These results confirm the viability of C,C-η2 bound intermediates in CTP of thiophenes, providing an electronic rationale for the stability of such species, and thus that such processes can proceed as living polymerizations. We further show that C,S-κ2 species may also be relevant in nickel-catalyzed CTP of thiophenes, providing new avenues for exploitation and optimization.Nickel-catalyzed catalyst transfer polycondensation (CTP) of thiophenes is an efficient strategy for the controlled synthesis of polythiophenes. However, a detailed view of its reaction mechanism has remained elusive with unresolved questions regarding the geometry and bonding of critical Ni(0) thiophene intermediates. Herein, we provide experimental and computational evidence of structurally characterized square planar  η2-Ni(0)–thiophene species and their relevance to the mechanism of catalyst transfer polycondensation.The mechanism for nickel-catalyzed catalyst transfer polycondensation of thiophenes to polythiophenes. Here the authors show experimental and computational evidence of structurally characterized square planar  Polythiophenes have been widely explored as building blocks in both electronic and optical molecular devices due to their efficient electronic and thermal conductivity as well as their high quantum efficiencies6. Among current strategies for polythiophene synthesis, nickel-catalyzed catalyst transfer polycondensation (CTP) has proven particularly effective in the production of polythiophenes with high-molecular weights, regioregularity, low polydispersities, and end-group control9. The mechanism of thiophene CTP has attracted significant attention since it was first shown that it proceeded via living chain-growth polymerization13, which requires that the metal catalyst must remain attached to the nascent polymer chain throughout the process  intermediates have been characterized (I and II)16, the identity of the postulated Ni(0) intermediate (III) has remained elusive.The much anticipated revolution from bulk to molecular electronics hinges upon efficient and controlled exploitation of appropriate molecular transistors, diodes, integrated circuits, and optoelectronic devices-η2 Ni(0) structures17. Such structures are consistent with computational18 and experimental20 data, but conclusive structural evidence is lacking. However, computational studies indicate the feasibility of alternative Ni–thiophene isomers and the available spectroscopic data are not inconsistent with other potential structures. In fact, there exist several reasonable bonding modes in metal thiophenes 23 and a variety of π adducts 27. A direct analogy between arene and thiophene binding modes is, therefore potentially problematic as has been noted previously28. Only two structurally characterized complexes are known to adopt the proposed C,C-η2 binding motif in thiophene complexes: the Harman group identified a saturated 18e− tungsten complex with a weakly bound C,C-η2 thiophene ligand29 and Agapie and coworkers isolated a unique palladium(I) dimer with a bridging cis-μ-η2:η2 thiophene30. Although these two reports demonstrate the feasibility of C,C-η2 binding for thiophene, they differ both structurally and electronically from the proposed CTP intermediates, and thus cannot directly address whether such bonding may be achieved in a low valent d10 system. For these reasons, elucidation of the geometric and electronic structure of species such as III remain an unsolved challenge until now32. Herein, we report the structure and properties of species that are directly relevant to the CTP process, providing an opportunity to evaluate the factors that allow for living polymerization of thiophenes.McNeil and coworkers have postulated that such Ni(0)–thiophene intermediates could, by analogy to known Ni(0)–arene complexes, be formulated as C,Csee Fig. , includi34. C–Br insertion can be avoided by choosing unsubstituted thiophene ligands and we anticipated that removing the bromine substituent would have a relatively minor effect on the electronic properties of the thiophenic ligand. This is supported by computational data (vide infra). Preventing C–S insertion, however, is a somewhat greater challenge. Given the more restricted geometry of the predicted C–S insertion product, we rationalized that increasing steric bulk near the metal centre could assist in minimizing C–S insertion. Density functional theory (DFT) calculations on the relative energies of the π complex relative to the C–S insertion product indicated a decreasing preference for C–S insertion on going from thiophene, bithiophene, and trithiophene  thiophene π complexes would be to limit oxidative addition via cleavage of the C–X bond or the internal thiophene C–S bonds as shown in Fig. 235, we used a bulky diphosphine Ni(0) benzene dimer [(dtbpe)Ni]2(μ-η2:η2-C6H6)  phosphinoethane) as a precursor from which to explore the formation of thiophene complexes in pentane at low temperature (~245 K). In the presence of a large excess of thiophene, an orange solid is obtained after in vacuo removal of solvent. The solid-state molecular structure of the recrystallized product (2), [(dtbpe)Ni]2(trans-μ-η2:η2-thiophene) indicates formation of a nickel(0) dimer with a bridging μ-η2:η2 thiophene ligand  implies a Ni(0) ground state configuration and additional splitting of the 31P signals are consistent with weaker JPP coupling across the bridging thiophene ligand , (dtbpe)Ni(η2-thiophene) and the C–S insertion side-product (4), (dtbpe)Ni is obtained  data for 3 are similar to that of 2 although the observed JPP is slightly lower in 3 (~82 Hz), which is consistent with stronger binding to the π ligand in the monomeric species. We have been unsuccessful in isolating and/or crystallizing 3, presumably due to a significant enthalpic preference for 4  π complexesrature ~25 K. In t5, bithiophene), and suppress formation of the insertion product 6  relative to the monothiophene. We attribute this to a modest enthalpic preference for the π complex in the bithiophene system as predicted computationally  CTP intermediates. The metric parameters for 5 indicate strong similarities to other Ni(0) π complexes. In addition, the metric and electronic properties of the complex are in good agreement with a DFT-optimized analog  that can receive electron density from the diphosphine ligand as shown in Fig. 536. This situation formally creates a three-centre–four-electron (3c–4e)38 bond involving σ donation from the phosphines through to the η2 thiophene ligand via π backbonding from the nickel centre. Computationally, we estimate that almost 75% of the backdonation comes from the trans-phosphine ligands indicating a large cooperative binding effect through this 3c–4e interaction. The nature of this backbonding interaction was confirmed using Ni K-edge X-ray absorption spectroscopy  intermediates, which should affect ligand dissociation during catalysis. We therefore suggest that this effect is critical in ensuring living polymerization of thiophenes with nickel diphosphine catalysts.The isolation and characterization of 14. Intriguingly, At 200 K, we observe an additional species in solution (7) in equilibrium with the dominant 5 and 6 . We are currently exploring the isomerization kinetics to provide insights into the dynamics of [Ni] walking along the polymer chain.The nature of bonding in the Ni(0) π complex is critical to the overall CTP mechanism as it represents the intermediate that is most weakly bound and thus most likely to release the polythiophene chain during turnover. Previous studies have indicated that the bond must be strong enough to prevent loss of the ligand but labile enough to allow for chain walking along the polythiophene backbone3H vs. 3BrX). Notably, the 3Br complex may adopt two different C,C-η2 isomers depending on whether the metal binds adjacent to  or remote to  the bromo substituent, these isomers are easily interconverted via a symmetric C,C,C,C-η4 transition state ; this reflects greater asymmetry in the acceptor π* orbital of the 2-bromothiophene ligand. In addition, there is a very modest concomitant increase in the C = C bond distance, which is consistent with greater π backbonding in 3BrX due to bromo substitution.Our results demonstrate the viability of specific structural motifs as intermediates in the catalytic mechanism for CTP. Importantly, our results provide validation of DFT-derived geometric and electronic structure models for such species and increases confidence in the computational models. We, therefore, expanded our computational studies to include the catalytically relevant 2-bromo-substituted bithiophene complexes to explore the impact of halide substitution on the nature of the intermediates. In computational models, we find only minor differences upon inclusion of a 2-bromo substituent. As summarized in Table η2 (8BrH) and C,S-κ2 (4BrH) species are in equilibrium, and that other possible isomers such as C,S-η2 isomers (8BrH and 8BrX) are much higher in energy (by >40 kJ/mol). Calculated barriers for isomerization are quite large due to the strong π-backbonding in the C,C-η2 structure. The barrier for C–Br oxidative addition (to form 9Br) is similar to that for chain walking and also significantly larger than that for C–S oxidative addition near the bromo substituent (to form 4BrX). We note that 4BrX is both the lowest energy species in the isomerization landscape (excluding formation of 9Br) and that with the lowest barrier to formation. We, therefore, suggest that 4BrX and similar species formed during polymerization may serve as off-cycle intermediates during turnover. Such species are clearly important in hydrodesulfurization40, and we are currently exploring their relevance in CTP.Given that the computational method is robust for the ground state species, we expanded our investigation of the 2-bromosubstituted nickel complex to evaluate