Titanium nitride (TiN) has recently emerged as an attractive alternative material for plasmonics. However, the typical high-temperature deposition of plasmonic TiN using either sputtering or atomic layer deposition has greatly limited its potential applications and prevented its integration into existing CMOS device architectures. Here, we demonstrate highly plasmonic TiN thin films and nanostructures by a room-temperature, low-power, and bias-free reactive sputtering process. We investigate the optical properties of the TiN films and their dependence on the sputtering conditions and substrate materials. We find that our TiN possesses one of the largest negative values of the real part of the dielectric function as compared to all other plasmonic TiN films reported to date. Two-dimensional periodic arrays of TiN nanodisks are then fabricated, from which we validate that strong plasmonic resonances are supported. Our room-temperature deposition process can allow for fabricating complex plasmonic TiN nanostructures and be integrated into the fabrication of existing CMOS-based photonic devices to enhance their performance and functionalities. Although its loss is yet to be comparable to conventional plasmonic materials represented by noble metals (gold and silver), TiN possesses tunable optical properties, and is cost effective, chemically and thermally stable, and compatible with modern silicon fabrication technologies. Recent studies have shown that TiN can enable plasmonic performance comparable to that of gold, and is a better material candidate for hyperbolic metamaterials, transformation optics, and photothermal energy conversion7. To date, plasmonic TiN has been employed in a wide variety of applications including broadband absorbers8, spectrally selective infrared thermal emitters9, hyperbolic metamaterials10, local heating sources12, self-powered plasmonic photodetectors14, and nonlinear optics15.Titanium nitride (TiN)16; however, most of the TiN film deposition has been adopting a high-temperature sputtering process2 to achieve the plasmonic character . Sputtering process represents a mature and low-cost deposition approach, but the high-temperature has limited potential applications of plasmonic TiN, and prevented its integration into existing CMOS device architectures. There has been much effort in enabling room-temperature deposition of plasmonic TiN films. Edlou et al.17 reported over two decades ago the preparation of plasmonic TiN thin films by a DC magnetron reactive sputtering process, without intentionally heating or biasing the substrate during deposition. However, their effort was mainly aimed at the development of TiN films with desirable properties for harsh environments  by varying deposition conditions. It was later shown that significantly increasing the sputtering power (7 kW)18 can result in golden-colored plasmonic TiN films, which can also be realized by applying a high impulse sputtering power (>4 MW)19 or a substrate bias voltage20, yet at the expense of lower crystalline order, increased surface roughness and more defects. Recently, Wang et al.21 also reported the realization of plasmonic TiN films by DC magnetron reactive sputtering at room temperature. Yet, without annealing at 700 °C or higher temperatures, their plasmonic TiN films, instead of having gold-like luster, appear to be red. The measured λps of the as-deposited TiN films, regardless of film thickness, is beyond 700 nm, indicating that they are unsuitable for plasmonic applications in the visible region.Low-temperature growth of TiN is highly desirable to make it compatible with the complementary metal-oxide-semiconductor (CMOS) fabrication. Low-temperature plasmonic TiN deposition has been recently reported by optimizing the plasma exposure per growth cycle during plasma-enhanced atomic layer deposition (PEALD) at 250 °Cε1 at near- and mid-infrared wavelengths, can be realized on various substrate surfaces, and their plasmonic characteristics can be tailored through varying the reactive gas flow ratio. We find that our TiN possesses one of the largest negative values of ε1 as compared to all other plasmonic TiN films reported to date. We further fabricate two-dimensional periodic square arrays of TiN nanodisks, and validate from transmittance measurements that they support strong plasmonic resonances in the visible and near-infrared regions. The room-temperature sputtering process of TiN could be employed to ease the fabrication of more complex TiN-based nanostructures and potentially be integrated into modern CMOS processes to expand the practical applications of plasmonics.Here, we report on the realization of plasmonic TiN thin films by a room-temperature, low-power, and bias-free reactive sputtering process. We show that highly plasmonic TiN films, i.e., TiN films with large negative real part of the dielectric function ε1) and imaginary (ε2) part of the extracted dielectric functions of TiN films (thickness ~50 nm) deposited on various substrate surfaces at Ar:N2 = 80%:20% (except for the TiN films on quartz deposited at Ar:N2 = 95%:5%) using Drude-Lorentz model. All TiN films are clearly plasmonic in the spectral region of interest. In particular, the large negative values of ε1 for TiN deposited on hafnium dioxide (HfO2), silicon (Si), and quartz indicate that these films are highly plasmonic. Both of their ε1 and ε2 exhibit similar characteristics, and the variation of their λps is less than 20 nm , showing that our room-temperature sputtered TiN films have a weak dependence for these substrate surfaces as those prepared by ALD22. Note that λps of these films occurs at wavelengths shorter than that of the ALD plasmonic TiN22, therefore enabling a broader plasmonic response in the visible region. TiN films on poly(methyl methacrylate) (PMMA) possess a quite distinct dielectric function from others and are less plasmonic, which may have been caused by the surface roughness of the PMMA films induced by the ion bombardment during the sputtering process. As can be seen in the atomic force microscopy (AFM) images  and Si (0.888 nm) surfaces. It is expected that the optical properties of TiN films on PMMA are different since their roughness is essentially on the same order of the film thickness. Nevertheless, our results show that the room-temperature sputtering process can be employed to fabricate plasmonic TiN on unconventional polymeric or elastomeric substrate for flexible plasmonics and nanophotonics applications23, which is not possible by either high-temperature sputtering3 or low-temperature ALD16.Figure aginary ε part of ε1 and ε2 of TiN films deposited by high-temperature sputtering, low-temperature ALD, and high-temperature epitaxial growth, in dashed lines denoted as ref 13, ref 216, and ref 35, respectively. Epitaxial TiN films grown on lattice-matched substrate to our knowledge possess the largest negative value of ε1 among all plasmonic TiN films reported so far due to their high crystalline quality and low roughness, although they need to be grown at a fairly high temperature (750 °C). Our room-temperature sputtered TiN films are apparently comparable to the epitaxial TiN and superior to films prepared by both high-temperature sputtering and low-temperature ALD in plasmonic character. We believe that the strong plasmonic character of our room-temperature sputtered TiN films is enabled by the close proximity of our samples to the plasma during deposition providing the necessary high energy reactive ions and activated species in lieu of substrate heating or biasing24. Note that the observed strong plasmonic character of our TiN is in sharp contrast to the dielectric properties of titanium oxynitride (TiOxNy)25, which usually exhibits double epsilon-near-zero (ENZ) characteristics without substrate heating and special chamber preparation. We also plot in the same figures the optical data of gold (Au) reported by Johnson and Christy26. Despite its several disadvantages for plasmonic applications4, Au still has better plasmonic character (more negative ε1) and a smaller loss  compared to TiN, which can be further verified by examining the general figure of merits (FOMs) for localized surface plasmonic resonance (LSPR) defined as − ε1/ε227, as shown in Fig. 29 where plasmons are resonantly excited by incident light and subsequently decay nonradiatively into hot carriers on an ultrafast time scale, it is desirable to have plasmonic materials possessing not only a large negative ε1 but also a large ε2 value. Our TiN films are clearly most suitable for hot-carrier applications in comparison with Au and all the other TiN films, allowing for both strong plasmonic coupling (due to larger negative ε1) and fast plasmon decay (due to larger positive ε2). In Fig. L) in the (200) orientation estimated using the Scherrer’s formula30 regardless of substrate surface is comparable to that for the reactive sputtered TiN films reported elsewhere31: L = 7.33, 7.89, 9.76, and 7.89 nm for the films deposited on HfO2, Si, quartz, and PMMA, respectively.We plot for comparison in Fig. ε1 (solid lines) and ε2 (dashed lines) of the TiN films (thickness ~50 nm) deposited on Si at various Ar:N2 gas flow ratios . As can be seen, the optical properties of the TiN films can be tuned by adjusting the Ar:N2 ratio in the sputtering process. The value of ε1 becomes more negative for the films deposited at higher N2 flow and is most negative when Ar:N2 reaches 80%:20%, but further increasing the N2 content in the reactive gas instead makes TiN films less plasmonic, essentially similar to the trend observed in the earlier work2. These findings are also in good agreement with high-temperature sputtered TiN films on Si and magnesium oxide (MgO) substrate, but are different from those on sapphire as well as TiN prepared in a room-temperature sputtering with a substrate bias voltage20. It could therefore be inferred that in addition to the Ar:N2 ratio, the plasmonic character of sputtered TiN films is strongly influenced by numerous other processing parameters including substrate material, deposition temperature, external biasing, and so on. In fact, we find in our experiments that the Ar:N2 = 80%:20% TiN films possess the most negative value of ε1 as compared to those deposited at other gas flow ratios for all substrate surfaces but quartz, for which the plasmonic character of the TiN films is optimized when Ar:N2 is 95%:5%. We also find that the λps of our TiN films  when the ratio of N and Ti in TiN films is near unity33. This suggests that our Ar:N2 = 80%:20% films are most likely to be stoichiometric, although others should still have close stoichiometry due to their comparable λps values. In Fig. 2 gas flow ratio has an impact on their crystalline structure. The Ar:N2 = 95%:5% films exhibit a predominant peak for the (200) orientation along with two small peaks for (111) and (220) orientations. The (111) peak intensity gradually increases with increased N2 flow, although both Ar:N2 = 85%:15% and Ar:N2 = 80%:20% films still show a (200) preferred orientation. Further increasing the N2 flow dramatically changes the crystalline structure of TiN, making the Ar:N2 = 70%:30% and Ar:N2 = 60%:40% films preferentially grown in the (111) orientation. These observations seem to suggest that our TiN films in the (200) preferred orientation possess better plasmonic character than (111) oriented films; however, other factors such as oxygen impurity content20 also need to be considered to fully elucidate the relationship between their structural properties and the corresponding plasmonic response.In Fig. 2 = 80%:20%, as shown in Fig. 35 have shown that the thickness dependent optical properties of plasmonic thin films arise from changes in surface-to-volume ratio, grain boundary, and carrier scattering from substrate surface as thickness varies. In particular, when films are within the ultrathin regime (thickness ≤10 nm), their plasmonic character significantly degrades due to island formation36, low mobility interface layers37 or quantum confinement effects38, and special thin film preparation methods such as high-temperature epitaxial growth as well as substrate treatment are thus required. However, in most of the practical plasmonic structures and devices, the thicknesses of their constituent plasmonic layers are within the thickness range of the present study . Our room-temperature sputtered TiN films can therefore be employed for a wide variety of practical applications for plasmonics due to their high plasmonic character. It is also worth noting that Sugavaneshwar et al.39 recently claimed the realization of the TiN films with the best plasmonic character reported to date by pulsed laser deposition (PLD). Despite the demonstrated high plasmonic character , their TiN films are in fact much thicker (thickness = 180 nm) than those reported in literatures. The plasmonic