The unilateral depletion region is verified via both the Fermi level and the infrared response measurements. The device demonstrates a pronounced photovoltaic behavior with a short-circuit current of 1.3 μA and a large open-circuit voltage of 0.61 V under visible light illumination. Especially, a high external quantum efficiency of 71%, a record high power conversion efficiency of 9% and a fast response time of 9 μs are achieved. Our work suggests an effective scheme to design high-performance photovoltaic devices assembled by 2D materials.Van der Waals (vdW) heterodiodes based on two-dimensional (2D) materials have shown tremendous potential in photovoltaic detectors and solar cells. However, such 2D photovoltaic devices are limited by low quantum efficiencies due to the severe interface recombination and the inefficient contacts. Here, we report an efficient MoS 2 and doped AsP heterojunction with unilateral depletion region reporting high external quantum efficiency of 71% under zero applied bias.Photovoltaic devices based on 2D materials still suffer from low quantum efficiencies due to interfacial charge recombination and inefficient contacts. Here, the authors design photovoltaic detectors and photodiodes based on MoS A variety of high-performance photodetectors with ultrahigh responsivity or gain, ultrahigh detectivity have been reported based on transition metal dichalcogenides (TMDs)11, III–VI group layered semiconductors14, black phosphorus16, and so on18, from visible to mid-infrared wavelength range22. Thus far, these 2D photodetectors with high responsivity are operated in photoconductive mode with high gain (including photogating mode), which suffer from either a large dark current or a slow response speed. Further, a bias is required for the device operation resulting in a static power dissipation, unsuitable for self-powered or portable battery-powered devices. A good photodetector should currently possess a high responsivity, a short response time, a high signal-to-noise ratio, and low or no power consumption for practical applications23. Photodiode detectors are the ideal choice for satisfying the above requirements. 2D-materials-based van der Waals heterostructures (vdWHs) offer an innovative avenue for this photodetection via the photovoltaic effect26. As compared with the conventional covalently or ionic bonded heterostructures, vdWHs possess an obvious advantage in terms of dangling bond-free interfaces that are free of lattice matching constraints, which opens up opportunities to exploit the numerous more combinations of materials with unique and widely disparate properties in a single vdWHs device27. Thus far, many artificial vdWHs aiming at photovoltaic detectors or solar cells have been assembled via the layer stacking or van der Waals epitaxy techniques26. Lee et al. reported the gate-tunable photovoltaic response in the atomically thin van der Waals p-n heterodiodes assembled by monolayer or multilayer n-type MoS2 and p-type WSe2, where an external quantum efficiency (EQE) of 34% was measured for multilayer p-n junctions28. Similar works were also reported independently by Cheng et al. and Furchi et al. at the same time30. In addition to WSe2/MoS2 vdWHs, various vertical vdWHs based on other 2D layered materials, such as BP/MoS232, MoTe2/MoS234, GaTe/MoS236, GaTe/InSe37, GaSe/GaSb38, and AsP/InSe39, have also been explored in photovoltaic detectors and photovoltaics. However, the EQE (or power conversion efficiency) of those 2D heterodiodes is still low, most below 55% (5%) due to the severe tunneling-assisted interface recombination of the photogenerated electron-hole pairs in such thin bilateral depletion regions as well as the inefficient carrier selective contacts because of the Schottky barrier at the semiconductor-metal interface28. In addition, in these heterodiodes with bilateral depletion regions, both the photogenerated electrons and holes need to cross the heterointerface where they are easily trapped, leading to a slow response speed35. To resolve these problems, we propose and design a heterodiode with a unilateral depletion region. The unilateral depletion region heterodiode is defined as a heterojunction where one side near the heterointerface is carrier-depleted while the other side is carrier-accumulated  layered materials have been widely studied since the discovery of graphene due to their atomic layer thicknesses, strong light-matter interactions and layer-dependent electronic and optical properties2) and black arsenic phosphorus (AsP) 2D layered semiconductors to construct the van der Waals heterodiode for realizing the unilateral depletion region to reduce the severe tunneling-assisted interface recombination and interface trapping effect. The elemental ratio of As to P in AsP alloy is about 1 ± 0.01 according to the element analysis in our previous work39. MoS2 is a promising 2D material for high-performance visible photodetectors11, and AsP is a typical p-type 2D semiconductor with a narrow bandgap of 0.2–0.3 eV in bulk40. Thick MoS2 flakes with weak p-type conduction and thick AsP flakes are exfoliated to build the van der Waals pp+ heterodiode. The MoS2/AsP heterodiode exhibits a gate-tunable backward-like diode behavior with a current rectification ratio over 6 × 103. Upon 520 nm laser illumination, the heterodiode exhibits a pronounced photovoltaic effect with a short-circuit current as high as 1.3 μA and a large open-circuit voltage of 0.61 V, enabling an ultrahigh photocurrent to dark current ratio over 1 × 106. Specifically, a high responsivity of 0.3 A W−1 and a high external quantum efficiency of 71% are obtained under zero bias. Remarkably, a high power conversion efficiency of 9% and a fast response time of 9 μs are achieved, which, to the best of our knowledge, are the highest values ever reported for photovoltaic detectors based on 2D vdWHs25.In this work, we choose molybdenum disulfide  is a typical n-type 2D semiconductor and AsP is an intrinsically p-type, a normal pn junction forward diode is often observed when these two materials are stacked to build the heterojunction. To understand the mechanism of this contrary electrical behavior of the MoS2/AsP vdWHs diode, the electrical transport of both the individual MoS2 and AsP field effect transistors (FET) are separately measured. As shown in Supplementary Fig. 2 FET exhibits an abnormal light p-type conduction behavior with a very weak gate modulation of the channel carrier density. The p-type conduction of thick MoS2 flakes (with thickness larger than 50 nm) is further verified via a systematic investigation of MoS2 FETs with different flake thicknesses 42. The weak gate control is likely due to the thick MoS2 channel. In addition, the nonlinear I–V curves of MoS2 FET indicate a Schottky contact between Cr metal and MoS2. The transfer curve of the AsP FET  technique is used to measure the surface potential of both layers, from which the Fermi level difference can be obtained. To accurately obtain the surface potential, we assembly a clean MoS2/AsP heterostructure with the similar layer thicknesses  at the MoS2/AsP interface. Meanwhile the electrons can hardly be injected into MoS2 because of the large Schottky barrier for electrons. Accordingly, the current is low at positive bias as shown in Fig. As above state, the relative position of Fermi level determines the operation of this pp2/AsP vdWHs pp+ heterodiode, an electrical I–V curve and infrared (IR) photovoltaic response method are used to distinguish the different band structures of the pp+ and pn heterojunctions. A normal MoS2/AsP pn heterodiode is fabricated as a control by choosing a thin MoS2 (thickness of 9 nm) and AsP (thickness of 21 nm) flakes , resulting in a built-in electric field with direction pointing to AsP side. Therefore, a regular forward diode behavior is observed, as shown in Fig. 2/AsP pn heterodiode, as shown in Fig. 2/AsP pp+ heterodiode with unilateral depletion region, since the MoS2 is hole-depleted and AsP is hole-accumulated, the direction of the built-in electric field is pointing towards the MoS2 side, opposite to that in MoS2/AsP pn heterodiode. Therefore, an abnormal backward-like diode behavior is observed, as shown in Fig. I–V curves and the IR photovoltaic response definitely confirm that our MoS2/AsP pp+ heterodiode has a unilateral depletion region in its band structure.To further confirm the unilateral depletion region is formed in this MoS2/AsP vdWHs diode with unilateral depletion region and evaluate its performance in photovoltaic detectors in visible wavelength range. All the photoresponse measurements are conducted at zero gate voltage (Vg = 0 V) unless otherwise stated. Figure Ids–Vds curves of the MoS2/AsP vdWHs diode under dark and 520 nm laser illumination, respectively. Obviously, the curve is upshifted under illumination, exhibiting a distinct photovoltaic response. A high short-circuit current Isc of 1.3 μA and a large open-circuit voltage Voc of 0.61 V are obtained under the power density of 108.8 mW mm−2 corresponding to 100× AM 1.5 intensity. The photocurrent to dark current ratio is over 1 × 106 at zero bias, showing an ultrahigh signal-to-noise ratio. As a comparison, no obvious visible photovoltaic response is observed in the normal MoS2/AsP pn heterodiode  and power conversion efficiency (PCE) can be calculated as follows: FF = Pmax/Isc·Voc, PCE = Pmax/Pin, where Pin is the incident optical power25. A Pmax of 0.39 μW, a FF of 0.5 and a PCE of 9% are achieved under the power density of 108.8 mW mm−2 or incident power of 4.35 μW. The large PCE, to the best of our knowledge, is the highest value ever reported for photovoltaic detectors based on 2D vdWHs25. We are aware that absorption efficiency is thickness-dependent, thus the quantum efficiency and power conversion efficiency are thickness-related. To investigate the effect of thickness on the device performance, six MoS2/AsP heterojunction devices with different layer thicknesses , whereas open-circuit voltage Voc shows a logarithmic relationship with optical power  are calculated at zero drain bias. The responsivity is defined as R = Iph/Pin·A, where Iph is the net photocurrent, A is the junction area of the vdWHs device. EQE is the ratio of collected charge carriers to incident photons and can be calculated by EQE = Isc·h·c/e·λ·Pin, where h, c, λ are the Planck’s constant, the speed of light and wavelength of the incident laser, respectively. A responsivity of 0.3 A W−1 and EQE of 71% are obtained at power density of 108.8 mW mm−2. Considering the large power variation range (three orders of magnitudes), the responsivity and EQE of the MoS2/AsP vdWHs photovoltaic detector is relatively robust, showing a good linear dynamic range. The gate modulated photovoltaic response of the MoS2/AsP vdWHs diode are also measured and provided in Supplementary Fig. To further investigate the photovoltaic response of the MoS2/AsP vdWHs diode. As shown in Fig. 2 channel due to the relatively thick MoS2 flake. Under the large built-in electric field in the MoS2 unilateral depletion region, the electrons freely move to the AsP side and are collected by the drain electrode, meanwhile the holes move to the MoS2 side and are collected by the source electrode. Thus, a negative Voc and positive Isc is observed in Fig. 2 unilateral depletion region. By this means, although a little part of energy (approximately 0.25 eV per one photon) is lost, the recombination of photogenerated electron-hole pairs are significantly suppressed and the carrier collection efficiency is greatly improved, thus the EQE and PCE are enhanced as we mentioned previously. In order to confirm the photovoltaic response is mainly induced by the heterojunction not the Schottky junction effect, scanning photocurrent mapping is used to acquire