the overall energy profile for all possible isomers of this species. The resulting energy landscape for Ni(dmpe) binding to 2-bromothiophene is shown in Fig. η2 thiophene complexes is strong evidence that 5 is highly relevant as a model for the actual intermediates in nickel-catalyzed CTP. In addition, our work supports the proposal that both the higher energy endo isomer (identified by NMR) and the exo-C,S-κ2 isomer (from DFT) are also relevant to the catalytic reaction mechanism. In fact, these results strongly support the idea that catalysis must proceed through both Ni(0) species as has been previously proposed17. Csp2–Csp2 coupling via reductive elimination necessarily generates an endo-η2CC species  C,C-η2CC-bound Ni(0) thiophene complex confirm the overall structure of previously proposed intermediates in CTP and yield new insights into the mechanism of action and the importance of the diphosphine ligand in ensuring strong metal–thiophene bonding via a dominant push–pull π-backbonding interaction. Living polymerization conditions may occur due to this surprisingly strong bond between the d10 metal centre and the growing polymer chain. A preference for the catalytically competent exo-η2CC isomer is also observed. These conclusions provide clarity into the mechanism of catalyst transfer polycondensation and hence provide the basis for further work on improving catalyst performance in the efficient synthesis of an important building block for molecular electronics.Our studies on the unsupported 6, C6D6, and toluene-d8 were purchased from Aldrich and dried over sodium/benzophenone before being distilled and degassed by three freeze–pump–thaw cycles. Thiophene was purchased from Aldrich, were dried over activated 4 Å molecular sieves, distilled and degassed by three freeze–pump–thaw cycles. Bithiophene was purchased from Matrix and degassed by vacuum. Complex 1 was prepared according to the literature procedures. NMR spectra were recorded on 300, 400 MHz spectrometers and are referenced to residual protio solvent  for 1H NMR spectroscopy, solvent peaks  for 13C NMR spectroscopy. 31P NMR spectra were referenced to 85% H3PO4 at 0 ppm. Mass spectra and elemental analyses were performed by the microanalytic services at the Department of Chemistry of the University of British Columbia. Details of specific synthetic procedures are included as Unless stated otherwise, all reactions were performed in a glovebox under an atmosphere of pure nitrogen using standard Schlenk techniques. Anhydrous pentanes, toluene, diethyl ether, and tetrahydrofuran were purchased from Aldrich, sparged with dinitrogen, and dried further by passage through towers containing activated alumina and molecular sieves. THF-doC. Data were collected in a of ϕ and ω 0.5o oscillations using 20.0 second exposures. The crystal-to-detector distance was 40.14 mm. Data were collected and integrated using the Bruker SAINT software package. Data were corrected for absorption effects using the multi-scan technique (SADABS). The data were corrected for Lorentz and polarization effects. The structure was solved by direct methods. In refinement of structure 5, the Ni–thiophene–Ni fragment is disordered and was modeled in two orientations with equal proportions. in refinement of structure 5. The material crystallizes with the bis-thiophene disorder in two orientations. The two orientations are related by a 180° rotation about an axis parallel to the C4–C5 bond. All nonhydrogen atoms were refined anisotropically. All hydrogen atoms were placed in calculated positions. The weighting scheme was based on counting statistics. Neutral atom scattering factors were taken from Cromer and Waber. Anomalous dispersion effects were included in Fcalc; the values for f' and f'' were those of Creagh and McAuley. The values for the mass attenuation coefficients are those of Creagh and Hubbell. All refinements were performed using the SHELXL-2016 via the OLEX2 interface. Additional crystallographic details are given in Supplementary Table All measurements were made on a Bruker APEX DUO diffractometer with a TRIUMPH curved-crystal monochromator with Mo-Kα radiation. The data were collected at a temperature of −183.0 ± 0.1 2-TZVP) for all atoms. Optimized geometry coordinates are given in Supplementary Table tert-butyl ligand to manage computational expense. Statistical mechanics calculations of entropic and thermal effects were performed using the rigid rotor and harmonic oscillator approximations at 298.15 K and 1 atm. Potential energy surface scan was applied to find the potential intermediates and transition states geometries, and intrinsic reaction coordinates was calculated to confirm the connection between the transition state and reactants/products and calculation determined intermediates. Accurate single point energies were calculated with solvation model based on density solvent effect using M06 functional. Computational efficiency was improved by applying the RI approximation (RIJCOSX) for the hybrid functional. All calculations were performed with integration grid (ORCA Grid4). XAS TD-DFT calculation were performed with a dense intergration grid (Gird6) for better implementing Scalar relativisitic effects by using ZORA corrections, and reduced by using MOanlayzer software. NBO calculations were calculated with Gaussian 09 program package, AIM41 and CDA42 calculation were performed in Multiwfn software by using NBO outputs using B3LYP/def2-TZVP level. All calculations were run on the UBC Chemistry Abacus cluster and on the Westgrid GREX cluster.DFT calculations initial geometries for all molecules were obtained from crystallographic coordinates (where available) or constructed from standard models. Geometry optimizations and numerical frequency calculations were performed using version 3.0.3 of the ORCA computational chemistry package. Molecular geometries were optimized using the B3LYP functional and all electron basis sets  and quickly frozen under liquid nitrogen environment. XAS Ni K-edges were acquired at the SSRL beamline 7-3, which is equipped with a Si(220) ϕ = 90° double crystal monochromator, a 9 keV cutoff mirror, and a He cryostat (at 20 K). Data were collected using a Canberra 30-element Ge solid-state detector with a 3 mm Co filter. Data averaging and energy calibration were performed using SixPack, The AUTOBK algorithm available in the Athena software package was employed for data reduction and normalization. [Ni(dtbpe)]2-arene complex and Ni(dtbpe)Cl2 were used as reference to evaluate the oxidation state. Specifics of XAS fitting parameters are given in Supplementary Table All the X-ray absorption spectroscopy (XAS) samples except Ni(dtbpe)bithiophene complex were analyzed as solids under anaerobic conditions and diluted in boron nitride (20–50% by weight). Ni(dtpe)bithiophene complex was prepared in dry toluene solvent and treated with extra bithiophene at RT to avoid the formation of S–C insertion byproduct Ni(dtbpe)(Supplementary Information


The in-gap states in sulphur line vacancies cause 1D electron-rich channels that are mapped experimentally and confirmed using density functional theory calculations. We show how electrostatic fields are sensitive in defective areas to changes of atomic bonding and structural determination beyond conventional imaging.Defects in materials give rise to fluctuations in electrostatic fields that reflect the local charge density, but imaging this with single atom sensitivity is challenging. However, if possible, this provides information about the energetics of adatom binding, localized conduction channels, molecular functionality and their relationship to individual bonds. Here, ultrastable electron-optics are combined with a high-speed 2D electron detector to map electrostatic fields around individual atoms in 2D monolayers using 4D scanning transmission electron microscopy. Simultaneous imaging of the electric field, phase, annular dark field and the total charge in 2D MoS 2 and WS2 systems by 4D STEM.Imaging electrostatic field around individual atoms or defective areas in monolayer 2D materials is crucial to understand their structural coordination. Here, the authors report local changes in specific atomic bonds and provide in-depth structural information of complex defective monolayer MoS This has been revolutionized by high-speed electron detectors, either in the form of 2D pixelated cameras (2D-PCs) or as segmented detectors6. These have enabled strain maps across samples with picometer precision, and deep sub-Angstrom spatial resolution using ptychographic reconstruction methods8.4D scanning transmission electron microscopy (4D-STEM) is gaining momentum for probing materials at sub-Angstrom resolution with the full electron−atom scattering interactions recorded in a convergent beam electron diffraction pattern (CBED)10. Phase data can be reconstructed using pytchographic methods, together with simultaneously recorded ADF-STEM images11. The intensity fluctuations of the CBED pattern are used to produce differential phase contrast images that relate to momentum transfer to the electron beam as it propagates through the samples’ electrostatic fields13. Atomic resolution images of electrostatic fields and charge distributions have been recorded for bulk crystals, such as GaN, where beam damage does not limit the long acquisition times3. Using 2D-PCs, this is done by measuring the intensity center of mass, while for quadrant detectors, the differential signal between opposite quadrants is used. Translating 4D STEM to the single atom level is more difficult because of the rapid sample damage at time scales faster than the acquisition speed and hence low beam dose is essential13. Furthermore, mapping features around single atoms in defects is challenging due to the low signal to noise15. However, 2D materials do offer a thin volume for direct interpretation in electron microscopy18, and