quality of their thinner films (thickness ~30–50 nm) remains to be verified to allow for a fair comparison.We then investigate the thickness dependence of the dielectric function and crystalline structure of the TiN films deposited on Si at Ar:N2 = 80%:20% films deposited on Si substrate at three different depths by in situ Ar ion milling: at the film surface, in the middle of the film, and near the substrate. The measured XPS spectra for Ti 2p, N 1 s, O 1 s, and C 1 s are plotted in Fig. p doublet lines  at the binding energies of ~455 and ~461 eV along with the nitrogen peak at 397.2 eV confirm the characteristic TiN phase40. The surface oxidation of the TiN films results in the formation of titanium dioxide (TiO2), manifesting as the other two peaks at 458.4 and 464.1 eV in the Ti 2p spectrum as well as the oxygen peak at 530 eV. The expected carbon signal detected at 284.6 eV accompanied by a small peak at 288.5 eV originates from the carbon contamination of the surface of the TiN films, as they are fully exposed to the atmosphere after growth. Moreover, the filling of the valley between the Ti 2p3/2 and 2p1/2 transitions near the binding energy of 457 eV indicates the existence of an intermediate chemical state between TiO2 and TiN, i.e., TiOxNy, which also appears as the shoulder at 395.7 and 531.4 eV in the N 1 s and O 1 s spectra, respectively. After etching halfway through the films, the characteristics of the TiN phase become clearly dominant while the features of the surface oxidation and carbon contamination are reduced, including the significant enhancement of the Ti 2p signals as well as the N 1 s peak for TiN, the strong suppression of both the TiO2 peaks in the Ti 2p spectrum and the oxygen peak in the O 1 s spectrum, and the complete elimination of the two carbon peaks in the C 1 s spectrum. The signals representing the TiOxNy state are also much weaker, suggesting that the titanium oxynitride forms mostly near the film surface. By comparing the measured Ti 2p spectra including the spectral line shape and peak positions to those reported in the previous XP spectroscopic studies of reactive sputtered TiN films41, we find that the bulk of our Ar:N2 = 80%:20% TiN films is likely to possess high purity with a small amount of oxygen impurity. This is evidenced by the elemental compositions of the films (see Table xNy (even for the TiOxNy films deposited at high temperature after careful chamber preparation)25, showing much stronger TiN characteristic signals and much less distinct features for TiOxNy.To verify the chemical compositions of our room-temperature sputtered TiN films, we perform x-ray photoelectron spectroscopy (XPS) measurements on the Ar:N2 = 95%:5%, obtained by averaging the measured p- and s-polarized reflectance/transmittance. The reflectance R first exhibits a minimum (R = 24%) at λ ~400 nm, and then significantly increases to R ≥ 70% as moving towards near-infrared wavelengths. The corresponding transmittance T instead reaches its maximum (T = 22%) at λ ~400 nm, and then decreases with increasing wavelength and remains <5% for λ > 900 nm. The observed characteristics of both reflectance and transmittance closely resemble those for Au films, validating that our TiN films are highly plasmonic/metallic. The reflectance minimum (or transmittance maximum) of TiN occurs on the high energy side where strong interband transitions prevail, whereas reflectance maximum (or transmittance minimum) in the longer wavelength region results from the high metallicity of TiN1. The high plasmonic quality of our TiN films is further confirmed by their Au-like luster42, as evident in the photograph inset to the Fig. In Fig. 2 = 95%:5%; thickness = 45 nm) on quartz substrate. The nanodisk diameter d is varied from 100 to 180 nm while the period is fixed to p = 250 nm. In Fig. d = 100, 140, and 180 nm are shown (solid curves), obtained by averaging the measured p- and s-polarized transmittance. A pronounced transmittance dip is observed for all d values, corresponding to a plasmonic resonance mode excited in the TiN nanodisk arrays. For d = 100 nm, the transmittance dip occurs at λ = 630 nm with T = 71%. When d is larger, the observed transmittance dip clearly redshifts: for d = 140 nm, the transmittance dip occurs at λ = 690 nm with T = 46%; for d = 180 nm, the transmittance dip occurs at λ = 760 nm with T = 27%. The redshift suggests that the excited resonance is a localized surface plasmon resonance (LSPR) mode. We perform full-wave numerical simulations for the transmittance of TiN nanodisk arrays using CST Microwave Studio to gain insight into our experimental results. The simulations are carried out in frequency domain with periodic boundary conditions using the dielectric functions of TiN extracted from variable angle spectroscopic ellipsometry (VASE) measurements and online optical data43 for quartz. The simulated transmittance spectra are shown in Fig. d = 100 nm, the transmittance dip occurs at λ = 615 nm with T = 66%. Increasing the diameter d to 140 and 180 nm shifts the dip to λ = 663 and 711 nm and reduces the corresponding transmittance to T = 39% and 20%, respectively. Note that all of these resonance dips appear to be slightly stronger  and occur at shorter wavelengths as compared to those observed in the experiments. This discrepancy can likely be reduced by minimizing the fabrication imperfections  in the structural model for the fitting of the ellipsometry data of TiN. The simulated electric field intensity at the plasmonic resonance for arrays with d = 100 nm is shown in the inset to Fig. 11.We fabricate two-dimensional periodic square arrays of TiN nanodisks using our room-temperature sputtered TiN films , while the pressure and RF power were held constant at 3 mT and 275 W respectively for all TiN films. Note that we found increasing the N2 content would reduce the film deposition rate, and the plasma in the sputtering process could not be sustained when further increasing the nitrogen content in the gas flow ratio to Ar:N2 = 40%:60%. It should also be noted that, although no intentional substrate heating was introduced, the substrate temperature during the sputtering process may have been slightly higher than room temperature, due mainly to the energy transfer from the sputtered species such as ions and neutrals from the target and the electrons from the plasma discharge. However, we expect this temperature increase should be insignificant, and the substrate temperature was much lower than that used in the low-temperature ALD work16. The dielectric function, thickness, transmittance , and reflectance (incident angle 20°) of the sputtered TiN films were measured using VASE at wavelengths from 300 to 2500 nm. XRD, XPS (VGS Thermo K-Alpha), and AFM (Bruker Innova) measurements were conducted to characterize the crystalline structure, chemical composition, and surface roughness of the TiN films, respectively. Arrays of TiN nanodisks (array area = 1 mm2) on quartz substrate were fabricated by electron-beam (e-beam) lithography and inductively coupled plasma (ICP) etching. Au nanodisk arrays of the same surface area were fabricated on quartz by e-beam lithography, metal evaporation, and liftoff. All of the fabricated nanodisk arrays were examined using high-resolution scanning electron microscopy (SEM), and their transmittance was measured at normal incidence using VASE.Sputtering of TiN thin films was carried out at room temperature in a radio-frequency (RF) reactive sputtering system (Kurt J. Lesker) using three-inch titanium targets (99.995%) on different substrate surfaces including Si, quartz, HfO


Hydrothermal activity in the crust results in the precipitation of large volumes of silica and often involves the formation of ore deposits, the shaping of geothermal systems, and recurring earthquakes. Pore fluid pressures fluctuate between lithostatic and hydrostatic, depending on seismic activity, and some models suggest the possibility of flash vaporization, given that fluid pressures can drop to the level of vapour at fault jogs during seismic slip. The phase changes of water could create extremely high supersaturations of silica, but the mechanisms of quartz vein formation under such extreme conditions remain unclear. Here we describe flash experiments conducted with silica-saturated solutions under conditions ranging from subcritical to supercritical. We found that amorphous silica is produced instantaneously as spherical nano- to micron-scale particles via nucleation and aggregation during the evaporation of water droplets. The nanoparticles are transformed to microcrystalline quartz very rapidly by dissolution and precipitation in hydrothermal solutions, with this process requiring less than one day under supercritical conditions because of the huge surface areas involved. We suggest that such short-lived silica nanoparticles have significant impacts on the dynamic changes in mechanical behaviour and hydrology of hydrothermal systems in volcanic areas. There are several mechanisms of quartz vein formation, including the upflow of deep-seated fluids along fracture networks and diffusion from the host rocks without significant fluid advection10. Mineralogical and fluid inclusion studies of epithermal or mesothermal gold–quartz veins have revealed seismically induced fluctuations in fluid pressure between lithostatic and hydrostatic conditions in the crust, thereby causing the boiling of hydrothermal fluids and silica precipitation12. Weatherley and Henley13 proposed a model in which gold–quartz veins form by the flash vaporization of fluids at the very instant of earthquake rupturing. In their model, the fluids could be decompressed to the level of vapour at a fault jog. They speculated that gold and silica particles could be co-precipitated by the flash vaporization. However, this interesting hypothesis has yet to be proven.Quartz occurs ubiquitously as a fracture-filling mineral, as silica is the most abundant component of the crust, and the solubility of quartz in water is strongly dependent on temperature and pressure14. During diagenesis, amorphous silica  in a deposit may transform into more stable phases, with cristobalite  and chalcedony changing to quartz over a period of >1000 years14. The question arises as to whether quartz veins may form via amorphous silica, even under hydrothermal conditions in the crust at higher temperatures of T > 200 °C. Some quartz veins are filled with crystals grown from the vein walls, but other types show mosaic (blocky) textures or textures composed of microcrystalline quartz10. The microcrystalline type is thought to have developed through various mechanisms, including plastic deformation of quartz grains and precipitation of amorphous silica (as a precursor) from highly supersaturated solutions17. Metastable silica phases usually contain water, which is easily lost during deformation and transformation into more stable phases. However, the metastable silica phases themselves are not preserved in most quartz veins.Despite quartz being thermodynamically stable at the Earth’s surface and in the crust, it is known that metastable silica polymorphs also occur commonly at the surface or in subsurface deposits at temperatures of <100 °C in association with hot springs and seafloor hydrothermal vents20 have revealed that various types of silica precipitation occur via a range of mechanisms under supercritical conditions including epitaxial quartz overgrowth, formation of metastable silica minerals, and 3-dimensional homogeneous (or heterogeneous) nucleation of quartz in fluids, depending on temperature and solution chemistry. In the field of engineering, silica nanoparticles have been synthesized by the rapid expansion of silica-dissolved solutions21 or the evaporation of silica gel dispersed droplets22, but their geological significance has not been considered.Experimental studies of silica precipitation remain limited. Flow-through experimentsThe aim of this study was to understand the mechanism of silica precipitation induced by flash vaporization, and to evaluate whether the formation of amorphous silica is a key step in the formation of quartz veins in the crust. We conducted novel experiments on quartz formation comprising two steps: flashing experiments under subcritical or supercritical fluid to air pressures with granite-dissolved aqueous solutions, and batch-type experiments to investigate the transformation of the flashing products under hydrothermal conditions.2O , 353 °C and 36.7 MPa (FL350), 400 °C and 37.8 MPa (FL400), and 450 °C and 35.8 MPa (FL450). All runs showed nearly isothermal decompression paths within the first 1.5 seconds, with or without a phase transition of H−1, indicating that the particles comprise amorphous silica23 . In our experiments, amorphous silica nucleated instead of quartz in all runs, because of the lower interfacial energy of amorphous silica with water (65 mJ m−2) compared with that of quartz . Equation 2O results in the formation of fine nuclei (100–200 nm in run FL250 and 30–200 nm in FL350), whereas during flashing from supercritical fluids (runs FL400 and FL450) the continuous nucleation of amorphous silica is expected to occur over a wide range of P–T conditions during gradual changes in water density, resulting in a wide range of nucleus sizes , and 450 °C and 36 MPa  to investigate the transformation of amorphous silica particles produced by flashing Fig.  and BT452 with minor amounts of Al2O3 (<1 wt.