Nanofabrication research pursues the miniaturization of patterned feature size. In the current state of the art, micron scale areas can be patterned with features down to ~30 nm pitch using electron beam lithography. Here, we demonstrate a nanofabrication technique which allows patterning periodic structures with a pitch down to 16 nm. It is based on focused ion beam milling of suspended membranes, with minimal proximity effects typical to standard electron beam lithography. The membranes are then transferred and used as hard etching masks. We benchmark our technique by electrostatically inducing a superlattice potential in graphene and observe bandstructure modification in electronic transport. Our technique opens the path towards the realization of very short period superlattices in 2D materials, but with the ability to control lattice symmetries and strength. This can pave the way for a versatile solid-state quantum simulator platform and the study of correlated electron phases. Focused-ion beam (FIB) lithography enables high-resolution nanopatterning of 2D materials, but usually introduces significant damage. Here, the authors report a FIB-based fabrication technique to obtain high quality graphene superlattices with 18-nm pitch, which exhibit electronic transport properties similar to those of natural moiré systems. Accordingly, the push to further improve nanofabrication techniques is an ongoing effort to fit more electronic components per unit area or to develop quantum devices and quantum bits, relying on quantum coherent control in the nanoscale regime3. Another important application is in quantum materials, where materials’ properties can be tuned in situ. Arrays of quantum dots with sufficiently large Coulomb interactions could lead to the observation of metal-Mott insulator quantum phase transitions and potentially even the d-wave superconducting phase4. Recent discoveries of correlated phases in twisted bilayer graphene due to the superlattice (SL) potential6 motivate further exploration of graphene SLs with more versatility in the lattice design and in situ control.Nanoscale fabrication is at the heart of the technological revolution of semiconductor technology and scientific breakthroughs in nanotechnology, progressing rapidly for more than five decades8, but is impractically slow for micron-scale lithography. Other techniques, such using self-assembled polymers, can achieve high resolution and yield9, at the expense of limiting design flexibility in what patterns can be implemented. A higher throughput alternative is ultraviolet optical lithography, the main lithography technique in the semiconductor industry, which is faster but is not suited for academic research and development.Nanofabrication techniques always involve a trade-off between patterning resolution, throughput and flexibility. For example, scanning tunneling microscopy, can be utilized to rearrange materials on the atomic scale12.In academic research, the most common nanopatterning technique is based on electron beam lithography (EBL), which can be thought of as a compromise offering reasonable throughput and nanoscale resolution. Forward scattered electrons limit the minimal patternable feature size, while secondary electrons due to (inelastic scattering) interactions with the resist limit the pitch resolution. As such, using high acceleration voltages and thin organic resists, patterns with 35 nm pitch can be obtained, but significant reduction of the feature size has not been demonstrated18. However, two major limiting factors affect He-FIB-based nanofabrication: (1) Milling of materials directly on a substrate is accompanied by deposition of He bubbles inside the substrate, contaminating it and affecting surface topography19. (2) Secondary collisions damage the quality and integrity of the patterned material20. It is possible to reduce the damage by milling suspended materials (where substrate scattering is absent) or using resist lithography21. However, suspending the material is complex and the amount of FIB induced unintentional damage is still expected to modify the material properties in the form of ion implantation and/or amorphization24. Here, we propose and demonstrate a nanofabrication technique that combines the high resolution of He-FIB milling with minimal damage to the patterned material.Another common nanofabrication approach uses focused-ion beam (FIB) milling to pattern the target material directly. In particular, helium (He) FIB routinely achieves single digit single feature resolution and pitch on the order of 10 nm25 or graphene/hexagonal boron nitride (hBN) heterostructures27), it is possible to engineer SLs by applying a periodic electrostatic potential28. When applied to low-dimensional materials, such as graphene, this modifies its electronic band structure when the SL period is much smaller than the electronic coherence length29. Several approaches have been explored to introduce such potentials: from patterning the graphene directly12, to inducing a periodic potential modulation by patterning the dielectric material31 or the gate electrode34. In all cases, it is essential to reduce damage to the patterned material, keeping it flat and free of external contamination. However, to realize the full potential of artificial SLs, a new lithographic technique is required, one which can create high quality periodic patterning on the sub-20-nm scale, approaching the moiré length in graphene aligned on hexagonal boron nitride or magic angle twisted bilayer grapheme . The resolution of our method is demonstrated directly by atomic force microscopy (AFM), whereas the quality is demonstrated by applying our technique to engineer SL potentials in graphene via patterned graphite electrodes. Graphene could be very sensitive to the presence of charge traps, impurities and patterning errors in the patterned gate electrode. However, transport data from our samples indicates minor unintentional damage to the patterned electrodes, even for highly dense patterns with an 18 nm pitch. Similar to moiré systems , which is commercially available on a large scale and high quality. Importantly, the suspended membrane can be removed from its supporting frame and transferred with a polymeric stamp Fig. . The memask Fig.  with a s nm Fig. .Fig. 2FaTo quantify the disorder of our lattices, we perform Fourier analysis of the AFM images Fig. , finding32. By tuning the silicon backgate voltage (Si BG) and the patterned gate voltage (PBG), one can modulate the strength of the SL and the carrier type in the graphene layer to observe the cloned Dirac cones, see Fig. Figure The second device studied in this work, Dev 2, has an 18 nm period square SL. To the best of our knowledge, there is no experimental work about electrostatically induced SLs with such a short periodicity. In this case, there is a top gate electrode (TG) apart from the PBG. This combination of gates allows us to control the SL strength and the total carrier density of the system independently. Therefore, we keep PBG fixed at certain voltages and sweep TG to change the carrier density. In longitudinal resistivity, we observe clear signatures of the emergence of satellite peaks at sed Fig. . Furthersed Fig. , i.e., nThe prominence of the SL signature is different in the two devices. To understand this, we must examine the specific characteristics of both devices. As a rough approximation for the electrostatic potential modulation along the graphene layer for the formation of the SL, we consider a periodic one-dimensional system with a patterned bottom gate, global top gate . Prior to milling in FIB, the membranes are annealed in a HThe membranes are mounted on a hole in the sample holder, to improve imaging contrast and loaded into the He focused-ion beam microscope (HIM). The HIM is then aligned to high precision on metallic features in the sample, with typical parameters being 5 After initial alignment, the membrane area is located in the HIM . The sample is usually allowed to rest for 30–90 min , to minimize the effects of piezo drift in the sample stage. Following this resting period and additional finer alignment, the membrane is milled using a preprogrammed point-by-point deflection list. For large features, a milling dose comparable to 0.9 In addition to the majority of the samples produced during our research, using He-FIB, we were also able to produce similar results (but with larger milling periods) using a Gallium focused-ion beam microscope (Ga FIB). These samples were processed similarly to the above described samples milled by HIM, with the exception being that the membrane was mounted with the window facing down (situated above a hole in the sample holder), in order to facilitate locating the intended milling area with the FIB. The resolution of the specific Ga FIB microscope used in our experiments was restricted by various technical issues, limiting the size of the spot effected by the Ga ions to about 20 nm and the lattice period to the order of 50 nm. With a more narrowely focused FIB, we estimate, based on our experience, that a ~10 nm single feature and ~30 nm lattice period should be achievable.2 substrate at a temperature of 158 °C to clean its interfaces, and subsequently temperature was increased to 180 °C to melt the poly (bisphenol A carbonate) (PC) film42. After dissolving the PC film in chloroform, the heterostructure quality is checked through AFM imaging and Raman spectroscopy. Finally, the heterostructure is again picked up and released following the same procedure on the patterned graphite gate. It is important to note that the hBN flakes between the graphene layer and the patterned gate electrode must be thin to allow for efficient doping modulation.The fabrication process of the patterned graphite gate is explained in the main text. After the cleaning process, we prepared an hBN/graphene/hBN (hBN/graphene/hBN/graphite/hBN in the case of Dev 2) heterostructure. We first dropped it on a clean Si/SiO43 to our heterostructure. In the case of the contacts, since they are right on top of the patterned graphite flake, we perform first an SF6 etching process44 to remove only the top hBN, followed by an O2/Ar etching to remove the graphene, but not the thin bottom hBN that will prevent leakage current between the bottom graphite gate and the electrodes. We deposit Cr(3 nm)/Au(40 nm) followed by a lift-off in acetone.Standard EBL is used to pattern the Hall bar geometry and make one-dimensional edge contactsElectrical measurements were performed in a He flow cryostat from ICE Oxford operating at To remove substrate height variations, we apply a high pass filter with a cut off frequency Supplementary informationPeer Review File