to study fluctuations of electrostatics around single atoms.Direct collection of CBED patterns on 2D-PCs provides rich information about phase and momentum transfer from the electron beam interactions with the samples’ electrostatic fields2 and WS2, form ultralong 1D channels by S sputtering at high temperature19. Density functional theory (DFT) calculations show that as the width of the S line vacancies increases from 1S to 2S, the band gap narrows from 1.9 to <0.1 eV, and becomes metallic at 4S width20. Theory suggests that these 1D conduction channels are due to the metal-rich bonding areas that form within the larger vacancy sections, but experimental verification of this has yet to be achieved with sufficient resolution to identify charge variations in regions of single atomic bonds. These W−W bonds create 1D sub-nm conduction channels in the 2D semiconductors with potential use in nanoscale electronics and devices. However, the detailed atomic structure of the ultralong 2S and 3S line vacancies is complex and difficult to accurately determine using only ADF-STEM or phase contrast images. Therefore, the multicomponent images obtained from 4D STEM, including total charge maps, are crucial to gaining a better understanding of the structure−property correlations. Furthermore, by using first principle calculations, we can determine the predicted electric fields and total charge values in these monolayer systems and quantitatively compare it to the experimental values. Prior work has primarily used image simulation methods to compare to experimental 4D STEM results.For semiconducting 2D monolayers, transition metal dichalcogenides (TMDs), such as MoS2 and WS2 2D crystals with qualitative agreement to the predicted values from DFT calculations. Experimental values are quantitatively half of the DFT predicted values and this stimulates further investigations. Measurements are performed on sulfur line vacancies that form 1D channels at high temperature through vacancy diffusion into ordered lines. Metal−metal bonding is present in the S line vacancies and is shown to lead to electron-rich channels that act as in-gap states for 1D conduction. More complex line vacancies with wider S vacancy regions are studied and show significant modulation of electric fields around atoms. Using a combination of ADF-STEM, phase imaging, electric field and total charge images, we are able to deduce the atomic structure of complex defective regions with a higher degree of certainty than using just one form of imaging contrast alone. The high sensitivity of the electric field maps to atomic bond coordination provides spatial information about nearest neighbor atoms that is not easily extracted from ADF-STEM images or phase maps.Here, we show that 4D STEM can directly image electrostatic fields, total charge and phase maps with atomic resolution in monolayer MoSE┴) is proportional to the averaged quantum mechanical expectation value for momentum, which is measured from the center of mass (ICoM) of the CBED intensity distribution10. The high stability of the 4D STEM setup is demonstrated by the HAADF image . These are directly compared to the DFT calculated values and we typically find the experimental values being half that of the DFT. A prior report that compared the experimental electric field values to image simulations found the experimental values to be half and suggested that residual aberrations, partial coherence, and defocus may be the cause18. Here we used a scaling factor of ~2 to the 4D STEM data to achieve a match to the DFT-calculated maps , whereas in MoS2  is only just slightly higher than that around 2S due to the similar total scattering cross section. The E┴ vector map 23. An SiO2 (300 nm)/Si substrate was used for growth and was sonicated in acetone, followed by an oxygen plasma treatment. CVD was carried out using 20 mg of MoO3 powder  and 500 mg of S  as the precursor with Ar as the carrier gas under atmospheric pressure. A 500 sccm of Ar gas was used to purge the tubes for 30 min, followed by heating the S powder to ∼180 °C for 10 min under an Ar flow rate of 150 sccm. The second furnace was heated at a rate of 40 °C min–1 to 800 °C, whereas the MoO3 powder reached a temperature of 300 °C. The reaction was conducted at 800 °C for 20 min with 10 sccm Ar. For WS2, CVD was performed using the same system, but with 200 mg of WO3 precursor and 300 mg of S powder, with a growth temperature of 1170 °C for 3 min. Samples were transferred using a PMMA support onto DENS in situ heating chips that contained slits in the thin SiN windows, cut by a focused ion beam. The PMMA was removed by acetone before TEM examination.MoS24, operated at an accelerating voltage of 60 kV. The camera length was 6 cm, when imaging MoS2, the aperture used was CL2-2 (40 μm), convergence semi-angle 39.1 mrad, beam current 48.5 pA; for WS2, the aperture was CL2-3 (30 μm), convergence semi-angle 30.6 mrad, beam current 28.3 pA. Each 4D STEM data set contains 256 × 256 6-bit CBED patterns, dwell time in each CBED pattern  is 0.5−1 ms. Simultaneous HAADF detector was used, with inner collection angle of 111 mrad and outer collection angle of 223 mrad. The ultrafast 2D pixelated detector used for the 4D STEM work is the Merlin for EM Hybrid Pixel Detector (HPD), developed by Diamond Light Source and is built around the Medipix3 ASIC5. It combines direct detection of electrons with rapid readout in pixelated form for dynamic imaging. Noise-less detection of single-electron events and near ideal DQE and MTF detector responses down to 60 kV energies make this system ideal for our proposed work.Scanning transmission electron microscopy was performed using an aberration- corrected JEOL ARM300CF equipped with a JEOL ETA corrector3N4 membranes using focused ion beam milling before transferring the MoS2. In situ heating to 800 °C was used to reduce surface contamination and to reduce hole opening from chemical etching. Long line defects are formed at this high temperature.This was performed using a commercially available in situ heating holder from DENS Solutions (SH30-4M-FS). Heating the sample was achieved by passing a current through a platinum resistive coil imbedded in the TEM chip (DENS Solutions DENS-C-30). The resistance of the platinum coil was monitored in a four-point configuration, and the temperature was calculated using the Callendar−Van Dusen equation . Slits were fabricated in the SiCodes written in Matlab were used to process all 4D STEM data and DFT data.x, Ixcom, and y, Iycom, components, which are proportional to the electric field components Ex and Ey, respectively of the electric field vector perpendicular to the electron beam direction, E┴ given by Eq. i and j are the unit vectors in the x and y directions and Ex and Ey are the magnitudes in the x and y directions. |E┴| is the magnitude of the total electric field perpendicular to the electron beam.Electric field maps were generated from the CBED pattern captured by the 2D pixelated detector by first measuring the intensity center of mass h is Planck’s constant, and λ is wavelength.For the 4D STEM data, we convert the center of mass shift (CoM) to moment transfer entclass1pt{minima10 showed that the measured Electric field (E⊥) is given by Eq. e is elementary charge and ν is the velocity and z the thickness.z, i.e. 15 being half the value of the simulated experiment. It is worth noting that our theoretical values come from first principles calculations, not from simulated 4D STEM experiments. The electric field mentioned here E⊥ is probe convolved (I) with the intrinsic electric field (ER⊥) by Based on Ehrenfest’s theorem, ref. ρ) in units of elementary charge per Å2, was calculated from Eq. 10, using electric field in units of (V). εo is the vacuum permittivity.E┴ in Fig. Ex and Ey values.The total charge  method developed by Maiden and Rodenburg18, and is likely due to residual aberrations or defocus as stated in ref. 18. In Fig. For quantitative comparison of DFT and experimental values we first smeared the DFT results to have spatial resolution similar to the experimental data, see supplementary note vz with initial momentum σ and the corresponding effective electric field is given by Eq. z integrated electric field can be obtained equivalently by solving for the planar charge z = 0 plane with the dielectric constant εo in space. We first expand the planar charge distribution into the Fourier components The shifts for the center of mass of probing electron beam are imparted by the microscopic electric field generated by the charges in the suspended layered material. Here we assume the effects of the probing electrons on the layer itself can be ignored, and the microscopic electric field can be determined by the stationary ground state charge distributions. With the Ehrenfest’s theorem, the probe electrons traveling at speed z-integration of the electric field and the convolution from the probing beam Gaussian smearing, the total effective electric field is given by Eq. with mentclasspt{minima2 crystal that shows excellent agreement between the two. Having justified the validity of pseudo-potential DFT that assumes frozen core electrons, we further perform the pseudo-potential DFT simulations for a supercell crystal with line defect. Below we give more details on numerical implementations.To simulate for the effective electric field and the charge density distribution in the layered compound compared with the experiment, we perform the ab initio density function theory (DFT) calculations for the pristine crystal and supercells with line defects. Electron charge density, being a core physical quantity in the formulation of DFT, can be handled differently depending on how core electrons are treated. In the all-electron-type DFT, the core electrons are treated as valence electrons that respond to the changes of the local environment, while in the pseudo-potential