the photocurrent distribution. As seen in Fig. 2 FET device . A MoS2 and narrow bandgap AsP 2D layers. We show that this kind van der Waals heterodiode can overcome the severe tunneling-assisted interface recombination of photogenerated electron-hole pairs and inefficient carrier selective contacts inevitable in previous 2D heterodiodes based photovoltaic detectors. Meanwhile the unilateral depletion region configuration can greatly reduce the interface trapping effect of the photogenerated carriers. As a result, the MoS2/AsP heterodiode exhibits a pronounced photovoltaic effect with a short-circuit current as high as 1.3 μA and a large open-circuit voltage of 0.61 V under 520 nm laser illumination. More importantly, a record high power conversion efficiency of 9% and a fast response time of 9 μs are achieved. Our work lays a foundation for the next generation of 2D vdWHs photovoltaic devices comprising a combination of a large bandgap semiconductor as the depleted absorption layer and a narrow bandgap semiconductor as the effective carrier selective contact.In summary, we have proposed and demonstrated a van der Waals heterodiode with a unilateral depletion region and a narrow bandgap semiconductor carrier selective contact for high-performance photovoltaic detectors, which was successfully fabricated by assembling thick weak p-type MoS2/AsP vdWHs investigated here were fabricated by a commonly used dry transfer method utilizing a polydimethylsiloxane (PDMS) carrier. First, relative thick AsP flakes were mechanically exfoliated from an as-grown AsP crystal using Scotch tape and transferred onto a silicon substrate (with 300 nm SiO2). Then, MoS2 flakes were exfoliated onto the PDMS film from the commercial bulk crystal  and transferred onto the AsP flake to form the heterostructure under the optical microscope assisted by an aligned transfer platform. The exfoliation and transfer processes were conducted in the N2-protection glovebox to minimize the oxidation of AsP. For device fabrication, electron-beam lithography  was employed to define the electrode patterns. A Cr/Au (15 nm/55 nm) metal thin film was deposited as the contact electrodes by thermal evaporation, followed by the standard lift-off process.The MoS2/AsP vdWHs devices were performed in a probe station (Lake Shore) using a semiconductor device parameter analyzer . The devices were wire-bonded onto a 24-pin chip carrier and a 520 nm laser was used for photoresponse measurements. A supercontinuum light source (YSL sc-pro) is employed to obtain the spectral photocurrent response. An oscilloscope  was used to record the transient photocurrent response to resolve the photoresponse time. In all the photocurrent measurements, the laser was focused on the device with a ×20 objective (NA = 0.45), and the laser spot is near flat with a size of about ∼20 μm, larger than the heterojunction area to make sure a uniform illumination. All the devices were measured at room temperature in an ambient environment.Raman spectra were achieved by a confocal Raman/PL system (LabRAM HR800) using 532 nm laser as an excitation source. The thickness and surface potential of the two layers were obtained by AFM and KPFM, respectively (Bruker Multimode 8). The electrical characterizations of the MoSSupplementary InformationSource Data


However, the possible engagement in vasomotion of cell metabolic oscillations of mitochondrial or glycolytic origin has been poorly explored. Metabolic oscillations associated with the production of ATP energy were previously described in cells, while limited studies have investigated these fluctuations in-vivo. Here, we characterised a low-frequency metabolic oscillator (MO-1) in skin from live wild-type and Nrf2−/− mice, by combination of fluorescence spectroscopy and wavelet transform processing technique. Furthermore, the relationships between metabolic and microvascular oscillators were examined during phenylephrine-induced vasoconstriction. We found a significant interaction between MO-1 and the endothelial EDHF vasomotor mechanism that was reduced in the presence of oxidative stress (Nrf2−/− mice). Our findings suggest indirectly that metabolic oscillations may be involved in the mechanisms underlying endothelium-mediated skin vasomotion, which might be altered in the presence of metabolic disturbance.Arterioles in the cutaneous microcirculation frequently display an oscillatory phenomenon defined vasomotion, consistent with periodic diameter variations in the micro-vessels associated with particular physiological or abnormal conditions. The cellular mechanisms underlying vasomotion and its physiological role have not been completely elucidated. Various mechanisms were demonstrated, based on cell Ca The physiological role of these oscillations is unclear, with contrasting studies supporting the development of vasomotion in both diseased and healthy conditions2. The origin of the oscillations is mainly determined by the local contraction and relaxation of vascular smooth muscle cells (VSMCs), which act as synchronised pacemakers in the vessels wall3. Although in several arteries (e.g. rat aorta) the oscillations may depend exclusively on the activity of VSMCs1, in other vascular beds (i.e. rat mesenteric artery) the most internal layer of blood vessels (endothelium) plays an important modulatory role during arterial motion5. Vasomotion is more prominent in small resistance micro-vessels2 such as skin arterioles that represent an optimal sample for the in-vivo examination of this phenomenon reflecting the general health conditions of the cardiovascular system6.The term vasomotion indicates rhythmic oscillations of blood vessels diameter responsible for changes in the vascular tone and blood perfusion to tissue4, or by indirect techniques such as Laser Doppler Flowmetry (LDF) based on monitoring the heterogeneous fluctuations of microvascular blood perfusion signal2. The combination of LDF with the wavelet transform signal processing analysis has revealed oscillations in the human cutaneous microcirculation that relate to physiological phenomena: myogenic (52–145 × 10−3 Hz), neurogenic (21–52 × 10−3 Hz), endothelial nitric oxide (NO)-dependent (9.5–21 × 10−3 Hz), and endothelial NO-independent (5–9.5 × 10−3 Hz)8.Non-invasive observation of skin vasomotion can be achieved by direct methods measuring vessels diameter, e.g. intravital video-microscopy2. The importance of the cooperation between cytosolic and membrane oscillators to enhance the VSMCs synchronisation responsible for vasomotion has been largely explored and recognised12. They act inducing Ca2+ fluctuations in VSMCs, respectively through the oscillatory release of Ca2+ from the sarcoplasmic reticulum11, and via the opening activity of voltage-dependent Ca2+ channels and large-conductance K+ channels of the plasma membrane12. Furthermore, Ca2+ and membrane potential oscillations have been described in endothelial cells (ECs), suggesting that in some vascular beds (i.e. rat mesenteric artery) in response to specific stimuli (e.g. α-adrenergic stimulation) a primary oscillation from ECs and the interaction with VSMCs might be essential for vasomotion15.For the explanation of the cellular mechanisms causing the oscillations of vessels diameter observed during vasomotion, three different kinds of cellular oscillators have been proposed as possible drivers of this phenomenon: cytosolic oscillator, membrane oscillator and metabolic oscillator1. This mechanism has never been considered important because of limited experimental evidence for the involvement of PFK in glycolytic and Ca2+ fluctuations2. However, recent studies have strongly suggested the participation of PFK in reactions responsible for glycolysis and Ca2+ oscillations18, with a possible driving effect exerted in-vivo by external glucose availability and uptake20. Furthermore, metabolic mitochondrial oscillations have also been reported, mainly associated with fluctuations of the mitochondrial membrane potential ΔΨm that may be mediated by calcium23. The external oxygen required for sustaining the electron transport chain has been proposed as the driving force for the mitochondrial oscillator18. All this evidence suggests that the energetic and oxygen tissue requirements might stimulate vasomotion phenomena associated with Ca2+ oscillations of metabolic origin, which may involve the intercellular communication between heterogeneous groups of cells .A third hypothesised driving mechanism is the presence of a metabolic oscillator represented by oscillations in the activity of the glycolytic enzyme phosphofructokinase (PFK) that cause fluctuations in glycolysis, ATP levels and in the activity of plasma membrane ion channels and membrane potentialSaccharomyces cerevisiae21, cardiac myocytes24, β-cells23, and can be detected indirectly by measuring the fluctuations in the autofluorescence of the intermediate product of energy metabolism NAD(P)H (nicotamide adenine dinucleotide). However, there are limited studies describing metabolic oscillations in live tissue. Mitochondrial fast oscillations have been reported in-vivo in rat, and detected by intravital two-photon (2P) microscopy measurements of NAD(P)H25. Nevertheless, to our knowledge, the slow metabolic NAD(P)H oscillations described in many in-vitro or ex-vivo cell studies23 have not been investigated in-vivo.Metabolic oscillations have been studied in different cell types, i.e. yeast α-adrenergic stimulation with the vasoconstrictor drug phenylephrine (PE). Data were processed by continuous wavelet transform (CWT) spectral analysis to characterise heterogeneous oscillations of NAD(P)H and LDF signals. We found low-frequency oscillations of NAD(P)H autofluorescence with periods of 2.4 min, 5 min and 10 min. To our knowledge, this is the first study in live skin tissue of slow NAD(P)H oscillations previously described in cells. Furthermore, we found relevant correlations between metabolic and vasomotion oscillations suggesting a link between these processes. Therefore, our results support the idea of vasomotion as a dynamic process in the context of a metabolically active microvascular network involving the cooperation of different cell types and vascular segments15, and a possible driving mechanism of metabolic origin.In the present study, we have utilised skin tissue from live mice to simultaneously measure NAD(P)H and microvascular blood flow respectively by laser fluorescence spectroscopy (LFS) and LDF, under physiological conditions and during −/− mice. The Nrf2 transcription factor is a key regulator of the cell redox state, involved in the resistance to oxidative stress by activating the cellular antioxidant defence26. Nrf2 knockout leads to abnormal mitochondrial activity, altered redox status, increased oxidative stress, and has an impact on cardiovascular dynamics27. We used the Nrf2−/− strain to investigate the differences in metabolic and vascular dynamics between normal mice and animals with impaired antioxidant defence that may favour the development of metabolic and cardiovascular disorders. Figure et al.5, this vasoactive agent is able to stimulate a vasomotion phenomenon mediated by endothelial cells. Thus, the use of PE provided the opportunity to study at the same time the role of the endothelium in vasomotion and its relationship with metabolic oscillations. Because of the small number of animals available in the study, the experiments were repeated in duplicate for each mouse and the average value of each variable between the two experiments was taken as final value for data analysis. Data of all the variables analysed in this study are summarised in Table Experiments were performed on 5 wild-type (WT) control and 6 Nrf22) are shown in Fig. −/− (p = 0.007) mice during PE-induced vasoconstriction, however, a stronger