Pre- and 2-h post-treatment blood samples were taken. AWS severity was recorded using the Glasgow Modified Alcohol Withdrawal Score (GMAWS). The primary outcome measure was 15% change in erythrocyte transketolase activity (ETKA) in group 3. Secondary outcome measures were change in plasma lactate concentrations and time to GMAWS = 0. 127 patients were recruited, 115 patients were included in the intention-to-treat analysis. Pre-treatment, the majority of patients had normal or high erythrocyte thiamine diphosphate (TDP) concentrations (≥ 275–675/> 675 ng/gHb respectively) (99%), low serum magnesium concentrations (< 0.75 mmol/L) (59%), and high plasma lactate concentrations (> 2 mmol/L) (67%). Basal ETKA did not change significantly in groups 1, 2 or 3. Magnesium deficient patients (< 0.75 mmol/L) demonstrated less correlation between pre-treatment basal ETKA and TDP concentrations than normomagnesemic patients (R2 = 0.053 and R2 = 0.236). Median plasma lactate concentrations normalized (≤ 2.0 mmol/L) across all three groups , but not among magnesium deficient patients in group 1 (n = 22). The median time to achieve GMAWS = 0 for groups 1, 2 and 3 was 10, 5.5 and 6 h respectively (p < 0.001). No significant difference was found between groups for the primary endpoint of change in ETKA. Co-administration of thiamine and magnesium resulted in more consistent normalization of plasma lactate concentrations and reduced duration to achieve initial resolution of AWS symptoms.Alcohol withdrawal syndrome (AWS) occurs in 2% of patients admitted to U.K. hospitals. Routine treatment includes thiamine and benzodiazepines. Laboratory studies indicate that thiamine requires magnesium for optimal activity, however this has not translated into clinical practice. Patients experiencing AWS were randomized to three groups: (group 1) thiamine, (group 2) thiamine plus MgSOClinicalTrials.gov: NCT03466528. Currently, one in five patients admitted into the UK hospital system satisfy criteria for diagnosis of alcohol use disorder (AUD)2. Previous reports estimate that 10% of patients who satisfy the criteria for diagnosis of AUD will develop alcohol withdrawal syndrome (AWS) during their hospital admission3.In 2018, the WHO reported that harmful alcohol use accounted for 5% of deaths worldwide and 10% of deaths per year in the European region5. In contrast, thiamine concentrations were in the normal range and not independently associated with poorer survival at 1-year5. This study also reported that > 60% patients attending the Emergency Department with AWS had plasma lactate concentrations > 2.0 mmol/L5. Thiamine and magnesium are recognised to be cofactors in a number of enzymes such that they may influence the formation of lactate5. Thiamine and magnesium act as essential co-factors for a family of three mitochondrial dehydrogenases  that orchestrate efficient mitochondrial aerobic ATP production6. Therefore, in the context of normal partial pressure tissue oxygen concentrations and in the absence of an inflammatory response or intense skeletal muscle activity , thiamine and/or magnesium deficiency mediated increase in metabolism of glucose to lactate may represent pseudo-hypoxic disruption of normal glucose metabolism (‘Dirty burn metabolism’) , which catalyses the reversible arm of the Pentose Phosphate Pathway (PPP). TK has a central role in the generation of reducing equivalents for the maintenance of the cellular redox state10. Furthermore, increased plasma lactate concentrations may occur with thiamine and/or magnesium deficiency15.Biochemical evidence of thiamine deficiency may manifest directly with reduced mass of thiamine in erythrocytes, or indirectly with reduced activity of the functional enzymatic marker of thiamine status, erythrocyte transketolase activity (ETKA)2+) is required for optimal absorption of thiamine from the gut, activation of free thiamine to active phosphorylated form (thiamine diphosphate (TDP)) and optimum activity of TDP dependent enzymes16. This biochemical interdependence has been understood for decades, however only one small study has examined the in vivo effect of concomitant administration of thiamine with magnesium17. A Cochrane review concluded that there was insufficient evidence as to the duration of treatment and dosages of thiamine required for the treatment/prevention of Wernicke’s encephalopathy (WE), while acknowledging that thiamine utilization in patients experiencing alcohol withdrawal syndrome is probably influenced by Mg2+ status18. Moreover, another Cochrane