DFT the core electrons are frozen leaving only the valence electrons as the ones being optimized in the simulation. Though all-electron DFT is expected to be more accurate in treating all the electrons equally in the solids, the computation is also more demanding than the pseudo-potential DFT. In our numerical strategy, we perform both all-electron and pseudo-potential DFT for the pristine MoS25. The local density approximation (LDA) exchange-correlation functional is used with a reciprocal k-grid size of 10 × 10 × 2 for a pristine MoS2 single layer crystal26. The muffin-tin radius between the atomic cores and the interstitial space is RMT = 1.29 (1.09)Å for a molybdenum (sulfur) atom. The maximum angular momentum used for the augmented-plane wave is 10 and the plane wave basis for the interstitial region has a cutoff 8RMT−1 (inverse of average Muffin-tin radius). The code supports the output of electron charge density and the microscopic electric field in space.For the all-electron DFT simulation, we employ a full-potential linearized augmented-plane wave (FP-LAPW) DFT calculation, implemented in the ELK code28. We use the PAW pseudo-potential formalism parametrized by PBE30. The k-grid sampling is 25 × 25 × 1 for the pristine 1 × 1 unit cell and 1 × 15 × 1 for the supercell with a line defect. The energy cutoff is 450 eV which sets the cutoff of this plane-wave-based pseudo-potential DFT code. The ground state charge density from the output is used to compute the effective electric field.In our work, we also performed the pseudo-potential DFT implemented in Vienna Ab initio Simulation Package (VASP) to compute the relaxed equilibrium crystal structure and the corresponding ground state electron charge density distributionSupplementary InformationPeer Review File


Assembly with TAEA results in highly ordered (MXene/TAEA)n multilayers where the TAEA expands the interlayer spacing of MXene flakes by only ~ 1 Å and reinforces the interconnection between them. The TAEA-pillared MXene multilayers show the highest electronic conductivity of 7.3 × 104 S m−1 compared with all reported MXene multilayers fabricated by LbL technique. The (MXene/TAEA)n multilayers could be used as electrodes for flexible all-solid-state supercapacitors delivering a high volumetric capacitance of 583 F cm−3 and high energy and power densities of 3.0 Wh L−1 and 4400 W L−1, respectively. This strategy enables large-scale fabrication of highly conductive pillared MXene multilayers, and potentially fabrication of other 2D heterostructures.We report Layer-by-Layer (LbL) self-assembly of pillared two-dimensional (2D) multilayers, from water, onto a wide range of substrates. This LbL method uses a small molecule, tris(2-aminoethyl) amine (TAEA), and a colloidal dispersion of Ti 3C2Tx MXene multilayers with a volumetric capacitance of 583 F/cm3.Layer-by-layer self-assembly method using small molecules holds promise to precisely combine atomically thin materials and enable fabrication of pillared structures with superior device performance. Here, the authors report controlled synthesis of supercapacitor electrodes based on 2D Ti The large-scale fabrication of such structures, however, remains an unsolved challenge. Recently a number of experimental5 and theoretical6 works have shown that thousands, and potentially many more, 2D materials can be exfoliated to form stable dispersions of 2D sheets. Exfoliated 2D sheets, especially in water, present a great opportunity for self-assembly of advanced 2D pillard or hetero-layered structures. Here we use 2D Ti3C2Tx, a member of 2D transition metal carbides and nitrides called MXenes7—a family of materials that have shown great promise for numerous applications15—to form 2D multilayers using an aqueous layer-by-layer (LbL) self-assembly technique. This method allows single flake assembly precision in each layer and sub-nanometer precision of the interlayer spacing.Two-dimensional (2D) materials are rapidly emerging as a class of materials and their most interesting applications arise when single layers of one or several 2D materials are stacked to form pillared 2D multilayers17. The aqueous etching and exfoliation process results in surface functionalized MXenes with a general formula of Mn+1XnTx, where M is a transition metal, X is carbon/nitrogen, n can be 1–3, and Tx represents the different O, OH, and F surface terminations8. The unique combination of metallic conductivity and functionalized surfaces has rendered MXenes as potential candidates for fast energy storage electrodes, because the metal carbide layers provide excellent charge transfer inside the electrodes and functionalized surfaces enhance the pseudocapacitive response of MXene electrodes19.MXenes are usually produced by a selective etching process in which “A” layer atoms of MAX phases, a large group of layered ternary carbides and nitrides, are removed in fluoride containing acidic solutions20. In addition, inevitable self-restacking of MXene flakes in such structures reduces the accessibility of electrolyte’s ions to the interior redox-active surfaces of MXenes thereby impeding the ionic transport channels for charge storage21. These issues have largely prevented full exploration of the charge storage capability of MXenes. Luckily, the high-aspect-ratio structure of MXene flakes and their highly functionalized surfaces enables them to be assembled into multifunctional pillared structures using LbL self-assembly1. LbL is typically a cyclical process in which two oppositely charged species are alternately deposited onto a substrate to form multilayer structures with a thickness that scales with the number of layers22. In LbL architectures, the different layers are held together by electrostatic interactions, covalent and hydrogen bonding, or ionic charge transfer1. LbL self-assembly could allow the different functional components—structural, ion conducting, and electron conducting phases—to be optimally arranged at the nanoscale and lead to an integrated network with a better interfacial strength, higher conductivity, and more accessible active surfaces23.Freestanding MXene films, fabricated by vacuum filtration of MXene dispersions consist of random stacks of the delaminated flakes and show poor resistance to strain because of the sliding between the stacked MXene flakes during the mechanical deformations24, poly (PDAC)25, or poly(sodium 4-styrene sulfonate)26/PEI-modified carbon nanotube27. The electrically insulating polymers in these multilayers, however, form large gaps between the adjacent MXene flakes which disrupt the electron conduction paths, thus preventing the formation of MXene-based LbL architectures with high electrical conductivity. The polymers are far larger than the thickness of individual MXene flakes, and therefore these systems do not enable LbL self-assembly with a single/few flake precision25. The addition of inactive polymers further increases the weight and volume of electrochemically inactive components in the electrodes28. It is thus still a challenge to achieve a highly conductive LbL structure using MXene or other 2D materials with single/few flakes precision in each layer, and with a small gap between the 2D layers.Recently, LbL self-assembly technique has been employed to assemble MXene multilayers with polyelectrolytes such as polyethyleneimine (PEI)3C2Tx MXene, to fabricate pillared multilayers of (MXene/TAEA)n in which the MXene flakes are assembled in a face-to-face quasi-intimate contact leading to a high packing density. The anchored pillars of TAEA create a small gap between the interface contact of MXene flakes resulting in a high electronic conductivity, and in a slightly expanded interlayer spacing between the individual MXene flakes which accelerates ion diffusion and provides facile access to the titanium atoms at the surface of MXene layers for fast pseudocapacitive charge storage. Benefitting from those features, the (MXene/TAEA)n multilayers show excellent electrochemical performance when used as supercapacitor electrodes, and they can also be self-assembled onto various non-metallic substrates including planar films, nonwoven fabrics, paper, foams, and even aerogels. We demonstrate that these multilayer films are conformal, conductive, and resilient to bending and compression.Here, we solve this problem by introducing a positively-charged triamino, small molecule, tris(2-aminoethyl) amine (TAEA), as the interlayer pillaring component for the LbL self-assembly of Tin multilayer films lms Fig.  by seque3C2Tx compared to its theoretical thickness (0.98 nm) is in agreement with literature reports and is believed to result from the confinement of water molecules29.Atomic force microscope (AFM) images showed that the thickness of individual flakes was around 2.5 nm, and their lateral size distribution was in the range of several hundreds of nanometers Fig. . The hig3C2Tx colloidal dispersion and the TAEA solution, and monitored the changes in the zeta potential of MXene and in the charge density of TAEA  under the same conditions, due to the protonation of all primary amino groups of TAEA molecule31. The increased charge density is in favor of the self-assembly processes amine (TAPA) as the counter phase for the LbL self-assembly of MXene  films, and glass slides ges Fig. , which sges Fig. . The linene Fig. . This sun multilayers  peaks, stemmed from the ordered stacking of MXene flakes’ basal planes, increased with the number of bilayers, n. We note that a more ordered and smooth LbL structure was obtained only when the counter phase was the small-molecule TAEA, compared with those of polymer-based LbL structures such as (MXene/PEI)n n26 films whose (0002) peak totally disappeared due to their less ordered structure. We attribute the ordered structure to the small size of TAEA which forms a sub-nanometer gap in between MXene flakes in the LbL films, leading to