In this letter, we propose a structure based on double-layer graphene-based planar chiral metasurface with a J-shaped pattern to generate asymmetric transmission for circularly polarized waves in the mid-infrared region. Asymmetric transmission of the double-layer structure can reach to 16.64%, which is much larger than that of the monolayer. The mechanism of asymmetric transmission is attributed to enantiomerically sensitive graphene's surface plasmons. Besides, asymmetric transmission can be dynamically tuned by changing the Fermi energy and is affected by intrinsic relaxation time. All simulations are conducted by the finite element method. Our findings provide a feasibility of realizing photonic devices in tunable polarization-dependent operation, such as asymmetric wave splitters and circulators. In this paper, we propose a structure based on double-layer graphene-based planar chiral metasurface with J-shaped pattern to generate asymmetric transmission, which can reach to 16.64%. Asymmetric transmission of chiral metasurfaces for circularly polarized waves can be defined as the total transmission difference between the same handedness waves propagating in the opposite directions or the opposite handedness waves propagating in the same direction.12 With the development of research on chirality with asymmetric transmission, electromagnetic devices applied for both linearly and circularly polarized waves have been designed, such as some polarization transformers, polarization analysers and polarization-controlled devices.13–16 They are widely used to promote the evolution of optical communication and photonics. The excitation of enantiomerically sensitive surface plasmons is a collective mode of electron oscillation at the interface of conductor and medium, which is in charge of asymmetric transmission for circularly polarized waves in planar chiral metasurfaces.17–19In recent years, chiral metasurfaces have become more and more popular in many fields such as optics. Chirality means that an object cannot be coincident with its mirror image. Existing studies of chiral metamaterials mainly focus on circular20–22 And for graphene metasurface, novel optical properties are dynamically tunable by changing the Fermi energy through voltage control or the changing intrinsic relaxation time through chemical doping.23–26 Compared with metallic surface plasmons, graphene surface plasmons have the advantages of low loss, and high localization.27 Thus, graphene will revolutionize metamaterials and metadevices, and promote the development of nano-optics.28 There are plenty of researches on metallic chiral metamaterials,29 but they are generally not tunable, which can be easily realized in graphene metasurface. Recently, people propose kinds of monolayer graphene chiral metasurface,30,31 and study the asymmetric transmission for circularly polarized waves. The researches of graphene planar chiral structure will deepen the understanding of the interaction between light and matter in planar chiral structure, which can be applied to tunable polarization sensitive devices, and circular polarizers. But the asymmetric transmission of monolayer graphene chiral metasurface is only up to 5%.As a kind of promising electrically tunable plasmonic material, periodical graphene metasurfaces can excite graphene surface plasmons in the mid-infrared and terahertz (THZ) region.T can reach a small value of 2.05%. When another J-shaped pattern, which is designed to be rotated by 90 degrees after being mirror symmetric operation with respect to the previous layer, is added, the asymmetric transmission ΔT can achieve 16.64%. The mechanism of asymmetric transmission can be explained with the enantiomerically sensitive graphene surface plasmons, which induce the charge-field excitations. Also, the peaks of asymmetric transmission blue shift with increasing Fermi level, and the values of peaks increase with increasing electron scattering time. Finite Element Method (FEM) simulations are conducted to verify our findings.In this letter, we propose a double-layer graphene-based planar chiral metasurface structure to achieve giant enhancement of asymmetric transmission for circularly polarized waves incidence in mid-infrared region. In the monolayer metasurface which is composed of J-shaped hollow graphene patterns, the asymmetric transmission Δp of the structure is set to be 270 nm, and the other parameters of the pattern are displayed in the ax = 230 nm and m = 125 nm, respectively. The length of J-shaped pattern is ay = 210 nm, and the width of hollow area is w = 20 nm. Periodic boundary conditions are set along x and y directions for our periodical structure and perfectly matching layers are set in z direction to achieve the minimal reflection for the outgoing electromagnetic wave.Firstly, we discuss the monolayer graphene metasurface, and one unit is shown in 32σintra represents intraband conductivity, it can be calculated as:σinter represents interband conductivity, it is characterized by:  Here,  can be expressed as:According to the Kubo formula, the conductivity of graphene consists of two parts: intraband and interband elements: can be expressed as:e is the electron charge, kb is Boltzmann constant, T (= 300 K) is temperature, ℏ is Planck constant, ω is the angular frequency, and Ef is Fermi energy. Here, the Fermi energy of the graphene is fixed to be 0.80 eV, and it can be changed by altering the gate voltage applied on the graphene planar metasurface. The relaxation time τ is defined as μEf/evF2. Here, the Fermi velocity and DC mobility are vF ≈ 106 m s−1 and μ = 10 000 cm2 V–1 S−1, respectively,22,33 thus τ is 0.8 ps.Ei = tijE0j, the indices i, j represents right-handed circularly polarized (RCP) or left-handed circularly polarized (LCP) components. The transmission matrix elements are defined as Tij = |tij|2. The circular transmission T can be applied to any dispersive optical system under the condition of coherent light incidence. The transmission matrix elements are the same for circularly polarized waves of opposite handedness propagating in opposite directions. It means that the LCP(RCP) waves incident forward equal to the RCP(LCP) waves incident backward. Thus, we can define the transmittances of the LCP waves with opposite propagation directions asTf and Tb represent the transmittances in the forward and backward incident directions for LCP. TLL and TRR are the direct transmission, and TRL and TLR are the circular polarization conversion transmission. For our chiral structure with non-C4 symmetry, the direct transmissions of planar chiral metasurface are insensitive to polarization or propagation directions (TLL = TRR). The asymmetric transmission ΔT is defined as the difference between Tf and Tb.LCPcirc is the asymmetric transmission for LCP incidence, and ΔRCPcirc is the asymmetric transmission for RCP incidence.The pattern of the J-shaped graphene metasurface has a character of chirality, which can reveal asymmetric transmission when the incident circularly polarized waves have different handedness or propagation directions. Thus, the asymmetric transmission is only decided by circular polarization conversion transmission.Ef is fixed to be 0.80 eV, for the monolayer J-shaped graphene metasurface. Black and red lines represent LCP incidence along the forward and backward directions, respectively. It is found that the direct transmissions (TLL and TRR) always keeps the same with each other with the wavelength changing, and reaches minimum when the incident wavelength is 14.83 μm, shown as TRL is always a little larger than TLR with the wavelength ranging from 13 to 17 μm, and the maxima of TRL and TLR are 15.07% and 13.02% at 14.83 μm, respectively. Thus, the asymmetric transmission for LCP waves reaches the maximum value of 2.05% at the wavelength of 14.83 μm, as shown in T is small, compared with that of metallic planar chiral metamaterial structure.34,35 To realize larger asymmetric transmission, a double-layer structure is proposed based on the monolayer graphene-based J-shaped metasurface structure. y-axis. The medium between the two graphene layers is quartz, and the thickness of the quartz is 125 nm. The LCP waves are still coming perpendicularly from the front and the back of graphene-based planar chiral metasurfaces, respectively.Although the asymmetric transmission has been obtained, the ΔEf is fixed to be 0.80 eV for the double-layer structure shown as TLL and TRR are still identical, which proves that asymmetric transmission is independent of direct transmission. In TRL is slightly higher than that of the monolayer chiral metasurface shown in TLR goes down sharply to almost zero at the wavelength of 14.90 μm. Compared with the monolayer J-shaped metasurface, the resonant wavelength of graphene surface plasmon redshifts a little. Due to the existence of the near to zero value of TLR, the asymmetric transmission ΔT can be increased a lot. As shown in the T can reach 16.64% at the wavelength of 14.90 μm, which is much larger than 2.05% of the monolayer graphene-based planar chiral metasurface. Similarly, the asymmetric transmission peak of RCP incident waves reaches −16.64%, just the opposite of LCP. The giant enhancement of circular asymmetric transmission suggests that the structure of double-layer J-shaped graphene metasurface can greatly improve chirality.36 The excitation level is decided by the direction and polarization state of the incident waves. Here, graphene localized surface plasmon is excited along the J-shaped edge because the electric field is enhanced around the J-shaped edge shown in Ey of the induced electric field for LCP(RCP) incidences can reflect the excitation level of localized plasmon, and explain the difference between TLR and TRL. Here, the yellow and green arrows was painted to demonstrate the components Ey along y-directions as shown in TLR is smaller than TRL, and an asymmetric transmission (ΔT = TRL − TLR) of 2.05% is achieved for the monolayer metasurface structure. Ey for the LCP incidence indicated by yellow arrows in TRL of double-layer increases to 17.13%, compared with 15.07% of the monolayer. As shown in the 17,36 Thus, TLR is near to zero, and the asymmetric transmission of the double-layer metasurface can be greatly increased and ΔT can reach 16.64%.In order to understand the physical mechanisms behind the asymmetric transmission, the induced electric field in the hollow area of the bottom layer of graphene metasurface is studied in Kspp = ħω2/2α0Efc ∝ 1/Lg37 where α0 = e2/ħc is the fine structure constant. The resonant wavelength λ can be written as:Lg represents the resonant characteristic length of the graphene hollow pattern. Consequently, it is clearly demonstrated that increasing Fermi energy Ef can cause blue shift of these resonant wavelengths. In addition, when Fermi energy increases, the peak amplitude of asymmetric transmission increases from 15.82% to 19.19%. τ and asymmetric transmission, when the Ef is 0.80 eV. Here, μ is usually in the range of 3000 cm2 V–1 S−1 to 10 000 cm2 V–1 S−1, so intrinsic relaxation time τ can vary from 0.24 to 0.80 ps. With the increase of intrinsic relaxation time τ, the peak value of the asymmetric transmission increases from 5.95% to 16.64%, and the peak position remains unchanged, always at the wavelength of 14.90 μm. This increase of the asymmetric transmission peak value with increasing electron scattering time τ is caused by the reduced free-carrier loss in graphene, which can lead to a increase in the circular polarization conversion transmission TRL but no change of TLR. Therefore, the frequency peak position of asymmetric transmission can be tuned because the Fermi energy can be changed by tuning the gate voltage, compared with the metallic chiral metamaterials. For tuning the Fermi energy of graphene in the potential experiments, we can cover the structure with a layer of ion gel following the procedure described in 39To study the dynamical tunability of asymmetric transmission, we changed the Fermi energy and intrinsic relaxation time of graphene layers. When increasing the Fermi energy from 0.76 to 1.00 eV, the peak of asymmetric transmission has a blue shift changing from 15.30 to 13.30 μm, as shown in Ef is fixed to be 0.80 eV. y-axis, respectively. TLR and TRL varies with wavelength. For the structures of λ locates at 15.1 and 14.8 μm, shown as Other double-layer structures of J-shaped pattern have been also studied, and circular polarization conversion are calculated, when In summary, we demonstrated and studied the double-layer graphene-based planar chiral metasurfaces structure. Through FEM numerical simulation, we chose the most suitable orientation of double-layer graphene pattern, and the asymmetric transmission can reach to 16.64%, which is much larger than that of the monolayer. In addition, the induced electric field below the graphene chiral metasurfaces are analyzed for understanding the physical mechanism of asymmetric transmission. We attribute the asymmetric transmission for circularly polarized waves to the enantiomerically sensitive plasmonic excitations. Besides, the peak of asymmetric transmission could have frequency shift and amplitude changes when the Fermi energy and intrinsic relaxation times of graphene are changed. We believe our findings can be applied to tunable polarization sensitive devices, circular polarizers and other fields.There are no conflicts to declare.