review concluded that there was insufficient evidence to currently support the routine administration of magnesium to patients suffering acute alcohol withdrawal syndrome19.Interestingly, magnesium  when patients (n = 36) with AWS were treated with combined thiamine and magnesium versus thiamine aloneTo better understand the clinical practicalities and biochemical effects of concurrent thiamine and magnesium supplementation on ETKA, plasma lactate concentrations and rate of resolution of alcohol withdrawal syndrome, an investigation of a cohort of AUD patients presenting to the Emergency Department (ED) with symptoms of alcohol withdrawal syndrome was undertaken. All participants were recruited to the study following presentation to the Emergency Department with symptoms or signs of alcohol withdrawal syndrome. Patients with a background history of heart failure or renal impairment were excluded.20. Patients were recruited in the ED at Glasgow Royal Infirmary by convenient enrollment between 1/1/2017 and 1/6/2018 and followed up for one year thereafter. The trial ended when recruitment met last patient/last visit criteria.The trial protocol was approved by the West of Scotland Research Ethics Committee (16/WS/0162) and registered with clinicaltrials.gov (NCT03466528) (15/03/2018). All methods and procedures were carried out in accordance with relevant guidelines and regulationshttps://www.sealedenvelope.com/simple-randomiser/v1/listsInformed consent was received prior to administration of questionnaire and venesection. Randomization was performed by the trial statistician using the sealed envelope programme: Opaque envelopes detailing the subject number and treatment allocation were prepared. Patients were sequentially assigned a subject number once eligibility was confirmed and the corresponding treatment assignment envelope opened. The opened enveloped was retained and attached to the patient’s case report form (CRF). Patients were blinded to randomization allocation.Patients who were unable to give informed consent or < 18 years of age were excluded. Patients with a history of chronic renal or hepatic failure, hepatic encephalopathy; known hypersensitivity or previous allergy to any of the active substances in either trial medication; or severe concurrent medical condition that would prevent participation in trial procedures  were also excluded.Male or non-pregnant or breastfeeding females ≥ 18 years of age who were capable of giving informed consent and confirmed to be experiencing alcohol withdrawal syndrome (AWS) were approached for trial participation.22.AWS was determined by the Glasgow Modified Alcohol Withdrawal Scale (GMAWS) and standardized benzodiazepine (BDZ) treatment was administered as per the GMAWS protocol4) 2 g supplementation over 30 min duration according to randomization status. Pabrinex® is a multivitamin preparation that is equivalent to the “banana bag” administered in U.S. Emergency Departments.Thiamine in its commercial form, Pabrinex® (U.K.), was administered intravenously to patients with or without magnesium sulphate  or (group 3) MgSO4 2 g . Blood samples were taken for analysis prior to, and 2 h post administration of thiamine+/MgSO4. Patients who received MgSO4 alone (Group 3) received standard treatment  immediately after the 2-h trial samples were drawn. Intravenous fluid administration was standardized for all patients .Patients were randomized to receive one of three treatments: (group 1) standard treatment  or (group 2) standard treatment plus MgSO23.Standard serum separating tubes were used for routine serum biochemistry, EDTA tubes were used for both routine and erythrocyte thiamine diphosphate sample collection and trial samples were collected in non-gel lithium heparin tubes for measurement of ETKA and erythrocyte magnesium concentrations. All samples were conveyed to the laboratory within 1 h of collection. Routine biochemistry and hematology samples were analyzed contemporaneously. Whole blood EDTA samples for thiamine diphosphate measurement were frozen at − 70 °C and analyzed within ten days of being drawn. Trial samples for measurement of ETKA and erythrocyte magnesium were centrifuged (500G for 10 min), and plasma was carefully removed. Erythrocytes were washed three times prior to storage. Both the separated plasma and packed erythrocytes were stored at − 70 °C until analysis. All samples from any given patient were assayed in a single batch for each of the analytes to minimize inter-batch analytic variation23. The percentage increase in