains towards graphene.The distribution of potassium ( KAl2O10(OH)2. The structure of mica consists of an octahedrally coordinated Al3+ sheet which is sandwiched between two tetrahedrally coordinated sheets of Al3+ and Si4+, see Si4+ by Al3+ in the tetrahedral sheet results in a negative charge. Interlayer potassium cations hold together these 2:1 layers and balance the negative charge of the mica crystal. After cleavage of the mica, half of the potassium ions are expected to remain on each of the two created surfaces in order to preserve charge balance. The atomic lattice periodicity of mica has been obtained by Atomic Force microscopy (AFM) and Friction Force Microscopy (FFM)24K+ ions. In many cases the large forces exerted on the surface result in significant damage and loss of lateral resolution.Muscovite mica has been the substrate of choice for a large number of scientific studies and technological applications owing to its atomically flat surface, high surface energy, low thermal and electrical conductivity and its perfect cleavage plane along the 0001) direction directioKAl2Al,SiO10(OH)2.et al.Although scanning probe techniques have been extensively used to study the surface of mica immersed in aqueous ionic solutions679K+ ions is unlikely due to the randomness of the cleaving process. Other studies suggest the formation of K+ islands, leaving thus ion-free mica regionsUnderstanding the complexity of the lateral organization of ions on air-cleaved mica surface is key in crystal growth and self-assembly of molecules on mica and justifiably has received intense scientific interestK+ distribution and the conductance of graphene. Immediately above the K+ ions, graphene is significantly less conductive than over ion-free locations. This result indicates charge transfer from the underlying surface to graphene and is in good agreement with previous studies152526Here we use graphene as an ultra thin coating for enabling the visualization of single ions and ionic clusters on the mica surface. Graphene has been shown to be able to accurately stabilize and conform to water layers13141516171819212223Ih-ice. A cartoon of the experimental system is shown in μm2), we are unable to determine the exact number of graphene layers. Nevertheless from the size of the observed fractals we do not expect the thickness to be larger than 4 graphene layersFreshly cleaved mica is reactive and immediately a thin water film is adsorbed and covers its entire surface14pm and 35 pm, respectively. The difference of the RMS roughness between the two regions is a result of the extra water layer at the environment of the ice crystals, which can smoothen the interface by filling atomic scale defects. The obtained RMS values are larger than those reported in earlier AFM studies14Small scale STM images, nN in order to avoid any irreversible damage of the surface. Lateral force images, nm. The protrusions unambiguously indicate the presence of K+ ions at the graphene-mica interface. Surprisingly, the ions appear to preferentially form rows and small clusters. A reference mica lattice can be reconstructed by FFT and overlayed with the locations of the K+ ions, Si4+ or Al3+. In agreement with our observations, Loh et al.K+ ions are stable  graphene-mica interface under low humidity conditions. Note that LFM can resolve atomic lattice periodicities of solid surfaces4able see , demonstK+ ions a histogram of nearest neighbours is extracted and reveals a preference for having 2 and 3 neighbours  is used. In C-AFM a conductive tip is maintained in mechanical contact with the surface while it measures the current generated by the applied bias voltage. K+ ions is found inside the fractal. The simultaneously recorded conductance map is given in 5c. As expected, the regions above the double layer of water are substantially less conductive than the graphene regions just above the ice crystals. Since the conductance depends on the local electron density, i.e. the density of states around the Fermi level, the doped graphene shows a higher conduction because of a higher density of states15In order to confirm the existence of an ice-like layer covering a nonuniform distribution of K+ rows (vacancies) exhibit higher electrical conductance as compared to graphene directly above the ions. The correlation becomes better visible by overlaying the two images, as shown in K+ ions is negatively charged, whereas the mica surface with K+ ions is neutral. At the positions where the K+ ions are located the negatively charged surface is induced by the polarized ice layer. However, at locations where the K+ ions are missing, the negatively charged surface is induced by the ice layer as well as the mica surface. Therefore the local electron density is higher and a higher conductance is measured at the locations were K+ ions are missing. The small overlap  due to repulsive ion-ion interactions by forming specific arrangements. The repulsive ion-ion forces are screened by the presence of ordered water layers. The ordered water/ice-K+ interface is studied by measuring the electronic properties of the graphene cover using conductive AFM. At locations where the water molecules arrange in a fractal-like crystalline structure, graphene has a higher electrical conductance due to the higher local electron density induced by charge transfer from the ice layer. High resolution conductance images reveal single ions and and show distinct variations of the conductance exactly above the ice layer. These variations show a direct correlation with the nonuniform distribution of K+ ions. The current results are of importance in crystal growth and self-assembly on mica surfaces and give new valuable information on charge transfer phenomena in the graphene cover.Using scanning probe microscopies in combination with graphene as an ultra thin cover, we have managed to obtain information on the local organization of ionic domains on air-cleaved mica surfaces. Lateral force images show that the N/m and resonance frequency of 30 kHz. The AFM scanner was placed inside an environmental glove box, in which the relative humidity (RH) could be controlled. The RH was tuned by an adjustable 2N flow and was measured using a humidity sensor , with an accuracy of 1.8% between 10–90% RH. Scanning tunneling microscopy (STM) was performed in an RHK Technology UHV3000 variable temperature STM operating at a base pressure of 1 × 10−10 mbar, using chemically etched W tips. All the STM images are aquired at room temperature. For the STM and C-AFM measurements the graphene flake was mechanically connected with a bigger graphite flake acting as an electrode.Graphene was obtained from highly oriented pyrolytic graphite  through the microexfoliation process and immediately deposited on a freshly cleaved mica surface . Optical Microscopy  and tapping mode Atomic Force Microscopy, AFM  were used to determine the thickness of grapheneHow to cite this article: Bampoulis, P. et al. Graphene Visualizes the Ion Distribution on Air-Cleaved Mica. Sci. Rep.7, 43451; doi: 10.1038/srep43451 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Shigella mainly resides in the use of a Type 3 Secretion System (T3SS) to inject several proteins inside the host cell. Three categories of proteins are hierarchically secreted: (1) the needle components (MxiH and MxiI), (2) the translocator proteins which form a pore (translocon) inside the host cell membrane, and (3) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an “off” state by the presence of a tip complex. We have previously identified a gatekeeper protein, MxiC, which sequesters effectors inside the bacteria probably by interacting with MxiI, the inner‐rod component. Upon cell contact and translocon insertion, a signal is most likely transmitted from the top of the needle to the base, passing through the needle and allowing effectors release. However, the molecular mechanism underlying the transmission of the activation signal through the needle is still poorly understood. In this work, we investigate the role of MxiI in the activation of the T3SS by performing a mutational study. Interestingly we have shown that mutations of a single residue in MxiI (T82) induce an mxiC‐like phenotype and prevent the interaction with MxiC. Moreover, we have shown that the L26A mutation significantly reduces T3 secretion. The L26A mutation impairs the interaction between MxiI and Spa40, a keystone component of the switch between needle assembly and translocators secretion. The L26A mutation also sequesters MxiC. All these results highlight the crucial role of MxiI in regulating the secretion and transmitting the activation