Graphene resonant sensors have shown strong competitiveness with respect to sensitivity and size. To advance the applications of graphene resonant sensors, the damage behaviors of graphene harmonic oscillators after thermal annealing and laser irradiation were investigated by morphology analysis and frequency domain vibration characteristics. The interface stress was proven to be the key factor that directly affected the yield of resonators. The resulting phenomenon could be improved by appropriately controlling the annealing temperature and size of resonators, thereby achieving membrane intactness of up to 96.4%. However, micro-cracks were found on the graphene sheets when continuous wave (CW) laser power was more than 4 mW. Moreover, the fluctuating light energy would also cause mechanical fatigue in addition to the photothermal effect, and the threshold damage power for the sinusoidally modulated laser was merely 2 mW. In this way, based on the amplitude-frequency surface morphology of the graphene resonator, the thermal time constant of the order of a few microseconds was confirmed to evaluate the damage of the graphene oscillator in situ and in real time, which could be further extended for those resonators using other 2D materials. Micro resonant sensors have been widely applied in aviation, aerospace engineering and automation due to their high sensitivity, stable performance and direct frequency signal output. The resonator is the key element of resonant sensors, which dominantly affects the performance of the whole system. To develop a high-performance resonant sensor, the material of the micro resonators should be stiff, robust and stable.9 cycles [Graphene, an atom-thick two-dimensional material with a single-layer thickness of 0.335 nm, has demonstrated excellent mechanical , optical9 cycles , which m9 cycles , actuate9 cycles . This pe9 cycles ,12,13. TThe damage of graphene resonators can be attributed to the manufacturing process and operating conditions. For the fabrication of resonators, suspended transfer of graphene is a common step that might cause damage to the thin suspended membrane. Generally, graphene transfer in micro-mechanical systems is performed with the help of the widely used polymethyl methacrylate (PMMA) , PolydimThe Raman spectroscopy technique has been used to evaluate the extent of damage by calculating the intensity of the D and G peaks . HoweverIn this section, the damage to the graphene harmonic oscillator after thermal annealing is presented. Graphene membranes annealed at a temperature of 300, 375 or 450 °C were first observed under an optical microscope and scanning electron microscope (SEM)  to analyse the breakage rate of the membrane, on micro- and nano-scales, respectively. Then, the resonant characteristics, including quality factor and resonant frequency, were investigated by the aforementioned all-fibre experimental system.After the annealing process at each temperature, the intactness of the graphene was observed by an optical microscope. It has been found that graphene with a more than 95% free-standing area has the potential to be fabricated as a well-behaved harmonic oscillator; thus, this kind of membrane was noted to be intact in this paper. To be specific, 300 graphene membranes after each annealing process were randomly selected, and the number of intact membranes was counted. The statistical results are listed in Besides the stress between the graphene and PMMA, there was also a thermal interfacial interaction between the MLG and substrate in the heating process, which would also cause damage to the graphene, especially at the edge of the entire membrane. A photo of a typical damaged graphene caused by thermal interaction between the MLG and substrate is shown in Furthermore, in order to graphically depict the damage to the graphene membrane on a nano scale, the SEM photographs of the suspended graphene are given in dark/numall, wherein numdark and numall represent the number of dark pixels and all pixels, respectively. It could be concluded that a higher annealing temperature T or a larger suspended radius R would result in a larger broken area. Taking the graphene , for example, the graphene exhibited perfect intactness (100%). However, once the suspended diameter was increased to 100 μm, the breakage rate increased to 2.5%. When the annealing temperature was increased to 450 °C, the breakage rate rose rapidly to 30.9%. In the aforementioned experimental set up, the graphene breakage rate showed higher sensitivity to the annealing temperature than the suspended diameter.In this way, these SEM images were further binarized to calculate the proportion of the damaged area. Thus, the microscopic broken rate of the membrane could be calculated as numω0/Δω, where ω0 is the natural frequency and Δω is the 3 dB bandwidth of the amplitude–frequency curve. Thus, the resonant characteristics of the graphene resonators were investigated at the aforementioned three annealing temperatures, as illustrated in Besides the breakage rate and the surface morphology of the membrane, which has been mentioned above, the resonant characteristics were also investigated, including the resonant frequency and quality factor. Among these, the resonant frequency indicates the fundamental frequency of the oscillator, and the quality factor represents the energy loss per oscillation cycle, which is calculated by f20 = 1254.1 kHz (D = 20 μm), f60 = 338.9 kHz (D = 60 μm) and f100 = 153.5 kHz (D = 100 μm), and the standard deviation (STD) of frequencies were confirmed as σ20 = 192.7 kHz (D = 20 μm), σ60 = 59.0 kHz (D = 60 μm) and σ100 = 46.2 kHz (D = 100 μm), respectively , σ60 = 2.50 (D = 60 μm) and σ100 = 3.34 (D = 100 μm), respectively  and f60 = 661.3 kHz (D = 60 μm), and the STD values were calculated to be 641.8 kHz and 348.4 kHz, accordingly. The Q factors were measured to be 8.93 (D = 20 μm) and 6.85 (D = 60 μm) with the corresponding STD values of 2.03 and 2.72, respectively , the ratio of the inner strain of 20 μm, 60 μm and 100 μm membranes were estimated to be S1:S2:S3 = 2.69:1.78:1. This strain possibly resulted from the Van der Waals force between the graphene and the copper sidewall. At different temperatures, different mechanical energy might be introduced via distortions of the graphene lattice [For a circular membrane under tension, the fundamental frequency can be expressed as (1)f=2.4 lattice . For theNote that when the annealing temperature increased to 450 °C, the fabricated resonators of all sizes were damaged with the damaged areas as shown in Damage not only occurs in the fabrication process, but also in the working process of the harmonic oscillator. In the previous study on the laser-induced damage, the graphene was often tested by a Raman spectrometer  or microAs the modulated laser was verified with a higher possibility of damaging the membrane, the effect of the modulated laser on the graphene resonant characteristics was further explored. Thus, the graphene membrane was excited with a modulated laser whose power was gradually increased from 1 mW to 5 mW. Meanwhile, the motion was recorded by a CW laser with an extremely small amount of power, so that this laser would barely damage the structure of the graphene. The experiment results were recorded in From the frequency domain, z in the frequency domain could be written as [τ is the thermal time constant, R is the thermal resistance, C is the thermal capacitance (RC = τ), α is an effective thermal-expansion coefficient and P is the heating power. After taking the derivative of Formula (2), the imaginary part of the response function reached the maximum amplitude when ωτ = 1. Thus, the thermal time constant τ of the graphene membrane was calculated. According to Metzger et al. , a thermitten as : (2)z(ω)τ started to deviate at about 3 mW  of the resonator showed no synchronous improvement compared to the counterparts annealed at 300 °C (Q20 = 9.8).From the perspective of the application of graphene resonant sensors, the effect of the temperature-dependent annealing treatment on the intactness of suspended multilayer graphene was investigated through surface morphology observation to further evaluate the resonant behaviours of graphene resonators after the thermal annealing process. The experimental results showed that an annealing temperature of 300 °C leaves a certain degree of PMMA residue, which can prevent the breakage of graphene while annealing. In contrast, when the annealing temperature rose to 375 °C, more cracks, or even a total collapse, occur in the suspended membrane. In this way, although resonant frequencies that are twice as high could be achieved, the atmosphere pressure quality factor (Besides the annealing treatment, the effect of the laser irradiation on the intactness of the suspended multilayer graphene was also investigated. The damage was mainly caused by the modulated laser, which would induce both a thermal effect and mechanical fatigue. The damage threshold power for the modulated laser was found to be about 2–3 mW, which is about half the CW laser. Thus, the modulated laser power should be controlled carefully in application. Moreover, it was found that the fluctuation of the thermal time constant could be applied to evaluate this damage in situ and in real time.