p-value was found in knockout mice. SO2, measured by reflectance spectroscopy (RS), was similar between groups and decreased slightly following vasoconstriction without statistical significance.Results of blood perfusion and oxygen saturation  and elastin (450 nm)  index and to normalise NAD(P)H fluorescence:LFS skin irradiation by UV light stimulates the autofluorescence emission of multiple fluorophores, including NAD(P)H (490 nm), flavin adenine dinucleotide (FADnm) Fig. . Althougindex is a measure of the mitochondrial redox state, reflecting the balance between the reduced NAD(P)H and the oxidised FAD+ coenzymes. Comparing this index between WT and Nrf2−/− mice should provide discriminatory data, confirming that the Nrf2−/− phenotype is characterised by altered mitochondrial function due to impaired antioxidant defence and higher oxidative stress. NAD(P)H autofluorescence was normalised by elastin’s fluorescence peak to reduce blood volume artefacts (see more details in the RRindex and NAD(P)Hnormalised values were estimated from each spectrum and used to reconstruct a time series Hnormalised did not show significant statistical differences between groups, while RRindex was significantly lower in Nrf2−/− mice (p = 0.016) confirming that knockout animals were affected by altered redox state and higher oxidative stress.During each experiment, 20 UV spectra were collected in 20 min at a rate of a spectrum every minute, 10 at baseline and 10 during PE administration. RRies Fig.  for appl29. We found oscillatory frequency intervals related to tissue/cell activity similar to those reported in human studies: myogenic (50–150 × 10−3 Hz), neurogenic (20–50 × 10−3 Hz), endothelial NO-dependent (9–20 × 10−3 Hz), endothelial NO-independent (5–9 × 10−3 Hz)  and respiratory (150–1350 × 10−3 Hz) components showed higher frequency ranges  Fig. . In contges Fig. , confirm−3 Hz oscillator and the endothelial NO mechanism has been demonstrated33, the origin of the 5–9 × 10−3 Hz oscillator  is ambiguous. However, our results and literature reports29 suggest indirectly an association with the endothelial-derived hyperpolarizing factor (EDHF) vasodilation mechanism, thus here we will refer to the endothelial NO-independent component as EDHF. The reasons supporting this hypothesis are explained in the CWT allows the identification of two oscillators reflecting ECs activity. While the biological link between the 9–20 × 10In-vivo metabolic oscillations were detected by CWT analysis of NAD(P)Hnormalised and RRindex reconstructed signals. Figure −3 Hz), metabolic oscillator-2 (MO-2) (2.5–5 × 10−3 Hz) and metabolic oscillator-3 (MO-3) (1.5–2.5 × 10−3 Hz). However, we will discuss only results of MO-1 because MO-2 and MO-3 frequency ranges fall largely at the edges of the cone of influence region in the CWT scalogram, where data might not be reliable34  by the total absolute energy Etot of the wavelet spectrum and the number of frequencies in the interval of the oscillator.Figure ei and frequency of the EDHF endothelial oscillator were significantly greater in WT mice at baseline . The EDHF energy and frequency changed significantly in both groups during PE stimulation following opposite trends. The values increased in Nrf2−/− mice  and decreased in controls . The endothelial NO oscillator displayed the same trends observed for the EDHF oscillation. However, we found statistical significance only for the decrease of energy during phenylephrine delivery in WT mice (p = 0.008). The neurogenic and myogenic components showed patterns similar to those of the endothelial oscillations. The energy was reduced in normal mice after PE administration, with a good p-value for the neurogenic oscillator (p = 0.04) and a p-value close to statistical significance for the myogenic component (p = 0.08). We evaluated also the relative maximal amplitude ai of the wavelet peaks, estimated by normalising the absolute amplitude Ai of each oscillator by the total amplitude Atot of the wavelet spectrum and the number of frequencies in the interval of the oscillator. The data of ai are summarised in Table ei but with lower statistical significance.The spectral −/− mice, especially for the endothelial components that are key factors determining microvascular function. This suggests that oxidative stress may affect endothelial function and the overall cardiovascular dynamics in the long term.These results outline opposite microvascular reactivity to PE in WT and Nrf233, thus the expected oscillatory pattern during mild vasoconstriction (1% PE) should be a decrease of ei, as observed in WT animals. In contrast, Nrf2−/− mice displayed an increase of LDF oscillators’ ei during phenylephrine administration. The reason for this behaviour might be the activation of an endothelium-mediated vasomotion resistance mechanism to attenuate vasoconstriction defined myoendothelial feedback15, consistent with the alternation of vasoconstriction and vasodilation induced respectively by simultaneous targeting of VSMCs with PE and EDHF/NO endothelial signalling. The cause for the activation of this mechanism may be a general major vasoconstriction in micro-vessels affected by oxidative stress. We cannot exclude that also WT mice may activate this mechanism in response to higher vasoconstriction stimuli (PE > 1%).In general, the increase of LDF oscillators’ spectral energy reflects vasodilation dynamicsei, ai and f of NAD(P)H MO-1 and RR MO-1 wavelet peaks are summarised in Fig. ei (p = 0.01) and RR MO-1 ai (p = 0.04) during PE delivery. The baseline RR MO-1 amplitude ai was lower in WT compared to Nrf2−/− mice (p = 0.097), while differences in frequencies were not relevant.Data of the spectral normalised and RRindex oscillations reflect the cellular oxido-reductive dynamics, consistent with the turnover between the oxidised and reduced forms of NAD(P)H and FADH2 coenzymes as part of the cyclic reactions for the production of ATP energy during glycolysis and oxidative phosphorylation (OXPHOS) processes. The results of the spectral analysis outline different dynamics of metabolic oscillators in WT and Nrf2−/− mice, especially for the RR MO-1. Considering that RRindex reflects mitochondrial function, the major discriminatory power of RR MO-1 may indicate abnormal mitochondrial energetic dynamics in Nrf2−/− mice due to increased amounts of reactive oxygen species (ROS) affecting the electron transport chain.NAD(P)Hϕ(ωk) between oscillations in a specific frequency range of two signals measured simultaneously. We used WPCO to explore the phase interaction between the endothelial EDHF oscillator and the MO-1 of NAD(P)Hnormalised (CϕNAD(P)H MO–1/EDHF) or RRindex (CϕRR MO–1/EDHF) signals, which fall in the same low-frequency interval (5–9 × 10−3 Hz). Because our analysis does not include the surrogate data testing described recently by Gruszecki et al.35, we cannot make reliable assumptions on the degree of coherence at rest due to bias affecting the low frequencies. However, we can cautiously compare differences between mice phenotypes and changes in response to PE stimulation. Non-significant differences were found for the CϕNAD(P)H MO–1/EDHF , and changed significantly in both groups during PE administration showing a decrease in WT animals (p = 0.01) and an increase in Nrf2−/− mice (p = 0.003)  Fig. . These rei and both NAD(P)H MO-1 ei  and RR MO-1 ai . NAD(P)H MO-1 ei showed also high negative correlations with the energy of endothelial NO , neurogenic  and myogenic  oscillators, and the RR MO-1 ai was negatively related to blood perfusion . None of these correlations were observed in Nrf2−/− mice. These results outline a robust association between the oxido-reductive fluctuations of NAD(P)H and FADH2 involved in ATP biosynthesis and the activation/inactivation of microvascular regulatory mechanisms. Indeed, the correlations probably indicate that in response to 1% PE the spectral energy of vascular oscillators decreases as the energy of slow metabolic oscillators increases. This may reflect a change in the turnover between NAD(P)+ and NAD(P)H promoting the inhibition of EDHF/NO endothelium-mediated vasodilation, and the enhancement of VSMCs-mediated vasoconstriction.Figure ei and the NAD(P)H MO-1 ei  or the RR MO-1 ai  in the PE subgrop. These observations suggest that the metabolic oxido-reductive dynamics may be involved especially in the modulation of the EDHF pathway during α-adrenergic stimulation. In contrast, the endothelial NO and myogenic ei showed a more significant correlation with NAD(P)H ei in the baseline subgroup .In spite of reduced statistical power, the analysis of correlations for the single baseline and PE subgroups provided useful additional information, indicating which are the correlations mostly associated with the baseline and PE time points. Significant negative correlations were found between the endothelial EDHF −/− mice. Also in this case due to the small number of animals (n = 6), the results are presented either as pooled group  and separate baseline and PE subgroups (n = 6). The main correlations found in the pooled group were related to the wavelet frequency f of the endothelial NO oscillator, which was positively correlated with NAD(P)H MO-1 ei  and negatively correlated with SO2 . These correlations might reflect the overexpression of the endothelial nitric oxide synthase (eNOS) enzyme described previously in Nrf2−/− mice27, leading to major production of NO probably to balance for dysfunction in other vasodilation pathways preserving vascular function in vessels affected by oxidative stress27. The correlations may be consistent with this evidence because both NADPH and molecular oxygen participate in the reaction for NO biosynthesis36. Therefore, the increase of endothelial NO frequency correlated with the decrease of SO2 and the increase of NAD(P)H MO-1 ei might reflect respectively growth of NO production, consumption of molecular oxygen and oxidation of NADPH in NADP+. We could speculate that a sustained NO production may compensate for dysfunction in the EDHF vasodilator activity that was found significantly lower at baseline in mice affected by oxidative stress compared to WT , while the endothelial NO frequency and the oxygen saturation were significantly correlated only during PE delivery , which may indicate that the two correlations are not linked to the same physiological process.Figure  WT Fig. . However5 was stimulated through administration of low-dose phenylephrine. Comparisons between Nrf2−/− mice affected by oxidative stress and WT controls revealed an opposite behaviour of vascular oscillations in the two models, indicating an effect of oxidative stress on microvascular reactivity. The most relevant differences were found especially for the endothelial EDHF oscillator, and vasomotion was more prominent in Nrf2−/− mice probably due to the activation of the myoendothelial feedback15 vascular modulation in response to oxidative stress.In this work, cutaneous vasomotion and cell metabolic oscillations were examined simultaneously from live mice. A vasomotion phenomenon specifically mediated by NO and EDHF endothelial mechanismsnormalised and RRindex signals that displayed different dynamics in WT and Nrf2−/− models, probably reflecting an alteration of the mitochondrial energetic processes due to oxidative stress. Relevant correlations were found in WT mice between metabolic and microvascular oscillators, highly suggesting an involvement of the cellular processes associated with fluctuations of NAD(P)H concentrations (i.e. OXPHOS and glycolysis) in microvascular reactivity. These correlations were absent in Nrf2−/− mice suggesting an influence of oxidative stress on the interaction/coupling between cell metabolic reactions and microvascular regulatory mechanisms.We characterised a low-frequency