a quasi-intimate interfacial contact between the flakes similar to pure MXene films. Additionally, (0002) peaks of (MXene/TAEA)n shifted from 6.89° for pure MXene films to 6.38°, which showed a uniform pillaring effect of TAEA in LbL process38. This corresponds to an increase of 1.0 Å in the average interlayer spacing from 12.8 Å up to 13.8 Å, which means an average interlayer distance of 1.38 nm between the MXene flakes in the multilayer films.XRD patterns of the (MXene/TAEA)ers Fig.  showed o20, MXene/CTAB38, or MXene/PANI films21).We note that this is in fact a very small increase in interlayer spacing even if we consider the small size of the TAEA molecule, which we believe results from complex interface interactions between MXene flakes and TAEA in a process which is yet not fully characterized and understood in this or other reported polymeric systems  bilayer. The sheet resistance of the LbL films then decreased sharply to 154 Ω sq−1 (7.3 × 104 S m−1) for 20 bilayers. This conductivity is similar to that of films fabricated using small Ti3C2Tx flakes (7.78 × 105 S m−1)39 and Ti3C2Tx MXene clay (1.5 × 105 S m−1)16 which were made by similar methods to those used here, i.e., combined the etching by LiF + HCl solution and delamination by sonication. This sheet resistance is notably lower than that of polymer-based LbL MXene films: 830 Ω sq−1 for (MXene/PEI)20 2025, and 1400–450 Ω sq−1 for (MXene-PVA/CNT-PSS)n26, because polyelectrolytes form big insulating gaps between adjacent MXene flakes, such as in (MXene/PEI)n with an 8.71 Å increase of interlayer spacing  aerogels and melamine foams as examples of porous 3D frameworks n multilayers, for example on the nonwoven fibers, increases linearly with the bilayer numbers with an average increase of 5.54 mg g−1 per bilayer tes Fig. , and enen multilayers is mainly attributed to the spray-LbL process, which aerosolizes the dispersions of Ti3C2Tx MXene flakes and the solution of TAEA and generates a strong convection and rearrangement between the interface of MXene flakes and TAEA35. We also provided a pressure gradient, using a vacuum suction on the back side of the substrates during the LbL self-assembly, to further force the aerosolized MXene or TAEA through the entire porous substrates n multilayers on the different substrates under various mechanical deformations. We first measured the changes of the sheet resistances of (MXene/TAEA)n on planar PET substrates as a function of the bending radius and cycle numbers. The results show a normalized resistance (R/R0) of 1.9 at a bending radius of 1.5 mm  mm Fig.  and reta mm Fig. . The n multilayers. Figure 6 on foams decreased almost linearly up to 50% compression. We could further compress the foam to 80% strain and return to the uncompressed state again retaining a normalized resistance of 1.23. To the best of our knowledge, this is the only report of compressible 3D MXene-based materials  curves ors Fig.  using 6 at a scan rate of 2 mV s−1  at the scan rate of 2 mV s−1 and 349 F cm−3 (108 F g−1) at 200 mV s−1, exhibiting the highest capacitance retention of 65.5% at higher rates compared to the electrodes with fewer bilayers 20  The expanded interlayer spacing improves ion movement with a small charge transfer resistance (Rct) of 2.3 Ω obtained from electrochemical impedance spectroscopy (EIS)  The assembly of MXene flakes with face-to-face quasi-intimate interface contact leads to an ordered dense packing which results in high volumetric capacitances and also generates higher electronic conductivity for fast charge transport at higher rates.The excellent electrochemical performance of our devices result from the LbL pillared multilayer structures, which we believe have three unique features: (i) The pillars of hydrophilic TAEA between the interlayer spacing of MXene flakes facilitates proton access to more shallow- and deep-trap sites inside MXene interlayers, enhancing the intrinsic pseudocapacitive response of the electrode5 S m−1 which is similar to that of a pristine MXene film, because the tunneling distance between the pillared MXenes in multilayers is very small. (iv) They can act as electrodes in solid-state supercapacitors and deliver a high energy density of 3.0 Wh L−1 at a high power density of 4400 W L−1. These values are higher than most previously reported carbonaceous electrode-based all-solid-state supercapacitors. These results stem, firstly, from the enhanced interlayer spacing which facilitates proton access to deep-trap sites inside MXene interlayers, and, secondly, from the dense packing of single flakes in multilayers which increases volumetric capacitance without compromising electronic conductivity during fast charge/discharge. v) They can be fabricated over large surface areas and various substrates such as nonwovens and even aerogels. When assembled onto these substrates, they exhibit the resistance to extreme deformations such as bending, twisting and knotting and even extreme compression (up to 80% strain). We attribute this to the pillaring effect of TAEA which reinforces the interconnection between MXene flakes.We have demonstrated an efficient strategy for fabricating pillared MXene multilayers using LbL self-assembly from water. These films have five noteworthy features: (i) They grow linearly for each assembled bilayer, which indicates our LbL self-assembly is a highly precise method. (ii) They have an ordered structure similar with the pristine MXene films, as shown by XRD data, but with an increased interlayer spacing of 0.1 nm. The formation of 2D ordered structures with LbL self-assembly, and this level of precision in pillaring is, to the authors’ knowledge, distinct from previous literature reports. (iii) Their conductivity approaches 1045, or electronic devices1.This self-assembly strategy should easily be applicable to other 2D colloidal building blocks such as graphene, layered transition metal oxides, and transition metal dichalcogenides. We believe that the combination of several 2D materials with this LbL method could also open the path towards self-assembled 2D heterostructures for energy storage2SO4) (≥97.5%), and the polyethylene terephthalate (PET) film were purchased from Sigma Aldrich. The anionic potassium polyvinyl sulfate (KVPS) was obtained from Wako Pure Chemicals, Osaka. A commercial nonwoven was obtained from ICA Sweden, and a Chinese traditional cellulose paper (XuanZhi) from Alibaba and melamine foams (MF) from Recticel. CNF aerogel was prepared as reported previously45.Tris(2-aminoethyl)amine (TAEA), branched PEI (60 kDa), poly (PVA) , sulfuric acid (H3C2Tx (d-Ti3C2Tx) MXene solution was prepared according to previous reports in the literature17. In a typical synthesis, 2 g of LiF powder was dissolved in 40 mL 6 M HCl solution. The solution was stirred for 5 min to LiF completely dissolve in the acidic solution. Then 2 g of Ti3AlC2 powder was slowly added to the etchant mixture (over 10 min). An ice bath was used to avoid excessive heat generation while adding the MAX phase powder to the etchant. The etching was carried out at 35 °C for 24 h under continuous stirring at 550 rpm. After etching was complete, the exfoliated powders were washed with DI water and centrifuged several times until the pH was about 6. The final MXene powders were dispersed in deaerated DI Water (in a 1 g to 100 mL ratio) and were probe sonicated in an ice bath for 1 h (35% power). The obtained solution was again centrifuged at 3500 rpm for 1 h and the supernatant was collected (referred to as Ti3C2Tx MXene solution). The concentration of the MXene solutions was measured by filtering a known volume of the solution over Celgard® membrane and weighing the obtained freestanding MXene film after it was completely dried.Delaminated-Ti3C2Tx MXene, PEI, TAEA, spermidine and tris(3-aminopropyl)amine (TAPA) solutions with a concentration of 1 g L−1. The treated substrates were first dipped into TAEA solution for 5 min and were then rinsed 3 times by Milli-Q water for 3, 2, 1 min per time to remove the weakly absorbed molecules. After that, the cation-coated substrates were dipped into the Ti3C2Tx MXene dispersion for 5 min and then rinsed with Milli-Q water as the same steps above. This cycle made one bilayer of (MXene/TAEA)1, and the bilayer was repeated to fabricate the desired multilayers, denoted as (MXene/TAEA)n where n is the bilayer number. The as-prepared multilayer films were dried at room temperature under a vacuum condition. The multilayers over 20 bilayers were dried twice, for example, (MXene/TAEA)30 was dried first after 20th bilayer and last after 30th bilayer.We used silicon wafers and PET films as the planar substrates for the spin-assisted immersive-LbL assembly of MXene. Prior to the spin-assisted immersive-LbL assembly of MXene, silicon wafer and polyethylene terephthalate (PET) were cut into desired strips and were treated with oxygen plasma  for 10 min at a high level under vacuum. In the process of spin-assisted immersive-LbL assembly, we used a dipping robot (nanoStrata Inc.) with a spinning model, and the Ti3C2Tx MXene and TAEA solutions were used with a concentration of 1 g L−1. All porous substrates used were cut into the desired dimensions and were laid on a cellulose membrane which had been fixed in an adjustable-flow vacuum system. Before the deposition of (MXene/TAEA)n bilayers, the vacuum-assisted system was opened to hold the substrates by vacuum. The airbrushes  were used to spray the atomized solutions which were driven by the compressed ultrapure Ar regulated to 20 psi and were held at a sufficient distance to reach the entire surface of substrates simultaneously. The substrates were first sprayed with TAEA solution for 3 s and were then rinsed by spraying Milli-Q water for 5 s to remove the weakly absorbed TAEA molecule. Subsequently, the TAEA-coated substrates were sprayed by the MXene for 3 s and then rinsed by spraying