Graphene-based microwave devices have enabled reconfigurability, thus paving the way to the realization of flexible wireless terahertz systems with featured performances. Despite great progress in the development of graphene-based terahertz devices in the literature, high insertion loss and wide tunable range are still significant challenges at such high frequencies. In this work, we introduce the use of graphene to implement a reconfigurable printed ridge gap waveguide (RPRGW) structure over the terahertz frequency range for the first time. This guiding structure is suitable for both millimeter and terahertz wave applications due to its supporting quasi-TEM mode, which exhibits low dispersion compared to other traditional guiding structures. The presented solution is featured with low loss as the signal propagates in a lossless air gap, which is separated from the lossy graphene elements responsible for the reconfigurable behavior. In addition, this guiding structure is deployed to implement a tunable RPPGW power divider as an application example for the proposed structure. Reconfigurable systems can be constructed through deploying sub-systems having the ability to reversibly change their functionality under some controlling process. One of the major revolutions of reconfigurability in electronics is the field-programmable gate array (FPGA), which has a powerful impact on digital design methodologies alongside wireless systems3. In a related context, few trials are initiated to develop reconfigurable microwave components providing an additional level of functionality for wireless systems. These components can be implemented using a traditional guiding structure such as a microstrip line, rectangular waveguide, and substrate integrated waveguide6. However, these guiding structures have high material losses and fabrication limitations over higher frequency bands. Therefore, the research community exerts more effort to develop innovative guiding structures such as ridge gap waveguide (RGW) to overcome the high frequency limitations and satisfy the technical requirements for future wireless technology9. RGW is considered as a promising guiding structure for millimeter and terahertz wave applications due to the recently reported advantages such as supporting the propagation of quasi-TEM (Q-TEM) mode with lower loss compared with other guiding structures14.Reconfigurable systems are gaining considerable attention as they are expected to play an important role in the development of terahertz wireless technologies. Reconfigurability can result in flexible, cost-effective, and smart transceivers overcoming the design challenges at such high frequencies and achieving the technological future demands19. One of the promising techniques to realize these switches are using graphene. Graphene has incited great interest due to its attractive features that lead to potential usage in different applications22. Graphene’s transport characteristics and conductivity can be tuned by either electrostatic or magnetostatic gating, or via chemical doping, thereby leading to the possibility of developing various electronic devices25. Based on the possibility of controlling the graphene conductivity (impedance) by field effects, several studies have been conducted showing a possibility to reconfigure the performances of microwave devices and antennas33. However, few studies have addressed the effect of tuning the graphene on the frequency response of guiding structures such as coplanar waveguide, parallel plate waveguide, rectangular waveguide, microstrip line, and substrate integrated waveguide40.Reconfigurability of microwave components can be achieved through various mechanisms such as switches, variable reactive loading, and mechanical and material variation. The most common technique used is deploying a switch such as MEMS, PIN diode, and field-effect transistor (FET), which can alter the performance of components in a discrete manner36. A reconfigurable Electromagnetic Band Gap (EBG) unit cell is investigated to construct the PRGW guiding structure. Simulated results show that inserting graphene in the EBG unit cells influences the higher frequency of the band gap. This is achieved by altering the chemical potentials of the loaded graphene. Thus, the operating bandwidth of the PRGW line can be varied corresponding to changing the chemical potentials of graphene. As an application of the proposed RPRGW line, a wideband reconfigurable PRGW power divider is simulated. The results verify that the proposed RPRGW can provide an effective guiding structure for reconfigurable terahertz devices.To our best knowledge, the use of graphene to implement a reconfigurable printed ridge gap waveguide (RPRGW) at terahertz is proposed for the first time in this paper. The PRGW structure offers the separation between the graphene and the signal path, which imply by nature the low loss characteristics compared with SIW graphene-based structure as the graphene exists in the signal pathThe aforementioned applications and challenges in the terahertz motivate the scientific community to develop novel solutions that can overcome significant losses and provide reconfigurable features. We introduce for the first time the deployment of graphene in the printed ridge gap waveguide technology to enable the reconfigurable behavior of the entire structure. We propose the placement of the graphene in the cell structure away from the signal path to avoid the loss of contribution of the graphene, which will be discussed in detail in the following section. Before going through the proposed structure, we will give a brief discussion about the graphene material characteristics and modes of operation.T is temperature in kelvin37. The chemical potential is determined by the carrier density, where varying the carrier density will affect the mobility of the electron-hole, thus changing the material conductivity. The carrier density can be controlled by the application of a gate voltage, wherein in the ungated case both the carrier density and chemical potential can be considered zero24. The conductivity of graphene e = 1.60217662 × 10Many articles have discussed the electronic model of graphene, which can be modeled as an infinitesimally thin surface characterized by a surface conductivity 41. It can be noticed that the imaginary part of the conductivity is negative, meaning that the graphene can be modeled by a surface impedance 42:The effect of chemical potential variation of the graphene’s real and imaginary conductivity is studied and plotted in Fig. The real and imaginary part of the graphene impedance is plotted in Fig. 9. The signal propagates in the form of a Q-TEM mode between the ridge and the top ground, while the cells prevent any leakage within the operating bandwidth43. The cell stop band represents the operating bandwidth of the entire structure. This structure has been investigated intensively in the past decade, where various components have been proposed based on this promising technology45. This technology provides a wide band operation with minimal dispersion, which is suitable for high-frequency applications. In addition, a printed version of the same structure has been proposed to provide the same advantages in a low-cost guiding mechanism. Different unit cell structures have been examined in the literature for (RGW) Ridge Gap waveguides and PRGW—Printed Ridge Gap waveguides14. In this work, we aim to utilize a novel unit cell structure, where some metallic parts can be replaced by graphene. Afterward, applying a controlling voltage will vary the conductivity of cell parts replaced by graphene. As a result, the effective cell shape will depend on the applied controlling voltage. Hence, the operating bandwidth of the entire structure can be adjusted through the controlling voltage.Ridge gap waveguide is considered among the emerging guiding structures, introduced for the first time in 2009First of all, we propose an innovative cell structure to ensure the operation at the target frequency band, where some metallic parts will be replaced by graphene in a later design stage. The proposed cell construction, row of cells, and a ridge surrounded by three successive cells are illustrated in Fig. 7. The dispersion relation extraction setup is shown in Fig. 7:l, and m is the mode number, while The proposed cell is modified by adding the graphene material in four rectangular cuts within the cell perimeter to provide tuning elements. It is worth mentioning that the band gap of the conventional unit cell can be determined using the eigenmode solver, which is a numerical technique available in many 3D EM analysis software packages such as CST and HFSS. However, the eigenmode solver packages do not support lossy materials solution, accordingly, they fail to calculate the dispersion diagram of the proposed graphene doped unit cell. Other solvers can be used to solve the lossy materials unit cell dispersion diagram however, a mathematical method based on the scattering parameters is proposed to extract the dispersion relation and study the effect of the graphene material on the band gap of the unit cellTo obtain the dispersion relation, the simulated scattering parameters are obtained using the model shown in Fig. mentclass2pt{minimThe same outcomes are obtained through the simulated response of the model presented in Fig. 47.The geometrical configuration of the proposed T-junction power divider implemented using the graphene-based terahertz PRGW line that has been discussed in the previous sections is shown in Fig. }% 0.8–1. THz at a}% 0.8–1. THz at aThe investigation of the use of graphene in implementing a reconfigurable printed ridge gap waveguide (RPRGW) at terahertz has been proposed in this article. A printed ridge gap waveguide has well-known advantages compared with other guiding structures including low radiation and material loss, which is suitable for very high frequencies. An electronic model of graphene at terahertz has been studied in detail, where an infinitesimally thin surface characterized by surface conductivity has been deployed to model the graphene. A novel unit cell structure has been proposed, where some metallic parts have been replaced by graphene to achieve a reconfigurable electromagnetic band gap by changing the chemical potential. In addition, the graphene is not allocated in the signal path, which leads to a featured reconfigurable configuration with minimal losses. An innovative mathematical methodology based on the scattering parameters has been proposed to extract the dispersion relation and study the effect of the graphene material on the band gap of the unit cell. As an application example of the proposed technology, a dynamic PRGW line, and power divider has been proposed, where the effect of altering the chemical potential on operating bandwidth has been studied. For the future extension, this work can be applied to different guiding structures such as groove waveguides and substrate integrated waveguides, however, more losses will occur.