signal of the T3SS.The virulence of  This disease is responsible for more than 1 million deaths per year globally, essentially among children under 5 years of age in developing countries  to inject virulence proteins into eukaryotic cells , Salmonella (PrgJ), Pseudomonas (PscI), or Burkhoderia (BsaK). Recently, MxiI homologous protein, PrgJ, has been shown to interact with the cytoplasmic part of proteins composing the export apparatus in Salmonella  is composed of more than 20 proteins assembled into four parts: (1) a cytoplasmic part called the C‐ring, (2) an export apparatus localized in the inner‐membrane ring, (3) a basal body spanning the inner (IM) and outer (OM) membranes, and (4) an extracellular needle , an inner‐membrane protein, undergoes a conformational change following its autocleavage into two fragments, called Spa40CC and Spa40CN  secretion of effectors  highlights the role of the needle subunits  leading to an “effector mutant” phenotype, which is also rescued by the mxiC‐like phenotype. We have demonstrated here that some mutations of MxiI (on T82 residue) affect its interaction with MxiC and lead to exactly the same phenotype than the mxiC mutant. Moreover, the charge of the residue seems to play a key role in the secretion control. We have shown that the C‐terminal part of MxiI (74–93 residues), probably located inside the secretion channel, is sufficient for MxiC binding.In this study, we have undertaken a novel series of point and random mutations within MxiI to analyze its role in signal transmission aiming to find MxiI mutations that can lead to an 22.1Shigella flexneri strains were derivatives of the wild‐type strain M90T (serotype 5a)  were transformed with pET30a. Shigella were phenotypically selected on Congo red (CR) agar plates and grown in tryptic soy broth (VWR) at 37°C with the appropriate antibiotics at the following concentrations: zeocin 50 μg/ml, kanamycin 50 μg/ml, streptomycin 100 μg/ml, ampicillin 100 μg/ml, and chloramphenicol 25 μg/ml for E. coli strains and 3 μg/ml for Shigella strains.2.2mxiI), used to complement the mxiI mutant, was constructed by inserting a BamHI/XhoI digested PCR fragment, carrying native mxiI gene, into the BamHI/XhoI sites of the low copy vector pSU18 . Directed mutagenesis was carried out according to the procedure of the Quick Change Mutagenesis kit (Stratagene). The use of each primer in PCR creates a restriction site  containing 200 μg/ml CR, and incubating for 20 min at 37°C. Bacteria were centrifuged at 13,000 g for 15 min at RT and proteins present in the supernatant were analyzed by SDS‐PAGE. Western blotting was performed on polyvinylidene fluoride (PVDF) membranes  and developed using chemiluminiscence . Immunodetection was carried out as described by Botteaux et al.  was transformed with pGEX4T1 or its derivatives expressing, respectively, GST alone or GST fusion proteins and cultured in 100 ml of lysogeny broth (LB) at 37°C. Protein expression was induced with 0.1 mmol/L isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) for 3 hr at 30°C. Bacteria were harvested, suspended in PBS, and then lyses by sonication in presence of 1% Triton X‐100. The lysates were then clarified by centrifugation and the supernatants mixed with glutathione‐Sepharose 4B matrix beads  previously equilibrated with PBS buffer during 1 hr at room temperature on a rotor shaker and then washed three times with PBS. Then the beads were incubated 16 hr at 4°C in a rotor shaker with cleared extract of E. coli strains (Rosetta DE3) expressing His‐tagged recombinant proteins. Beads were washed eight times and proteins eluted by incubating beads for 10 min with elution buffer . The eluted proteins were resolved by SDS‐PAGE and analyzed by Coomassie blue staining or Western blotting.2.52. Briefly, cells were seeded at 1 × 105 cells/well in 24‐well plates 24 hr prior infection. Shigella strains were grown at 37°C to mid‐log phase, washed once with PBS, and suspended in DMEM. Bacteria were then centrifuged onto plates (MOI of 100) at 2,000 g for 10 min and further incubated 45 min at 37°C. Infected cells were washed three times and incubated 1 hr with gentamicin (50 μg/ml). Finally, cells were lysed with PBS‐Triton 0.1% and intracellular bacteria were diluted and plated on TSB agar Petri dishes for colony‐forming units (cfu) counting. Hela cells invasion was defined as 100% for the wild‐type strain (M90T).Bacteria ability to invade HeLa cells was tested with a gentamicin protection assay  and grown at 37°C to mid‐log phase, washed once with PBS, and suspended at a concentration of 1,010 bacteria/ml. Horse red blood cells (Oxoid) were centrifuged at 2,000 g for 10 min at 4°C and washed twice with cold PBS. Then 50 μl of each sample was mixed in 96‐well flat bottom and centrifuged at 2,000 g for 10 min. After 1 hr incubation at 37°C, the reaction was stopped by the addition of 100 μl of cold PBS. Cells were suspended and further centrifuged at 2,000 g for 10 min. Optical density of the supernatant was measured at 540 nm. Red blood cells lysis was defined as 100% for the wild‐type strain (M90T).The contact‐mediated hemolysis assay was performed as previously described  in the same buffer. After serial dehydration samples were dried at critical point and coated with platinum by standard procedures. Observations were made in a Tecnai FEG ESEM QUANTA 200 (FEI) and images were processed by SIS iTEM (Olympus) software.Whole bacterial cells were applied to glow discharged carbon‐coated Formvar copper grids. Bacterial cells were negatively stained with 4% ForMol. Observations were done on a Tecnai 10 (FEI) microscope coupled to a Veleta charge‐coupled device (CCD) camera (Olympus iTEM), and images were captured and analyzed using SIS Olympus iTEM software. Whole bacterial cells were applied to glow discharged carbon‐coated Formvar copper grids and negatively stained with 4% Uranyl acetate. Observations were done on a Tecnai 10 (FEI) transmission electron microscope coupled to a Veleta CCD camera (Olympus iTEM), and images were captured and analyzed using SIS Olympus iTEM software. For SEM, samples were fixed overnight at 4°C in glutaraldehyde 2.5%, 0.1 mol/L cacodylate buffer (pH 7.2), and postfixed in OsO3mxiI which could result in an mxiC‐like mutant phenotype. As MxiI shares 18% of sequence identity with MxiH, which is also implicated in signal transmission, we first generated 8‐point mutations of conserved residues between these two proteins , and to invade HeLa cells. As shown in Table mxiI mutants show a phenotype similar to the wild‐type strain for colony color on CR plate, proteins secretion, hemolysis, and cell invasion, one presents exactly the same phenotype as the mxiI mutant (mxiIL63A), and another presents a global reduction in proteins secretion (mxiIL26A). Nevertheless, none of all the mutations led to a hyper‐red‐colony phenotype on CR plate suggesting that none of them is able to abolish the interaction between MxiI and MxiC.We decided to perform site‐directed mutagenesis within 3.1mxiIL26A strain  and YscI (Yersinia), play a role in substrate specificity switching and functional needles formation . In contrast to unmodified GST‐MxiI, GST‐MxiIL26A did not co‐elute His‐Spa40CT nor His‐Spa40CN (205–258)  even if produced at a similar level  were subsequently analyzed for their constitutive secretion phenotype. Two of them, harboring a mutation of the T82 residue, into a lysine (T82K) or an arginine (T82R), presented the same phenotype than the mxiC mutant as they constitutively secrete early and late effectors  and a GST‐MxiC , we thought that charge of the residue could influence its capacity to bind MxiC and to control secretion. To answer this question, we have replaced the T82 residue by a negatively charged (glutamate) and a hydrophobic nonpolar  residue on the pSM6. The proteins secreted by the different mxiI mutants (expressing MxiIT82E and MxiIT82A) under both constitutive and induced conditions were analyzed. Unlike mxiIT82K and mxiIT82R strains, mxiIT82E and mxiIT82A strains allow proteins secretion under induced and noninduced conditions like the wild‐type strain  or a positively charged one (mxiIQ67K). We found that the mxiIQ67E, like the mxiIQ67A, presents an “effector mutant” phenotype while the mxiIQ67K allows proteins secretion like the wild‐type strain  leads to a mxiC‐mutant phenotype in terms of early effectors secretion . To do so, we have first mutated conserved residues between MxiI and MxiH as the latter is also implicated in signal transmission  harboring an mxiC‐like phenotype (hyper‐red colonies). As expected these mutants were no longer able to interact with