%), Na2O (1.0–1.5 wt.%) and K2O (<1 wt.%)  Table . By day %) Table . By day tra Fig.  suggest ca Table .c-axes. These crystals developed crystal forms typical of quartz . The peak at ~185 cm−1 is broad , the silica transformation occurred more rapidly. Even on day 1, hemispherical polygonal quartz aggregates (5–10 μm in size) were commonly present, with individual quartz crystals of 1–2 μm in size Fig. . These prtz Fig. , with so2O Table . Raman sm−1 Fig. . Spectram−1 Fig.  occur atoad Fig. , probablK2O Fig. .In the experiment under vapour conditions (run BT350vap), the spherical amorphous silica particles remained even after day 28 Fig. , althougmSi = 9.19 mmol kgH2O−1) was lower than the solubility of quartz under the conditions of run BT350 , but higher than the solubility of quartz in run BT450   by day 3, then decreased gradually to 15.51 mmol kgH2O−1  . Al and K concentrations  decreased, with the decreases being more pronounced in run BT450  than in run BT350 , which is largely consistent with the formation of Al-Na-K silicates  Fig. . In run O−1 Fig. . In run −1) Fig. . The pH −1) Fig. . Na conctes Fig. . Fe conc−1 Table , indicat26 and natural silica sediments27, but transformation rates have not been determined precisely because solid products rather than solutions were analysed. The change in Si concentration in run BT350 is explained by the coupled dissolution of amorphous silica and precipitation of cristobalite . The dissolution of silica occurred more rapidly than cristobalite precipitation for the first three days, with the Si concentration increasing to a level exceeding cristobalite solubility (intermediate between those of quartz and amorphous silica). Then, with the increase in volume and surface area of the cristobalite, its precipitation rate exceeded the dissolution rate of amorphous silica, causing Si solubility to decrease towards that of cristobalite , is simply expressed by the first order kinetic equation31 as follows:k_ is the precipitation rate constant (mol m−2 s−1), and AQtz is the reactive surface area of quartz (m2). Q and KQtz,eq respectively represent the activity product and its equilibrium constant for quartz. With using k_ = 2.2 × 10−4 mol m−2 s−1 at 450 °C18, when we consider the quartz precipitation from the 110 ml input solution (mSi = 9.19 mmol kgH2O−1) on quartz surface with AQtz of 31 mm2 , it takes >50 days at 450 °C for the Si concentration to decrease to near the solubility of quartz (<5 mmol kgH2O−1). This means that quartz precipitation via amorphous silica, as observed in our two-step experiments, is much more rapid than has been estimated in models of quartz overgrowths on pre-existing crystals in vein walls, probably due to huge surface areas of amorphous silica and microcrystalline quartz  increases with increasing pressure and decreasing temperature. Along a cold geothermal gradient (~10 °C km−1) such as a subduction zone interface, the solubility of amorphous silica is 3–5 times higher than that of quartz in temperature range of 200 °C–450 °C. Such a condition makes it difficult for amorphous silica to form even by the change from lithostatic to hydrostatic pressure39, except in extreme cases such as flash vaporization. In contrast, along a high geothermal gradient, such as in volcanic and geothermal areas where the gradient may exceed 100 °C km−1, the solubility of amorphous silica is only 1–2 times higher than that of quartz, which means that the formation of amorphous silica can be realized by subtle fluctuations in fluid pressure from lithostatic to hydrostatic, as well as boiling processes12. The scales of silica in the pipelines of geothermal power plants provide direct evidence for the formation of amorphous silica by flashing under such high temperatures. We suggest, therefore, that amorphous silica forms more commonly than expected, even within hot crust (T > 300 °C). Aggregates of nano-scale amorphous silica are short-lived, and are transformed to microcrystalline quartz with colloform or banded structures , as commonly observed in epithermal or mesothermal quartz veins.Since amorphous silica is not preserved in hydrothermal quartz veins, it remains unclear how common the formation of amorphous silica is within the crust. One simple constant regarding the formation of amorphous silica is that the fluid, which was originally in equilibrium with quartz, should exceed the solubility of amorphous silica at the time of vein formation. The solubility ratio of amorphous silica with respect to quartz Fig. ; ref.38 41 and continuous records of groundwater levels42 indicate that a redistribution of fluid pore pressures and fluid flow is induced by earthquakes, and that a dynamic oscillation of permeability is induced by distant earthquakes42. The permeability oscillation is often explained by the clogging and removal of clay mineral particles at pore throats on fault surfaces43. In the hydrothermal systems of volcanic areas, the flow of silica nanoparticles is easily self-organized as the result of oscillations in fluid pressure. We speculate that these silica nanoparticles are easily transported and can clog faults effectively, thus affecting the dynamic behaviour of the crust.One notable characteristic of silica nanoparticles produced by flashing is their high mobility with fluid flow, which is quite different from the silica that is precipitated as quartz overgrowth from vein walls. Recent seismological observations−1. The temperature was 370 °C and the fluid pressure was regulated by the back-pressure valve to be 41 MPa. The solution was cooled and stored in the plastic tank until each experiment. The concentrations of Si, Al, Na, K, Ca, Mg, and Fe in the input solutions and the solutions after the batch experiments were determined by inductively coupled plasma–atomic emission spectrometry  at Tohoku University, Sendai, Japan. The solubility of quartz used in this study was calculated as a function of water density following Manning7. The solubility ratio of amorphous silica with respect to quartz is from Karásek et al.38. The pH of the input solution was 6.8 at room temperature (Table We used a single high-Si solution as the input solution for the flash and batch experiments. The solution was prepared by the dissolution of quartz (100 g) and granite sand  with a grain size of 1–2 mm in the flow-through apparatus. Distilled water entered the cylindrical autoclave at a constant flow rate of 0.7 mL minre Table .A cylindrical autoclave (volume 110 mL) was used for the flash experiments, and its inner wall and inner caps were made of a titanium alloy (Ti-6Al-4V) Fig. . The autTwo series of batch experiments were conducted at 350 °C and vapour-saturation pressure (run BT350), and 450 °C  at Tohoku University. Due to the rough surfaces of the products, the semi-quantitative analyses of chemical compositions were conducted by EDXS. Silica minerals were identified by micro-Raman spectrometry (Horoba XploRA PLUS) at Tohoku University, with a 532 nm laser and a 2400 grooves/mm grating. pH was measured at room temperature before and after the batch experiments.Supplementary Information


In summary, nonuniformly spaced echo trains introduce a new approach to molecular imaging of J-coupled species, such as lipids, which may have implications diagnosing metabolic diseases.Previous work has highlighted the complicated and distinctive dynamics that set signal evolution during a train of spin echoes, especially with nonuniform echo spacing applied to complex molecules like fats. The work presented here regards those signal patterns as codes that can be used as a contrast mechanism, capable of distinguishing mixtures of molecules with an imaging sequence, sidestepping many challenges of spectroscopy. For particular arrays of echo spacings, non-monotonic and distinctive signal evolution can be enhanced to improve contrast between target species. This work presents simulations that show how contrast between two molecules: (a) depends on the specific sequence of echo spacing, (b) is directly linked to the presence of J-coupling, and (c) can be relatively insensitive to variations in B0, T2 and B1. Imaging studies with oils demonstrate this phenomenon experimentally and also show that spin echo codes can be used for quantification. Finally, preliminary experiments apply the method to human liver  Specifically, neglecting T2 decay (spin-spin relaxation), signal from a standard Carr-Purcell-Meiboom-Gill (CPMG) sequence yields signal at the nth echo peak, S(nτ), equal to:m and ωm are constants, which depend on the J and Δω of each coupled spin pair as well as the echo spacing of the pulse sequence, τ. While this equation describes a simple sum of sinusoids, the complication is that cm and ωm are not straightforward functions of J, Δω and τ. In general, calculating these parameters requires knowledge of off-diagonal terms in the density matrix using average Hamiltonian theory or a numerical simulation. Analytic equations have only been published for small spin systems and a few extreme limits in echo spacing4.The behavior of coupled spins in a spin echo train have been known since the early days of MR (magnetic resonance) to depend on both spectroscopic parameters specific to the molecule and also on experimental parameters specific to the sequencem = J of the spin pair. At the other limit, when τ ≪ 1/J, ωm approaches zero, so all J-modulation disappears. The importance of this latter case resurfaced in MRI in the early 90 s, when it was noted that fat tissues appeared hyperintense on fast spin echo images, despite being relatively dark on conventional T2 weighted images7. Debate ensued over the origin of this phenomenon, but it is now generally accepted that the primary cause is suppression of J-modulation, which otherwise reduces spin echo intensity in complex molecules like lipids.Two approximations applicable at the extreme limits are widely known. When τ ≫ 1/J, ωet al. performed extensive simulations studying more complicated spin systems (A3B2 and A3B2C2) in echo train experiments of different echo spacing3. Those studies documented a wealth of erratic behavior, including a narrow range of τ where dynamics transitioned from relatively smooth evolution, where S(nτ) contains just a few frequency components, to “erratic” where a complex and variable number of frequency components contributed to the signal evolution. Furthermore, modeling the initial signal decay as a monoexponential T2, the authors found that “apparent T2” could vary by two orders of magnitude for τ between 10 and 40 ms. A study of effective T2 as a function of coupling strengths suggested that dependence on spectral parameters is similarly dramatic. Notably, this profound variability was observed even for these studies of uniformly spaced CPMG pulse sequences.Typical fast spin echo (FSE) experiments applied to fats are not easily described by any simple formula; they involve echo spacings that do not fall at either simplifying extreme, as well as relatively complicated molecules. Stables 9. Several studies in the Warren group, extending quantum computing work by Uhrig11, highlighted the changes in dynamics that occur in going from a uniformly spaced sequence, like the classic CPMG, to a sequence with nonuniform spacing between echoes. From a T2 perspective these two sequences should yield identical echo magnitudes at the last echo. But theoretical work from quantum computing suggests they may differently refocus particular frequency components in the relaxation that lead to decoherence. However, in vivo experiments found the most dramatic differences between these two sequences in fat tissues, due to modulation of the J-coupling by the different spin echo trains8.Previously reported studies have demonstrated distinctive dynamics that shape the signal evolution observed during a nonuniformly spaced train of spin echoes14. The work presented here tests whether this evolution is sufficient to identify and quantify different molecules. Unlike traditional MR spectroscopy methods to identify chemical components, static magnetic field (B0) inhomogeneity is unimportant in a spin echo train, which may have advantages for distinguishing species in vivo, especially those with similar chemical shift17. Furthermore, by tailoring the echo