transketolase activity following addition of TDP represents the patient’s thiamine status (> 15% and > 25% indicating moderate and severe thiamine deficiency respectively). For the present trial, in addition to routine ETKA measurement (in vitro TDP enhanced ETKA measurement), pre- and post-treatment basal ETKA has also been reported to examine the relative change of ETKA in response to the randomized treatment administered.Routine ETKA measurement (in vitro TDP enhanced ETKA measurement) is a functional marker of thiamine status that measures the percentage change in ETKA when TDP is added to the assay, relative to basal ETKA23.Measurement of thiamine diphosphate in erythrocyte involved HPLC isocratic separation with post-column derivatization using sodium hydroxide and potassium ferricyanide and fluorescent detection. Results were expressed as nanogram of thiamine diphosphate per gram of haemoglobin (ng/gHb)24. Briefly, ETKA was determined by a two-step coupled enzymatic reaction both with and without the in vitro addition of TDP . The reduction of NADP+ to NADPH was measured spectrophotometrically at 340 nm over 30 min to calculate ETKA in U/gHb. Haemoglobin for both thiamine diphosphate and ETKA methods were determined by the cyano-methaemoglobin assay25:Erythrocyte transketolase (ETKA) was measured by adapting the method developed by Bayoumi and Rosalki26. Haemoglobin concentration in the sample was derived using the 57Fe concentration and the above equation26.Erythrocyte magnesium was measured using inductively coupled plasma mass spectrometry on the Agilent 7900 ORS-ICP-MS . Erythrocyte magnesium was reported as mmol per gram of haemoglobin (mmol/gHb) to mitigate pipetting errors that are commonly associated with pipetting packed erythrocytes due to their viscosity23.Serum C-reactive protein (CRP), albumin and magnesium were measured in accordance with the manufacturer’s instructions, by routine laboratory procedures, on an automated analyzer . For C-reactive protein, the limit of detection was 1 mg/L. The inter-assay CV was < 5% over the sample concentration range for the analytes measured17, the primary endpoint of 15% change in ETKA in the magnesium only arm was chosen for the present trial. A three-group trial was designed to detect a minimum biochemically significant difference of 0.16 U/gHb change from baseline between the groups, with 80% power and a 5% significance level. This required 37 participants to be randomised to each arm of the trial. A total sample size of 120 was chosen to account for any dropouts or missing data.Based on pilot study data that showed significant change in thiamine dependent enzyme activity (ETKA) when patients (n = 36) with AWS were treated with combined thiamine and magnesium versus thiamine alones). All analyses were done using SPSS software  at a 5% significance level. Owing to the number of statistical comparisons carried out (~ 45), a p value ≤ 0.01 was considered statistically significant.Data are presented as medians and inter-quartile ranges (IQR). Categorical data were analysed using chi-squared and McNemar’s tests; continuous data were analysed using Mann–Whitney or Kruskal–Wallis tests. Wilcoxon’s signed ranks test was used to analyse the pre- and post-treatment difference between paired samples within randomization groups. Repeated measures ANOVA was used to analyse the pre- and post-treatment difference between randomization groups and post-hoc comparisons were done using Tukey and Dunnett’s tests. Correlations were calculated using Spearman’s correlation (r27.All patients (n = 127) recruited to the trial fulfilled the criteria for inclusion with age, sex, BMI and objective evidence of alcohol withdrawal syndrome or scored positively on FAST questionnaire indicating Alcohol Use Disorder. Twelve patients were recruited more than once and therefore excluded from the analysis after the first attendance resulting in a total of 115 patients included in the baseline analysis; 3 patients opted out of the trial prior to administration of the trial drug and self-discharged from the Emergency Department. All 115 patients are included in the intention to treat analysisThe clinicopathological characteristics are shown in Table Of the laboratory analysis, the majority of patients had bilirubin (58%), alkaline phosphatase (68%), AST/ALT ratio < 2 (65%), albumin (77%), CRP (57%), urea (68%), sodium (71%), potassium (82%), MCV (51%) and platelets (70%) within the laboratory reference interval.p = 0.064)  in the outpatient setting. It was of interest