MxiC, supporting our initial hypothesis. Moreover, the loss of MxiC–MxiI interaction leads to an earlier secretion of MxiC (before induction) and explains why MxiC is no longer able to ensure its role in promoting translocators secretion in these backgrounds. Indeed, unlike the MxiCF206S variant, the MxiIT82K and MxiIT82R show the same defect in translocators secretion after CR induction than the mxiC mutant. Interestingly, the T82 residue is conserved with PrgJ, the MxiI homologue from Salmonella, which can interact with the MxiC counterpart, InvE  and shown that they are effectively sufficient to bind MxiC, supporting the conservation of the regulatory mechanism. As the C‐terminal part of MxiI harboring the T82 residue is not well conserved with MxiH  or negative (E) residues. Moreover, the charge seems to impact directly MxiC binding as MxiIT82K and MxiIT82R totally abolish MxiC binding. Thus, we can postulate that this deregulated phenotype is due to the loss of interaction between the MxiI variants and MxiC which results in MxiC early secretion. Interestingly, the change in negative charges on the surface‐exposed residues of MxiC by positive ones also leads to a deregulated secretion phenotype by an unexplained mechanism  and is rescued by mxiC inactivation, but the mxiHK69A needles are shorter than the wild‐type strain  that decrease, even abolish, translocators and effectors secretion. As we failed to detect the MxiI variants in Shigella background by Western blot, we cannot exclude a lack of expression in the mxiIL63A strain explaining the absence of the needle, especially given that the MxiIL63A counterpart in Yersinia (YscIL80A) is not expressed . Indeed Spa40CT is known to control substrate specificity switch between needle components and translocators secretion. Interestingly MxiIL26A is no longer able to interact with Spa40CT and Spa32 seems weakly secreted. So, as previously shown for MxiI homologous proteins (Marlovits et al., Salmonella strain‐expressing PrgJL29A but InvJ, the Spa32 counterpart in Salmonella, is secreted and the needle complexes are similar to wild‐type ones in this strain (Lefebre & Galán, In the course of our study, we also found two mutations within MxiI (MxiImxiI can lead to the same phenotype than the mxiC mutant by impairing their mutual interaction. The domain responsible for this interaction was also identified and its localization in a highly conserved domain within MxiI homologous proteins suggests that this mechanism is probably conserved among others T3SSs. Nevertheless, further structural and electrostatic studies of the inner rod would allow a better understanding of the mechanism of signal transmission through the T3SS needle.The results presented here strengthened our previous model in which the MxiC–MxiI complex regulates the effectors secretion. In fact, we have shown that point mutation in None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


In this paper, reduced graphene oxide functionalized with cobalt ferrite nanocomposites (CoFe@rGO) as a novel type of electromagnetic wave (EW) absorbing materials was successfully prepared by a three-step chemical method including hydrothermal synthesis, annealing process and mixing with paraffin. The effect of the sample thickness and the amount of paraffin on the EW absorption properties of the composites was studied, revealing that the absorption peaks shifted toward the low frequency regions with the increasing thickness while other conditions had little or no effect. It is found that the CoFe@rGO enhanced both dielectric losses and magnetic losses and had the best EW absorption properties and the wide wavelength coverage of the hole Ku-Band when adding only 5wt% composites to paraffin. Therefore, CoFe@rGO could be used as an efficient and lightweight EW absorber. Compared with the research into traditional absorbing materials, this figures of merit are typically of the same order of magnitude, but given the lightweight nature of the material and the high level of compatibility with mass production standards, making use of CoFe@rGO as an electromagnetic absorber material shows great potential for real product applications. In recent years, electromagnetic wave (EW) absorbing materials have attracted substantial attention due to the widespread use in military and civil applications, such as electromagnetic protection, anechoic chambers, radiation-proof materials12456789107122O3, γ-Fe2O3 and Fe3O4 have good EW absorption effect within a certain frequency range  due to their high magnetization intensity and magnetic anisotropy1516192022232 hybridization of 3 carbon atoms with σ-bonds on the 2D plane and π-bonds orthogonal to them. Reduced graphene oxide (rGO), a widely-known derivative of graphene obtained from chemical reduction of graphene oxide by thermal, chemical or electrical treatments, possesses good dielectric properties, which can be used to prepare absorbing materials with dielectric loss mechanism121825Metallic nano-oxides are an important type of absorbing materials; therefore, developing magnetic nano-materials with large magnetic loss and high magnetic permeability is a promising approach for absorbing materials2O4 mixed to rGO nanocomposites (CoFe@rGO) were prepared by facile hydrothermal synthesis. It was found that when adding only 5 wt% composites to paraffin, the prepared CoFe@rGO with a thickness of 2.3 mm had the best EW absorption property. Compare with previous studies on the Fe3O4/graphene and CoFe2O4/graphene, this material had much lower amount (only 5%) and much wider bandwidth (7.17 GHz), which is much more suitable as a lightweight broadband electromagnetic wave absorbing coating. Considering its simple synthetic method and controllable ratio in the final composites, this kind of synthesized materials would be highly valuable in commercial use.Based on the above considerations, CoFexFe2-xO3 and CoFe@rGO. xFe2-xO3 particles were short rod-like or rice-like shaped particles, densely packed together and exhibiting localized agglomeration. The rice-like particles were about 400 nm in length with a diameter of about 180 nm. 2O4 particles were unevenly distributed in the graphene substrate, presenting pronounced localized agglomeration. The rice-like particles were about 400 nm in length with a diameter of about 200 nm. 2O4 are well-dispersed on RGO sheets. The high-resolution TEM image of the CoFe2O4 particles exhibits a lattice spacing of 0.295 nm corresponding to the (311) planes.−1 and G-band at 1593 cm−1, and 2D-band at 2670 cm−1. For graphene, the D-band and G-band are the Raman characteristic bands. The D-band was due to the random arrangement of graphite or induced by lattice defects of C atoms. The higher the intensity of the D-band, the more the lattice defects in the crystals. The G-band is due to the stretching vibration of C atoms in the sp2-hybridized plane  while the 2D-band is due to the secondary Raman scattering of regional boundary phonon. The intensity ratio of D-band to G-band, ID/IG, could characterize the degree of crystal disorder. The intensity ratio of D-band to G-band (ID/IG) for graphene oxide was 0.88. The red curve represents the Raman spectrum for the prepared CoFe@rGO. The D-band, G-band and 2D-band of the CoFe@rGO were at 1345 cm−1, 1589 cm−1, and 2704 cm−1; the corresponding intensities were 86.26, 100.49, and 11.14. The ratio of D-band to G-band (ID/IG) was 0.86, slightly lower than that of the graphene oxide. This indicates that CoFe2O4 eliminated defects during the preparation of CoFe@GO nanocomposites, which affected the Raman peaks of graphene. There were weak Raman peaks for CoFe2O4 in the spectrum of CoFe@rGO at 520 cm−1, and 533 cm−1 in the inset of 2O4 in the Raman spectrum.xFe2-xO3. This may be due to the overlap of the diffraction peak of the reduced graphene oxide and that of the ferric oxide, which indicates that during the heating process and annealing process of the facile hydrothermal synthesis, the ordered graphite crystal structure was reduced to some extent, that is, the graphene oxide was reduced into reduced graphene oxide. Moreover, apart from the reduced graphene oxide in the composites, we can detect the presence of CoFe2O4, Fe2O3, and Fe. The diffraction peaks of CoFe2O4 were at 30.08°, 35.24°, and 43.06°, corresponding to the crystal planes (220), (311), and (400). These peaks matched the standard PDF CARDS of CoFe2O42O3 were found at 24.14°, 33.15°, 40.85°, 49.48°, 54.09°, 57.43°, 62.45°, and 63.99°; these peaks corresponded to the crystal plane reflection of the (012), (104), (113), (024), (116), (122), (214), and (300) planes respectively. The diffraction