spacing, it may be possible to maximize contrast between species of interest, thereby providing a new contrast mechanism for molecular imaging in vivo.This previous work on irregularly spaced refocusing pulses focused on the magnitude of the last echo following a spin echo train preparation, but we hypothesized that the full evolution across the train of echoes may deserve further analysis. Since modulation across the echo train depends on spectroscopic parameters, signal evolution across a spin echo train may constitute a type of molecular fingerprint22. We present results demonstrating experimental reproducibility of these signals, as well as the potential for quantitative analysis of the species present in each voxel. Finally, we present an initial demonstration in two human livers, one lean and one steatotic. In human studies, the echo train signal is nonmonotonic only in the steatotic liver, suggesting that the unusual signal evolution is driven by the coupled spin networks present in lipids.The following studies present preliminary simulations and experiments demonstrating the idiosyncratic nature of behavior under irregularly spaced spin echo trains. Simulations show the power of echo spacing to tune contrast between two species, as well as effects from imperfect B0 (main magnetic field), T2, or B1 (radio frequency magnetic field). The oils tested experimentally with this method, diacetin and triacetin, provide simplified models of the glycerol backbones of diglycerides (DAG) and triglycerides (TAG), which have high clinical significance for the development of metabolic and liver disease11.Figure Furthermore, in addition to changing the signals from an individual molecule, the contrast between two molecules can be modified by the spacing of an echo train, as seen by comparing panels . The correlation between signal from Mol A vs Mol B in these two different (nonuniform) echo spacings, is, respectively, 0.84 vs 0.96, suggesting these very similar experiments provide very different contrast. Both uniform spacings give a correlation of 0.95 between molecules, which provide little basis for distinguishing them. Finally, we consider off-resonance and the potential for imperfect refocusing of spins in panels , which show changes in the signal pattern, but little change in the correlation and contrast . However, this additional degree of freedom to manipulate signal pattern across echoes suggests this is another potential source of contrast, though it is not fully explored in the presented work.max with some marginal dependence on mean echo spacing (ESP). This hypothesis is tested in Fig. With a conventional MRI view of spin echo trains, one might expect contrast depends primarily on TESince ESP arrays taken from the same distribution may still differ significantly in their actual μ(ESP), we also plot signal correlation vs actual μ(ESP), not just μ(ESP) of the source distribution, for each sequence. Notably, this is equivalent to a plot of correlation vs actual maximum echo time, so this plot captures both the factors typically associated with contrast in a fast spin echo experiment. As in the previous panels, there is no simple trend between μ(ESP) and contrast. These results are exciting in that they show echo spacing is a powerful dimension for tuning J-coupling contrast, but they highlight the need to test many candidate spacings. These simulations suggests that contrast in nonuniformly spaced spin echo trains, measured as correlation between signals, is not a simple function of mean echo spacing or maximum echo time.Finally, simulations were performed to investigate the effects of B0, T2 and B1 variability. For each contrast generating pulse sequence from Fig. As expected from theoretical considerations, signal evolution and contrast are wholly unaffected by global B0 shifts, an appropriate model for B0 in a refocused spin echo image. Since apparent T2 is easily dominated by J-coupling dynamics, changes in the underlying T2 also have minimal effect. Errors in B1 can be more significant, but for most sequences, the signal from each species changes very little. Furthermore, by using crusher gradients that select for a pure |Δp| = 2 coherence transfer, and by limiting analysis to region of interests (ROIs) with uniform B1, the influence of this confound can be reduced.The first experimental validations of this approach were tested on a series of oil mixtures made of diacetin and triacetin , which respectively contain the same glycerol backbone structures as diglycerides and triglycerides. Since commercial diacetin is a mixture of diacetin, triacetin, and monoacetin, simulation results could not be directly translated and echo spacings were tested empirically. Figure Signal vectors from pure liquids, whether arising from spin echo trains or spectroscopy, can be regarded as basis functions that can be used to decompose the bulk signal into its constituents. This approach, using the pure diacetin/triacetin signals shown in Fig. Finally, the same nonuniformly spaced echo train which distinguished diacetin and triacetin was initially tested in two subjects with prospective acquisition correction (PACE) navigator self-gating. These studies aimed only to establish feasibility of the sequence, testing both a nonuniformly spaced 16 echo sequence (a 25-6-6 pattern found useful in diacetin/triacetin studies) and a comparable CPMG (ESP = 16 ms). Figure in vitro experiments, and even preliminary in vivo experiments, the basic phenomenon that irregularly spaced echo trains can generate complicated and distinctive echo patterns that reflect chemical composition. This result is expected from long established theory, but this work is the first to explore this as a means of chemical identification. An echo spacing optimized to distinguish particular lipids or diseases could potentially generate echo patterns that, via pattern matching analysis, could generate a disease score to aid diagnosis.These studies demonstrate with simulations, 24. One potential application is in the measurement of TAG vs DAG in fatty liver. Extensive work in mice and humans concludes that while TAGs are a relatively neutral form of lipid storage, DAG accumulation in liver activates protein kinase C isoforms, directly triggering hepatic insulin resistance, which eventually leads to more serious diseases such as Type 2 diabetes and liver disease21,27, Liver biopsy samples show a stepwise trend in DAG/TAG ratios between healthy liver, benign steatosis, and nonalcoholic steatohepatitis (NASH)28. Therefore, the characterization of fat chemistry via J-coupling contrast could be an important component of a multiparametric MRI exam to distinguish benign steatosis from NASH29.Given the global rise in obesity there is increasing recognition of the role of fat in health and diseaseOne potential concern is whether fatty acid chains will complicate or overshadow the phenomenon demonstrated for glycerol backbones. Though it is subject to the same basic phenomenon, this component of the molecule has a far more complicated J-coupling structure that may be more difficult to tease apart through J-coupling dynamics. However, since the spins in the fatty acid chain primarily resonate below 4 parts per million (ppm), a crusher pulse suppressing that part of the spectrum could improve contrast based on glycerol backbones.31.Another significant concern may be whether B0 or B1 variations will confound results. Most B0 variation does not change Δω between coupled spins and is not expected to affect spin echo signal intensity at all, as we have verified with simulations. Additional signal loss due to diffusion through B0 gradients should be comparable within a given disease state and thus would be built into the signal model, though an additional diffusion or T2 map could also be used as a correction factor. Simulations do suggest that a relatively small subset of potential echo spacings exhibit significant B1 sensitivity. For human studies, an additional scan may be needed to generate a B1 map so that flip angle can be tuned to high accuracy over the ROI targeted for analysisIn summary, this work introduces a new approach to the molecular characterization of J-coupled species, such as fat. The approach has the advantage of being insensitive to B0 and easily incorporated into an imaging sequence. Irregularly spaced echo trains could be used to generate a signal vector which can be decomposed into relevant basis functions, or they can also be used as a contrast preparation module to target the echo of greatest contrast. This approach to distinguishing fats could ultimately find a broad range of biomedical applications, such as distinguishing DAG and TAG in liver, imaging lipid based therapeutics, or detecting other lipotoxic subtypes.33. Simulated experiments used a 16 echo train with echoes always acquired symmetrically about the 180. More specifically, each pulse sequence was defined by a 16 element array of delays (τi). Following the initial 90° excitation pulse, each sequence consisted of 16 concatenated modules of the form: τi /2 − 180° RF − τi /2 – echo, with the acquisition window centered around the echo, thus starting slightly before τi /2. Each echo consisted of 128 points acquired over 1.3 ms to minimize the effect of chemical shift. To test off-resonance effects on B1, the broadband refocusing pulse was replaced with a 170° pulse on H3 of Mol B and a 180° pulse on all other protons. For each pulse sequence, the array of delays was generated by a random selection from a Gaussian distribution with the indicated mean and standard deviation, taking absolute values to avoid negative delays. Each pulse sequence was simulated on each molecule, and the magnitude of the two resulting 16 echo signals was analyzed for correlation. Low correlation between the signals indicates potential for good contrast.Density matrix simulations were performed using version 1.6.2782 of Spinach using spin parameters shown in Table To test whether signals depended primarily on average echo spacing in an irregularly spaced sequence, density matrix simulations were performed for sets of ten sequences, all with 16 echoes whose spacings were randomly chosen from a single Gaussian distribution , and standard deviation, with absolute values taken to avoid negative delays). Each sequence was simulated on two 5-spin molecules modeled based on the glycerol backbones of DAG and TAG, referred to as Mol A and Mol B, respectively. Contrast between the species was quantified as correlation between the two echo-train signals. The CMPG sequence was included as the case where StdDev(ESP) = 0. For every sequence that generated correlation <0.9, the sequence was simulated again assuming a global B0 shift of +/−0.5 pp, as well as with global T2 multipliers of 0.7 to 1.3 in increments of 0.1 and with B1 discrepancies of +/−10%.All MRI experiments were performed on a 3 T Siemens Trio magnet with a birdcage receiver coil. Bottles were prepared with arrays of diacetin/triacetin oil percentages of 100/0, 50/50, 10/90, 5/95 and 0/100 and arrayed along either the x or y axis of the magnet, so that bottles would not overlap in a 1D projection. Bottles were imaged with a spin echo train with 16 echoes, repeated with 64 steps of 1D phase encoding corresponding to a 20 cm field of view (FOV). Therefore, the entire timeline of each freely evolving echo (128 points acquired over 1.3 ms per echo) was acquired from each bottle. Repetition time (TR) was 2 s, and echo time (TE) spacing was either 16 ms across the echo train, or else followed a pattern of 25-6-6. Single voxel spectra were also collected from each bottle using the single voxel spectroscopy sequence after shimming on each volume individually. Other scan parameters for the spectroscopy experiment were TR/TE/Ns/BW/FOV = 2 s/30 ms/512/1 kHz/2 cm × 2 cm × 2 cm. The entire experiment was repeated on a different day with bottles placed in a different order and orientation. Signals from each bottle were then quantified by decomposition using the signals from pure diacetin and triacetin acquired in the first experiment.For human images, subjects were imaged on the same 3 T scanner with PACE navigator gating monitoring the diaphragm lung interface. Images were acquired with a Siemens spin echo train imaging sequence, modified to acquire 16 contrasts with nonuniform timing. Intensity in each image is expected to reflect magnitude of the echo peak at each echo time. All experimental protocols were approved by the Yale Institutional Review Board (Protocol #1109009027), and informed consent was obtained from all subjects, who were adults, as described in the protocol. All procedures in these studies followed the relevant guidelines and regulations. Scan parameters were TR/SliceThickness/FOV/Resolution/BW = 3000/5 mm/38cmx30cm/128 × 51/781, using echo spacing 25-6-6 or a uniform spacing of 16 ms.