Milli-Q water. The cycle was repeated to fabricate the (MXene/TAEA)n films with the desired thickness.We used a vacuum-assisted spray-LbL assembly process to coat the MXene multilayer films onto the different non-metal porous substrates. In the process, the Ti1. Briefly, the Ti3C2Tx MXene and TAEA solutions were poured sequentially on the top of the aerogel or foam and then were forced through by applying a vacuum pressure on their bottom. The samples were rinsed with Milli-Q water after each step. The cycle was repeated to fabricate the (MXene/TAEA)n films with the desired bilayer numbers.To further show the LbL assembly of MXene onto the larger surface of 3D CNF aerogel and melamine foam, we used our previously reported rapid-LbL assembly method® mode. The contact angles were measured on contact angle meter with CAM200 model (KSV Instruments LTD). QCM-D  was used to investigate the formation of (MXene/TAEA)n multilayers. XRD spectra were obtained from the MXene multilayers assembled on silicon wafers and were conducted on a PANalytical X’Pert PRO powder diffractometer in the range of 4–30° (2θ). The interlayer spacing d (nm) of MXene multilayers was calculated according to the following equation (λ (λ = 0.15406 nm) is the wavelength of X-ray used, and θ0002 is the scattering angles of the (0002) peak of MXene multilayers.SEM images and EDS spectra were taken by a high-vacuum field emission scanning electron microscope with . AFM images were captured by a Multimode 8 Atomic Force Microscope (AFM) with a NanoScope V controller  in the ScanAsystequation :1\documen multilayers.The thickness of MXene multilayers was obtained from the cross-sectional SEM images, by averaging the values from 10 different sites. The mass loading of MXene multilayers was obtained from 5 parallel samples, using a balance (±0.01 mg) to weigh the mass change in a given area before and after the assembly of (MXene/TAEA)3C2Tx MXene dispersions at different pH was tested using a Zetasizer ZEN3600 . The charge density of 1 g L−1 TAEA at different pH was titrated using a 716 DMS Titrino, Metrohm, with a standard chemical of KVPS (a charge density of −0.379 μeg mL−1 at 0.05 g/L) as the titrant and ortho-toluidine blue as the indicator. The charge density Q (μeg mL−1) of TAEA was calculated according to the following equation  and conductivity −1) of MXene multilayers was calculated according to the following equations:R is the resistance measured, W and L are the width and length of the real area measured, and d is the thickness of MXene multilayers.The sheet resistances of MXene multilayers were measured by SourceMeter 2401 KEITHLEY, Beaverton through a 2-point probe technique. The samples were cut into defined dimensions of 1.5 cm length and 1 cm wide, and the short edges were coated with silver paste to avoid the contact resistance between the film and metal probe. The sheet resistance −1 in compression and extension was chosen.Electromechanical properties of MXene multilayers on melamine foams was measured by placing the samples with flat surfaces against two plates of aluminum foils placed inside of an Instron 5594 universal testing machine , and connecting the foils to a multimeter to monitor the resistance change with the compressive strain. The back sides of aluminum foils were protected by insulating plastic sheets, and a velocity of 10% min2SO4 was added to the above solution and then stirred vigorously for 1 h at room temperature.1 g of PVA was added in 10 mL of Milli-Q water. The whole mixture was then heated up to 85 °C under stirring until the solution turned clear, and then cooled under ambient conditions. Subsequently, 3 g of concentrated Hn on PET through tailoring the films to the desired shape (1 cm × 2 cm) and coating one end with the silver paste to decrease the contact resistance. The electrodes of (MXene/TAEA)n on nonwoven, and CNF aerogels were prepared as follows: The composites were first tailored to the desired shape, and then one end was connected with a strip of nickel foil using the silver paste. The contact point was protected using paraffin wax to prevent the electrolyte from the contact point.We fabricated the electrodes of the (MXene/TAEA)2SO4 gel electrolyte was slowly poured on the working area of the (Ti3C2Tx/TAEA)n electrodes, and then they were dried under vacuum condition for overnight to vaporize the excess water and to remove the trapped oxygen from the electrolyte. After that, the two identical electrodes were pressed face-to-face together, leading to a structure in which the solid electrolyte on each electrode constructed a thin separating film.To assemble the solid-state devices, the PVA/HWe used a VMP3 potentiostat  to conduct all electrochemical characterizations. We measured the EIS spectra over a frequency range of 10 mHz to 200 kHz with a perturbation amplitude of 10 mV.n multilayers were used as the working electrode, and a Pt foil and an Ag/AgCl electrode were used as the counter electrode and the reference electrode, respectively. A 1 M H2SO4 solution was used as the electrolyte. The areal capacitance CA (mF cm−2) was calculated from the CV curves according to the following equation:As is the effective area of working electrodes, ν is the scan rate, ΔV is voltage window, i(V) is the current, Vi is the initial potential and Vv is the vertex potential.In the three-electrode configuration, (MXene/TAEA)CA (mF cm−2) per electrode was calculated from the CV curves according to the following equation:A is the effective geometric area of the two electrodes.In the solid-state supercapacitors, the areal capacitance Cs (F g−1) and Cv (F cm−3) per electrode were calculated according to the following equation:S is the mass loading of (MXene/TAEA)n multilayers and d is the thickness of (MXene/TAEA)n multilayers.Gravimetric capacitance CA (mF cm−2) per electrode from charge-discharge profiles according to the following equation:i is the current, Δt is the discharge time, ΔVd is the discharge voltage window after the IR drop.We also calculated the Ev (Wh L−1), and power density Pv (W L−1), based on two electrodes, was calculated according to the following equations:Volumetric energy density Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


However, the difficulty of access and the complex cyto-architecture of the organ of Corti has made interrogating the individual metabolic states of the diverse cell types present a major challenge. Multiphoton fluorescence lifetime imaging microscopy (FLIM) allows label-free measurements of the biochemical status of the intrinsically fluorescent metabolic cofactors NADH and NADPH with subcellular spatial resolution. However, the interpretation of NAD(P)H FLIM measurements in terms of the metabolic state of the sample are not completely understood. We have used this technique to explore changes in metabolism associated with hearing onset and with acquired (age-related and noise-induced) hearing loss. We show that these conditions are associated with altered NAD(P)H fluorescence lifetimes, use a simple cell model to confirm an inverse relationship between  Acquired sensorineural hearing loss (SNHL) accounts for approximately 90% of these cases2, with the most common being the noise-induced (NIHL) and age-related (ARHL) conditions. The vulnerability of cochlear sensory hair cells to trauma and, more recently, the associated loss of cochlear nerve fibres are together believed to be the major causes of SNHL3. The contribution of cellular metabolism to this vulnerability is now becoming established10, guiding attempts at therapeutic intervention11. Given the highly heterogeneous cell population present in the sensory organ of the cochlea, the organ of Corti, conventional whole-tissue analyses will fail to reveal which cell types are critical to the pathophysiology. A full understanding of the role of altered metabolism in hearing loss therefore requires experimental approaches capable of interrogating intact cochlear preparations with cellular resolution. Here, we have used fluorescence lifetime imaging microscopy (FLIM) to observe cell-specific metabolic responses in the reduced pool of the metabolic cofactor nicotinamide adenine dinucleotide (NADH) and its phosphorylated analogue NADPH in response to hearing development, maturation and its noise-induced and age-related loss. The measurements motivate the development of a framework for interpreting the results of NAD(P)H FLIM experiments in terms of oxidative stress phenotypes, raising novel questions regarding the metabolic roles of individual cell types in the health and disease of the auditory system.The World Health Organization estimates that over 5% of the world’s population suffer from a disabling hearing impairment12. The spectrally-identical autofluorescence of NADH and NADPH is often used for this purpose17. Both cofactors carry reducing equivalents within the cell but are involved in distinct metabolic pathways18. The NAD pool participates primarily in ATP-generating catabolic processes, linking the citric acid cycle to the electron transport chain (ETC) in the mitochondria and regulating the rate of glycolysis in the cytosol. In contrast, NADP is involved in anabolic reactions such as lipid and nucleic acid synthesis and, importantly, in maintaining the glutathione and thioredoxin reductase antioxidant defence systems15. The widespread links between altered metabolism and disease have driven the use of NAD(P)H FLIM to investigate these processes in a variety of living cell and tissue models23.FLIM extends conventional laser scanning techniques to measure the rate of excited state depopulation at each pixel of an image. This photophysical property, known as the fluorescence lifetime, is altered by changes to the nanoscale local environment of a molecule, thus making FLIM a highly sensitive probe of intracellular biochemistry when applied to the intrinsic fluorescence of living