The gadolinium, gallium, aluminum garnet doped with cerium and co-doped with dysprosium ions were prepared using sol gel method. The SEM images show that after synthesis, the grains are below 100 nm. The powders were ultrasonically mixed with graphene nanoflakes and ceramics were prepared using the high pressure low temperature sintering technique. A series of the ceramics was prepared using different graphene content. The structure of the samples was examined using X-ray diffraction (XRD), scanning electron microscope (SEM) and Raman techniques. The spectroscopic properties were checked using conventional and persistent luminescence spectra measurements. The thermoluminescence glow curves and fading time of persistent luminescence measurements were performed to check how the graphene presence affects the electron traps number and depth. It was found that the addition of graphene improved the thermal conductivity of co-doped samples. This resulted in faster release of deeper traps and an increase in fading of persistent luminescence. The possibility of releasing energy from deep traps without additional stimulation may allow the use in different applications, the matrices and luminescent ions, which so far did not show persistent luminescence at room temperature. Persistent luminescence is a phenomenon where material emits light for a long time after ceasing the irradiation. This type of luminescence as an irradiation source can be used for visible light, gamma, X-ray or UV radiation. The long-time luminescence decay is the effect of releasing the electrons from the traps with the help of thermal energy . The eneDifferent additives can increase thermal conductivity of the composites, but the most well-known and recently used is graphene. The allotropic form of carbon, called graphene, consists of one plain layer of carbon atoms that are organised in a honeycomb lattice. In this structure, the low-lying electrons behave like massless relativistic Dirac fermion, which are the cause of many interesting properties such as conductance quantization feasibilities of inducing a band gap through the lateral quantum confinement and unusually high room temperature carrier mobility. One of the most interesting properties of graphene is its large thermal conductivity. The diamond-like carbon value of thermal conductivity is about 1.7 W/mK, and for graphene this value increases to 5300 W/mK ,5 or met3Al5O12) [3Al5O12 (LuAG) [3+/Gd3+ ions by other lanthanide ions  [3Al5O12 emits in the green/yellow range after excitation with blue radiation. The most desirable result is to obtain perfectly white light, so we need to use a phosphor, which has a wide emission band covering green and red regions after a blue light excitation. By doping the garnet lattice with some cations, we can modulate the emission colour. The smaller ions shift the emission band towards blue, and larger ones shift the emission band towards red [3Ga3Al2O12 garnet (GGAG), doped with Ce3+, focuses the attention of researchers due to the possibility of using this matrix as a good quality scintillator unique light yield where energy resolution can reach up to 50,000 photons/MeV. Because of the strong crystal field splitting, the emission of Ce3+ doped GGAG has a maximum of about 565 nm and can be used for warm white LEDs [Garnets are the most common materials used for lightening or various optical applications due to their important physical properties such as good thermal conductivity, large thermal stability and optical isotropy. In recent years, two of the most widely used garnets are yttrium aluminium garnet Y3Al5O12)  and Lu3A  and Lu32 (LuAG) ,14,15, b+, Eu3+) ,17,18. I+, Eu3+) . For exaards red . Gd3Ga3Aite LEDs ,22.This work was conducted to find out whether the addition of graphene flakes will change the thermal conductivity of the ceramics and in consequence their persistent luminescence properties. It was checked whether the modification of the thermal conductivity has an impact on the release of electrons from shallow and/or deep energy traps. Better thermal conductivity should reduce the amount of energy needed to release charges from energy traps and thus modify the properties of persistent luminescence. The ability to control persistent luminescence is of great importance in many applications. First of all, such emission is currently used in emergency marking and bioimaging. For such materials, it is important to obtain emissions in the red or infrared range. Unfortunately, some matrices and luminescent ions that could be useful here have traps too deep to observe emission at room temperature. In order to allow their emptying and to observe luminescence at the same time, various actions were taken that could release the trapped energy without additional stimulation. To verify our assumption, ceramics showing persistent luminescence and composites containing an admixture of graphene flakes were prepared and characterized. For all materials, their structure, morphology, spectroscopic properties, thermoluminescence and thermal conductivity were examined. Based on the obtained results, the mechanism of persistent luminescence was constructed, taking into account the impact of the graphene flakes addition and its relationship with the thermal conductivity of composites.2O3, powder, 99.995%, Stanford Materials Corporation, Lake Forest, IL, USA), dysprosium oxide , hydrated gallium nitrate (Ga(NO3)3∙xH2O, Onyxmet, Olsztyn, Poland), aluminium nitrate nonahydrate (Al(NO3)3∙9H2O, 98%, Alfa Aesar, Haverhill, MA, USA) and cerium nitrate hexahydrate (Ce(NO3)3∙6H2O, 99,99%, Sigma Aldrich, Saint Louise, MO, USA), citric acid  and ethylene glycol  were used to synthesize garnet powders.The gadolinium oxide (GdThe powders were prepared by the Pechini sol-gel method . The fir1 radiation (1.5406 Å) and step 0.03°. The morphology and homogeneity of the samples were checked using SEM images taken using FEI Helios G4 PFIB CXe DualBeam microscope . All measured samples were placed on aluminum holders using copper tape. The elemental composition was determined and mapped using an EDS detector: Bruker Nano GmbH XFlash Detector 630 . The excitation spectra of Gd3Ga3Al2O12 doped with cerium and co-doped with dysprosium were measured using FLS980 Fluorescence Spectrometer from Edinburgh Instruments  equipped with a holographic grating of 1800 lines/mm, flashed at 300 mm focal length monochromators in Czerny Turner configuration. To obtain the excitation and emission spectra a 450 W Xenon lamp was used and as a detector the R928P side window photomultiplier tube from Hamamatsu  was used. The persistent luminescence (PersL) spectra of the GGAG were measured at room temperature after 5 min laser diode irradiation  with a 445 nm wavelength using SilverNova CCD spectrometer  with 200 µm slit and 1 s integration time. The same system was used for conventional luminescence measurements. The thermoluminescence (TL) and PersL decay curves were measured using a lexsygresearch TL/OSL reader . The signal was detected by a R13456PMT detector  monitoring emission in whole spectral range (from 185 to 980 nm) with an integration time of 0.1 s. The TL curve was registered from room temperature to 300 °C. As an irradiation source, a blue laser diode PL 450B  was used. Before measurements, each sample was heated to 350 °C and kept for 1 min to traps cleaning. Thermal conductivity measurements were carried out using Physical Property Measurement System (PPMS®) operating in the Thermal Transport Option. The temperature range was from 2 K up to 295 K, the thermal conductivity coefficient was determined in a continuous mode of the device with an accuracy of ±5%.The XRD were collected between 10 and 60 2θ degrees at room temperature by an X’PERT PRO PANalytical  diffractometer using CuKα2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 powders and ceramics . This peak is assigned to the graphite structure whose presence is related to the effect of high pressure on the graphene flakes [2O4) [2.994Ce0.006Ga3Al2O12@xGraphene and Gd2.964Ce0.006Dy0.03Ga3Al2O12@xGraphene ceramics.The XRD diffraction technique was used to study the structure of the Gdceramics . Resultee flakes . The abs2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics with different graphene content are shown in The representative SEM images of the Gd2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics were taken (see To check how the graphene (carbon—“C”) is distributed through the ceramics volume, the EDS maps of the Gdaken see . It can 2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 nanoceramic samples with different graphene content show two broad bands at 344 and 441 nm  observed due to 4f-5d2 and -5d1 transitions of Ce3+ and sharp peaks at 275 and 310 nm  assigned to transitions from 6IJ and 6PJ excited level to the 6S7/2 ground state of Gd3+, respectively (21) and their redshift is a result of a higher disorder of the surrounding of Ce3+ ions and displacement from the cubic polyhedron to disordered square anti-prism (dodecahedron) [2 and redshift for 5d1 levels was observed (2 and 5d1 bands (Δ21) increases when the graphene concentration increases. It was also observed that full width at half maximum (FWHM) of the bands for the pure ceramic are broader in case of 5d1 and shallower in case of 5d2 level compared to the samples with graphene addition. A large value and a deviation from the FWHM change tendency observed for the sample co-doped with Ce3+/Dy3+ with the highest concentration of graphene (1.6%), is related to the fact that it was difficult to register the excitation spectrum for this sample with the same slits as for other samples and therefore in this case they were opened wider. It can also be noticed that FWHM of 5d1 decreases with an increase in graphene content and for the 5d2 level the tendency is the opposite. As in the GGAG matrix, the optically active ions may occupy several positions [The excitation spectra of Gdectively . For allahedron)  that theobserved . The dis2.994Ce0.006Ga3Al2O12 nanoceramics doped and