peaks of Fe2O3 matched the standard PDF CARDS2/(H2 + Ar) = 8/100 at 360 °C for 3 h, CoFe2O4 and Fe were generated by reducing the cobalt ferrite. There were also diffraction peaks identifiable with the presence of Fe2O3 in the XRD result, implying that the cobalt ferrite was not fully reduced. The remaining cobalt ferrites existed as CoxFe2-xO3 in the CoFe@rGO.To clarify the microwave absorption properties, the reflection loss (RL) can be calculated according to transmission line theory and using the relative complex permittivity and permeabilityZin) is calculated by:While the normalized input impedance  were measured by the coaxial reflection - transmission network method. The results are presented in The complex permittivity of GF-15 was too high, leading to poor matching with the complex permeability, such that the EW absorption properties had not been improved. For GF-5, its complex permeability was similar to that of GF-15, whereas the complex permittivity was obviously reduced, which is promising to improve the impedance matching characteristics of the composites. Therefore, the EW absorption properties of CoFe@rGO were enhanced.According to the following equations:3O4, the dielectric loss is increased and the magnetic loss is reduced with the inclusion of Co element into ferrite. In the frequency range of 2~14 GHz, with increasing the amount of CoFe@rGO to paraffin, the magnetic loss tangent gradually increased. Compared with samples GF-5 and GF-10, the magnetic loss tangent of sample GF-15 was improved in the range of 2~18 GHz, exhibiting a higher magnetic loss. Therefore, the impedance matching characteristics of the composites could be improved by carefuly controlling the addition of CoFe@rGO to paraffin.and using the eletromagnetic parameters of samples GF-5, GF-10 and GF-15, the dielectric loss tangent and the magnetic loss tangent were calculated. The relationship of the dielectric loss tangent vs. frequency and the magnetic loss tangent vs. frequency were plotted, as shown in 2O4 was 1:1, after annealing in the mixed gas of H2/Ar for 3 h, the prepared CoFe@rGO by adding 5 wt% composites into paraffin had the best EW absorption property.The EW absorption properties of CoFe@Rgo  were tabulated in 2O4 particles. For sure, the contribution of CoFe2O4 to the impedance matching of the composites and the EW absorption cannot be neglected. The microstructure of the composites also greatly promoted the absorption of the incident wave. In addition, other factors such as interface scattering and the polarization were as well beneficial to EW absorption. As a dielectric loss material, rGO was the main absorbent for enhancing the dielectric permittivity. The EW was absorbed by interacting with the electromagnetic field, while its degradation depended on the dielectric relaxation and interface polarization. When the EW entered into the composites, the directional movement of the carriers in the reduced graphene oxide formed a dispersion current, leading to dielectric relaxation and polarization at the interface. Thereby, the electromagnetic energy was consumed by being converted into heat. Meanwhile, the Fe in the composites acted as a dielectric material and could also absorb the EW by the polarization relaxation effect. By adding magnetic CoFe2O4 particles into the rGO dielectric material, the composites were formed. On the one hand, the impedance matching of the composites was improved. The impedance difference between the composites and air was reduced, which was beneficial to letting more EW into the inside of the composites. On the other hand, the composites were magnetized, generating natural resonance etc. The composites had a dual nature of magnetic and dielectric loss, which was conducive to the EW absorption and to broaden the absorption bandwidth. The interface polarization in the composites could facilitate the electromagnetic wave absorption. rGO had the characteristics of large specific surface area, layered structure and so on. When mixing with CoFe2O4 particles, a large number of interfaces were generated, like the interface between CoFe2O4 particles and rGO. The scattering and the polarization at the interface could induce interaction between electromagnetic waves, thereby facilitating the EW absorption by the composites.According to the EW absorption properties of CoFe@rGO with different additive amounts, it is found that the complex permeability of GF-5, GF-10 and GF-15 were almost the same, whereas there were big differences in complex permittivity. GR-5 had the highest complex permittivity and showed the best EW absorption property, indicating that the additive amount of composites to paraffin could influence the dielectric loss significantly shown in . However2O4 contributed primarily to the EW absorption. The good absorbing property of the composites was due to the dielectric loss of CoFe2O4 and the dielectric relaxation and polarization of rGO. This novel and lightweight absorbing material has good prospects for commercial applications.In summary, a novel absorbing material reduced graphene oxide functionalized with cobalt ferrite nanocomposites (CoFe@rGO) was prepared. When the additive amount was 5 wt% to paraffin, the sample showed the best EW absorption property. In this case, when the thickness was 2.3 mm, the reflection rate reached the lowest at 16.63 GHz by −25.66 dB, and the bandwidth corresponding to RL less than −20 dB was 2.03 GHz. When the thickness was 2.7 mm, the bandwidth corresponding to RL less than −10 dB was 7.17 GHz from 10.87 to 18.04 GHz, covering the entire Ku band. The EW absorption mechanisms of CoFe@rGO were studied. The EW absorber was mainly attributed to the dielectric relaxation and polarization of rGO. Besides, the interface scattering and the hysteresis loss of CoFexFe2-xO3 by hydrothermal reaction. Existing literature indicated α-Fe2O3 nano rings could be prepared by hydrothermal reaction by adding FeCl3. In a similar manner, the hydrothermal reaction for preparing CoxFe2-xO3 was undertaken for 12 h at a furnace temperature of 220 °C by controlling the mole fraction of Co and Fe and adjusting the quality of FeCl3•6H2O and CoCl2•6H2O. After the reaction was completed, the reactor was cooled to room temperature in air. The solution was then centrifuged, and the precipitates were dried, giving the brick-red solid powder intermediate CoxFe2-xO3;Preparation of Co2O3/rGO nanocomposites by facile hydrothermal synthesis with the prepared CoxFe2-xO3 and graphene oxide (GO). The literature showed that graphene oxide GO could turn into reduced graphene rGO by hydrothermal reactionxFe2-xO3 (mass ratio of 1:1) were uniformly dispersed in a solution of ethanol and glycerol (ratio of 3:1), followed by placing the mixed solution in the reactor for hydrothermal reaction at a furnace temperature of 180 °C for 12 h. After the reaction was completed, the reactor was cooled to room temperature in air. The solution was then centrifuged, and the precipitates were dried, giving brown CoxFe2-xO3/rGO nanocomposites;  Preparation of CoFexFe2-xO3/rGO nanocomposites from step (ii) were annealed at 360 °C in mixed gas of H2/Ar = 8/92. Finally, CoxFe2-xO3/rGO nanocomposites were reduced into CoFe@rGO. Preparation of CoFe@rGO. The prepared CoCoFe@rGO were prepared by facile hydrothermal synthesis. The preparation process had three steps:The different mixture proportions of the measured samples are illustrated in The samples were characterized by field emission scanning electron microscopy (FESEM) , X-ray diffraction (XRD) (Rigaku DMAX-RB), and Raman spectroscopy . The electromagnetic parameters of the samples were measured by a vector network analyzer system (HP722ES) from 2 to 18 GHz. Reflectivity loss values were calculated by Matlab software according to the complex permittivity and complex permeability. The CoFe@rGO prepared with the above process were mixed with paraffin to make a paste that could be shaped into coaxial samples. The electromagnetic parameters of the samples were tested by vector mesh analyzer to calculate the absorption properties. The sample had an inner diameter of 3.0 mm, an outer diameter of 7.0 mm, and a thickness of 2.0 mm. Three different sets of samples were prepared for comparative experiments, in which, the adding proportion of the composites were 5%, 10%, and 15%, respectively; while the amount of paraffin was 0.5 g for all three samples.How to cite this article: Ding, Y. et al. Reduced Graphene Oxide Functionalized with Cobalt Ferrite Nanocomposites for Enhanced Efficient and Lightweight Electromagnetic Wave Absorption. Sci. Rep. 6, 32381; doi: 10.1038/srep32381 (2016).