that only one of the patients recruited to the trial had erythrocyte thiamine concentration below the reference range < 275 ng/gHb) < 1%) prior to randomisation and treatment. Prior to randomisation and treatment, the majority of patients (59%) had erythrocyte thiamine diphosphate concentrations within the normal reference range (≥ 275–675 ng/gHb) and 40% of patients had erythrocyte thiamine diphosphate concentrations above the reference range (> 675 ng/gHb)  had circulating magnesium concentrations that could be considered to be low (< 0.75 mmol/L) Fig.  and had Following treatment, patients randomised to group 1  and group 2 (combined thiamine and magnesium treatment group) were observed to increase erythrocyte thiamine diphosphate concentrations   (see Table p < 0.001) , was not observed to change significantly in group 1 , group 2 (thiamine and magnesium group) or group 3     demonstrated less correlation between pre-treatment basal ETKA and erythrocyte thiamine diphosphate concentrations than patients who had serum magnesium concentrations in the normal range (≥ 0.75 mmol/L) (pre-treatment R mmol/L dp = 0.843).Of the laboratory analysis of substrates and products of intermediary metabolism, the median plasma glucose concentration for majority of patients (68%) was within the normal range  prior to randomisation and treatment (Table p < 0.001), GMAWS max ≥ 4 (n = 110) (p = 0.009) and length of hospital stay > 5 days (n = 104) (p = 0.020).It was of interest that pre-treatment plasma glucose ≥ 7.0 mmol/L was significantly associated with pre-treatment plasma lactate concentration > 2.0 mmol/L (n = 105)  were found to have plasma lactate concentrations > 2 mmol/L, and the median concentration of plasma lactate was > 2 mmol/L across all three randomization groups. Prior to treatment, 64%, 72% and 67% of patients in groups 1, 2 and 3 respectively had plasma lactate concentrations > 2 mmol/L. Following treatment, 44%, 26% and 42% of patients in groups 1, 2 and 3 respectively had plasma lactate concentrations > 2.0 mmol/L   across all three randomisation groups (p = 0.192). On secondary analysis, the median time to achieve a GMAWS score = 0 for groups 1, 2 and 3 was 10, 5.5 and 6 h respectively (p < 0.001) . The median time for patients with pre-treatment serum magnesium concentrations < 0.75 mmol/L to achieve a GMAWS score = 0 in groups 1 (n = 19), 2 (n = 19) and 3 (n = 19) was 10, 6 and 6 h respectively (p = 0.013).Prior to randomisation and treatment, the majority of patients (54%) n = 61) were found to have GMAWS ≥ 4  at time of recruitment to the trial (Table  were foup = 0.060) . The median diazepam equivalent dose of benzodiazepine required for patients with pre-treatment serum magnesium concentrations < 0.75 mmol/L to achieve a GMAWS score = 0 in groups 1 (n = 19), 2 (n = 19) and 3 (n = 18) was 60, 50 and 40 mg respectively (p = 0.130).The median diazepam equivalent dose (mg) of benzodiazepine required to achieve the first GMAWS score = 0 for patients in groups 1, 2 and 3 was 50, 40 and 30 mg respectively , however it has not replicated the significant enhancement of ETKA that was measured in the pilot trialIn the present trial, group 1  and group 2 (thiamine and magnesium group) were observed to demonstrate increases in erythrocyte thiamine diphosphate concentrations. It was of interest that only one of the patients was thiamine deficient (< 1%) prior to randomisation and treatment. Following treatment, the majority of patients in group 1 (97%) and group 2 (94%) had erythrocyte thiamine diphosphate concentrations above the reference range (> 675 ng/gHb). In group 3, 30% of patients had pre-treatment erythrocyte thiamine diphosphate concentrations above the reference range and this remained unchanged following randomisation and treatment.In contrast to thiamine status, the majority of patients (59%) were observed to be magnesium deficient (serum magnesium concentration < 0.75 mmol/L) and the median serum magnesium concentration was observed to be below the lower limit of the reference interval across all three randomisation groups. Patient groups 2 and 3 who received magnesium alone or combined treatment with thiamine and magnesium increased their serum magnesium concentrations and it was of note that neither of these groups reached serum magnesium concentrations in the toxic range.One potential explanation, for the difference between the pilot study and the present trial in ETKA results, is that some of the patients recruited to the pilot study may have been thiamine and/or magnesium