samples24. For either molecule, two species with distinct lifetimes of 0.3 ns and 0.7 ns are observed in aqueous environments, which likely result from alternate configurations of their nicotinamide ring25. Inside cells, NAD(P)H emission with a lifetime of 0.4 ns is typically observed, likely reflecting an equilibrium between these two freely-diffusing states, alongside a longer lifetime component which arises through binding of the cofactors to enzymes15. NAD(P)H FLIM measurements therefore report three fluorescence decay parameters at each pixel of an image: the shorter lifetime of free NAD(P)H in solution τfree, the longer average lifetime of the enzyme-bound species present at that pixel τbound, and the proportion of the NAD(P)H at that pixel which is enzyme bound αbound13. The quantities τbound and αbound have been observed to differ in response to metabolic perturbation and the onset of disease, prompting diagnostic tools to be designed based on time-resolved autofluorescence methods26. Indeed, these techniques have previously been applied to investigate the metabolism of the cochlea27. However, their full potential has yet to be realised as the interpretation of the nanoscale properties reported by the NAD(P)H fluorescence decay in terms of the underlying physiological or biochemical state of the tissue has not yet been unambiguously defined17.Free in solution, the fluorescence lifetimes of NADH and NADPH are identicalτbound19. We demonstrated that this parameter reflects the ratio of [NADPH] to [NADH], as NADPH exhibits a longer fluorescence lifetime than NADH when bound to enzymes within cells. This has allowed us to separately interrogate these functionally-distinct pools using fluorescence techniques for the first time30. In immature cochlear explant cultures, we measured significantly larger values of τbound in pillar cells, suggesting locally increased [NADPH]/[NADH] ratios, and we showed that this was associated with enrichment of reduced glutathione (GSH) in those cells31. The present study is a direct extension of that work, now applying NAD(P)H FLIM in the intact cochlea to investigate metabolic contributions to the pathophysiology of NIHL and ARHL.In our previous work, we sought to understand the biochemical relevance of changes in 32. We segmented the metabolic responses into the primary functional cells of the organ of Corti shown in Fig. 33.We compared NAD(P)H FLIM measurements in intact bulla preparations from adult mice (3 weeks old) that had or had not been subject to 100 dB octave-band (8–16 kHz) noise for 2 hours, a noise exposure protocol that elicits a temporary threshold shift (TTS) that persists for at least a day following treatmentτbound under control conditions were significantly (P = 10−19) longer in pillar cells than in hair cells, at 3.24(±0.07) ns in OPCs (57 cells across 4 repeats) and IPCs (52 cells) compared to 3.00(±0.10) ns and 3.12(±0.09) ns in OHCs (90 cells) and IHCs (33 cells) respectively. The mild noise exposure caused a significant decrease in τbound in all four cell types  and IPCs  than the OHCs  and OPCs .In line with our previous work, we observed that values of pes Fig. . The ext34 allowed the differing responses of the NADH and NADPH pools to be determined. Using this approach, we found that the large decrease in τbound in the IHCs and IPCs upon noise exposure reflected a significant increase in NADH, rather than a decrease in NADPH, relative to the OPCs H FLIM on cochlear preparations of one week (1 W), two weeks (2 W), three weeks (3 W), one month (1 M), one year (1 Y) and two years (2 Y) of age. These data are shown in Fig. In rodents at birth, the organ of Corti is not fully mature, supporting cells are not fully differentiated and sensory hair cells are not fully functional. Hearing onset begins after ~12 days, at which point the hair cell synapses are well formed, the endocochlear potential is established and the tunnel of Corti, bounded by the inner and outer pillar cells, has formed19, the pillar cells did not exhibit the highest τbound. At 2.90(±0.08) ns, this parameter was equal in OHCs, OPCs and IPCs, with a significantly larger value in the IHCs at 3.00(±0.20) ns (P = 10−5). As hearing developed between 1 W and 2 W, τbound changed in the opposite direction in the hair cells and pillar cells, decreasing to 2.78(±0.06) ns in OHCs (P = 10−7) and to 2.91(±0.09) ns in IHCs (P = 0.008) but increasing to 3.03(±0.08) ns in OPCs (P = 10−6) and 3.01(±0.08) ns in IPCs (P = 0.002). From 2 W to 3 W, the enzyme-bound lifetime increased in all cell types (P = 10−5), establishing the pattern we observed previously of larger τbound in OPCs and IPCs than in the OHCs and IHCs. In contrast, τbound decreased in all cell types between 3 W and 1 M (P = 0.02), indicating possible redox adjustments upon maturation, before remaining constant between 1 M and 1 Y. Development therefore appeared to be accompanied by a significant metabolic switch36, before the redox state settled throughout adulthood.At 1 W, in contrast to our previous work in immature rat cochlear explant culturesτbound decreased in OPCs, IPCs and IHCs by an average of 6% (P < 0.005) between 1 Y and 2 Y but remained constant in OHCs. However, the validity of the OHC data at this time point in these cells is debatable due to their significant loss by this time in the C57BL/6 strain38, resulting in only 11 surviving cells being imaged across 10 repeats. For other cell types and ages, the number of cells included in each reported mean are included for reference in Supplementary Material Table Calculating the relative concentrations of NADH and NADPH in each cell type at each age Fig. , OHCs co19, changes in τbound have been interpreted as changes in the relative levels of NADH and NADPH. However, it is the NAD +/NADH and NADP +/NADPH redox ratios, rather than the absolute levels of their reduced forms, which are the drivers of the biochemical pathways they regulate39. As such, it would be more convenient to be able to understand changes in τbound in terms of the wider metabolic phenotype.In accordance with our previous workτbound values measured in the pillar cells and increased GSH levels19. Corresponding GSH measurements at 2 W, 1 M and 1 Y showed a similar relationship, with an R2 correlation coefficient of 0.6 across the four cell types measured H lifetime may thus reflect the balance between ROS defence and production, an established metabolic phenomenon known as oxidative stress15.A further cellular-level property that may determine the enzyme bound NAD(P)H lifetime can be surmised by considering the contrasting principal roles of NADH and NADPH. The systems for neutralising reactive oxygen species (ROS) inside cells are regulated by NADPHτbound and oxidative stress, we performed experiments in simple cell models. Oxidative stress can be increased by increasing the rate of ROS generation or by depleting antioxidative capacity42. In HEK293 cells, chronic low-dose rotenone treatment was used to increase the production of ROS by the ETC43, and BSO was used to deplete GSH44. DHE staining was used to confirm that both treatments increased oxidative stress, with the baseline rate of oxidation increasing by 16(±5)% (P = 0.04) and 23(±6)% (P = 0.02) in the presence of rotenone and BSO respectively  ns  and BSO a decrease of 0.15(±0.05) ns  as shown in Fig. To investigate the possibility of an inverse relationship between ely Fig. . With boτbound values correlated with decreased oxidative stress, we repeated the DHE measurements on HEK293 cells in which the enzyme NAD kinase had been overexpressed45. We previously observed this perturbation to cause greatly enhanced enzyme bound NAD(P)H fluorescence lifetimes of 3.81(±0.09) ns19. In line with our hypothesis, the rate of DHE oxidation in these cells was greatly decreased, by 39(±2)% relative to control (P = 0.001).Conversely, to investigate whether increased 45, increasing the antioxidative capacity of the cells and the values of τbound measured from them19. Using the fluorescence decay parameters and NAD(P)H fluorescence intensity measurements, we calculated the corresponding changes in NADH and NADPH concentrations following rotenone and BSO treatment % increase in NADH (P = 0.0003), whereas it decreased following BSO due to a 23(±4)% decrease in NADPH (P = 0.002). These data provide the first direct evidence of a convenient relationship between changes in the relative concentration of these two cofactors and the resulting impacts on both the physiological state of oxidative stress and the NAD(P)H fluorescence decay parameter τbound.NAD kinase overexpression increases the level of NADPH but not NADH47, although recent data has shown that mild noise exposure can also result in partial loss of auditory nerve fibre (ANF) connections with IHCs48. The mechanical activity associated with the sound-induced movements of the organ of Corti is likely to put both the sensory hair cells and structural supporting cells under significant metabolic stress during prolonged noise exposure (i.e. “auditory ageing”). Such metabolic effects are consistent with our observed changes in the enzyme bound NAD(P)H fluorescence lifetime measured in noise-exposed excised bulla preparations in each cell type. This could indicate a common stress mechanism, perhaps as a result of mechanical trauma or due to the propagation of changes in cellular redox state from hair cells to support cells, possibly via intercellular calcium signalling that we have previously described51. Moreover, intercellular metabolite exchange has previously been demonstrated between different cells in the brain52 in the export of GSH by astrocytes for uptake by neurons53. This has recently been shown to be mirrored in the cochlea, where supporting cells are thought to contribute to the detoxification of cochlear ROS by releasing GSH through Cx26 hemichannels54.The sensory hair cells and phalangeal