co-doped with cerium and dysprosium ions were measured under 445 nm laser diode excitation  may lead to a reduction in residual Ce4+ to Ce3+ and, as a result, a shift of the emission wavelength toward lower wavelengths. The decrease in the emission intensity is related to the dark gray color of the ceramics after the addition of graphene and their strong light absorption.The emission spectra of the Gdcitation . An inte of Ce3+  can be o ligands ,32. Herr ligands  showed t2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics were registered after ceasing 445 nm laser diode irradiation  of the decay was assigned to the bright emission in the initial time, and was observed due to releasing of the electrons from the shallow traps. As the carriers from deeper traps need more energy to be released, they are transferred more slowly to the excited states of Ce3+ and, therefore, in the fading exponent a long component (τ2) is also observed. It can be seen that for both types of ceramics, the decay related to the shallow traps became longer with the increase in the graphene content and at the same time the longer component dropped instantly  for 5 min  limits the thermal conductivity by three @RT. Below 140 K the difference between different graphene concentration on thermal conductivity is negligible. An entirely opposite process is observed for the Gd2.964Ce0.006Dy0.03Ga3Al2O12 sample. For the cerium/dysprosium co-doped case, adding the graphene enhances the heat flow as desired. Graphene acts here as a catalysator. The 0.1% graphene addition results in a third increase in thermal conductivity from 1.5 W/mK up to 2 W/mK at room temperature. The highest graphene addition (1.6%) lifts the thermal conductivity to 4.25 W/mK. The examined ceramics have demonstrated that improvement of heat flow cannot be straightforwardly achieved by just a graphene addition. The most important factor is the way that the graphene is incorporated into the matrix structure, and as a result, if it acts as a scatterer or carrier of heat.The impact of the graphene content in Gdmeasured . For allty value . The mec3Ga3Al2O12 matrix has relatively small activation energy [3+ through CB (4). As the energy of the traps is relatively small (below 1 eV), the captured electrons may already be released at room temperature and due to improved thermal conductivity and greater traps distribution, it is also easier to release electrons from deeper traps. When the charge carriers are released, they move back to the emission center and then to the ground state giving persistent luminescence (4). As the charge carriers are gradually released from trapping centers, the persistent luminescence may take up to tens of minutes.The mechanism of persistent luminescence is proposed by adopting the energy transfer diagram already described by us  supplemen energy  the rema2.994Ce0.006Ga3Al2O12 and Gd2.964Ce0.006Dy0.03Ga3Al2O12 ceramics was investigated. The XRD measurements show that in the ceramics containing graphene, the structure is maintained, but small changes in the crystallographic environment of luminescent ions may appear. SEM and EDS maps and their analysis show that the graphene added to the ceramics does not form a uniform network, which in some cases may hinder the interpretation of the results. In the ceramics excitation spectra, an increase in the splitting of the 5d states (Δ21) with the changing content of graphene can be observed, due to displacement from the cubic polyhedron and an increase in disorder in the environment of Ce3+ ions. The fading time of the persistent luminescence shows that after graphene addition, the shallower traps are emptied slower, while carriers from deeper traps are released faster, which shows that the presence of graphene mainly affects the traps with higher energy. These observations are confirmed by thermoluminescence measurements, where a shift of the glow curve towards higher temperatures (higher energies) is observed. The relationship between the depth of the traps, the presence of the Dy co-dopant and the changes observed in persistent luminescence are related to the increase in thermal conductivity after the addition of graphene, which was confirmed by thermal transport measurements. It can be concluded that the addition of graphene can improve the process of released electrons from deeper traps and have an influence on persistent luminescence, mainly in the materials where the charge carriers binding traps are deeper. However, for the best effect, low concentrations of graphene (about 0.1–0.2% by weight) and its homogeneous distribution between the grains should be preserved.The influence of graphene addition on the persistent luminescence of the Gd


In contrast, females show higher capacity for remodelling, with peak plasticity corresponding to highest estrogen levels during the estrous cycle. Estrogen manipulation through ovariectomies and estrogen replacement with 17β estradiol in vivo was found to significantly alter spine density and mitigate disease severity. Collectively, these findings reveal that synpatic plasticity is reduced in ALS, which can be amelioriated with estrogen, in conjuction with improved disease outcomes.Amyotrophic lateral sclerosis (ALS) attacks the corticomotor system, with motor cortex function affected early in disease. Younger females have a lower relative risk of succumbing to ALS than males and older females, implicating a role for female sex hormones in disease progression. However, the mechanisms driving this dimorphic incidence are still largely unknown. We endeavoured to determine if estrogen mitigates disease progression and pathogenesis, focussing upon the dendritic spine as a site of action. Using two-photon live imaging we identify, in the The online version contains supplementary material available at 10.1007/s12035-022-02742-5. Amyotrophic lateral sclerosis (ALS) exists on a disease spectrum with frontotemporal dementia (FTD), sharing pathological and genetic features, and clinical overlap –3. In ALALS presents with sex differences in incidence, severity, and progression. Younger females appear to be more protected than males to ALS onset ; howeverNeuroplasticity of dendritic spines is critical for processing information, with changes in synaptic structure and function being determinants of cognition, behaviour, and circuit integrity . The genprpTDP-43A315T (TDP-43) mouse  mice were all purchased from Jackson Laboratories, with all lines maintained on a C57/BL6 background. To capture high-resolution fluorescence of layer V pyramidal neurons residing in layer V and extending into layer II/III the TDP-43 model was crossed with Thy1-YFPH mice. For tissue collection, animals were injected with an intraperitoneal (i.p.) terminal anaesthesia  and transcardially perfused with 0.01 M phosphate-buffered saline (PBS). The cortex was immediately dissected and frozen in liquid nitrogen, and stored at − 80 °C prior to experimental processing. Male and female mice were included.Experimental procedures utilised male and female mice and were approved by the Animal Ethics Committee of the University of Tasmania (A0016593). Experiments were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013) and in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) Guidelines. Mice were housed in individually ventilated cages at 20 °C with littermates unless otherwise stated, on a 12-h light–dark cycle, with access to food and water ad libitum. C57/BL6 , Thy1-YFPH  , 32, andThe mouse estrous cycle is divided into four stages  cycling every 4–5 days . For exp2, 2 mM MgSOa) applied regularly. Dexapent  was topically applied to the dura surface after removal of the bone island. A coverglass  was placed over the dura flush with the skull, and Loctite® adhesive (Henkel Adhesives) applied around window edges. The cranial window was sealed with dental cement  covering the exposed cranium, wound margins, and lip of the coverglass. At the time of the first 2PLSM imaging round, a titanium steel bar  was fastened with Loctite® adhesive to the dental acrylic over the intact hemisphere for animal stabilisation. Mice were subsequently monitored for 2 weeks for any adverse effects, as a standard for all surgical interventions.Open skull cranial windows were surgically implanted over the motor or somatosensory cortices of male and female WT and TDP-43 mice at post-natal day (P) 50, following the protocol previously described by Holtmaat et al. . Mice wez-plane coordinate was saved, and objective changed to a W Plan-Apo 40 × /1.0 water immersion objective (Nikon), after which the same z-plane coordinate was initiated to capture a high-powered region of interest to identify the same position in consecutive imaging sessions. The MaiTai laser captured blue/green emissions (Channel 1 PMT 550–565 nm) at 910 nm/1700mW of power at a maximum of 25% equivalent to 70 mW [z-stacked images (20 μm thick at 1-μm slices). To locate layer II/III for in vivo imaging, z-stacks were begun at a depth of 150 μm from fluorescence first visible from the cortical surface to a maximum depth of 300 μm. Landmark dendrites were identified during the initial session and returned to in consecutive sessions, to follow the same dendritic spines over time.In vivo two-photon microscopy was performed with a custom-built microscope (Scientifica) equipped with a Ti–sapphire laser  and a × 20 water immersion objective , controlled by ScanImage software underMATLAB® software (MathWorks) . Power dto 70 mW . The denTo capture dendritic spine turnover and survival, mice were anaesthetised with an i.p. injection of 0.12 mg/g ketamine to 0.01 mg/g xylazine  and imaged every 24 h. Approximately 10 dendrites from each dendritic compartment per animal (for density) or 4 dendrites per animal , per group were quantified and manual counts were performed under blinded conditions in Neurolucida™, with layer II/III spines annotated along the segment as mature or immature according to their morphology . BrieflyN(t)/N0, where N0 is the number of spines at 0 h and N(t) is the number of spines