We aimed to assess the relationship between levels of a cerebrospinal fluid (CSF) marker of pericyte damage, soluble platelet-derived growth factor receptor β (sPDGFRβ) and CSF markers of blood-brain barrier (BBB) integrity  and disease pathology  in Alzheimer’s disease (AD).n = 23) in a separate neurologically normal group for which the CSF/serum albumin ratio had been determined.sPDGFRβ and albumin were measured by sandwich ELISA in ante-mortem CSF from 39 AD and 39 age-matched controls that were grouped according to their biomarker profile . sPDGFRβ was also measured in matched serum and CSF samples (p = 0.0038) and correlated positively with albumin , total tau  and phosphorylated tau  in AD but not in controls. CSF sPDGFRβ did not correlate with Aβ42. Serum and CSF sPDGFRβ were positively correlated  in the independent neurologically normal CSF/serum matched samples.CSF sPDGFRβ level was significantly increased in AD (We provide further evidence of an association between pericyte injury and BBB breakdown in AD and novel evidence that a CSF marker of pericyte injury is related to the severity of AD pathology.The online version of this article (10.1186/s13195-019-0534-8) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) and vascular dementia (VaD) together account for most cases of dementia. Cerebral hypoperfusion, neurovascular uncoupling and blood-brain barrier (BBB) breakdown are defining features of VaD, but there is now compelling evidence they are also major contributors to cognitive decline and disease pathology in the early stages of AD . PerPDGFRβ is shed from human pericytes (but not vascular smooth muscle cells) when they are cultured under hypoxic conditions (1% oxygen for 48 h) or exposed to Aβ peptides . Cell meNation et al.  reported181P and Aβ42  had previously been determined using commercially available sandwich enzyme-linked immunosorbent assays (ELISAs) . All AD patients had abnormal CSF levels of the core AD biomarkers , while controls had normal levels . The cut-off values used in this study are in line with current clinical practice and closely resemble those outlined by Hansson et al. [APOE status were not recorded, and matching serum samples were not available for this cohort.CSF sPDGFRβ and albumin were measured in 39 AD cases and 39 controls for which CSF t-tau, p-taun = 23) spanning a larger age range (23–84 years). The CSF/serum albumin ratio had previously been determined by an immunoturbidimetric albumin method on Elecsys . The demographics of this cohort are presented in Table sPDGFRβ was, however, also measured in an independent CSF and matched serum sample cohort from neurologically normal controls n = 3 spanninCSF and serum sPDGFRβ levels were measured by sandwich ELISA . CSF samples (100 μL undiluted) and serum (diluted 1 in 40 in proprietary dilution buffer supplied with the kit) were loaded. Standards, samples and blanks were added in duplicate. Absorbance was read at 450 nM in a FLUOstar OPTIMA plate reader . Reproducibility reported in the datasheet indicates an inter-assay CV < 12% and intra-assay < 10%, with spike recovery between 90 and 110% for serum, plasma and cell culture media. PDGFRβ concentration in samples was calculated by interpolation against the standard curve for each case, derived from serial dilutions of recombinant PDGFRβ . The mean values from duplicate measurements are presented.CSF albumin level was measured by sandwich ELISA (Cat no 108788) . CSF samples were diluted 1 in 2000 in proprietary dilution buffer supplied with the kit. Standards, samples and blanks were added in duplicate. Absorbance was read at 450 nM in a FLUOstar OPTIMA plate reader . Albumin concentration in each sample was interpolated from a standard curve derived from serial dilution of recombinant human albumin (200–3.125 ng/mL). The mean values are presented.t tests or ANOVA with Bonferroni post hoc correction was used for comparisons between groups, and Pearson’s test was used to assess linear correlation. Data values outside the 99% confidence interval of the linear regression line were considered to be outliers and removed prior to statistical analysis. The analyses used SPSS version 16  and GraphPad Prism version 6 . p values < 0.05 were considered statistically significant.As the CSF sPDGFRβ and albumin levels were normally distributed, unpaired two-tailed p = 0.0038)   but not in controls   Fig. c. Inspecr = 0.50, p = 0.0017) but not in controls   but not in controls   or AD cohort   or AD  groups   Fig. a. CSF sP85) Fig. b.Fig. 2We have confirmed that CSF sPDGFRβ is increased in AD, to an extent that correlates with CSF albumin level (a marker of BBB leakiness) and with the neurodegeneration biomarkers CSF t-tau and p-tau. We have also shown that the level of sPDGFRβ in CSF correlates with that in serum in an independent cohort of matched CSF/serum samples. Our findings support recent reports of pericyte injury and BBB leakiness in AD, as demonstrated in histological and biochemical studies of autopsy tissue  and in bF7/F7 mice, which have disrupted PDGFRβ signalling, have a more pronounced vascular phenotype associated with vascular loss and BBB leakiness [+/− heterozygous mice were crossed with Tg-APP mice, there was accelerated Aβ accumulation and clinical disease progression [Post-mortem studies have confirmed pericyte loss and BBB leakiness in AD , 10. Redeakiness , and whegression , supportgression .Compromised BBB integrity that can be demonstrated in the hippocampus in normal ageing and is exacerbated in MCI, as revealed by advanced dynamic contrast-enhanced MRI, is associated with elevated CSF sPDGFRβ . Nation APOE genotype [The mechanisms of pericyte injury in AD remain unclear, but both Aβ and hypoperfusion are probably contributors. Aβ peptides at supraphysiological concentrations were shown to be toxic to human brain pericytes , dependegenotype . The toxgenotype . In a bigenotype . The preAPOE genotype, that would be of interest for further analysis. This is also an observational cross-sectional study and does not provide clues as to the timing and regional changes in pericyte loss in relation to onset and progression of disease. The extent of vascular burden within our cohort is also unclear, and as in other clinical studies, it is likely to include a spectrum of AD and mixed dementia cases. Other variables that related to the collection of the samples and might theoretically impact on the measurements  were also not available for analysis. Nonetheless, our data together with recent studies suggest that markers of vascular dysfunction, including pericyte loss and BBB leakiness, are related to cognitive impairment in MCI and disease pathology in AD and can potentially be monitored by analysis of CSF and possibly also serum. The inclusion of vascular biomarkers as part of an AD research framework, as suggested by Sweeney and colleagues [This study has several limitations including the small number of AD cases and controls and the paucity of clinical details relating to the de-identified AD cases, such as dementia severity, disease duration and lleagues , would iAdditional file 1:Figure S1 CSF-sPDGFRβ is not altered in relation to age or gender. (a) Scatterplot showing no statistically significant relationship between CSF-sPDGFRβ level and age in AD (red) and control (green). The best-fit linear regression line and 95% confidence interval for each group are superimposed. (b) Bar chart showing CSF-sPDGFRβ level in control and AD group stratified for gender. No significant differences were observed. Bars represent the mean ± SEM. (DOCX 209 kb)


Weak materials in seismic slip zones are important in studies of earthquake mechanics. For instance, the exceptionally large slip during the 2011 Tohoku-Oki earthquake has been attributed to the presence of smectite in the fault zone. However, weak fault rocks cannot accumulate large amounts of elastic strain, which is thought to counter their ability to enhance seismic rupture. It is well known that if the permeability of a weak fault is low enough to allow friction-induced thermal pressurization of interstitial fluid, the fault strength decreases dramatically. However, whether intrinsic weakness of fault material or thermal pressurization more efficiently produces large slip on faults bearing weak materials has not been determined. To investigate the role of weak materials in earthquake rupture dynamics, we conducted friction experiments and dynamic rupture simulations using pure smectite and pure graphite to represent weak fault materials. Even when we assumed no thermal pressurization, simulated faults in both media were able to trigger large slip because their extremely low friction was insufficient to arrest the inertial motion of rupture propagating along the fault. We used similar rupture simulations to investigate the cause of the huge slip near the trench during the 2011 Tohoku-Oki earthquake and demonstrated that it can be attributed to thermal pressurization, although our findings suggest that the presence of smectite in the plate-boundary fault may also be required. Because weak materials such as phyllosilicates and graphite have low frictional resistance4, their presence is thought to weaken faults in the brittle regime7 and to account for fault creep such as that observed along the San Andreas Fault in California7. The exceptionally large fault slip near the axis of the Japan Trench (50–80 m) during the 2011 Tohoku-Oki earthquake9 has been attributed to the presence of low-friction smectite12. However, there is evidence that clay minerals cannot accumulate sufficient elastic strain/stress to cause such a large fault slip13 and that their presence also stabilizes the slip14. The lack of seismicity and fault rupture along the shallow parts of plate subduction boundaries  has been attributed to the presence there of subducting clayey sediments16. However, recent friction experiments simulating subduction driving rates of centimetres per year revealed unstable slip behaviour in clayey faults that would favour earthquake rupture17. Despite numerous studies during recent decades on the effect of weak materials on earthquake mechanisms, it is still not well understood.Our knowledge of the frictional strength of faults, which is the predominant control of rupture dynamics and earthquake magnitude, has benefited from almost half a century of laboratory experiments on rocks18) , we discovered a graphite-bearing slip layer within a fossil subduction-boundary megathrust in the Shimanto accretionary complex, Japan  in earthquake rupture dynamics. To investigate a smectite-bearing slip zone we numerically modelled a plate-boundary thrust in the Japan Trench (ca. 40–80 wt.% bulk smectite content18) Fig. . Althougpan Fig. . The slitra Fig. . The cartra Fig. . In conttra Fig. . Thus, bτ) was plotted versus slip (D) under 1.5 MPa normal stress (σn)  by averaging the values of τ/σn, and those at the fast slip rate by power-law fitting according to the relationship τ = (µd − (µd − µp)exp(−D/Dc))σn, where µd and µp are dynamic and peak friction coefficients, respectively, and Dc is critical slip distance . The resultant shear stress (σn) Fig. . We deteτ) on normal stress (σn) showed high linearity. We determined the friction coefficients, µi, µp, and µd of smectite to be 0.055, 0.064, and 0.018, respectively  to a realistic fault geometry of the Japan Trench  we used the values we obtained in our friction experiments on smectite and graphite. An earthquake nucleation procedure was manually invoked at 11 km depth (see Methods) and the results of the computation showed the direction of rupture propagation and the evolution of slip along the fault. The dynamical model parameters are summarized in Supplementary Fig. To understand how low friction of weak materials affects the process of seismic rupture and the amount of slip, we simulated two-dimensional dynamic rupture propagation on the basis of a slip-weakening friction power lawnch Fig. , a unifoµi were assumed to be lower than those of µd , which are higher than those for our pure smectite, probably because the fault rocks in the Japan Trench contain high-friction components such as quartz and feldspar (ca. 20 wt.