The present study aims to enhance the understanding of the tumor microenvironment of lung cancer patients who underwent surgery, by means of analysis of PD-L1 expression in tumor cells and in intratumoral immune cells (IICs). It was found that PD-L1 expression was more frequent in tumor cells than in IICs. Collective analysis by Tissue Microarray Assay (TMA) for PD-L1 expression in tumor cells and IICs did not reproduce the findings for separate individual analysis of tumor tissues. Patients with past history of smoking were more likely to express PD-L1 in tumor cells than those who never smoked. Patients with past history of smoking were less likely to have PD-L1 positive IICs compared to those who had never smoked. The immunohistochemical expression of PD-L1 in tumor cells and IICs did not correlate with survival.Lung cancer is the leading world cause of cancer-related death, in both genders, and smoking is the main etiological factor. The discovery of immune checkpoints corroborates the hypothesis that ligands presented in tumors modulate the mechanisms of carcinogenesis and the immune activity of tumor microenvironment. Among the most studied coregulatory molecules, PD-1 Lung cancer remains the leading cause of cancer death worldwide, for both men and women. Over half of people diagnosed with lung cancer die within one year of diagnosis and the 5-year survival is less than 18% [In Brazil, according to 2016 estimates of INCA , the incidence rate for tracheal, bronchus, and lung tumors will be 17330 new cases  for men and 10890 cases  for women [Despite major advances in the personalized medicine, non-small-cell lung cancer is still related to poor prognosis.Until recently, non-small-cell lung cancer was considered a nonimmunogenic tumor, but there is now evidence highlighting the integral role played by both inflammatory and immunological responses in lung carcinogenesis . The disRecent studies show that tumor cells and antigen presenting cells modify tumor microenvironment through PD-1 receptor activities involving their ligands, PD-L1 and PD-L2 , 6. PD-1 CD274 gene [Programmed cell death 1 ligand 1 (PD-L1) or B7 homolog 1 (B7-H1) is a type I transmembrane protein encoded by the274 gene .The PD-1 function occurs primarily in peripheral tissues, where the T cells can contact their immunosuppressive ligands PD-L1 (B7-H1) and PD-L2 (B7-DC), which are expressed by tumor cells, stromal cells, or both –15. It hMore than that, PD-L1 expression may also vary according to different tumor microenvironments  and evenOne recent study showed that PD-L1 expression was not associated with gender, histology, differentiation status, or lymph node metastasis. However, PD-L1 expression was increased in stage III NSCLC compared with stage I/II .Other studies showed that PD-L1 expression was significantly associated with smoking –21, gendThe role of PD-L1 expression neither as a prognostic nor as a predictive factor is controversial, being suggested in several studies , 23, 24.Indeed, recently, the KEYNOTE-001 trial of pembrolizumab (a humanized antibody that targets the programmed cell death 1 receptor) for advanced NSCLC showed a significantly favourable survival in patients with that PD-L1 expression greater than 50% in comparison to those with expression lower than 50% .PD-L1 seems to be a mutable biomarker, with variable expression patterns related to heterogeneity in different areas within primary or metastatic lesions.Histopathological material aging or the interval between tissue collection and treatment may also influence PD-L1 expression –30.Positivity criteria for PD-L1 expression  are not standardized yet and that certainly may contribute for all these discrepancies.This study attempts to elucidate PD-L1 expression in lung tumor microenvironment. Possibly it may help to understand different clinical outcomes in lung cancer and establish more effective therapeutic measures.Tumor tissue samples and data were obtained from 177 cases. These patients underwent surgical resection of primary lung cancer between 2003 and 2014 at Barretos Cancer Hospital, Brazil. H&E stained sections were reviewed by a pathologist (CSN) and histological subtyping was assessed using the current World Health Organisation 2015 classification. Staging was undertaken according to the 7th edition AJCC tumor, node, metastasis (TNM) classification. The ethical use of human tissue for research was approved by the Institutional Review Board, and the design of this study followed the principles of the Declaration of Helsinki and also complied with the principles of good clinical practice.Inclusion criteria include patients older than 18 years of both genders, diagnosed with non-small-cell lung cancer , who underwent pneumonectomy, lobectomy, segmentectomy, or nodulectomy with available slides and paraffin blocks for histopathological analysis in the Pathology Department of Barretos Cancer Hospital.Exclusion criteria include inappropriate histopathological material by either poor quality or shortage due to its use in other studies and patients with a current diagnosis of another primary malignancy in any location of the body other than nonmelanoma skin cancer or in situ carcinoma of the cervix.PD-L1 immunohistochemistry membranous expression was assessed in TMA (Tissue Microarray Assay) with 2 cores of 1 mm in diameter of each tumor and also the expression was evaluated in whole tissue section of the same tumors .μ thick and the sections were transferred to positively charged glass slides.TMA blocks were made with MTA I  device , according to manufacturer's specifications. TMA and all the paraffin blocks were sectioned with 4At least one section per paraffin block was stained with hematoxylin-eosin in order to confirm the histology and to certify of tumor tissue availability in the slides by a pathologist and another slide was submitted to PD-L1 immunohistochemistry test.Four micra thick sections (TMA and whole tissue sections) were dewaxed at 80°C for 20 minutes and then were transferred to Ventana BenchMark Ultra Autostainer . Antigen retrieval was performed using Ventana Cell Conditioning solution 1 (pH: 8.5) for 64 minutes at 97°C. Anti-PD-L1 antibody  at titration of 1 : 25 was incubated for 60 minutes at 36°C. The detection of antigen antibody reaction was performed using UltraView DAB Universal Detection kit . Strong membranous trophoblastic staining in the placenta was used as positive control. Tissue expression of PD-L1 was categorized dichotomically into negative (<5% membranous tumor cells expression) or positive (≥5% membranous tumor cells expression)  and alsoData were described on the average, standard deviation, minimum, maximum, and quartiles for quantitative variables and frequency tables for qualitative variables. The agreement between the collective analysis technique or TMA (tissue microarray) and individual analysis was done using the Kappa coefficient to assess the reproducibility.p value than 0.2 in the previous test were selected and subsequently adjusted into a logistics multiple regression model. For modeling, one feature (variable) was removed each time by prioritizing those with higher p value, up to a set of significant variables.The correlation between clinical and demographic characteristics and PD-L1 expression in tumor cells and IICs was determined using the chi-square test (or Fisher's exact test) for the qualitative characteristics. To check the overall association between covariates and PD-L1 marker, those that had lower p value less than 0.2 were selected in simple analysis and adjusted in multiple Cox regression model. Then, modeling was continued as the same description in the logistic regression. To estimate the overall survival curve for PD-L1 expression the Kaplan-Meier method was used.For overall survival, the time between diagnosis and death for any reason (which is the event of interest) or last objective information of individuals who have not died yet was considered. To compare each feature and check the list of features with survival (one at each time), simple Cox regression was used. For joint evaluation between variables, those with For survival analysis only overall survival was considered, because it is a retrospective study and there was no standardization in the follow-up of these patients in the past. In this study we considered the statistical significance of 0.05 and SPSS 21.0 software was used for statistical analysis.In regard to demographic characteristics, as seen in The most common histological type was adenocarcinoma  with II degree of differentiation . Most of tumors had TNM Classification of Malignant Tumours