metabolic oscillator (MO-1) of NAD(P)H2+ oscillations at the basis of vasomotion. Despite ECs seemed the microcirculation component mostly associated with slow metabolic oscillators, metabolic fluctuations were also correlated with neurogenic and myogenic components indicating a global cooperation of multiple factors during vasomotion with higher/lower contributions depending on the type of stimulus and vascular bed. In the context of skin microcirculation, primary metabolic Ca2+ oscillations coming from ECs and the EDHF may represent the key drivers and regulatory factors for vasomotion. Based on our results, vasomotion can be considered an adaptive mechanism directed by the energetic requirements/stimuli in the environment surrounding blood vessels aimed at ensuring an optimal intake of nutrients (i.e. glucose) and oxygen for the production of ATP energy, rather than a process leading exclusively to the spontaneous pacemaker activity of VSMCs. This definition could help addressing better several controversial aspects, i.e. the observation of vasomotion in both healthy and diseased conditions2, or the primary involvement of ECs or VSMCs in different vascular beds2. These contrasting observations may be the result of variable energetic requirements and molecular signals in the parenchymal tissue surrounding the micro-vessels, depending on the degree of nutrition/oxygenation, the presence of a particular pathology, and the cell types involved. According to the literature, the factors released by microvascular ECs  can diffuse to the underlying parenchymal tissue and modulate the mitochondrial metabolism, the production of ROS and inflammation37. Thus, considering that the autofluorescence signals measured in this work have a prevalent epidermal origin, the correlations found between metabolic oscillations and vasomotion might also reflect the communication between skin cells and the microvascular network to fulfil the cutaneous metabolic needs in response to the α-adrenergic stimulus.Our findings indicate indirectly that cell metabolic oscillators may have an important role in modulating vasomotion in response to specific stimuli at least for oscillatory processes endothelium-mediated. Therefore, this raises interest in the study of cell mitochondrial and glycolytic oscillators to elucidate further the mechanisms driving the Ca−/− models. As future perspective to increase the applicability on a broader basis, further studies are required on a larger number of animals and on additional mouse models. In particular, testing a larger number of Nrf2−/− mice after challenging with high fat diet or after inducing diabetes would be useful to characterise the behaviour of the nonlinear biomarkers in the presence of an extensive oxidative stress load or a metabolic disease, and testing the biomarkers in a knockout model for the eNOS enzyme would be useful to elucidate better the role of NO.A shortcoming of the present research is the impossibility to extend the results on a general basis due to the small-scale design of the study and the restriction of the experiments exclusively to the WT and Nrf2−/− model). As future perspective, a longitudinal study using more homogeneous aged animals could be also of interest to characterise the patterns of the nonlinear biomarkers for different age ranges.The experiments in the present work were restricted to female mice due to limited availability of animals, thus another point to address in the future is also the extension of the study to male animals. The groups of tested mice were perfectly matched for an heterogeneous age range (36–60 weeks), which allowed making only genotype-related comparisons. However, this does not represent an issue because the main purpose of the current study was exploring the relationship between metabolic and microvascular oscillations in normal physiology and then use an example where metabolic function was disturbed H, i.e. fluorescence lifetime-based methods40 and multiphoton microscopy43, allow a better monitoring and discrimination between glycolytic and mitochondrial NAD(P)H with high spatial-temporal resolution at cell level. Combining these methods with CWT can help studying both slow and fast NAD(P)H oscillators. Nevertheless, these technologies still require further implementation for the application on humans and for providing continuous NAD(P)H recordings, they are expensive and lacking of portability and device design for combining with the LDF probe to study simultaneously metabolic and microvascular oscillators.The methodology used in this work can be easily translated for the concurrent study of metabolic and microvascular dynamics in humans and the exploration of metabolic oscillators as cardiovascular risk factors for diagnostic purposes. The use of LFS to monitor NAD(P)H provided some advantages, i.e. label-free minimally invasive nature of the measurements, convenient combination of LFS and LDF probes for simultaneous recordings, portability of the technology. However, there are limitations leading to the discrete nature of LFS measurements, necessity to reconstruct NAD(P)H−/− (n = 6) female mice on a C57Black/6 background aged 36–60 weeks. Mice were maintained under a 12:12-h light/dark cycle at 22.0 ± 1.0 °C and 50% humidity, and were fed ad libitum on a standard rodents chow diet regime and water.Animal work was conducted at the School of Medicine of the University of Dundee, after gaining approvals by the local ethics committee . All the experimental procedures were performed in accordance with the UK Home Office relevant guidelines and regulations under the auspices of Project Licence PPL No. 60/4265. Animals included in the research were WT (n = 5) and Nrf244. A light anaesthesia was maintained by delivering 1.5–2% isoflurane (Abbott Laboratories) in oxygen (1.5 L/minute) through an inhalation nose cone . Before collecting measurements, mice were anaesthetised through a standard Boyle’s Apparatus to prevent movement artefacts and were laid on a heat mat at 37.0 °C, as described previously by usone Fig. .45. We used an iontophoresis chamber  of 20 mm internal diameter attached to the mouse flank using double-adhesive tape, and a reference electrode placed underside of the animal. The chamber was filled with a 2 ml 1% phenylephrine solution, and LFS and LDF probes were placed at two adjacent sites on the skin area inside the chamber  connected to the electrodes.Iontophoresis allows the local delivery of vasoactive drugs in the skin microcirculation without inducing systemic effects. The drug is transferred transdermally by the unidirectional movement of ions in a solution, through a continuous current generated by a reference electrode and a platinum electrode incorporated in a ring-shaped chamberber Fig. . LFS/LDFin-vivo excitation of cell endogenous fluorophores, i.e. NAD(P)H, allowing the detection of autofluorescence emission peaks proportional to the tissue concentration of the biomarkers48. We assessed skin NAD(P)H autofluorescence with a single-point LFS probe  provided with irradiating and detection sensors at a distance of ~1 mm for targeting a tissue volume of ~1 mm3. Discrete 10 seconds autofluorescence spectra were sampled from the mouse flank by a 365 nm UV excitation light (1 mW) at a rate of one spectrum every minute during iontophoresis. The spectra were processed by Matlab R2015a (The MathWorks Inc.) to extract the amplitudes of NAD(P)H (490 nm), FAD+ (550 nm), and elastin (450 nm) peaks H autofluorescence normalisation are provided in the LFS employs a low-power laser 1–5 mW) for the aks Fig. . The red–5 mW fornts Fig.  using th45. We measured skin blood flow using a laser Doppler flowmeter  provided with a probe identical to the LFS probe described above, which was calibrated with a fluoroplastic oscillating disk simulating Brownian motion. A 20 min LDF signal was measured during iontophoresis  according to the reflectance spectroscopy principles51. The technique is based on the analysis of the signal backscattered from the skin to detect the differential absorption of red and green visible lights by the oxygenated (oxyHb) and deoxygenated (deoxyHb) haemoglobin (Hb). The absorption is proportional to the relative molar concentrations of Hb fractions in the tissue, which are extracted from the backscattered spectrum by application of the Beer-Lambert law52:εi(λ) is the molar extinction coefficient of each absorber, Ci is the molar concentration of each absorber, and μa(λ) is the absorption coefficient. The percentage SO2 is finally estimated as follow52:LDF allows the indirect measurement of blood perfusion by sensing the Doppler-shift in wavelength generated when a monochromatic and coherent light is backscattered by moving blood cellssis Fig.  using a et al.52.More details and references on how the terms of equations 29, providing optimal frequency resolution for low frequencies and good time resolution for high frequencies. We employed this method to investigate heterogeneous oscillations of LDF, NAD(P)Hnormalised and RRindex signals, using a CWT with scaling factor s, time t, Morlet wavelet function ψ and central frequency 128:The CWT method is largely employed to analyse blood perfusion vasomotion dynamics in the time-frequency domainEi28, amplitude Ai28 and frequency f28 from the time-averaged wavelet peaks were performed using Matlab R2015a (The MathWorks Inc.). Figure normalised signals.CWT and the extraction of the spectral energy ϕ(ωk) between oscillators in the same frequency interval of two signals recorded simultaneously53. The analysis returns values between 0 and 1, where Cϕ(ωk) ≈ 0 indicates absence of coherence, Cϕ(ωk) ≈ 1 complete coherence, and 0 < Cϕ(ωk) < 1 partial coherence53. We investigated the coherence between MO-1 of metabolic signals and the LDF endothelial EDHF oscillator that are located in the same frequency range. The analysis was performed through the WPCO code provided in the Lancaster University website http://py-biomedical.lancaster.ac.uk by Professor Aneta Stefanovska research group, which estimates Cϕ(ωk) according to the principles described by Bandrivskyy et al.53 and Clemson et al.54.The WPCO analysis provides information on the phase relationship Cr, differences between baseline and PE time points were tested by paired t-test, and differences between groups were assessed by unpaired t-test. The correlations were considered relevant for p ≤ 0.05 and values of r > 0.5 or < −0.5. The t-test was considered significant at p < 0.05 with a statistical power of 0.8.Statistical analyses were performed by R-Studio software. The Shapiro-Wilk test revealed normal distributions for all the variables tested in the study. Therefore, the correlations were evaluated by calculating the Pearson’s correlation coefficient Supplementary information