surviving after 24 h [Ngained + Nlost)/(N(t1) + N(t2)) where Ngained is the number of spines gained over 24 h; Nlost is the number of spines lost over 24 h; and N(t1) is the total number of spines present at the first time point and N(t2) is the total number of spines present at the second time point [Nlost) and a mature spine ‘gained’ (included in Ngained) (and vice versa). Ex vivo two-photon microscopy was used to quantify dendritic spine densities (p/μm) in layer II/III from layer V pyramidal neurons of the motor cortex within whole brains at P60. Layer II/III corresponds to the depth which in vivo 2PLSM was undertaken, with our previous studies having identified that spine pathology is present in the male TDP-43 motor cortex in this region in 20-μm fixed sections [The same dendritic segments were selected and imaged for analysis over the experimental timeline. Spines were identified on the dendritic segments and tracked over time. Survival fraction  was calculated as ter 24 h . Turnoveme point , 39. In sections .Tissue stored at − 80 °C was weighed and placed in RIPA buffer  with EDTA free protease inhibitor tablets (Roche), then homogenised using a T10 basic Ultra-Turrax homogeniser (IKA) for 3–5-s bursts. The homogenate/lysate was centrifuged at 13,000 rpm at 4 °C for 10 min. Protein concentration of whole brain/motor cortex protein fractions was determined using the DC Protein Assay (Bio-Rad). Western blots were blocked and probed for antibodies listed in Table z-spacing of 0.5 µm were collected. Standard excitation and emission filters were used.Mice were terminally anesthetised with an intraperitoneal injection overdose of sodium pentobarbitone (Jurox USA) and transcardially perfused with 4% (w/v) paraformaldehyde  in 0.01 M phosphate-buffered saline . Coronal sections containing primary motor cortex were collected from each animal and a blocking solution containing 1% normal goat serum and 0.3% triton-X (Sigma-Aldrich USA) in 0.01 M PBS was added for 1 h. Sections were incubated with anti-GFP and anti-ERß (Table −1 O2), mice were maintained on isoflurane (1.5–2%) and lay supine on a heatpad kept at 37 °C for cleaning using chlorhexidine (4%) on the dorsal portion. Using scissors, a small incision was made laterally into the skin, approximately above the site of the body cavity and ovarian fat pad. After blunt dissection of the fascia, a second small incision was made in the muscle, creating an opening into the body cavity. The fat pad of the ovaries was located using blunt forceps, and pulled out of the incision to be clamped and lain over the back of the mouse. The ovarian bursa was torn using fine-tipped forceps, releasing the fallopian tubes and the ovary. A low-heat cauteriser was then used to remove the ovary from the fat pad, without creating large bleeds or damage to the fallopian tubes. Once removed, the fat pad was released and carefully reinserted into the body cavity. These steps were then repeated for the other side of the mouse, removing both ovaries. After ovarian excision, the muscle incision was closed with a single surgeon’s knot using absorbable synthetic sutures and the skin incision closed using Michel clips. Mice were given a post-surgical s.c. injection of metacam (meloxicam 5 mg/ml dosed at 1 mg/kg) and 1 ml of saline, and were monitored for normal activity upon recovery. Mice with slower healing incisions were housed individually.Female WT and TDP-43 mice underwent ovariectomy surgery at P40, with female mice typically becoming sexually mature by P42-56 . SurgeryOsmotic mini pumps (ALZET® Model 2004) were inserted at the time of ovariectomy in female WT and TDP-43 mice at P40, containing either saline in 50% dimethyl sulfoxide (DMSO) (vehicle control) or 10 mg/ml 17β Estradiol  in 50% DMSO under blinded conditions [Disease severity was assessed using a neurological scoring system to evaluate phenotypic progression of hind limb weakness (Supplementary Table 1) –46. The t-tests, one-way, two-way, or three-way ANOVA, and Spearman’s correlation coefficient. For western blot and pathological analysis, each point on a graph represents a separate animal. For spine plasticity over time, each data point represents an individual dendrite imaged over time. P < 0.05 was considered significant and data reported as mean ± standard error of mean (SEM) unless otherwise stated. Where appropriate, neurological scoring and quantification of dendritic spine data was performed blinded.Statistical analyses were performed in GraphPad Prism (GraphPad Software) using tests of normality prior to The ability of spines to be plastic is crucial for network function, and disruption to their finely tuned structural dynamics can induce pathological conditions. The balance between dendritic spine persistence and dendritic spine turnover is influenced by many factors, and is in constant flux. The TDP-43 mouse carries a familial ALS mutation, and develops an ALS-FTD spectrum phenotype of motor and cognitive dysfunction , 44, 47,F = 12.55, p < 0.001), dendritic spine turnover was significantly decreased in TDP-43 males in comparison to WT males (p = 0.0019) and HE TDP-43 females (p < 0.001). Spine turnover in the TDP-43 BE female motor cortex was significantly decreased in comparison to WT BE female (p < 0.001). However, TDP-43 HE female spine turnover did not demonstrate the loss that TDP-43 BE, and TDP-43 males did, with no significant differences between TDP-43 HE and WT HE. There were no significant differences in spine dynamics in the somatosensory cortex  = 6.733, p = 0.0016), dendritic spine stability was significantly increased in TDP-43 male mice in comparison to WT male mice (p = 0.0021) and the TDP-43 HE female (p < 0.001). Spine stability in the TDP-43 BE female motor cortex was significantly increased in comparison to WT BE female (p = 0.0002) and TDP-43 HE female (p < 0.0001). However, TDP-43 HE female spine stability was not significantly different to WT counterparts. There were no significant differences in spine stability in the somatosensory cortex , and analysed using a two-way ANOVA with Tukey’s post hoc. In the motor cortex  = 20.07, p < 0.0001) and sex- and spine-type interaction  = 3.142, p = 0.043); yet, not a spine-type and genotype interaction  = 0.3960, p = 0.5296). Tukey’s multiple comparisons tests identified that the percent of both immature and mature dendritic spine turnover was significantly decreased in the male TDP-43 in comparison to WT (immature—p = 0.0031 and mature—p < 0.0001). Immature and mature dendritic spine turnover was also significantly decreased in the TDP-43 BE in comparison to WT BE (immature—p = 0.0003 and mature—p < 0.0001). TDP-43 HE was significantly increased in comparison to TDP-43 male (immature—p = 0.0020 and mature—p < 0.0001) and TDP-43 BE (immature—p = 0.0135 and mature—p < 0.0001). There were also significant differences in the percentage of immature to mature spine turnover between WT females (p = 0.0285) and TDP-43 HE females (p < 0.0001). This data indicates that mutant TDP-43 impacts both immature and mature spines' plasticity, and that whilst the protective effect of estrogen was not restricted to a certain morphological type, it was more pronounced in the mature spine types.To elucidate whether altered dendritic spine dynamics differentially impacts morphological subsets in the motor cortex, the turnover rates for spines identified to have a mature (mushroom or stubby) or immature (thin) morphology were quantified Fig. A. DendriTo investigate the relationship between estrogen levels and structural changes at the dendritic spine, we correlated dendritic spine survival and turnover to estrous cycle stage during imaging in the female WT and TDP-43 motor cortex. The stages of the estrous cycle were given a score of 1–4—with 1 being the lowest estrogen levels and 4 being the estrogen peak—based on previous work demonstrating the levels of estrogen present over the cycle  = 136.0, p < 0.0001) was revealed. Tukey’s multiple comparisons test found significantly increased global hTDP-43 in both BE (p = 0.0002) and HE (p = 0.0001) TDP-43 females, as compared to WT BE and HE females, with WT females found to express negligible disease protein levels  protein, taken at P60. Following a two-way ANOVA a significant main factor effect of genotype  = 7.45, p = 0.0259), Tukey’s multiple comparisons test did not reveal any significant differences in ERβ between specific groups  and beta (ERβ) are widely distributed throughout the brain and their differential activation influences neuronal physiology and neuroprotective genes –51. We uups Fig. J, K. ThuF = 18.08), Tukey’s multiple comparisons test revealed that whilst there were no significant differences in WT, WT + OVX, and TDP-43 spine density, TDP-43 + OVX females displayed a significant reduction (p < 0.0001) in dendritic spine density compared to WT, WT + OVX, and TDP-43 females n = 6 and + OVX with vehicle control pump) had a significantly increased disease severity compared to intact TDP-43 females. The vehicle control pump had no effect on disease score compared to the + OVX mice alone  and long-term depression (LTD) studies , 90, 91.In summary, disturbances in excitatory transmission have long been associated with ALS pathogenesis. The motor cortex plays an important role in this excitability dysfunction, as evidence now indicates that excitability alterations are in fact initiated in this brain region and move through the corticomotor system. Here, we demonstrate that TDP-43-altered dendritic spine plasticity specifically affects the motor cortex, and we have uncovered a mechanism for how females are conferred protection in the FTD-ALS spectrum—with a role for estrogen in structural plasticity. Future research into targeted estrogenic pathways directed at plasticity are relevant not only to the ALS-FTD, but to neurodegenerative diseases as a whole, with the potential to be harnessed as potent neuroprotectants in these devastating diseases.Supplementary file1 (DOCX 756 KB)Below is the link to the electronic supplementary material.