lower than III. Stages I and II were observed in 62 patients (35%) and 53 patients (29.9%), respectively. Lobectomy was performed in 147 cases (83.1%). Adjuvant chemotherapy was performed in 34 cases (19.2%) .PD-L1 was expressed in tumor cells and in IICs. Tumor cells had coarse chromatin, increased nuclei with irregular nuclear membrane, and abundant cytoplasm with ill-defined borders. However, intratumoral immune cells had loose chromatin, smaller fold nuclei without atypia, and variable cytoplasm, sometimes with dendritic expansions .In Figures In p < 0.001) when the immunohistochemical expression of PD-L1 in tumor cells in conventional histological slides  and TMA slides  was evaluated. Similar results were observed with PD-L1 expression in IICs in conventional histological slides  and IICs slides  with Kappa index of 0.328 (p < 0.001).As noted in Once the immunohistochemical expression of PD-L1 was not reproducible between TMA and whole tissue sections, all the analyses (univariate and multivariate) of correlation between PD-L1 expression and covariates  considered PD-L1 expression in whole tissue section.p value = 0.044.As noted in As seen in As seen in p value = 0.022).According to p value = 0.008)  .After logistic regression, smoking was the only variable that remained significant related to PD-L1 expression in antigen presenting cells.p value = 0.029). The chance of positive PD-L1 expression in intratumoral immune cells was also lower for patients with active smoking when compared to patients who had never smoked but without statistical significance   .p value = 0.254. Also, there was no statistically significant difference in the median overall survival when comparing PD-L1 expression in IICs: 49.50 months (95% CI 23.37 to 75.64) for patients with positive PD-L1 expression versus 41.51 months (95% CI 29.99 to 53.04) in patients with negative PD-L1 expression with p value = 0.795 . Considering PD-L1 expression in tumor cells of conventional histological slides , there was a higher median overall survival for patients with tumors that had positive PD-L1 expression; however this difference was not statistically significant: 98.75 months (95% CI 21.26 to 176.24) versus 41.51 months (95% CI 30.05 to 52.98) with According to this study, the frequency of PD-L1 expression in tumor cells was 37.9% of total cases, a little less than a recently published study that showed 53.1% of positivity , althougThis evaluation can occur in different ways: continuous distribution and proportion of PD-L1 positive cells at any intensity , percentThere is no standardized cutoff for positivity. Some studies have used 1%, 5%, 50%, or more to consider positive PD-L1 expression .Therefore, it becomes extremely difficult to establish any relationship between studies as a consequence of no standard positivity criteria for PD-L1 expression, which involves the whole process, from chemical reactions machinery to dilutions of reactants and its reading interpretations. As seen in Another important issue to be discussed is the feasibility of PD-L1 biomarker in order to be measured and analyzed in histopathological slides. Garon et al. noted PD-L1 deterioration in tumor samples cuts more than 6 months before staining . AccordiA recent study showed that tumor microenvironment cells  do not express PD-L1 in a uniform way . Smyth eTumor microenvironment has a wide range of different cell types mainly represented by myeloid-derived suppressor cells, macrophages, dendritic cells, and T lymphocytes beyond tumor cells . There iThis study analysis showed a fair (low) agreement between the two methods of immunohistochemistry , for PD-L1 expression evaluation in lung tumors. The medical literature has pointed to the importance that lung tumor heterogeneity may have regarding PD-L1 expression in small biopsies when compared to surgical specimens.Small isolated samples from a particular tumor region gathered by biopsy or punches for TMA construction, for example, cannot represent PD-L1 expression for the tumor as a whole. This expression cannot be generalized, because of its intrinsic regional variability. Immunohistochemistry assessment of slides covering a tumor in its largest possible area, with the largest represented diameters, seems to provide a more representative description of real PD-L1 expression in lung tumors.In this study, tumors of patients with smoking history were more likely to positively express PD-L1 in tumor cells and they were also more likely to not express this biomarker in IICs. Although the influence of smoking in PD-L1 expression in tumor cells  has alreIt is worth noting that changes in PD-L1 expression can also occur related to previous exposure to chemotherapy, a fact already demonstrated in urothelial carcinomas and lung cancer , 45. TheThere was no statistical significance related to PD-L1 expression and overall survival as previously described. Other studies show apparently not to be a predictive or prognostic value regarding PP-L1 expression , 46, 47.In summary, this study shows that, in a population of patients with non-small-cell lung cancer, the pattern of PD-L1 expression was heterogeneous, represented by two distinct cell types: the tumor cell (higher frequency) and IICs (lowest frequency). Positive PD-L1 expression was observed in 37.9% of tumor cells and in IICs of 24.3% of tumors.p value < 0.001, proving that TMA is not an adequate method to evaluate PD-L1 expression.TMA techniques  and conventional histological slides  showed fair agreement when they evaluated the immunoreactivity of PD-L1 expression in tumor cells (Kappa = 0.307) and in antigen presenting cells (Kappa = 0.328) of patients with non-small-cell lung cancer, Former smokers had a higher PD-L1 expression in tumor cells when compared to those who had never smoked. In contrast, former smokers had a lower PD-L1 expression in IICs when compared to those who had never smoked. There was also no correlation between patterns of PD-L1 expression and survival in this population.


The aim of this study was to explore first‐line nurse managers’ perceptions of the challenges involved in decision‐making concerning older patients who wish to die.A descriptive qualitative design in three communities in Norway between January 2016 and June 2016.Qualitative focus group interviews with eight first‐line nurse managers.One main theme and two themes were identified: Struggling to make the right decision, The challenge of social isolation and loneliness and When life becomes too painful and problematic. The sub‐themes revealed that: Arranging social meeting places where the older patients can talk to other older people is crucial. The participants explained that it was not an easy task to gain an overview of the older patients situation. The right decision could be encouraging the patients to talk about their problems by giving them more time, thereby showing that somebody cares about them. In the literature the term FLNM has been used interchangeably with “head nurse”, “ward sister”, “ward manager, “nursing manager” and “ward leader”  to obtain a range of perceptions  described some of the challenges faced by FLNMs in caring for older patients who wish to die. The 12 communities were contacted by e‐mail in December, 2015. Four communities answered in a positive way but wanted more information about the project, which was sent in February, 2016. Unfortunately, three of the communities declined, thus a new request was sent to two of the other communities on the first e‐mail list. Finally, three communities agreed to participate, thus the focus group comprised eight participants: three first‐line nurse managers from community A, two from community B and three from community C.2.3The study was based on qualitative focus group interviews Morgan, . Focus g2.4This study used a thematic analysis , an anaesthesia nurse (AKB) and as researchers. It can be assumed that their pre‐understanding influenced the interpretation of the themes and sub‐themes Gadamer, . Ideally4.2The role of FLNMs has changed a great deal and future research should focus on the different challenges they encounter in decision‐making. The participants emphasized their everyday work as well as responsibility for their staff (Skytt, Ljunggren, Sjödén, & Carlsson, However, FLNMs in Norway seem to find it difficult to decide whether to prioritise their role of clinician, manager or leader and do not appear to know what to do or who to turn to when they experience challenges in decision‐making in the organization Lageson, . They apALH, Study design; ALH, AKB, data collection; ALH, AKB, AB, ES, data analysis, discussion and preparation.