Cadmium (Cd) is a toxic heavy metal ubiquitous in the environment. Maternal exposure to Cd is associated with fetal growth restriction, trace element deficiencies, and congenital malformations. Cd exposure during adulthood is associated with cardiovascular disease (CVD); however, the effects of maternal Cd exposure on offspring cardiovascular development and disease are not well-understood. Utilizing a mouse model of maternal Cd exposure, we show that offspring born to Cd-exposed mothers have increased heart weights at birth and susceptibility to hypertension during adulthood. Despite inefficient maternal-fetal transfer of Cd, maternal Cd alters fetal levels of essential trace elements including a deficiency in iron, which is required for cardiovascular system development, oxygen homeostasis, and cellular metabolism. RNA-seq on newborn hearts identifies differentially expressed genes associated with maternal Cd exposure that are enriched for functions in CVD, hypertension, enlarged hearts, cellular energy, and hypoxic stress. We propose that a maternal Cd exposure-induced iron deficiency leads to altered cellular metabolic pathways and hypoxic conditions during fetal development; this stress may contribute to increased heart weight at birth and the programming of susceptibility to hypertension in adulthood. These studies will give insights into potential mechanisms through which maternal Cd exposure impacts cardiovascular development and disease. Maternal exposure to heavy metals2, endocrine disruptors3, and over- or under-nutrition4 during gestation have all been shown to negatively affect the development and future health of offspring, even after cessation of exposure.The environment during development can impact health in later life, a concept called developmental programming6. Cd is naturally found at relatively low levels in the soil (0.1–0.5 parts per million (ppm))7, but higher levels can be found in clustered geographical locations due to natural sources or pollution9. Exposure to Cd has dramatically increased over the last century due to human activities such as fossil fuel combustion, metal mining and refining, tobacco smoking, and municipal waste incineration10. Humans are exposed to Cd through inhalation or ingestion. Cd inhalation occurs predominantly through smoking cigarettes, but can also occur through inhaling contaminated dust or through occupational exposure. Cd ingestion is the main source of exposure among non-smokers and occurs through the consumption of contaminated water or food9, particularly crop plants that absorb Cd from the soil. Produce that is recommended as part of a healthy diet especially during pregnancy, such as whole grains and leafy greens, is often the primary source of dietary Cd11. Cd is not required by any processes in the body and is poorly excreted, resulting in long-term storage in several target organs7. Females tend to absorb and accumulate more Cd in their bodies than males with Cd absorption being the highest during pregnancy, likely due to increased requirements for iron12.Cd is a toxic heavy metal found throughout the environment and is designated as one of the ten chemicals of major public health concern by the World Health Organization (WHO)15. Several epidemiological studies have demonstrated an association between blood and urinary Cd levels and cardiovascular disease , which is the leading cause of death and disability in the United States (US)16. Cd exposure increases oxidative stress2, a critical event in the pathogenesis of hypertension18. Uncontrolled hypertension and smoking are two of the three risk factors for CVD19.Chronic exposure to Cd during adulthood has been shown to negatively impact cardiac health20 and altered heart morphology and endothelial function during adulthood in rats2. We recently showed in a human cohort that higher maternal blood Cd levels during pregnancy are associated with changes to cord blood DNA methylation at genes involved in cardiometabolic functions21, suggesting that maternal Cd exposure could affect their regulation and potentially influence cardiac development and health. However, it has not been established in humans or rodents whether these molecular and physiological changes are associated with impaired cardiac function or biomarkers of CVD.While the association between Cd exposure during adulthood and CVD has been well-demonstrated in human and animal studies, only a few studies have examined the impacts of early-life Cd exposure on cardiac development and the programming of long-term CVD. Maternal exposure to Cd has been associated with fetal congenital heart defects in humans24. We generated F1 mice from matings between two genetically divergent inbred strains to study allele-specific epigenetic and transcriptional responses to maternal Cd exposure. The results of these allele-specific analyses will be presented elsewhere. In the current study, we use this mouse model to show that maternal Cd exposure causes increased heart weight in the offspring at birth and that maternal Cd exposure alone is sufficient to program susceptibility to hypertension in adulthood. To inform on molecular mechanisms that may contribute to the observed phenotypes, we identify altered gene expression pathways and essential trace element levels at birth caused by maternal Cd exposure.To address these outstanding questions, we have established a mouse model of maternal Cd exposure Fig.  using a 0 females through their drinking water: 0 ppm (control), 1 ppm , and 50 ppm (high dose previously used in other rodent studies). F1 mice were sacrificed at birth or aged to 6 months. For ease of annotation, the generation of the mice and the Cd dose to which they were exposed will be herein referred to as: F(generation)(Cd dose). For example, F01ppm refers to mothers exposed to 1 ppm Cd and F150ppm refers to offspring maternally exposed to 50 ppm Cd. A hybrid mating scheme between two genetically divergent inbred strains of mouse, C57BL/6J (B) and CAST/EiJ (C), was employed to facilitate a separate study of allele-specific DNA methylation and gene expression changes in response to maternal Cd exposure, the results of which will be presented elsewhere. F1 mice will be herein referred to as B × C (B mother × C father) or C × B (C mother × B father).The exposure model and mating strategy are outlined in Fig. 2,49 = 1.250, p = 0.295 (B × C) and F2,26 = 0.377, p = 0.690 (C × B), one-way analysis of variance (ANOVA) comparing the three Cd doses; sex ratio: p = 0.943 (F10ppm), p = 0.115 (F11ppm), p = 0.633 (F150ppm), chi-square test based on expected 1:1 male:female ratio). However, 50 ppm Cd was found to significantly increase the number of problematic deliveries for C mothers , iron (Fe), potassium (K), magnesium (Mg), sodium (Na), copper (Cu), selenium (Se), and zinc (Zn) were quantified in 6-month-old B × C female blood and liver tissue  were measured in newborn blood and liver tissue to quantify all essential trace elements in the mouse that were feasible with our spectrometry methods. Strikingly, maternal Cd exposure resulted in significant differences in levels of all elements tested . No Cd was detected in newborn blood or liver tissue for any of the treatment groups given our reporting limits, consistent with other studies showing that the placenta acts as a barrier to the efficient maternal-fetal transfer of Cd12.Due to the absence of any overt biomarkers of hypertension in adulthood, we focused on newborn Fd Tables  and 4. I1 pups may be a consequence of perturbed trace element homeostasis in mothers. 50 ppm Cd significantly affected maternal blood levels of Ca, Fe, K, Mg, Na, P and S; additionally, 50 ppm Cd significantly affected maternal liver levels of Fe and Zn .Levels of these 13 elements were also quantified in maternal blood and liver tissues at the time of newborn pup dissections to determine if the levels observed in Fn Tables . The dir10ppm and F150ppm B × C female mice at birth. Of the 14,698 genes with aligned reads passing our quality control filtering, 302 genes were found to be significantly differentially expressed between the two treatment groups . Among the differentially expressed genes, there was a significant enrichment for phenotype or disease terms related to hypertension, abnormal heart and cardiovascular system development, and nutritional disease  were significantly differentially expressed due to maternal Cd exposure, consistent with the RNA-seq data. Most of these showed a dose-dependent response to maternal Cd exposure, although the differences between F10ppm and F11ppm mice were significant only for two genes   Fig. .16. Although there are genetic risk factors for CVD, increased CVD prevalence has been linked to environmental factors including exposure to Cd and other toxic metals15. Most research in humans and animal models has focused on the significance of chronic Cd exposure during adulthood. Although Cd inefficiently crosses the placenta12, there is growing evidence that maternal exposure to Cd can impact child development and health44. However, molecular mechanisms of Cd toxicity in this context have been challenging to study in humans.CVD is the leading cause of death and disability worldwide7. However, geographic clusters can contain higher levels of Cd as shown by a study in North Carolina identifying regions with up to 1.6 ppm Cd in the soil8. Therefore, exposure to 1 ppm Cd can be considered a realistic level of human exposure. The mean level of blood Cd in the US population has been reported to be 0.337 μg/L8; this level is below our reporting limits and consistent with our inability to quantify Cd in the blood of F01ppm mice. The mean level of Cd detected in the blood of F050ppm mice was approximately 15.5 times the mean blood Cd levels of the US population; however, people living in Cd-polluted areas demonstrate blood Cd levels similar to or higher than the levels quantified here in F050ppm mice46. Additionally, fetal growth restriction was observed primarily in the F150ppm mice, a phenotype consistent with Cd exposure in humans47, providing further support that the high dose is relevant to the study of Cd-associated human health outcomes.To better understand the impact and mechanisms of action of developmental Cd exposure on cardiovascular health, we established a mouse model of maternal Cd exposure. Two doses of Cd were chosen to reflect a low and a high dose relevant to human exposure levels. Cd naturally occurs in the environment at low levels; the average amount of Cd in the Earth’s crust is estimated to be roughly 0.3 ppm49. Hypertension was observed in maternal Cd-exposed B × C, but not C × B females; this discordance could be explained by the small sample size of F150ppm C × B female mice, a consequence of the negative impact of 50 ppm Cd on C female breeding performance. Alternatively, our results could reflect differences in gene-by-environment interactions between the parental inheritance of the B and C alleles, such that the maternally-inherited B allele and/or the paternally-inherited C allele is more susceptible to developmental Cd exposure. Our hybrid mouse system will provide a valuable model for dissecting the genetic basis of differences in susceptibility to Cd-induced hypertension.Using this model, we have shown that maternal Cd exposure is associated with increased heart weight at birth and can program hypertension during adulthood. Our data showing that maternal Cd-induced hypertension is observed only in females is consistent with sex-specific responses to Cd reported in other studies150ppm B × C mice. We did not detect any differences compared to F10ppm controls at this age, suggesting that molecular changes in early life may be sufficient to program this phenotype. We therefore assayed trace element levels at birth and, strikingly, demonstrated perturbation of all elements measured. Many of these elements are important for cardiovascular development and function. For example, Na was significantly increased in pup blood and livers due to maternal Cd exposure; excess dietary Na is a significant contributing factor to hypertension50. Na concentrations also correlate with left ventricular hypertrophy51, which was previously seen in rats maternally-exposed to 30 ppm Cd2. Additionally, Se levels were significantly decreased in pup tissues in response to maternal Cd exposure; Se is a vital component of antioxidant enzymes, and diseases due to Se deficiency present with enlarged hearts, congestive heart failure, and other altered cardiac features52. Fe deficiency may be one of the most significant contributing factors to the reduced birth weight, increased heart weight at birth, and hypertension observed in our study. Fe deficiency is associated with increased oxidative stress that may damage cells in the heart and endothelial lining of