deficient, whereas 99% of patients recruited to the present trial were thiamine replete.28, however no consideration had been given to the potential role of co-existent magnesium deficiency when this assertion was made.Nonetheless, it was of interest that ETKA was activated > 15% in some of the patients in the present trial who had normal direct measurements of pre-treatment erythrocyte thiamine diphosphate concentrations Table a. This oThe present randomised trial gives an insight into the activity of ETKA in the presence of low and normal magnesium status in both in vitro and in vivo settings. When pre-treatment basal ETKA results were analysed in the context of magnesium status, it was apparent that the activity of ETKA following stimulation with TDP was significantly less among the magnesium depleted groups as compared to the magnesium replete groups Fig. a.30, such measurements are currently batch analysed and therefore not available to the clinician when deciding whether or not to administer thiamine.ETKA fell out of favour as a measure of thiamine status in the late 1990’s as it had been reported to be difficult to standardize, resulting in significant inter-laboratory variability. This difficulty in standardization and significant inter-laboratory variability may have been attributable to variation in baseline magnesium status in individual samples. Although routine ETKA measurement has been superseded by the advent of high performance liquid chromatography (HPLC) that enabled reliable and direct measurement of TDP in erythrocyte samples31 and some have suggested that it was the activity of thiamine, which was relevant, rather than the finite mass available32. On this basis, some authorities continue to recommend the measurement of baseline and TDP enhanced ETKA as the gold standard for the accurate quantification of intracellular thiamine status33. However, to date this laboratory based knowledge has not translated into evidenced based clinical practice.Furthermore, it is well established in the biochemistry laboratory setting that the activity of transketolase is dependent on the presence both TDP and magnesium4, the results of the present trial may offer an insight into one potential mechanism by which low circulating magnesium status may result in suboptimal thiamine dependent enzyme activity and deranged intermediary metabolism.In the present trial, when ETKA results are analysed in the context of magnesium status, it is apparent that basal ETKA relative to erythrocyte thiamine diphosphate concentrations is significantly less among the magnesium depleted groups as compared to the magnesium replete groups  had plasma lactate concentrations > 2.0 mmol/L and the median plasma lactate concentration was > 2.0 mmol/L across all three randomization groups. This observation persisted when patients who had experienced a seizure in the 12-h period prior to the trial related presentation were excluded from the analysis. Furthermore, it was of interest that there was a significant normalization of plasma lactate concentrations in those patients who were initially magnesium deficient in the thiamine and magnesium (Group 2) and the magnesium alone (Group 3) groups. In contrast, plasma lactate concentrations did not normalise among magnesium deficient patients in the thiamine alone group (Group 1) and median plasma lactate concentrations were observed to remain above the upper limit of the reference interval (> 2.0 mmol/L) among magnesium deficient patients in this group (Group 1) see Fig. . The con34, the consistent normalization of plasma lactate concentrations demonstrated among patients in the present trial who received combined thiamine and magnesium treatment is worthy of investigation in other clinical conditions (e.g. Systemic Inflammatory Response Syndrome).This trial presents the largest cohort of AWS patients to have non-seizure related elevated plasma lactate concentrations recorded to date and as such elevated plasma lactate concentrations may have further use as a predictor of likelihood of development of severe AWS. Furthermore, given that sustained elevation of plasma lactate concentrations are widely recognized by clinicians as an adverse prognostic indicator in both general and critical care clinical settings36. The activity of these receptors is interdependently balanced. NMDA activity is mediated by glutamate38. Alcohol directly enhances GABA receptor complex activity and chronic alcohol consumption results in down-regulation of GABA receptors36. Chronic excess alcohol