Pathologic vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury promotes the development of occlusive vascular disease. Therefore, an effective chemical agent to suppress aberrant proliferation and migration of VSMCs can be a potential therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. To find an anti-proliferative chemical agent for VSMCs, we screened an in-house small molecule library, and the selected small molecule was further validated for its anti-proliferative effect on VSMCs using multiple approaches, such as cell proliferation assays, wound healing assays, transwell migration assays, and ex vivo aortic ring assay.Among 43 initially screened small molecule inhibitors of kinases that have no known anti-proliferative effect on VSMCs, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606) showed significant anti-proliferative effect on VSMCs. Further experiments indicated that BAY61 attenuated the VSMC proliferation in both concentration- and time-dependent manner, and it also significantly suppressed the migration of VSMCs as assessed by both wound healing assays and transwell assays. Additionally, BAY61 suppressed the sprouting of VSMCs from endothelium-removed aortic rings.The present study identified a Syk kinase inhibitor as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its underlying molecular mechanisms, such as its primary target, and to validate its in vivo efficacy as a therapeutic agent for restenosis and atherosclerosis. Normal vascular smooth muscle cells (VSMCs) maintain a minimal proliferation rate and active contractility . HoweverIn the present study, we screened number of small molecule inhibitors of six major kinase subfamilies, to find an alternative therapeutic agent for suppressing VSMC proliferation and migration based on the results of multiple experiments, such as cell proliferation assays, wound healing assays, and transwell migration assays, that assessed the anti-proliferative and migratory effect on in vitro. The effect of the selected small molecule on the sprouting of VSMCs from endothelium-denuded aortic rings was also examined. The results indicated that the selected small molecule, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606), is an effective agent for suppressing proliferation and migration of VSMCs in vitro and ex vivo and that has therapeutic potential for treating occlusive vascular disease involving aberrant proliferation and migration of VSMCs.For initial screening, through a literature search, 43 small molecules Table , whose eTo examine the concentration-dependent effect of the Syk inhibitor (BAY61), VSMCs were treated with increasing concentrations of BAY61  for 24 h. The cellular proliferation results indicated that BAY61 had a significant anti-proliferative effect at all concentrations tested, although the effect was more profound with concentrations of 5 μM or higher  remained about 50% unclosed Fig. a. FurtheTo further investigate the effect of BAY61 on VSMC proliferation, an ex vivo aortic ring assay was performed. The endothelium-denuded thoracic aortas of rats were cut to prepare aortic rings with thickness of 1 mm. Matrigel-embedded aortic rings were cultured in 10% FBS-supplemented DMEM with either vehicle  or BAY61 (10 μM) for 7 days. VSMC sprouting from the endothelium-denuded aortic rings was inhibited by BAY61 even in the presence of 10% FBS in the culture medium Fig. .Fig. 4BAIn the present study, through a screening of small molecule inhibitors of kinase subfamilies, we identified a Syk inhibitor as a potent anti-proliferative and migratory small molecule for VSMCs. Protein kinases are known to play critical roles in signaling pathways implicated in development, differentiation, death and proliferation of cells . TherefoSyk, along with zeta-chain associated kinase of 70 kDa (Zap-70), is a member of the Syk family of kinases that are activated downstream of Src kinases . Syk is Our data also clearly demonstrated that the Syk inhibitor BAY61 significantly suppressed VSMC proliferation and migration. Previous study had reported that suppression of Syk activation led to subsequent inhibition of extracellular signal-regulated kinas (ERK) 1/2 signaling and p38 mitogen-activated protein kinase signaling . TherefoOur ex vivo aortic ring assay result showed that BAY61 prevented sprouting of VSMCs, and furthermore, suppression of Syk has been suggested to be an effective way to control atherosclerosis via subsequent suppression of endothelin-1 production from endothelial cells . TherefoIn summary, as an effort to identify anti-proliferative agent to control occlusive vascular disease, this paper describes the anti-proliferative and migratory effects of BAY61, a Syk inhibitor, on VSMCs for the first time. The selected Syk inhibitor significantly suppressed VSMC proliferation and migration in vitro and ex vivo. These data suggest that the BAY61 may be a potent therapeutic agent for the treatment of occlusive vascular disease and warrants further studies to evaluate its in vivo efficacy and detailed underlying mechanism.2 flasks at 37 °C in a humidified atmosphere of 95% air and 5% CO2 .All experimental procedures for animal studies were approved by the Committee for the Care and Use of Laboratory Animals, Yonsei University College of Medicine, and were performed in accordance with the Committee’s guidelines and regulations for animal care. Rat aortic VSMCs were isolated and cultured using methods previously published . The thohttp://www.ncbi.nlm.nih.gov/pubmed). For primary screening, VSMCs were seeded in 48-well culture plates using DMEM with 10% FBS and then treated with 10 μM of each candidate compound for 24 h. The anti-proliferative effects of the candidate compounds were determined by a cell proliferation assay.From an in-house small molecule library mainly composed of inhibitors of six major kinase subfamilies, 43 candidate small molecules with no known anti-proliferative effects on VSMCs were selected based on a published literature search using Pubmed  was conducted following protocol previously published , and Abcam (β-actin:ab8227).Aortic ring assay to monitor the outgrowth of VSMCs was conducted following a published protocol with modifications . Brieflyp value of less than 0.05 was considered statistically significant.Quantitative data were expressed as the mean ± SEM (standard error of measurement). For statistical analysis, one-way ANOVA with Bonferroni correction was performed using OriginPro 8 SR4 software  if there were more than three groups. A


This article contains data related to the research article entitled “Atomistic simulation on nanomechanical response of indented graphene/nickel system” . There are five sets of data obtained by molecular dynamics simulations for nanoindentation of five different graphene/nickel systems, which are single nickel system, monolayer graphene on nickel system, double-layer graphene on nickel system, three-layer graphene on nickel system and four-layer graphene on nickel system. The calculated load-displacement of the five different indented systems is also listed. Specifications tableValue of the data•Data presented here provide the nanoindentation mechanical properties of nickel substrate with or without graphene coverage on.•We show the nanoindentation mechanical properties of different number of graphene layers suspended on nickel system.•The enhancement effect of graphene on the contact stiffness and load bearing capacity of graphene/nickel system can be calculated from the data presented here.1The data presented in this article show the displacement and related load of the five different indented graphene/nickel systems , and the22.1In our simulations, models with a graphene sheet suspended on a face-centered cubic (fcc) Ni (111) surface and diamond spherical indenters were established, as shown in 2.2The accuracy of a potential function determines the reliability of Molecular dynamics (MD) simulation. Up to now, there are many types of force fields available, which have been parameterized to describe a variety of systems. The embedded-atoms method (EAM) potential developed by Mishin et al. 2.3An energy minimization process was carried out to avoid overlaps in the positions of the atoms after models completed. The periodic boundary conditions were employed in the transverse directions (XY directions). A Nose/Hoover thermostat was applied to maintain the temperature of 300 k. The equations of motion were integrated using the Verlet algorithm