vasculature53, possibly contributing to the susceptibility to hypertension. The heart responds to chronic stress by increasing in size to increase blood flow, oxygen, and nutrient delivery to tissues54, potentially explaining the increased heart weight at birth. Not surprisingly, a severe Fe deficiency is associated with an increased risk of CVD in humans53.To gain insight into the molecular basis of the hypertensive phenotype, we measured a marker of circulating ROS and essential trace element levels in adult female F050ppm tissue appeared to correlate with fetal levels, but were not statistically significant, likely due to small sample sizes. Thus, many of the essential trace elements seen perturbed in the pups may be due to existing Cd-induced perturbations in the mother. It is also possible that the trace element levels are further exacerbated at the placental level, as Cd can interfere with nutrient transport across the placenta29.We propose that perturbed trace element homeostasis could represent one of the earliest events in the mechanisms leading to the phenotypes observed in this study. The direction of changes of many of the trace element levels in fetal tissues correlated with those seen in maternal tissues; this correlation highlights the importance of including a pre-conception exposure window in animal models of maternal exposure, as maternal exposures in reality often begin prior to pregnancy. Additional trace elements in F55. One study showed that 50 ppm maternal Cd exposure in rats reduced Fe in maternal liver and whole fetuses, yet increased Zn in maternal liver29, consistent with the findings in our study. The study also quantified Zn and Cu in whole fetuses, but found no significant differences as a result of 50 ppm maternal Cd exposure. Here, we show a significant increase in blood Zn and Cu, yet a significant decrease in liver Zn and Cu, which could explain why no overall differences were observed in whole rat fetuses.Zn and Fe are the most well-studied elements affected by maternal Cd exposure due to their similar chemical properties to Cd and shared mechanisms of absorption56; this is likely due to the heart needing to adapt to a new environment .Our RNA-seq data from hearts at birth provide further support for early life molecular changes programming susceptibility to hypertension in adulthood. To our knowledge, this is the first time that a global transcriptomic analysis has been performed in the heart in the context of Cd exposure. We show changes in expression in genes implicated in hypertension, abnormal heart and cardiovascular system development, and increased heart size. Our RNA-seq data are further enriched for multiple mechanistic pathways that contribute to these diseases such as hypoxia, altered cellular energy, ROS, nitric acid homeostasis, and altered metal homeostasis, consistent with our trace element analysis. There was a significantly larger proportion of genes that increased in expression in response to maternal Cd exposure, consistent with a previous study in rats that found an overall increase in RNA synthesis in cardiac muscle of juvenile offspring maternally-exposed to 50 ppm Cd59. However, there was a significant enrichment for the term ‘Cadmium sulfate’ among differentially expressed genes identified by RNA-seq, indicating that maternal Cd exposure may act through similar mechanisms in the offspring as those in response to Cd exposure during adulthood.Mechanisms through which maternal and adult exposure to Cd affect health may differ as the placenta acts as a barrier to Cd, but no such barriers exist when exposed to Cd postnatally. Considering that little (if any) Cd enters fetal circulation, maternal Cd exposure may exert its effects indirectly, such as impacting placental development and function60. Measurements are also susceptible to fluctuations in the environmental temperature, although mice were maintained on a heat pad and under a blanket during the procedure to control for this. Another limitation of our study is the small sample size of F150ppm C × B mice, due to the effects of Cd on C female breeding performance.Blood pressure was measured using the non-invasive tail cuff method to limit stress associated with surgery and recovery. The technique has been extensively used and is appropriate for measures of obtaining overall arterial pressure rather than instantaneous pressure in response to a stimulus. There are limitations to this technique; for example, tail cuff arterial pressure does not always mirror central arterial pressureIn summary, based on our assessment of trace element levels and transcriptomic analysis of newborn hearts, we propose that maternal Cd exposure leads to a nutrient-poor environment in the offspring during development, which leads to increased heart weight and susceptibility to hypertension during adulthood. Further studies using our mouse model will be required to test this hypothesis empirically. Our observations provide novel insights into mechanisms underlying Cd-associated CVD.0 generation. Animals were maintained on a standard 14-hour light, 10-hour dark cycle61 in Green Line IVC Sealsafe cage housing systems (Tecniplast) and fed LabDiet 5001 rodent diet (Granville Milling Company) ad libitum. 5- to 7-week old female F0 B or C mice were provided unrestricted access to filtered drinking water  containing 0 ppm, 1 ppm, or 50 ppm Cd in the form of CdCl2 . Multiple cohorts of females were exposed to Cd for 5 weeks. The study was not blinded, but females from all groups were treated simultaneously within their cohorts. After 5 weeks, females were mated with previously unexposed males of the opposite strain and of similar age. Cd exposure continued throughout mating and pregnancy, and was discontinued once a litter was observed  and CAST/EiJ (‘C’) mice were obtained from the Jackson Laboratory and bred within their respective strain to generate the Fved Fig. . Cd expo1 offspring were generated to enable analyses of allele-specific gene expression and DNA methylation for a separate study . The genotypes of F1 mice are referred to as ‘B × C’ (B mother × C father) and ‘C × B’ (C mother × B father)  or −80 °C .Hybrid Fer) Fig. . F1 animSample sizes are presented in Supplementary Table 1 mice by a noninvasive tail-cuff method using a CODA 2-channel system  as described62. Mice were brought to the testing room and remained in their cages for 30 minutes to acclimate. Animals were restrained on a heating pad and covered with a towel to maintain body temperature. Animals were restrained for 5 minutes before measurements were taken. Blood pressure data used for analysis were taken on the fourth or fifth consecutive day of testing. Only readings in which a heart rate was detected were used. Sample sizes are presented in Supplementary Table Blood pressures were measured on a random subset of 5.5-month-old F1 mice was collected immediately after cervical dislocation and decapitation in 1.7 mL microtubes, allowed to clot for at least 15 minutes at room temperature, then centrifuged at 4 °C for 15 minutes at 2000 rcf to separate the serum. Serum was transferred to a clean microtube, then stored at −80 °C until analysis.Trunk blood of 6-month-old FSerum was thawed on ice, then subjected to a BCA assay to determine protein concentration . Briefly, serum was diluted 1/30 in 1X autoclaved PBS  to be within the working range of the BCA kit. Standards for the BCA kit were also diluted in 1X autoclaved PBS. 3 μL of the standards and serum samples were each tested in triplicate according to kit instructions. Absorbance at 562 nm was measured on a FLUOstar Omega microplate reader (BMG Labtech). Protein concentration was calculated according to kit instructions.The diluted serum samples were further diluted in 1X autoclaved PBS to reach a final concentration of 10 μg/mL, then subjected to a protein carbonyl ELISA kit according to the manufacturer’s instructions . Absorbance at 450 nm was measured on a FLUOstar Omega microplate reader (BMG Labtech). Sample sizes are presented in Supplementary Table 1 dissections and then stored at −20 °C was used for this purpose. Blood was weighed after collection as measuring volume was not technically practical. All maternal and F1 samples were digested in high-purity nitric acid  overnight at room temperature, then submitted for analysis through the NCSU Environmental and Agricultural Testing Service Laboratory. Trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer Elan DRCII, while macro elements were determined by ICP-Optical Emission Spectrometry (ICP-OES) using a Perkin Elmer ICP-OES Model 8000. Trace elements (ICP-MS) were run under standard conditions and data were normalized to non-analyte internal standards (115In or 103Rh) to correct for small differences due to instrumental drift, sample/standard solution matrices, and sample transport. A laboratory-check standard of a different stock solution was prepared and used to verify the calibration standard solution (RSD +/− 10%). A continuing laboratory check standard was analyzed every 12 samples, and the calibration curve was repeated if the value was +/− 10%. All samples were run against a 5 or 6 point multi-calibration curve with a linearity R2 ≥ 0.999. Macro elements run on the ICP-OES were run using a meinhardt nebulizer/cyclonic spray chamber using the same quality assurance/quality control procedure above except without internal standards. Multi-calibration standard solutions were purchased through Spex Certiprep or Inorganic Ventures, Inc. and used within the expiration date. Elements and their detection and reporting limits are listed in Table 10–30 mg of blood or liver tissue collected during maternal and F1 tissues, sex and direction of cross were determined to not significantly influence the detected levels of elements, so B × C and C × B male and female data were pooled within tissue type and treatment group. Male and female pups were equally represented in each treatment group. Maternal strain was determined to not significantly influence the levels of elements, so B and C F0 data were pooled within tissue type and Cd dose. Initially, only Cd, Zn, and Fe were quantified in F0 and F1 newborn tissues, but upon encouraging preliminary data, additional F0 B and F1 newborn B × C samples were tested for these three elements and all other elements described above. Sample sizes are presented in Supplementary Table For newborn FRNA was extracted from 24 newborn B × C female whole hearts using the AllPrep DNA/RNA/miRNA kit . Eight 0 ppm females representing 6 litters, eight 1 ppm females representing 4 litters, and eight 50 ppm females representing 5 litters were used. Hearts stored at −80 °C were homogenized in the kit’s lysis buffer using a microtube homogenizer . RNA was extracted from the hearts following the manufacturer’s protocol for 10–30 mg of starting material. RNA was suspended in nuclease-free water and quantified using a Nanodrop 2000. RNA purity and size integrity were determined at the NCSU Genomic Sciences Laboratory (GSL) using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies). All RNA samples had an RNA Integrity Number (RIN) ≥ 9.9.10ppm and four F150ppm B × C female mice  was submitted to the NCSU GSL for indexed library construction and sequencing. Purification of messenger RNA (mRNA) was performed using oligo-dT beads provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs [NEB]). Complementary DNA (cDNA) libraries for Illumina sequencing were constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) using the manufacturer-specified protocol. Following adapter ligation, the samples were selected for a final library size (adapters included) of 400–550 bp using sequential AMPure XP bead isolation (Beckman Coulter). Library enrichment was performed and indexes for each sample were added during the protocol-specified PCR amplification. Amplified library fragments were purified and checked for quality and final concentration using an Agilent 2200 Tapestation  combined with a Qubit fluorometer (ThermoFisher). Libraries were pooled in equimolar amounts for clustering and sequencing on an Illumina HiSeq. 