consumption also generates increased glutamate production and mediates up-regulation of NMDA receptor activity39. Hence, when a ‘relative deficiency’ of alcohol occurs in a chronic alcohol user, NMDA activity predominates causing symptoms of alcohol withdrawal to manifest and, if left untreated, seizures to occur40. The data presented in the present trial would suggest that those patients who received magnesium (Groups 2 and 3) experienced faster initial resolution of AWS than those in Group 1 who received thiamine alone. Hence, the combined administration of thiamine and magnesium to patients with AWS related elevated plasma lactate in the context of low circulating magnesium concentrations may have therapeutic potential in ameliorating the duration/severity of AWS experienced by patients. However, despite the significant reduction in time to initial resolution of AWS, no significant benzodiazepine sparing effect was shown in the data. This raises the possibility that other micronutrients that influence the stability of the NMDA receptor may also be deficient in this patient group e.g. zinc.Inhibitory gamma-aminobutyric acid (GABA) and excitatory N-methyl-D-aspartate (NMDA) neurotransmitter receptors are implicated in the pathogenesis of alcohol withdrawal syndrome and seizures41. Thus, reduced GABA production may be attributable to both direct and indirect mechanisms of alcohol related compromise of Krebs cycle. However, to the best of our knowledge, there are no studies reporting an association between parenteral thiamine administration and treatment of AWS symptoms. While it is possible that the delayed administration of thiamine to patients in treatment Group 3 may have delayed enhanced GABA production by optimisation of thiamine dependent enzyme function, this is not reflected in the data. Moreover, NMDA receptor channels are reported to exhibit blockade by physiological concentrations of magnesium and a high affinity voltage dependent magnesium-binding site has been shown to regulate the activity of this receptor channel42. NMDA receptors therefore require magnesium to maintain stability and integrity44, and low magnesium concentrations are known to up-regulate NMDA receptor activity, lowering seizure thresholds45. Hence, the reduction in time to achieve initial GMAWS = 0 in the groups that received parenteral MgSO4 (Groups 2 and 3) compared to the group that received thiamine alone (Group 1) may be attributable to the direct stabalizing effect of Mg2+ on the NMDA receptor channel.It is also of interest that essential neurotransmitters  are produced by Krebs cycleOverall, this therapeutic approach warrants further exploration as it remains to be conclusively determined if co-administration of thiamine and magnesium will result in faster resolution of elevated plasma lactate concentrations, reduced duration and/or severity of AWS symptoms and better patient outcomes for patients with alcohol use disorder who have deficiency of these cofactors and experience AWS.Pabrinex® is a multivitamin preparation, containing thiamine, riboflavin, pyridoxine, ascorbic acid, nicotinamide, and glucose. Given the role of riboflavin as a co-factor for pyruvate dehydrogenase, a potential limitation of the present trial is that pre- and post-treatment riboflavin concentrations have not been measured as this is a potential confounding factor for the interpretation of plasma lactate concentrations.Only Group 1  demonstrated significant enhancement of ETKA (the primary endpoint), however post hoc analysis demonstrates significant enhancement of ETKA among patients with serum magnesium concentrations > 0.75 mmol/L relative to patients with serum magnesium < 0.75 mmol/L. The findings of low serum magnesium concentrations (< 0.75 mmol/L) and elevated plasma lactate concentrations have implications for patients with AWS. As such, these investigations should form part of the routine investigations undertaken for patients presenting to the Emergency Department with alcohol use disorder. Overall, co-administration of thiamine and magnesium resulted in more consistent normalization of plasma lactate concentrations and faster initial resolution of AWS symptoms. It remains to be determined if this treatment strategy may translate into meaningful clinical outcomes for patients experiencing alcohol withdrawal syndrome.Supplementary Information.