The complex refractive index of few-layer graphene, n − ik, was extracted from the reflectance contrast to the bare substrate. It was found that each few-layer graphene possesses a unique dispersionless optical index. This feature indicates that the optical conductivity does not simply scale with the number of layers, and that inter-layer electrodynamics are significant at visible energies.We have experimentally studied the dispersion of optical conductivity in few-layer graphene through reflection spectroscopy at visible wavelengths. A laser scanning microscope (LSM) with a supercontinuum laser source measured the frequency dependence of the reflectance of exfoliated graphene flakes, including monolayer, bilayer and trilayer graphene, loaded on a Si/SiO Optical response of graphene has recently emerged as an important subject of research. This interest is partly fueled by the promising prospect of utilizing graphene as a photonic material for device applications12345691015 cm−2).The observation of highly confined and low-loss plasmons at mid-infrared wavelengths121415low-energy approximation breaks down, and the linear band structure and a universal optical conductivity are violated. Presuming that transition from a linear energy dispersion (at IR) to a non-linear one (at UV) occurs smoothly, it is plausible that tangible deviations from the former should begin to appear in the visible regime.On the other hand, the observation of saddle-point excitonsIn this regime, the optical response of thin films may be probed by several methods including near-field microscopy, ellipsometry, and transmission/reflection measurements. The use of near-field probes for interrogating visible graphene plasmons is limited due to the interband transitions and high dissipation under conventional doping levels, as highlighted earlier. Moreover, ellipsometry techniques normally suffer from a low spatial resolution, hindering measurement of small graphene flakes. Therefore, transmission/reflection experiments have constituted the most widely used means to characterize few-layer graphene in the visible spectrum.H and E are the magnetic and electric fields, respectively, ε0 = (n–ik)2 is the permittivity of free space, εc is the complex relative permittivity, σ is the optical conductivity, and ω is the angular frequency of the incident monochromatic optical field. Clearly, if the refractive index, and, consequently, dielectric function εc are independent of frequency, then σ must necessarily be a linear function of ω. Although the precise complex value of the deduced refractive index varies among different experimental determinations, they are closely clustered within a small range: 2.1 < n < 2.7 and 1.0 < k < 1.7.Early optical reflection measurements18192021222324Nonetheless, the best-known transmission measurement was performed on suspended few-layer graphene1728This paper reexamines the optical response of few-layer graphene over the visible range by means of a refined reflectance spectroscopy technique. In particular, we compare the two hypotheses of universal optical conductivity and dispersionless optical index. Note that the latter implies a dispersive optical conductivity varying linearly with frequency.Since the thickness of graphene is several orders of magnitude smaller than an optical wavelength, transmission through graphene essentially samples only the real part of the optical conductivity. The imaginary part, however, manifests in the reflection. The negligible amplitude of the reflection from suspended graphene, thus, renders transmission measurements insensitive to the imaginary part. The same disadvantage persists for reflection measurements where graphene films are directly laid on a substrate. Therein, the reflected power predominantly originates from the reflection at the substrate interface. In order for both the real and imaginary parts of the optical conductivity/index to affect the measured signal, we have coupled the graphene flakes to a Fabry-Pérot resonator. The Fabry-Pérot cavity may be simply realized by deposition of a thin dielectric film on the substrate, whose thickness determines the resonant frequency. Given that the overall reflectance from a Fabry-Pérot structure results from multiple reflections, the effect of the interaction with graphene is magnified. While the phase retardation induced by graphene may be negligible for a single reflection, its accumulation for many reflections, however, yields a measurable effect. This fact has been previously exploited to identify different few-layer graphene flakes2 over-layer. This particular SiO2 thickness provides a third-order Fabry-Pérot absorption resonance corresponding to an antinode at the graphene layer around a (free-space) wavelength of 550 nm. The sample was then annealed in H2 and Ar at a temperature of 500 °C for 3 hours to remove adsorbed contaminants. A graphene flake was exfoliated onto a commercially available Si substrate with a nominally 300 nm-thick SiORegions of mono-layer, bi-layer, and tri-layer graphene were identified by means of Raman spectroscopyμm diameter over the sample using a set of two galvano-mirrors. An X20 Mitutoyo infinity-corrected long working-distance objective lens, with a NA of 0.42, was used to focus the incident light onto and gather the reflected light from the sample. Given the flake dimensions (less than 60 μm on a side) and the working distnace of 2 mm, the deviation of the incidence angle from normal is less than 0.9°. The reflected beam was directed to a Si photodiode which measured the reflected power at each pixel.Reflectance measurements were performed by a laser scanning microscope (LSM)A Fianium SC400 supercontinuum laser was used as the LSM light source whose wavelength was varied from 545 nm to 700 nm by means of an acousto-optic tunable filter with a bandwidth of <2 nm.2 near the graphene flake was determined, after the reflectance measurements, through selectively removing the oxide by hydrofluoric acid etching followed by AFM, which gave a value of 308 nm ± 0.5 nm for the oxide thickness adjacent to the exfoliated graphene flake.The thickness of the oxide layer enters to the data analysis (below) as an input parameter and its accurate determination is essential to the accuracy of the ultimate results. Therefore, despite the fact that ellipsometry and scanning electron microscopy (SEM) were performed on the substrate, the actual thickness of the SiOAs RG, normalized by that of the bare substrate, R0. The measured reflectance for each graphene few-layer followed a Fabry-Pérot lineshape centered around ~600 nm. The contrast to the substrate monotonically increased for thicker graphene few-layers. This is qualitatively anticipated since thicker flakes absorb more and appear darker. Interestingly, the reflectance spectra underwent a slight red shift for thicker flakes. The resonance shift evidently demonstrates the loading effect of graphene on the Fabry-Pérot cavity. This shift is a manifestation of the significance of the imaginary  part of the optical conductivity (index).2 were obtained from ref. In the literature, reflection/transmission analyses on graphene usually involved applying thin-film approximations, which neglect the phase retardation associated with transmission through and reflection from graphene. Here we used a complete Fresnel analysis on the graphene-loaded Fabry-Pérot multilayer cavity. The substrate was modeled as a semi-infinite medium. A 3.3 Å thickness was assumed for each layer of graphene, corresponding to its van der Waals interlayer distance. The refractive indices of Si and SiOThe dielectric functions of graphene were modeled based on: first a universal optical conductivity and, second, a complex constant over the wavelength range of interest. These two forms were incorporated into the Fresnel theory in order to explain the measured data, as shown in The deduced optical refractive indices were, further, used in order to calculate the opacity of suspended few-layer graphene based on the Fresnel theory. The fact that each graphene few-layer possesses a different optical index is also interesting. It has been previously argued that the equal steps in the opacity of few-layer graphene indicate that the electrodynamics of graphene is predominantly determined by its in-plane response. However, the result that each few-layer graphene exhibits a unique optical index suggests that inter-layer electrodynamics significantly affect the optical response of graphene in the visible regime. Moreover, the difference in the imaginary parts imply that optical conductivity does not simply scale with the number of layers.2 substrate by applying the Fresnel theory of reflection/refraction to the air/graphene/SiO2/Si multilayer. We found that the reflectance spectra can be well explained by a dispersionless effective optical refractive index rather than a constant optical conductivity. Furthermore, it was found that each few-layer possesses a unique optical index, as tabulated in We performed reflectance spectroscopy on exfoliated monolayer, bilayer, and trilayer graphene coupled to a Fabry-Pérot resonator by means of a laser scanning microscope and a supercontinuum laser over the wavelengths of 545 nm to 700 nm, corresponding to photons of energy 1.77 eV to 2.28 eV. The optical index of the few-layer graphene flakes were determined based on their contrast to the bare Si-SiOHow to cite this article: Ghamsari, B. G. et al. Measuring the Complex Optical Conductivity of Graphene by Fabry-Pérot Reflectance Spectroscopy. Sci. Rep.6, 34166; doi: 10.1038/srep34166 (2016).