2500 DNA sequencer, utilizing a 125 bp single-end cycle sequencing kit (Illumina). The software package Real Time Analysis (RTA) was used to generate raw bcl, or base call files, which were then de-multiplexed by sample into fastq files using bcl2fastq Conversion Software v2.17 (Illumina).Total RNA from hearts of four F63 was used for indexing, aligning, read counts, and differential expression analysis; all analyses were performed using the recommended parameters. The HISAT2-index-align-2.1 app was used to index and align reads to the mouse reference genome (GRCm38.6 assembly). Aligned reads were counted using the HTSeq-count-0.6.1 app. Differential gene expression analysis was performed using the DeSEQ2  app. A statistical report of this analysis is presented in Supplementary File Raw sequencing reads were transferred to the Cyverse Discovery Environment (DE) using Cyberduck. FastQC was performed using the FastQC 0.11.5 (multi-file) application (app). The Trimmomatic-programmable-0.33 app was used to trim the reads using the parameters HEADCROP:10, SLIDINGWINDOW:4:20, and MINLEN:20. FastQC was performed again to verify the quality of trimmed reads. The Mouse RNASeq DeSEQ2 pipeline published by Cyverse65. The KEGG, GO biological process, GO molecular function, GO cellular component, DSigDB, and MGI mammalian phenotype database results were used from the Enrichr analysis. Only enrichments with significant adjusted p values (<0.05) were considered. 10 genes that frequently occurred across multiple relevant terms and categories were chosen for qRT-PCR validation. Supplementary Fig. 3 was generated using the 302 significantly differentially expressed genes using http://www.heatmapper.ca/ using average linkage clustering method and Pearson distance measurement method.The 302 genes that were significantly differentially expressed  between 0 ppm and 50 ppm maternal Cd exposure groups were used as input for enrichment analysis using Ingenuity Pathway Analysis  and EnrichrPolr2a was not significantly differentially expressed between treatment groups (data not shown) and was used as a reference gene. Quantification of expression was calculated using the ΔΔCt method66.100 ng of total RNA extracted as described above from 24 B × C female newborn pup hearts was used as a template to synthesize first strand cDNA. DNase I treatment was performed prior to reverse transcription as part of the Qiagen AllPrep kit protocol. Random primers  were annealed to the DNase I-treated RNA, then the RT reaction was performed according to the manufacturer’s protocol . qRT-PCR was performed on a Real-Time 7300 machine (Applied Biosystems) using SsoAdvanced Universal SYBR Green Supermix . The 20 μL RT-qPCR reaction mix per well was prepared according to the manufacturer’s protocol for SYBR Green. 1 μL of the cDNA for the standards or the tested samples was included in the 20 μL reaction. cDNA of the tested samples was diluted 1/10, while the standards were diluted 1/5, 1/10, 1/20, 1/40, and 1/80. Standards, the no template control (NTC), and all other tested samples were each run in triplicate. The cycling conditions were as follows: 95 °C for 30 seconds; 40 cycles of 95 °C for 15 seconds, 60 °C for 30 seconds; dissociation curve of 60.0 °C–95.0 °C. The primer sequences are provided in Supplementary Table 67  followed by Dunnett’s multiple comparison test using R software, comparing animals from the 1 ppm and 50 ppm groups to 0 ppm controls. Outliers, as defined by a point which falls more than 1.5 times the interquartile range above the third quartile or below the first quartile, were omitted from the analysis. B × C litters under n = 6 and C × B litters under n = 3 were omitted from raw or proportional weight analyses. Data are presented as the mean ± standard error of the mean. Modeling the data using JMP software (SAS Institute Inc.) showed no significant effects of litter on the measured endpoints.Supplementary File 1Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6


Circuit quantum electrodynamics has proven to be a powerful tool to probe mesoscopic effects in hybrid systems and is used in several quantum computing (QC) proposals that require a transmon qubit able to operate in strong magnetic fields. To address this we integrate monolayer graphene Josephson junctions into microwave frequency superconducting circuits to create graphene based transmons. Using dispersive microwave spectroscopy we resolve graphene’s characteristic band dispersion and observe coherent electronic interference effects confirming the ballistic nature of our graphene Josephson junctions. We show that the monoatomic thickness of graphene renders the device insensitive to an applied magnetic field, allowing us to perform energy level spectroscopy of the circuit in a parallel magnetic field of 1 T, an order of magnitude higher than previous studies. These results establish graphene based superconducting circuits as a promising platform for QC and the study of mesoscopic quantum effects that appear in strong magnetic fields. A transmon qubit insensitive to magnetic fields is a crucial element in topological quantum computing. Here, Kroll et al. create graphene transmons by integrating monolayer graphene Josephson junctions into microwave frequency superconducting circuits, allowing them to operate in a parallel magnetic field of 1 T. A transmon qubit consists of a Josephson junction (JJ) shunted by a large capacitance, coupled to a high quality factor superconducting resonator. In conventional transmon devices, the resonator is fabricated from Al and the JJ is fabricated from an Al/AlOx/Al tunnel junction1, both of which cease operation above the critical magnetic field of bulk Al, ~10 mT. Even when considering alternative type II superconductors such as NbTiN or MoRe that can sustain superconductivity beyond B = 8 T7, when subjected to a strong magnetic field the superconductor will experience detrimental effects such as reduction of the superconducting gap, increased quasiparticle generation8 and the formation of Abrikosov vortices that cause resistive losses in a microwave field. In addition to disrupting the superconductivity, magnetic flux penetrating the JJ produces electron interference effects that reduce the Josephson energy EJ and strongly suppress the transmon energy spectrum. If the transmon is to be used for fast quantum gates, fast charge-parity detection and long range quantum state transfer in QC schemes10 we are compelled to consider alternatives to conventional Al based JJs. Proximitised semiconducting nanowires, acting as gate-tuneable superconductor-normal-superconductor JJs11 have been used successfully in a variety of microwave frequency superconducting circuits, allowing for studies of Andreev bound states13, electrically tuneable transmon qubits15 and transmons that exhibit substantial field compatibility16. Graphene JJs are an attractive alternative as they exhibit ballistic transport, high critical currents18 and the atomic thickness of the graphene junction greatly reduces flux penetration, protecting the JJ from orbital interference effects that would suppress EJ in high parallel fields. When combined with geometric techniques that protect the superconducting film, such as critical field enhancement19 and lithographically defined vortex pinning sites21, the transmon circuit can be protected at magnetic fields relevant to these proposals, which approach and in some cases exceed 1 T24.A superconducting transmon qubitB|| = 0 T to resolve a linewidth of ≃ 400 MHz. Although the large linewidths prevent coherent qubit control, we demonstrate the device is insensitive to the applied magnetic field up to B|| = 1 T.In this work we report the integration of ballistic graphene JJs into microwave frequency superconducting circuits to create graphene based transmons. Using dispersive microwave spectroscopy we resolve the characteristic band dispersion of graphene, and observe coherent electronic interference effects that confirm the ballistic nature of our graphene JJs. We perform energy level spectroscopy at λ/4 coplanar waveguide (CPW) resonators multiplexed to a common feedline. Each resonator is capacitively coupled to a graphene transmon, with the graphene JJ being shunted by capacitor plates that provide a charging energy EC ≃ 360 MHz. The resonators and capacitor plates are fabricated from 20 nm NbTiN due to its enhanced critical magnetic field19, and we pattern the resonators with a lattice of artificial pinning sites to protect the resonator from resistive losses due to Abrikosov vortices21. The van der Waals pickup method is used to encapsulate monolayer graphene (G) between two hexagonal boron nitride (hBN) flakes and deposit it between the pre-fabricated capacitors plates  gives a large EJ, placing ft above the resonator and giving χ a small negative value  fr Fig. . This be1 T Fig.  and calc1 T Fig.  again re1. The readout tone is set to fr whilst a second tone fd is used to drive the circuit. Excitation of the system results in a state dependent shift of the resonator frequency, and is detected by measuring the change in the complex transmission S21 at fr. At VG = 0 V (p-regime), two-tone spectroscopy at B|| = 0 and 1 T  that can be fitted to extract ft and γ, giving a minimum linewidth of 166 MHz , then deposited between pre-fabricated capacitors using a PMMA based van der Waals pickup method. Contact to the graphene stack is made by etching in a CHF3/O2 environment, followed by sputtering MoRe (t = 80 nm). As shown in Supplementary Fig. 3/O2 plasma to be 1000 × 300 nm2 in size. Device B was contacted to provide a junction length of 500 nm. The long thin leads were geometrically restricted in two dimensions, making it less favourable for vortices to form, protecting the superconductivity of the contacts proximitising the junction. The junction is then shaped in a CHF3/O2 plasma to be 500 × 500 nm2. A SiNx/Ti/Au top gate stack is then sputtered to give full junction coverage, giving greater control of μ in the junction.To fabricate the two devices (A and B) 20 nm of NbTiN is sputtered onto intrinsic Si wafers in an Ar/N atmosphere. The resonators, feedline and transmon are reactive ion etched in an SFπ and copper powder filters. The line used to drive the feedline input was heavily attenuated to reduce noise and thermal excitation of the cavity, allowing the single photon cavity occupancy to be reached. The output line of the feedline was connected to an isolator (Quinstar QCI-080090XM00) and circulator (Quinstar QCY-060400CM00) in series to shield the sample from thermal radiation from the HEMT amplifier (Low Noise Factory LNF-LNC4-8_C) on the 4 K stage. Resonator spectroscopy of device A was performed using circuit (a) to measure the amplitude and phase response of the complex transmission S21 as the frequency was varied. Resonator and two-tone spectroscopy of device B was performed using circuit (b), with a splitter used to combine the readout and excitation tones. This allows the complex S21 to be measured, but only at fixed resonator readout frequency otherwise only |S21| can be recorded.All measurements were performed in a dilution refrigerator with a base temperature of 15 mK. The samples were enclosed in a light tight copper box, and thermally anchored to the mixing chamber. An external magnetic field is applied to the sample using a 3-axis vector magnet. The two different measurement configurations used in this manuscript are depicted in Supplementary Fig. Supplementary InformationPeer Review File