Nanoscale thin film coatings and surface treatments are ubiquitous across industry, science, and engineering; imbuing specific functional or mechanical properties . Non-destructive nanoscale imaging of thin film coatings across large (ca. centimetre) lateral length scales, crucial to a wide range of modern industry, remains a significant technical challenge. By harnessing the unique nature of the helium atom–surface interaction, neutral helium microscopy images these surfaces without altering the sample under investigation. Since the helium atom scatters exclusively from the outermost electronic corrugation of the sample, the technique is completely surface sensitive. Furthermore, with a cross-section that is orders of magnitude larger than that of electrons, neutrons and photons, the probe particle routinely interacts with features down to the scale of surface defects and small adsorbates (including hydrogen). Here, we highlight the capacity of neutral helium microscopy for sub-resolution contrast using an advanced facet scattering model based on nanoscale features. By replicating the observed scattered helium intensities, we demonstrate that sub-resolution contrast arises from the unique surface scattering of the incident probe. Consequently, it is now possible to extract quantitative information from the helium atom image, including localised ångström-scale variations in topography. Neutral helium microscopy is a completely nondestructive, surface-sensitive imaging technique. Here, the authors demonstrate sub-resolution contrast using an advanced facet scattering model to reconstruct the topography of technological thin films in the ångström range. As the probe cannot penetrate the bulk at all, the micrograph generated is exclusively of the surface under investigation5. The scanning helium microscope (SHeM) collimates a free-jet expansion of neutral helium via a simple pinhole aperture, defining the lateral resolution of the instrument6. Currently, resolution is primarily limited by detector efficiency, with state-of-the-art detectors enabling resolutions of the order of 40 nm7. By rastering the sample underneath the beam and collecting the backscattered helium signal, an image may be generated.Neutral helium microscopy exploits the inherent properties of its probe particle8 dictates the contrast mechanisms available in the generated micrographs. Neglecting resonant state trapping , the various interactions can be categorised as either elastic or inelastic processes. The dominant contrast mechanism is topographic in nature, which is itself a subset of the possible elastic scattering trajectories. The localised tilt of the sample surface with respect to the detector determines the collected signal, thus allowing the surface morphology to generate image contrast through ‘masking’ and ‘shadowing’ 10. Previous work has focused on surfaces with feature sizes greater than the lateral resolution of the instrument (supra-resolution), whereby the image formation mechanism results in the direct observation of these features6. However, the same scattering phenomena must also occur for feature sizes smaller than the lateral resolution of the instrument (sub-resolution). In addition, the reflected intensity can also be influenced by other elastic processes  or inelastic processes whereby the composition and local atomic character of the sample surface can also give rise to differences in the helium reflectivity13; resulting in distinct changes in Michelson contrast. Therefore, in principle, the SHeM is capable of generating contrast from sub-resolution topographic features in circumstances where this contrast channel dominates. The sensitivity of neutral helium to low concentrations of surface defects and adsorbates is well-documented in helium atom scattering (HAS) literature14. However, to date there have been no systematic investigations into the sensitivity of SHeM to such sub-resolution features.The nature of the atom–surface interaction2 layer) and imaged in the SHeM (see Methods for complete fabrication and imaging details). It should be noted that there was no subsequent sample surface modification  post evaporation. Figure 6. The averaged linescans in Fig. As a first step, model thin film samples were prepared to establish whether the SHeM can generate sufficient contrast to image sub-resolution structures at ultralow concentrations. Utilising a thermal evaporator, patterned ultrathin gold films of different thicknesses were deposited onto n-doped silicon surfaces  and growth conditions (for diamond samples) were used to generate model surface topographies spanning a broad range of feature sizes below the lateral resolution of the instrument; i.e., the nano to micro