%)11. However, if thermal pressurization occurred during frictional slip, fault strength would be dramatically weakened26. Coefficients of friction of µp = 0.170 and µd = 0.005 for fault rocks in the Japan Trench have been determined on the basis of thermal pressurization modelling and the in situ physical properties of the rocks18. Undrained friction experiments, in which thermal pressurization is likely to occur, have also shown ultralow friction: µp = 0.1 and µd = 0 (ref.11), µp = 0.12 and µd = 0.07 (ref.27). Thus, thermal pressurization can play an important role in fault slip, independent of the intrinsic frictional properties of the rocks. We therefore evaluated the evolution of slip near the Japan Trench with and without thermal pressurization.On the other hand, friction coefficients previously determined for plate-boundary fault rocks in the Japan Trench are 28, we focus here on the evolution of slip near the trench because the material properties of rocks at depths of around 24 km are unknown. By applying the spectral-element method21 to a realistic fault geometry of the Japan Trench  estimated by the Japan Meteorological Agency for the 2011 Tohoku-Oki earthquakench Fig.  together9). However, the case without thermal pressurization showed almost zero slip along the fault 11 was higher than µi (0.090)29. If the dynamic shear stress is higher than the initial shear stress, forward propagation of the rupture front becomes difficult30. Thus, we suggest that thermal pressurization  was the principal mechanism that produced the large slip near the trench during the 2011 Tohoku-Oki earthquake. The resulting frequency spectra for slip rate near the trench  indicated that unstable large slips can occur in segments that usually creep23. However, our study provides the first clear evidence that, in addition to coseismic weakening, extremely low friction of fault materials can be a principal mechanism for producing exceptionally large fault slips. Differences in the amounts of weak materials in localized slip zones within a fault might determine whether fault slip is enhanced or reduced, because more weak material promotes lower friction .Fault creep has been attributed to the presence of weak materials in fault rocks, for instance, talc-bearing serpentinite or smectite in fault gouge in the San Andreas Fault22 for a range of slip rates including ultralow subduction-driving slip rates17, and more comprehensive modelling is needed taking into consideration both nucleation physics and rupture propagation simulations similar to those presented here.The results of our integrated analyses of frictional properties of fault materials and dynamic rupture simulations will contribute to future quantitative evaluations of near-trench slip at plate boundary faults and may be applicable in future earthquake disaster mitigation studies in subduction zone settings. To further our understanding of the entirety of earthquake mechanics  in faults bearing weak materials, further research is needed on frictional stability as expressed by the rate-and-state friction law11. Time series of shear stress profiles (slip) were recorded at slip rates of 2 × 10−4 (slow) and 1 m s−1 (fast) after correction for friction between the sandstone cylinders and the sleeve. Axial loads applied during rotary shearing were 0.5, 1.0, 1.25, or 1.5 MPa.A rotary shear apparatus was used for our friction experiments , established by experimental rock friction results at relatively low slip rates22, has been thoughtfully described in previous research18. Although RSF has been applied to explain fault behaviour during shallow, slow earthquakes along plate interfaces35, dramatic weakening  can dominate RSF weakening during earthquake nucleation at slip speeds in excess of roughly 10−4 to 10−2 m s−1 (ref.36). During dynamic rupture, such high slip rates are rapidly exceeded soon after the passage of the rupture front, so RSF weakening is not dominant during the dynamic phase of seismic slip on crustal faults. Therefore, we applied a slip-weakening friction law in our modelling.We used the spectral element method (SEM2DPACK)18. All model parameters are summarized in Supplementary Figs To manually nucleate rupture at 11 km depth, we used negative values of strength excess : −5 and −10 MPa for the smectite and graphite faults, respectively. The strength excess of −10 MPa we used at 11 km depth was the same as that used at that depth in a previous analysis of megaquakes in the Japan TrenchSupplementary Information


Low-loss waveguides are required for quantum communication at distances beyond the chip-scale for any low-temperature solid-state implementation of quantum information processors. We measure and analyze the attenuation constant of commercially available microwave-frequency waveguides down to millikelvin temperatures and single photon levels. More specifically, we characterize the frequency-dependent loss of a range of coaxial and rectangular microwave waveguides down to  Interconverting the quantum information stored in stationary qubits to photons and faithfully transmitting them are two basic requirements of any physical implementation of quantum computation . CoherenIn the field of circuit quantum electrodynamics, experiments show the ability to use single itinerant microwave photons , 16 or jIn addition, studying the reduction of loss of superconducting waveguides has the potential to contribute to improving the fidelity of qubit state measurements  by minimPrevious studies of the attenuation constant were performed for different types of superconducting coaxial cables down to urements –31. In tIn this paper, we study the loss of coaxial cables and rectangular waveguides using a resonant-cavity technique from which we extract attenuation constants down to μm on the inner surface. All three rectangular waveguides are manufactured by Penn Engineering Components, Inc. [We evaluate the attenuation constant of coaxial and rectangular waveguides made by a number of different manufacturers from a range of materials, see Table poration  used a lmentclass2pt{minimax, Inc. . As a reax, Inc. . We invets, Inc. . Table 1We construct resonators from coaxial cables and rectangular waveguides as shown in the photographs and schematics of Figure At room temperature (RT) we use a vector network analyzer (VNA) and a through-open-short-match (TOSM) calibration to set the measurement reference plane to the input of the coupling ports of the waveguide according to the schematic presented in Figure mentclass2pt{minimThe waveguides are characterized at a nominal temperature of mentclass2pt{minimFor the measurements at base temperature BT  resonances.Under this condition it is sufficient to extract ckground  most reln. The fundamental frequency l, the relative permittivity of the dielectric c. The internal quality factor αα can be written as a sum of conductor loss δ. For a coaxial line a and the inner radius of the outer conductor b. To characterize the conductor loss of coaxial cables combining different materials for the center and outer conductors we introduce an effective surface resistance δ from measurements of To determine the frequency dependence of the attenuation constant ument}Qi 3\documeThe surface resistance of a normal conductor ment}1/σ . The thement}1/σ –42 showsn (Table The measured external quality factors at RT (Figure mentclass2pt{minimThe frequency dependence of the measured quality factors for CC141Al presented in Figure entclass1pt{minimaFollowing the same measurement procedure, we extract the quality factor of low-loss superconducting cables (e.g. see Figure nductors –42. Measδ from fitting Eq. (δ of the Keycom ldPTFE from the fit assuming δ of the ldPTFE of Micro-Coax and Keycom differ by a factor of ∼10. We extract the relative permittivities entclass1pt{minimating Eq.  to the mentclass1pt{minimal. The frequency dependent internal quality factor is [We performed similar measurements with three different rectangular waveguides of type WR90 (see Table mentclass2pt{minimmentclass2pt{minimactor is 8\documeWe have presented measurements of the attenuation constant of commonly used, commercially available low-loss coaxial cables and rectangular waveguides down to millikelvin temperatures in a frequency range between 3.5 and nductors , 40. We Our results indicate that transmitting signals on a single photon level is feasible within laboratory distances, e.g. 95% of the signal can be transmitted over distances of δ strongly dependents on the PTFE composite where Comparing our results to recent measurements of high quality 3D cavities  with quaeratures , 47 abou


Unwanted cells within cell population are targeted with MMWs in order to kill them by overheating. The instrument rapidly (within 2–3 seconds) heats a cell culture area of about 500 µm in diameter to 50 °C using a low-power W-band (94 GHz) MMW source. Heated cells in the area detach from the substrate and can be removed by a media change leaving a bare spot. Hence we named the instrument “CellEraser”. Quick, local and non-contact heating with sharp boundaries of the heated area allows elimination of the unwanted cells without affecting the neighboring cells. The instrument is implemented as a compact microscope attachment and the selective hyperthermic treatment can be done manually or in an automated mode. Mammalian cells heated even momentarily above 50 °C will not survive. This “temperature of no return” does not compromise cellular membranes nor does it denature proteins. Using the CellEraser instrument we found that the key event that determines the fate of a cell at elevated temperatures is whether or not the selectivity of its nucleus is compromised. If a cell nucleus becomes “leaky” allowing normally excluded (cytoplasmic) proteins in and normally nuclear-localized proteins out, that cell is destined to die. Quick heating by MMWs to higher temperatures (70 °C) denatures cellular proteins but the cells are not able to detach from the substrate – instead they undergo a phenomenon we called “thermofixation”: such cells look similar to cells fixed with common chemical fixatives. They remain flat and are not washable from the substrate. Interestingly, their membranes become permeable to DNA dyes and even to antibodies. Thermofixation allows the use of western blot antibodies for immunofluorescence imaging.As an alternative to laser-based methods, we developed a novel  In these methods a high power laser beam  is used to either sweep over the surface to lethally illuminate the unwanted cells3, or to cut out the cells of interest and physically separate them from the remaining cells5.Existing methods for targeted elimination of anchorage-dependent cells generally utilize laser technology such as laser ablation and laser microdissectionin situ photothermal laser processing was briefly commercialized in the 1980s6 but is no longer available. In this approach the cells were grown on a thin heat-absorptive adhering film. A laser was used either for direct cell irradiation and photothermal elimination or as a “cutter” when the desired cells were circumscribed with the laser to cut the film, followed by peeling of the film to remove undesired cells, leaving behind an island of desired cells on the surface. Thus the undesired cells were eliminated, and the selected cell population remained on the incised film disks adhered to the culture chamber where growth and proliferation can continue.One promising cell isolation technology based on in situ cell isolation called LEAP™  was commercialized by Cyntellect, Inc. in early 2000s7. Depending on the laser power and wavelength used, the instrument could irradiate and eliminate the unwanted cells either by photothermal or photochemical mechanisms. The phototermal approach required the presence of Allura Red AC dye in the cell culture to enhance the light energy absorption and resulted in immediate protein coagulation and cell necrosis. There are other examples of the use of laser irradiation in the presence of a chromophore to destroy cells in vitro2 and in vivo9. The dye-free photochemical approach used more laser power but did not lead to immediate destruction of targeted cells - instead it induced apoptosis over a period of 4 to 24 hous. The LEAP instrument is currently not on the market.Another laser-based instrument for 10. The LMD system is based on an upright microscope and focused laser is used for cutting plastic films where adherent cells are growing. The dissected piece of plastic with living cells on it falls by the force of gravity into a collection container where subsequent recultivation is possible as well as the downstream analysis of the extracted cells. The LMD is claimed by the manufacturers to be a contact- and contamination-free method for isolating specific single cells or areas of tissue from tissue samples.The only laser-based technique commercially available currently is a laser microdissection method, also known as LMD or LCM (laser capture microdissection)11. Also there is a leakage of cellular content into the culture medium. These aggressive byproducts harm the desired cells. The microdissection approach is not sterile and requires special consumables. All laser-based methods need special and expensive equipment, precise laser optics adjustment and in some cases, addition of special light energy absorbing dye to the cell media to intensify the cell damage. The majority of these methods cannot be used on a standard microscope and cannot be performed by an inexperienced operator.The shortcoming of the laser ablation approach is that a lot of free radicals and products of oxidation are formed during the ablation processin situ cell isolation method where selective hyperthermic damage is used to target and eliminate the unwanted cells12. The damage is done by local heating of adherent cell culture using millimeter wave (MMW) radiation. Cells heated above 50 °C round up, detach from the substrate and can be washed out by a media change as if the culture was “erased” – that is why the instrument is called CellEraser™. The CellEraser treatment could be repeated until all unwanted cells are removed from the culture without affecting the desired cells. Implemented as a compact microscope attachment, the instrument provides an easy, convenient, quick and inexpensive way for cell culture manipulation and cell isolation.As an alternative to LEAP or LMD techniques, we developed a novel 12 employs millimeter waves - MMW, electromagnetic radiation with the half-wavelength of about a millimeter or about 150 GHz in frequency. We used MMWs in the W-band that ranges from 75 to 110 GHz. The method is based on unique properties of MMW radiation exhibited upon interaction with water. When a millimeter wave meets a water phase, it cannot penetrate into the bulk of the water. Instead, all the MMW energy is released as heat in a narrow surface layer – for example, 94 GHz MMW have water penetration depth of about 0.36 mm13. The frequency of 94 GHz was chosen as it is most efficiently absorbed by water  of a long-chain alcohol (LCA) with a known melting point temperature is used as a local temperature sensor: melting of the film indicates that the LCA melting point temperature was reached. Table 17). The LCA film melts when the culture medium intercalated into or directly above the film is heated via application of the millimeter wave radiation. Dry film could not be melted by MMWs as absorption of millimeter wave radiation by LCA, higher alkanes or oils is negligible13.The calibration procedure is performed under conditions identical to the conditions at which the cells would be treated: the same type of cultureware is used, the internal surface of its bottom is coated with the LCA film, and the same volume of the same culture medium is added  abruptly lost the selectivity in their permeability, i.e. the cell nuclei became leaky. We used fluorescent proteins (FP) fused to a nuclear localization sequence (NLS) as reporters to detect the loss of NE selectivity similarly to previously published workasm Fig. . On the asm Fig. . Supplem24, remain nuclear even after RFP-NLS equilibrates between the nuclear and cytoplasmic compartments in the heated area  started to round up and to lose their anchorage to the substrate, and finally detached from it within 1–2 hours . These cells never divided and eventually died through apoptosis. The effect was reproducible in a number of immortal and stem cell lines (data not shown).27 . At 42 °C mammalian cells can survive long term28. Figure Rising temperatures was shown to change cell membrane fluidity and permeabilityHowever, DNA bound, cytoskeletal or other FP fusion proteins that were not free-floating inside the cell and could not leak out of it, were still fluorescent after brief exposure to 60 °C that caused membrane rupture and leakage of cytosolic proteins. For example, GFP-53BP1 did not lose its fluorescence and was localized in the nuclei while RFP leaked out  in a cell can be detected by monitoring GFP-53BP1 distribution pattern because as a part of the DNA repair machinery this protein forms bright foci at the sites of DSBsFigure 30. When cells were induced to detach by trypsin or EDTA, Trask phosphorylation started to occur at the onset of cell rounding before detachment, peaked when cells were completely detached and was maintained for prolonged periods as long as cells were kept in suspension29. If Trask phosphorylation is forcibly maintained by overexpression, it leads to the loss of cell adhesion and a detached phenotype30. We hypothesized that the heat-induced nuclear leakage followed by the cell rounding and detachment may also include Trask tyrosine phosphorylation consistent with its role in the negative regulation of cell adhesion.We noticed that the heat-induced cell rounding and detachment from a substrate is similar to the detachment of adherent cells during mitosis – a process during which adherent cells must rapidly sever cell–surface and cell-cell engagements to execute mitotic functions. It was reported that a transmembrane glycoprotein Trask (CDCP1) is tyrosine phosphorylated by src kinases during the cell-detachment phase of mitosis. The phosphorylation of Trask was tightly linked to cell retraction from the substrate and associated with anchorage loss in epithelial cells32 or 74333 of Trask. The only commercially available p-Trask antibodies  were developed for western blot applications that require denaturing of the protein sample by heating it to 95–100 °C for 5 min to expose the epitope. We tried to immunostain p-Trask in trypsin-induced or heat-induced rounded/detached cells using pY743-Trask western blot antibody after a standard paraformaldehyde fixation and Triton X-100 permeabilization but could not detect any signal (data not shown). Next we decided to repeat the experiment using “thermofixation” instead of the chemical fixation to denature p-Trask and make the pY743 epitope sequence accessible to pY743-Trask antibody.Published methods of phosphorylated Trask (p-Trask) detection were based on custom p-Trask antibodies that were made by immunizing rabbits with a phospho-peptide immunogens containing sequences centered around phosphorylated tyrosine 734Figure Our data confirmed Trask phosphorylation in cells heated to 50 °C for 2–3 seconds and suggest that Trask plays an important role in cell retraction, rounding and loss of anchorage in response to heat. We also demonstrated that “thermofixation” enables the use of western blot antibodies for immunofluorescence.We have developed a novel and unique cell manipulation technology that uses millimeter wave radiation for local heating of adherent cell cultures. It allows elimination of unwanted cells in the cultures by brief local heating to 50 °C that leads to selective hyperthermic damage first seen as cell detachment. The technology is implemented as a device, CellEraser - a compact microscope attachment that can remove or “erase” the unwanted cells from an adherent cell culture in an automated manner.in situ elimination of unwanted cells in adherent cell culture while it is propagatingAllows Controls the temperature and the size of the heated spotDoes not heat the bulk culture medium or the cultureware materialNon-invasive: does not compromise culture sterility, does not require addition of chemicalsDoes not affect the desired cells (no shear stress), does not induce immediate lysis of unwanted cellsWorks consistently among different mammalian cell typesCan be used for cells grown in standard cultureware34, etc.)Inexpensive alternative to other cell isolation systems , limiting dilution37, and microfluidics38. All of these techniques have limitations especially when applied to extremely rare or fragile cells or cells that exhibit low efficiency of outgrowth from a single-sorted cell. In situ cell isolation by CellEraser could overcome these issues as the desired cells are not affected in any way during the “erasing” process. The unwanted cells could be temporary left “unerased” on the periphery of the culture, away from the desired cells, to constantly condition the media and help the desired cells reach densities high enough when it is safe to eliminate all of the unwanted cells.There are no other commercially available systems/techniques that can non-invasively eliminate unwanted cells from adherent cell cultures. Current cell isolation and cell cloning approaches are based on flow sorting (FACS - fluorescence activated cell sorting), laser microdissectionPurification of induced pluripotent stem (iPS) cells and embryonic stem (ES) cells cultures from differentiating cells or isolation of differentiated cellsSingle-cell cloning by eliminating all unwanted colonies and leaving only one colony in a wellGeneration of tumor cell cultures originated from cancer patient tumor biopsies by removing the non-cancer cells to prevent them from taking over the cultureIf combined with a high content imager equipped with environmental control, the instrument can allow manipulation of the growing cultures in a high throughput modeA novel gap-creation method for wound healing assaysHeating a culture area to sub-lethal temperatures (40–43 °C) for studies of thermo-activated cancer drugs, hyperthermia effects on cancer vs normal cells in culture, etc.Possible applications of the instrument:We also showed that CellEraser provides a simple and fast alternative to conventional chemical fixation - “thermofixation”, a short-term MMW heating of a small area in a live cell culture to 70 °C Fig. . Conventin situ cell isolation, such technique could be used for local temperature-activated enzymatic sampling of desired cells (e.g. genotyping of multiple