We show that individual thiophene units and the lattice of semicrystalline spin-coated films of polythiophenes (PTs) may be resolved using AFM under ambient conditions through the low-amplitude (≤ 1 nm) excitation of higher eigenmodes of a cantilever. PT strands are adsorbed on hexagonal boron nitride near-parallel to the surface in islands with lateral dimensions ~10 nm. On the surface of a spin-coated PT thin film, in which the thiophene groups are perpendicular to the interface, we resolve terminal CH Semiconducting polymers are widely used in optoelectronic devices, in which their microstructure informs function. Here, the authors are able to resolve the molecular and sub-molecular ordering of polythiophene strands and thin films using atomic force microscopy, a significant step towards correlating polymer structure with device performance. These considerations are particularly important for conducting and semi-conducting organic polymers8, which are used widely in organic photovoltaics10, light-emitting diodes12 and other flexible optoelectronic devices16. The polymer microstructure plays a crucial role in the transport of charge carriers and excitons, thus determining device performance17. The close relationship between microstructure and carrier mobility is well established through X-ray diffraction studies of, e.g., polythiophene (PT) and its derivatives, which show that domains with typical dimensions of a few tens of nanometres are formed18. However, the nature of the boundaries between domains, which are crucial to an understanding of carrier transport at a macroscopic level17, cannot be determined from reciprocal space methods. In principle, scanning probe microscopy (SPM)21 is ideally suited to such structural studies, but the resolution which is routinely available using conventional atomic force microscopy (AFM) under ambient conditions is a significant constraint to the acquisition of images, which reveal ordering of polymers at the molecular and sub-molecular scale.Polymers, both synthetic and natural, are ubiquitous materials whose properties are strongly influenced by packing, conformation, and monomer composition of individual macromolecules. The ability to acquire real-space images of the microstructure of these materials with molecular-scale resolution is required to advance the understanding and control of their local ordering, a key element in the precise engineering of polymer properties22 and UHV26, images revealing single polymer strands may be acquired. This body of work also includes several studies of polymers formed by on-surface synthesis on noble metal surfaces30. AFM has also been used to acquire images of single strands of synthetic polymers37, whereas the introduction of torsional tapping-mode AFM imaging38 led to the acquisition of images of single molecules and semicrystalline domains of polyethylene39 achieving resolution down to 0.37 nm in air. This approach, however, requires specialized T-shaped ultrasharp cantilevers with carbon-whisker tips and a modified AFM with improved noise characteristics of the lateral deflection signal39. Previously, we have shown that higher eigenmodes can provide a route to higher resolution on molecular films and two-dimensional (2D) materials under ambient conditions41 and, in addition, Proksch and colleagues31 have recently shown that bimodal tapping imaging, where two eigenmodes are excited simultaneously, can provide high spatial resolution of semicrystalline polypropylene and polyethylene alongside their mechanical properties. A comprehensive overview of significant advances in polymer research achieved with AFM has been published by Wang and Russell1.In many examples of the high-resolution structural characterization of artificial polymers using SPM, scanning tunnelling microscopy (STM) is the preferred imaging modality, as, under a range of operating conditions including liquidIn this study we show that images with ultra-high resolution can be achieved in a conventional tapping mode (AC mode) using standard silicon nitride probes in conjunction with a commercially available instrument by exciting higher-order resonances of the cantilever; the resulting images provide new insights into the molecular-scale structure and order of semi-conducting polymers. The polymers chosen for our study are two commercial PTs, materials that are widely used to fabricate organic solar cells and field effect transistors, and we demonstrate this level of resolution for both single polymer chains deposited on the atomically flat surface of hexagonal boron nitride (hBN) and at the surface of a spin-coated thin film with a roughness of a few nanometres.PTs are formed from thiophene units that are covalently linked at the 2- and 5-positions of the heterocycle  and the lone pair of electrons localized on the that atom. A model structure of quaterthiophene is overlaid on Fig. p orbitals of sulphur atoms producing an exaggerated width for the PT backbone.We have further imaged individual P3DT molecules using a combination of small oscillation amplitude ) and the third eigenmode of the Multi75Al-G cantilevers to reveal the intramolecular structure of the PT backbone see Fig. . We have49 dispersion correction  surface. However, in its crystalline form, terthiophene exhibits a twisted trans-geometry with a torsional angle of about 171°–174°46. Bithiophene is known53 to have a completely flat  structure in the solid state and a twisted structure in the gas phase with a torsional angle of 148° ± 3°. Our calculations predict that bithiophene should have a twisted trans-gauche geometry when adsorbed on hBN with the same torsional angle of 173° calculated for terthiophene. Therefore, our calculations predict a twisted geometry with protruding sulphur atoms, thus accounting for the observed contrast in AFM scans of P3DT on hBN. In addition, the calculated S–S separation (0.78 nm) and molecule–substrate spacing (0.33 nm) are in good agreement with our measured values.To strengthen the assignment of the high-contrast features in Fig. 73° Fig.  and sulpWe also show in Fig. 54. In such images, we clearly resolve a square lattice with approximate dimension of 0.55 nm × 0.55 nm. These regions can be resolved in large area images  poly (P3HT). For these investigations, the P3HT film was deposited on a mica substrate on which a PEDOT:PSS layer had been previously deposited by spin coating (see Methods for more details). P3HT films formed in this way are expected to have similar morphological properties as the active layers in photovoltaic devices. Figure ges Fig.  where we58 from diffraction and spectroscopic studies that regioregular P3HT thin films form semi-crystalline domains in which the plane of the PT backbone is perpendicular to the substrate, forming an in-plane π–π stacked organization. In this arrangement, neighbouring sulphur atoms in a P3HT chain point alternatively up (away from the surface) and down (towards the surface). Neighbouring π–π stacked P3HT chains are arranged so that an ‘up’ sulphur atom is aligned with a ‘down’ sulphur in the adjacent chain. This leads to a crystal structure with an in-plane square lattice with dimensions determined from diffraction measurements to be b = c = 0.78 nm  within the film. In Fig. 59. The high-resolution AC-mode phase-channel image in Fig. x and y directions across the entire image and a 2DFFT is calculated at each position  analysis of the P3HT image in Fig. see Fig. . A typicsee Fig.  shows insee Fig. , revealisee Fig.  and showsee Fig.  that ovesee Fig. .Fig. 4Fo17 of disorder in semi-crystalline semiconductor polymers and represents a simple way in which orientational order is randomized; neighbouring domains have clearly distinct order and well-defined boundaries. In contrast, the boundary region in Fig. To highlight further the differences between the boundaries in the upper left of Fig. 17); although we see domains with local order that is preserved only over ~10 nm, in agreement with diffraction measurements5, the orientation of neighbouring 10 nm-scale domains is correlated within a super domain with much larger dimensions.The slowly varying lattice vectors in Fig. 26, whereas the compatibility of our method with solution deposition and technologically relevant spin-coated thin films offers some potential advantages. The real-space images are in good agreement with previous studies5 but provide new insights into the nature of the boundaries between domains that are not easily determined using reciprocal space methods. Our results highlight the important role for high-resolution AFM in determining the properties of polymer strands and thin films of technological relevance, and we anticipate future progress in correlating device performance with structural properties at the sub-molecular scale based on this technique.Overall, we have demonstrated that the use of higher eigenmodes in tapping-mode ambient AFM can be successfully employed to characterize both individual polymer strands down to a single-atom level and also the ordering of a semi-crystalline polymer with technological relevance. The combination of AFM and solution deposition provides a simple and high-resolution approach to characterizing the structure of polymers. This approach is complementary to the acquisition of images using a combination of electrospray deposition and SPM under ultra-high vacuum conditions2 layer with thickness of 300 nm. Following exfoliation, the substrate was cleaned by brief flame annealing with a butane torch. The polymer was deposited from a toluene solution (2.5 µg/ml); deposition times ranged between 10 and 30 s .The hBN flakes were transferred by exfoliation onto a silicon wafer terminated by a SiO−1) was dissolved in chlorobenzene and stirred overnight in darkness at 60 °C. PEDOT:PSS  was filtered through a polyvinylidene difluoride filter (0.45 μm pore size) and deposited onto mica substrates by spin casting . The films were annealed on a hot plate for 10 min at 130 °C. P3HT was deposited by spin casting . The samples were imaged before and after annealing. Annealed films were heated on a hot plate for 15 min at 130 °C; we observed no significant differences between annealed and non-annealed films.P3HT (regioregular > 95%) was purchased from Sigma Aldrich and used without further purification; PEDOT:PSS (HTL Solar) was purchased from Ossila. Spin-cast devices were prepared using a POLOS spin150i spin coater. P3HT (23 mg mlb/√2 = 0.55 nm lattice constant of the square P3HT lattice), a modulo 90° was then applied to the peak angle due to the square symmetry of the lattice. If the strongest intensity peak fell outside of this periodicity range, then 45° was also subtracted from the angle value before the modulo 90° operation, as this is associated with spatial frequency components along the b = c = 0.78 nm direction of the P3HT lattice. The angles of the strongest Fourier peaks were then plotted at the position of the kernel centres using a cyclic colour scale in the range 0–90°. Raw AFM data were extracted from.ibw files using Gwyddion60, and further Fourier processing and data presentation was performed using a Python script and presented using Matplotlib61.Following the sliding 2DFFT operation on each kernel position (see main text), the power spectrum at each position was rotationally interpolated at 1° increments and only spatial periodicities in the range 0.4–1.5 nm were retained to remove periodic noise due to scan artefacts and high-frequency noise along the raster direction . Maximum intensity values were then extracted as a function of rotation angle along with the spatial frequency at the position of the most intense peak. For peaks with a spatial period in the range 0.4–0.6 nm (i.e., the Supplementary InformationPeer Review File