Objective: The aim of this study is to review current evidence regarding the correlation between GERD and IPF and to evaluate the current studies regarding treatments for GERD-IPF. Methods: A review to identify research papers documenting an association between GERD and IPF was performed. Results: We identified several studies that have confirmed the association between GERD and IPF, with an increased acid exposure, risk of gastric aspiration and bile acids levels in these patients. Few studies focused their attention on GERD treatment, showing how antiacid therapy was not able to change IPF evolution. Conclusions: This review investigating the correlation between GERD and IPF has confirmed the hypothesized association. However, further large prospective studies are needed to corroborate and elucidate these findings with a focus on preventative and treatment strategies.Background: Idiopathic pulmonary fibrosis (IPF) is a rare and severe disease with a median survival of ∼3 years. Several risk factors have been identified, such as age, genetic predisposition, tobacco exposure, and gastro-oesophageal reflux disease (GERD). Prevalence of GERD in IPF is high and may affect 87% of patients, of whom only half (47%) report symptoms.  Idiopathic pulmonary fibrosis (IPF) is the most common form of progressive and irreversible interstitial chronic fibroinflammatory pneumonia, accounting for approximately 50–60% of all idiopathic interstitial pneumonias (IIPs) ,2. The eThe pathogenesis of IPF is highly complex and still not fully clear. IPF pathogenesis is correlated by an increase in the synthesis and release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1, as well as some fibrous factors such as transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) ,7,8,9,10IPF usually occurs in adults over 50 years old. IPF prognosis is poor, with a median survival of usually 2 to 3 years, which is comparable with that of some aggressive cancers ,13,14,15Traditionally, IPF treatment was based on immunosuppressants, glucocorticoids, oxygen therapy, and palliative measures. However, the PANTHER-IPF study showed that treatment with azathioprine, N-acetyl cysteine, and prednisone was associated with increased hospitalizations and mortality ,7,8,9,10The purpose of this study is to review the current evidence regarding the correlation between GERD and IPF by examining a group of studies treating the topic. Recent literature was also evaluated for an update on current treatments for GERD-IPF.Currently, antifibrotic therapies  are neither able to cure nor abort or reverse the progression of the disease, although they have proven effective in slowing down the progression, independently of age, sex, and the stage of the disease ,9. BenefHowever, there is little information on the pharmacological interactions of these two agents in IPF patients who are usually polymedicated ,13,14,15Pirfenidone belongs to the group of immunosuppressive agents. Pirfenidone (5 methyl-1-phenyl-2-[1H] pyridone) is an agent that combines anti-inflammatory and antifibrotic effects, acting on the regulation of TGF-5 activity, TNF-α and -β pathways, and cellular oxidation ,7,8,9,10Pirfenidone reduces the accumulation of inflammatory cells and attenuates the proliferation of fibroblasts, the production of cytokines and proteins related to fibrosis, and the increased synthesis and accumulation of extracellular matrix ,7,8,9,10Nintedanib is an intracellular tyrosine kinase inhibitor developed for the treatment of various types of cancer , as well as an antifibrotic agent ,7,8,9,10The most common adverse events (AE) of antifibrotic therapy involve the gastrointestinal tract ,7,8,9,10Gastroesophageal reflux disease (GERD) can be described as the symptoms complex (occurring at least twice a week) or complications due to the ascent of gastric contents into the esophagus and higher parts of the digestive system ,16,17,18Helicobacter pylori infection could provoke reflux esophagitis [In the last few years, the prevalence of GERD has increased all over the world, including 27.8% of the general population of the USA and 25.8% of European citizens. The reasons for the apparent increase in the prevalence of GERD symptoms and erosive esophagitis are not clear ,20. Incrphagitis ,22. Morephagitis .There isphagitis ,22,23.Patients with IPF, systemic sclerosis (SSc), asthma, and COPD are affected by GERD with higher prevalence than in the general population ,28,29,30Common symptoms of GERD are chest or epigastric pain, nausea, regurgitation, and bloating. We also mention cough, throat pain or burning, wheezing, and sleep anomalies among extraesophageal symptoms. GERD can be completely asymptomatic and coincidentally diagnosed. Therefore, an accurate (and timely) diagnosis of GERD remains difficult. There are a range of available diagnostic tests , although they all have important limitations  ,26,27,28is GERD a consequence of IPF or is it a risk factor for it? Some studies have hypothesized that GERD in IPF is more frequent because of potential confounding factors, such as advancing age and smoking [The number of newly diagnosed GERD patients included a steady increase in those with IPF in the last few years; we can also say the same for the prevalence of hiatal hernia, associated with erosive esophagitis ,42,43,44 smoking ,46,47,48Although knowledge of the pathogenesis of IPF remains incomprehensive, various individual genetic and epigenetic factors have been correlated with the development of fibrosis and potential contributions of some environmental exposures  have been presumed. In particular, several studies have hypothesized that GERD-associated microaspiration may lead to persistent inflammation impairing lung infrastructure, thereby possibly accelerating the progression of IPF. Repeated microinjury to alveolar epithelial tissues has been revealed as the first trigger of an aberrant repair process in which several lung cells develop abnormal behaviors that promote the fibrotic process ,46,47,48Currently, antacid therapy  for IPF patients with GERD symptoms has only a conditional recommendation according to IPF guidelines ,38,49,50Laparoscopic fundoplication is another treatment option for patients with IPF and GERD because theoretically it may help to control reflux and avoid aspiration, with the same effects mentioned above on the progression of the disease. ,53,54,55Hiatus hernia (HH) is an alteration of the integrity of the LES, a frequent finding by both radiologists and gastroenterologists, which can be associated with episodes of microaspiration, LES pressure reduction, and an increased risk of esophagitis . As for The prevalence of HH increases with both age—particularly the 6th decade—and obstructive airway physiology, like asthma. The risk factors of HH include increased abdominal pressure , coughing, and tobacco smoking ,60,61.A barium esophagram, esophageal endoscopy, and high-resolution CT can help to describe the anatomy of the esophagus in relationship to the diaphragm and find the location of the LOS ,64,65,66It is fundamental to understand whether HH in IPF patients is a comorbidity, a consequence, or a driver of IPF . Probably, extensive fibrosis causes a reduction in lung compliance, shifting the diaphragmatic pillars and increasing the prevalence of HH in IPF patients . CT fibrIt is still not clear whether HH can be considered a predictor of mortality from respiratory causes in IPF patients . Indeed,The therapeutic options to address GERD in adults include: diet and lifestyle changes  ,69; mediFocusing our attention on GERD treatment in patients with IPF, in 2015 an American preclinical study analyzed the effect of PPIs in mitigating lung injury and fibrosis in IPF, suppressing acid reflux, and arresting cytokines release. This study started from the evidence that PPI use is associated with increased longevity in IPF patients and analyzed esomeprazole action in reducing lung inflammation and fibrosis in rats, concluding that probably through GERD treatment it is possible to find a therapeutic effect on pulmonary fibrosis ,103,104.Nevertheless, at the same time, a systematic review  publishe® in order to identify relevant full-text manuscripts treating the GERD-ILD relationship from different points of view. The search was performed using the following keywords or synonyms: ‘GERD ILD’, ‘GERD IPF’, and ‘GERD Idiopathic pulmonary fibrosis’, without limitation on the date of publication, up to the 12th of March 2022.We performed a review of the literature in PubMedn > 20) of patients examined. We included reviews, meta-analyses, case studies, and retrospective studies, without any limitation on the language of the original text.Records were selected only if they were strictly treating the specified topic and in particular those with a focus on pulmonary perspective and with a larger number (p < 0.01) oesophageal acid exposure and risk of gastric aspiration. In particular, IPF patients had more bile acids ) and a more severe GERD—which can be a reason for lung damage and fibrotic evolution, the same evolution described by Baqir et al. [We selected 14 papers, of which 6 were original studies. Four were case–control studies; the two others were a retrospective study and a meta-analysis. We chose these six papers for their different designs, for the large number of examined patients , and in order to show that, if the association of GERD-IPF is well known, clinical implications still need to be discussed. r et al. —with a 1(n = add), identified an association between GERD and IPF ; homogeneity < 0.0001), although the potential for confounding exists, e.g., smoking. It is also remarkable that this meta-analysis was based exclusively on case–control studies, so the confidence in the estimate of association is low, as the authors themselves clarified.The 2019 meta-analysis,  which inThe retrospective study conducted from the analysis of the IPF Australian Registries  showed tThe possible etiologic role of reflux in pulmonary fibrosis has been advanced on the basis of the results of several studies that have demonstrated the high frequency of GERD in the IPF population. A major challenge is that gastroesophageal reflux is frequently asymptomatic, but a diagnosis is typically based on the presence of common symptoms and therefore often inaccurate.A major unresolved issue is to elucidate whether GERD is a direct driver of IPF or a simple consequence or innocent bystander, potentially due to a decreased pulmonary compliance during respiratory ventilation secondary to fibrosis. Manometric study of esophageal motor function in IPF patients has not demonstrated particular patterns of body dysmotility, and with only a few studies indicating a higher frequency of ineffective peristalsis ,55,56. RFrom a therapeutic point of view, the role of proton pump inhibitor (PPI) in GERD was studied particularly in the last decade ,50,55,56r=0.50, p=0.03) and FVC. The authors concluded that low distal and proximal MNBI, as well as an abnormal PSPW, are predictors of a more severe decline in lung function over 1 year on pulmonary function tests among pre-lung transplant IPF patients [Regarding GERD diagnosis, esophageal MII-pH is the current gold standard. Two abstracts have shown the correlation between mean nocturnal baseline impedance (MNBI) and postreflux swallow-induced peristaltic wave (PSPW) values and changes in pulmonary function testing parameters over 1 year in IPF patients ,50,55,56patients ,50,55,56patients ,50,55,56Another possible application of EndoFLIP could be the identification of those patients presenting EGJ (esophagogastric junction) dysfunction that could benefit from surgery. All these new diagnostic tools, in addition to a more diffuse and frequent use of MII-pH, should improve our ability to establish a more accurate cause–effect relationship between IPF and GERD and to assess the real effectiveness of both medical and surgical antireflux therapies ,50,55,56The examined data strongly support an association between GERD and IPF. Therefore, all IPF patients should be objectively screened for GERD by pathophysiological studies, mainly using HRM (high-resolution manometry) and potentially MII-pH. Moreover, bronchoscopy with analysis of BALF by dosing pepsin and/or bile acids could represent a diagnostic option in order to increase diagnostic accuracy, but it is seldomly performed in routine clinical practice for IPF due to an increased risk of exacerbations; therefore, it cannot be considered in the diagnostic routine for GERD-IPF. Furthermore, the efficacy of PPI therapy, criticized in the latest IPF guidelines, should be evaluated in prospective and randomized clinical trials against placebo, aimed to assess lung function parameters and the clinical response of respiratory symptoms and to improve oesophageal pH and impedance values. Antireflux surgery, particularly LARS, appears to be an effective and safe option for these patients. In conclusion, our current uncertainty about the GERD-IPF relationship and, consequently, about its therapeutic management, is due to the poor knowledge of the real pathogenic mechanisms of the pulmonary damage in IPF . Several