single-cell colonies grown in the same well/dish).We are planning to facilitate the cell “erasing” by treating the cell culture with MMW radiation at the presence of cold (room temperature) trypsin or other digestive enzymes whose activity is low at 20–22 °C. MMW heating a spot in the cell culture to 38–40 °C should result in a constant supply of active (hot) trypsin to the spot due to the convection flow. This will accelerate cell detachment in the heated area and should enable observing the “erasing” in real time, maybe within tens of seconds. The detached cells will not be killed in the process. They can be collected, analyzed (if necessary) and propagated. In addition to We used MMW radiation to study the effect of quick and brief heating on mammalian cells. To control the temperature we applied a simple and reliable method of thermometry based on melting of LCA films formed on the cultureware bottom: melting of the film indicated that the LCA melting point temperature was reached Fig. .We demonstrated that nuclei of the cells heated to 50 °C for 2–3 seconds became “leaky” Fig.  though t39. The authors relied on standard live-dead assays that indicate the enzymatic activity and/or the plasma membrane integrity, and the viability of the cells was assessed shortly after the heat exposure. The problem is that the heat effects could result in a delayed cellular damage and could take hours to develop. So the cell viability should be checked next day to judge about the effects of elevated temperatures.It was reported that mammalian cells can survive temperatures of about 65 °C with exposure time not exceeding 5 secondsOur main observation was that the cells were unable to recover after a thermal stress if such stress led to a loss in nuclear envelope selectivity – the earliest indicator of irreversible cell damage. To our knowledge, this finding was never reported before. The heat-induced NE leakage was observed as a loss in compartmentalization of intracellular proteins causing mislocalization of both nuclear and cytoplasmic protein components Fig.  within tThe main NE component is the nuclear double membrane that encapsulates the nuclear genome, regulates all nuclear trafficking of RNAs and proteins and prevents the passive diffusion of macromolecules between the nucleoplasm and the cytoplasm. Though we did not investigate the mechanisms of the NE selectivity loss, there are at least two possibilities: 1) rupture of the NE itself ; 2) loss of the nuclear pore complex (NPC) selectivity.40. Authors showed that NE of cells underwent repeated ruptures that allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. Nuclear membrane protrusions (“blebs”) typically formed at sites where the nuclear lamina, particularly the lamin B1 network, was weak or absent. Depletion of the lamins A, C and B2 significantly increased the likelihood of NE rupture, which together with previous studies42 suggest that lamins are important for stabilizing the NE. Authors also showed that DNA double-strand breaks (detected as rapid formation of new RFP-53BP1 foci) coincided with the NE opening events.The NE rupture was observed in cancer cells during cell migration through confining spacesOur observations of cells expressing FP-tagged lamin B1 (BFP-lamin B1) did not reveal any significant changes in BFP-lamin B1 organization in cells whose nuclei became “leaky” after heating to 50 °C compared to the neighboring unheated cells with non-leaky nuclei Fig. . Monitor43. Authors showed that NPC scaffold members did not turn over and had life-long residence at the NPC in non-dividing cells suggesting an idea that the presence of the same nucleoporins during the entire life span of post-mitotic cells comes at the physiological cost of ageing-related pore defects that lead to a loss of nuclear integrity43. A leakage of cytoplasmic proteins into the nucleus was also observed when NPCs were partially dismantled during mitosis44.Then the hypothesis of heat-induced NPC damage looked more probable. It assumed that the mislocalization of nuclear and cytoplasmic proteins happened through “leaky” nuclear pores. It was reported that ageing-related deterioration of NPCs weakened the nuclear permeability barrier and lead to leakage of cytoplasmic proteins into the nuclear compartment45. In our studies the cells were heated to higher temperatures that induced the irreversible loss of NE selectivity. We hypothesize that these temperatures triggered a rapid loss of the NPC functions that collapsed the macromolecular gradient across the nuclear membrane allowing the cytoplasmic proteins to enter the nuclear compartment and the nuclear proteins to exit into the cytoplasm. Since the biogenesis of the NPCs is restricted to the mitotic stages and ceased upon cell cycle exit43, the heated cells were not able to repair the leakage of the heat-damaged nuclear pores to restore the macromolecular gradient and self-terminated through apoptosis.It was shown that mammalian cells could survive incubation at 43 °C for 1 hour but their conventional nuclear import was suppressed due to nuclear accumulation, nuclear retention, and recycling inhibition of importin α29, respectively). The phosphorylation of Trask leads to an anchorage loss and a detached phenotype30. Although we did not study the mechanisms involved in heat-induced cell detachment, we hypothesize that initial cell reaction to heat have common features with mitotic detachment in terms of changes in cell physiology. However, the fate of heat-detached cells is quite different from dividing cells: they are not able to reattach back to the substrate, the lost NE selectivity never gets restored , and the cells eventually die through apoptosis : DAPI (D9542), YO-PRO™−1 Iodide , Calcein AM (C1359), Etoposide (E1383). Description of long-chain alcohols (LCAs) used is summarized in Table Human B lymphoblast RL (CRL-2261), lung carcinoma A549 (CCL-185), and osteosarcoma U2OS (HTB-96) cell lines were obtained from American Type Culture Collection (ATCC). Triple-tagged U2OS cell line  was obtained from Sigma-Aldrich. RL and A549 lines were grown in RPMI-1640 medium (R0883), U2OS lines – in McCoy′s 5 A medium (M8403. Both mediums were supplemented with 2 mM L-glutamine (G7513) and 10% fetal bovine serum (F2442). Cells were grown in 5% CO2 in air atmosphere, 37 °C. Trypsin-EDTA solution (T3924) was used for sub-culturing.BFP and TagBFP, GFP and TagGFP2, and RFP and TagRFP are synonymous for the fluorescent reporter genes/proteins in this manuscript. The BFP, GFP and RFP are monomeric fluorescent proteins originated from Evrogen and referred to as TagBFP, TagGFP2 and TagRFP respectively. Vectors pTagRFP-N (FP142) and pTagBFP-N (FP172) were obtained from Evrogen. NLS-RFP construct was made by inserting the NLS sequence (APKKKRKVGIHG) into the pTagRFP-N vector. In the case of GFP-53BP1, GFP is referred to as eGFP originated from eGFP-53BP1 plasmid .For transient transfection, the plasmids (1 µg) were nucleofected into 1 million cells with the Amaxa® Nucleofector® device . Nucleofector® Kit T (VCA-1002) for A549 cells and Kit V (VCA-1003) for U2OS cells were used along with program X-001 according to the product manual.26.We used standard live/dead dyes to assess two recognized parameters of cells viability: intracellular esterase activity  and plasma membrane integrity (impermeable DAPI). Leakage of expressed NLS-RFP also served as an additional confirmation of any damage to the membrane. A green-fluorescent YO-PRO-1 stain was used as an early marker of apoptosisFor standard fixation, cells were fixed for 15 minutes with 4% Formaldehyde prepared from 16% stock  diluted 1:4 volume per volume (v/v) with 1x PBS  to 4% Formaldehyde, then the cells were permeabilized for 15 minutes with 1x Permeabilization Buffer . For “thermofixation”, cells were quickly heated to 70 °C for 2–3 seconds using MMW radiation. The next steps were common for both types of fixation. The cells were blocked for 30 minutes with 1x Block Buffer . Primary phospho-Tyr743-CDCP1 (D2G2J) rabbit antibody  1:300 (v/v) diluted in 1x Block Buffer was applied to the cells and incubated for 1 hour at room temperature. Next the cells were washed 2 times with 1x Block Buffer and goat anti-rabbit IgG H&L conjugated with Alexa488  diluted 1:200 (v/v) was used as the secondary antibody for 1 hour incubation together with 0.5 µg/ml DAPI at room temperature. Then the cells were washed 2 times with 1x PBS and imaged.In Vivo Scientific, Product No: CH.HC5.SAT) was used to control the environment. Filtersets were DAPI (Ex 340–380/em 435–485); BFP (ex 395–410/em 430–480); YO-PRO-1, Calcein, GFP, Alexa488 (ex 450–490/em 500–550) and RFP (ex 530–560/em 590–650).Images were acquired in brightfield, phase contrast, differential interference contrast (DIC) or epi-fluorescence modes either on a Leica DM2700 upright microscope equipped with a Leica MC120 HD color camera or on a Nikon Eclipse Ti inverted microscope equipped with a Photometrics CoolSNAP ES2 cooled CCD camera. Leica 4×/0.10 objective was used on the upright microscope and Nikon 4×/0.13, 10×/0.30, 20×/0.75 dry and 40×/1.30 oil objectives were used on the inverted microscope. The camera exposure time did not exceed 1 s for epi-fluorescence and 50 ms for all other imaging configurations. Micro-Manager or MetaMorph® software was used for image acquisition and automation. For imaging, cells were grown in Ibidi cultureware (ibidi GmbH): µ-Slide 8 Well  without (Cat.No: 80826) or with 500 µm grid (Cat.No. 80826-G500); µ-Dish 35 mm, high glass (# 1.5 H) bottom without (Cat.No: 81158) or with 500 µm grid (Cat.No: 81168). Cells were imaged live (unless specified otherwise) in Hanks balanced salt solution (H8264) supplemented with 2% fetal bovine serum (F2442). Full microscope enclosure incubator . To control the distance between the output waveguide and the bottom of the cell cultureware, the waveguide was moved upwards until it touched the cultureware bottom. The touch position was detected via microscopy as image movement. Then the waveguide was withdrawn about 200 µm downwards and that distance was maintained during the calibration procedure and all subsequent experiments.The instrument is available from Cells exposed to elevated temperatures (50–55 °C) start to round up and lose the attachment to the substrate (similarly to cells in late stage of division). Those cells can be removed by a simple washing procedure: cell medium is removed by aspiration, Hanks balanced salt solution (H8264) is added and aspirated, and the medium is returned. Depending on the cell type, the washing step can be repeated twice to be sure that all cells are removed from the MMW treated area.Supplementary Figures and Supplementary Movie captions.Supplementary Movie 1 : Convection induced by CellEraser: application of millimeter wave radiation to a cell suspension (RL) imaged on an upright microscope.Supplementary Movie 2: Melting a spot in 1-HXD film using the upright microscope version of the CellEraser.Supplementary Movie 3: Melting a spot in 1-HXD film using the inverted microscope version of the CellEraser.Supplementary Movie 4: Automated ring melting in a 1-HXD film using the upright microscope version of the CellEraser.Supplementary Movie 5: Automated ring melting in a 1-HXD film using the inverted microscope version of the CellEraser.Supplementary Movie 6: “Erasing” a spot in a confluent A549 cell culture by local MMW mediated heating to 50°C for 2 seconds with washout 2 hours later.Supplementary Movie 7: Gap closure in a confluent A549 culture after CellEraser treatment.Supplementary Movie 8: Long-term cell fate after 50°C exposure.