pillar cells are traditionally known as the prime targets of noise exposure31, with τbound decreasing between the first and second postnatal weeks. The cochlear data herein are consistent with our proposal that a decrease in enzyme bound NAD(P)H fluorescence lifetime reflects an increase in oxidative stress in cochlear cells. Firstly, as τbound decreased in all cell types from 3 W (maturity) to 2 Y (aged), this correlation would be consistent with theories of ageing based on depletion of antioxidant systems and accumulation of ROS mediated damage55. Secondly, the steady increase in τbound in the pillar cells during post-natal maturation of the cochlea is consistent with an increasing expression of antioxidant enzymes observed in brain tissue over the course of postnatal development58. Finally, as sound exposure causes mechanical and metabolic effects in both hair cells and supporting cells, the acute and marked decrease in τbound in hair cells exposed to sound could reflect the lipid peroxidation, and resulting excess ROS generation, described upon acoustic overstimulation59. NAD(P)H FLIM may therefore provide a suitable technique for the assessment of antioxidant therapies for arresting SNHL.In hair cells, pathological noise exposure caused similar changes as observed around the onset of hearing, i.e. when sound first enters the cochleaτbound to oxidative stress can also provide further insight into existing NAD(P)H FLIM studies. For example, the frequently-observed shortening of the enzyme bound lifetime in cancer relative to healthy tissue62 could reflect the increase in oxidative stress thought to drive oncogenesis63, and our recent observation of diabetic pancreatic islets exhibiting a shorter τbound at low glucose levels than healthy equivalents supports suggestions of increased oxidative stress playing a role in the pathophysiology of this disease64. Further, a significant recent advance linking NAD(P)H FLIM measurements to intracellular biochemistry was Schaefer et al.’s report of correlations between the mean NAD(P)H lifetime and the pH of the mitochondrial matrix65. Specifically, the amplitude-weighted mean lifetime was observed to increase linearly with oxygen consumption rate (OCR), with the constant of proportionality between the two parameters dependent on the matrix pH. Given the definition of the mean lifetime, this constant of proportionality can itself be estimated to be proportional to τbound H fluorescence decay, strongly suggest changes in τbound as a new biomarker for changes in oxidative stress. Such a relationship would open new avenues of research in the NAD(P)H FLIM field, such as in the label-free identification of subsets of cell populations that may be resistant to pro-oxidant therapies72.This work demonstrates the application of NAD(P)H FLIM for monitoring changes in redox-associated metabolism in complex, living tissue samples. We show that metabolic modifications can be identified in different cochlear cell types within 2 hours of mild noise exposure, prior to any overt morphological change of the system, demonstrating the sensitivity of the technique. A decrease in the fluorescence lifetime of enzyme bound NAD(P)H occurred in HCs following noise exposure in both pathological and developmental (onset of hearing) contexts. 32. An RX6 processor  generated octave-band noise (8–16 kHz) for 2 hours, attenuated (TDT PA5) and amplified (TDT SA2) to a sound pressure level (SPL) of 100 dB. The speaker was calibrated prior to each use to ensure a flat (±2 dB) frequency response over this range. Pedal reflex and breathing rate were checked every 30 minutes. Control animals (n = 4) were not exposed to any additional sound beyond the normal background noise in the animal unit.C57BL/6 mice  were anesthetized with ketamine  and medetomidine  and positioned in a sound proofed booth on a heated pad 45 cm beneath the centre of a speaker (Stage Line PA Horn Tweeter MHD-220N/RD) as described previously31, approved by the UCL Biological Services Animal Ethics Committee. In addition to the noise exposure experiments, cochleae from 1 week old , two week old , one month old , one year old  and two year old  C57BL/6 mice were collected. Auditory bullae were isolated and transferred into Leibovitz’s L15 medium (Thermo Fisher Scientific) and then a window was opened in the apical turn. Reissner’s membrane was peeled away and the stria vascularis removed in order to gain visual access to the organ of Corti31. While C57BL/6 mice are well known to have a mutation in the cadherin23 gene (Cdh23ahl) that results in an accelerated form of ARHL38, we nevertheless consider that the relative changes we describe here for the first time are still particularly relevant as an initial internal description of changes in cellular metabolism with age in both sensory and non-sensory cells in the cochlea.Animals were sacrificed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 using a protocol described previously34 using an upright LSM510 microscope (Carl Zeiss) equipped with a 40× (NA 1.0) water-dipping objective. The basal end of the auditory bullae were secured to the bottom of a 35 mm plastic dish with a small drop of superglue and bathed in L15 medium. NAD(P)H was imaged in the apical coil of the preparation using a Ti:sapphire laser  tuned to 720 nm. Fluorescence was detected by a hybrid photomultiplier tube  after passing through a 435–485 nm emission filter. Emission events were registered by a time correlated single photon counting module . Scanning was performed continuously for 2 minutes with a pixel dwell time of 1.6 μs, resulting in 256 × 256 (125 µm × 125 µm) FLIM images containing between 101 and 103 photons per pixel. 5 × 5 binning (2.5 × 2.5 µm) was applied to increase pixel signal to noise and least squares fitting of the biexponential decay function19. Laser powers at the back aperture of the objective were 17(±1) mW. To account for variations in power at the imaging plane, due either to beam drift or depth of tissue in the beam path, it was necessary to normalise the NAD(P)H concentrations in each image to one cell type in the image window. The outer pillar cells (OPCs) were chosen, being the most metabolically-stable cell type present based on the smallest changes in both τbound and αbound following noise exposure.All experiments were performed using protocols described previously73. The bullae were then washed three times with PBS and incubated for 2 hours at room temperature in blocking solution containing 4′,6′-diamidino-2-phenylindole  and phalloidin Alexa Fluor 647 nm (33 nM). The quality of the excised bullae preparations were then evaluated by immunofluorescence  for 2 hours44. After opening, bullae at ages 2 W (n = 3), 1 M (n = 9) and 1Y (n = 8) were incubated in 50 μM MCB (Sigma-Aldrich) for 30 minutes. A subset of this now-expanded dataset has been published previously31. GSH-MCB was imaged on a Zeiss 510NLO Axioskop using multiphoton excitation from a Chameleon-XR Ti:Sapphire laser (Coherent) tuned to 780 nm and fluorescence emission was captured using a 435–485 nm bandpass filter. Image stacks were acquired at 2 μm intervals using a 40× (NA 0.8) water immersion objective. All experiments were performed at room temperature (20–23 °C) keeping all confocal imaging parameters constant between experiments.Monochlorobimane (MCB) passes across the cell membrane and forms a fluorescent adduct when combined with GSH in a reaction catalyzed by glutathione S-transferase. Conjugated GSH-MCB fluorescence can therefore be used as a readout of GSH levels−1 penicillin and 100 mg ml−1 streptomycin . Additionally, NADK+ cultures19 were grown in the presence of 0.1 mg ml−1 G418 (Gibco). Cells were grown as monolayers in sterile 75 cm2 tissue culture flasks (Thermo Fisher) in a 37 °C, 5% CO2 incubator. For imaging, a 22 mm diameter coverslip was placed in each well of a six well plate (Thermo Fisher) before adding 3 × 105 cells per well. Media was changed after 24 hours, when rotenone  or buthionine-sulfoximine  was added if required. Both stock solutions of the treatments were made up in DMSO, so an equivalent amount of DMSO was added to untreated wells (1 μl in 2 ml of growth media) as a vehicle control. Coverslips were imaged 24 hours later, held in a custom-made stainless steel ring and bathed in DMEM free of phenol red (Sigma) and buffered by 10 mM HEPES.HEK293 cells were grown in Advanced Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM GlutaMAX, 100 U ml74. DHE was excited at 351 nm and imaged using 435–485 nm (blue) emission filters. Oxidised DHE was excited at 543 nm and imaged using a 560 nm (red) long pass filter. 512 × 512 images were taken every minute and subsequently analysed in ImageJ. Blue and red fluorescence images were each normalised to their initial values before the blue to red ratio time series was calculated. Three repeats were performed for each treatment and seven repeats were performed under vehicle control conditions. The average red to blue (DHEox/DHEred) ratio time series was determined for each condition by taking a mean across repeats and its rate of increase was calculated using a weighted least squares straight line fit in OriginPro 2015 .For oxidative stress assessment, coverslips were loaded with 5 μM dihydroethidium (DHE) for 10 minutes before being imaged on an inverted LSM510 confocal microscope (Carl Zeiss) using a 40× (1.3 NA) oil immersion objective. DHE exhibits blue cytosolic fluorescence until it is oxidised, whereupon its localisation changes to the nucleus and absorption and emission spectra shift to the redth and 75th percentiles for the boxes and standard deviations for the whiskers. Horizontal lines correspond to the median value and the datapoint within the box reflects the mean.Statistical sampling considerations for the FLIM analysis protocol above are described in Supplementary Material Appendix Supplementary Information