length scales (see Methods section for full details). The Hurst parameter H  above the lateral resolution of the SHeM.Figure Sq). Figure Sq as a function of length scale for both glass and diamond systems, respectively. The relative SHeM contrast was confirmed using complementary HAS measurements . In addition, any large variations in modelled intensity between different input AFM datasets for the same sample also indicate that supra-resolution features are the dominant contributor to contrast. These conditions were only observed for the sandblasted glass sample (intensity differences >700% between regions) and consequently this sample exhibited the poorest agreement between simulation and experimental SHeM contrast. Second, when a 2D averaging filter or matrix downsampling was applied to the AFM maps prior to running the simulations , the predictive power of the model was destroyed in every case. This image processing is analogous to collecting AFM micrographs with a blunt tip. We therefore conclude that whilst neutral helium (and thus the SHeM) sees surface topography at least to the scale that is probed by AFM, it is the very fundamentals of image formation from elastically scattered helium—in particular shadowing, masking and facet angle—that are the dominant factors in determining SHeM contrast.2 via thermal evaporation is a complicated thin film growth system17. The growth exhibits distinct primary and secondary grain formation stages19. Initially, growth occurs through the formation of primary grains with a size of the order of the film thickness, with the irregular open channels between grains gradually filling in as deposition continues. Large secondary grains then form spontaneously once the first layer is completed. To determine the capability of the SHeM to probe sub-resolution topography, a series of polycrystalline gold films of mean thickness 1 Å  up to approximately 800 Å were deposited onto SiO2 substrates via thermal evaporation (see Methods). The gold films were shadow masked, leaving regions of uncovered SiO2 substrate for contrast measurements. The gold films were comprehensively characterised using SHeM, AFM and scanning electron microscope (SEM) imaging with the corresponding micrographs—as well as the experimentally determined SHeM contrast values—for each thin film thickness shown in Fig. 18).Having validated the facet-scattering model in simulating SHeM contrast from sub-resolution topographic features in single component model surfaces, it could now be applied to SHeM analysis of the thin gold film systems such as those shown in Fig. 21 channels as well as gold adatoms adhering to the AFM tip23. Consequently, facet-scattering model simulations based on the AFM height maps were incapable of describing the experimental SHeM contrast in the primary grain growth regime . The non-destructive imaging of thin film coatings across large lateral length scales is a major technical challenge for modern industry30. In particular, nanoscale defect control is critically important to the lithographic processes that have driven the 50-year history of semiconductor scaling, requiring detection of nano to micro scale defects across large wafer areas31.The unique understanding of surface topography offered by neutral helium microscopy has widespread application to nanoscale thin film coatings and surface treatments  working within appropriate constraints  allows a unique solution to be provided.The extreme sensitivity of SHeM contrast to nanoscale topographic features, in combination with its non-damaging nature, enables sub-resolution defect detection across large areas. In particular, for a well-characterised material system, inversion of the derived image contrast function Fig.  can be u8. By comparison, a corresponding AFM image would require stitching of 250 000 individual (20 μm × 20 μm) images and (assuming 10 s per image) stable imaging for 29 days (694 h). Furthermore, as discussed earlier, AFM imaging in this gold coverage regime (10 ± 2 Å) is unreliable due to gold adatoms adhering to the AFM tip.A basic confirmation of such a reconstruction was performed using standard AFM calibration gratings  the current lateral resolution of the instrument. The contrast observed in SHeM micrographs is driven by the processes of image formation: shadowing, masking, and facet angle. Incorporating these specific effects, a bespoke facet-scattering model was shown to describe the trends in the reflected intensities for multiple material systems, with feature sizes spanning orders of magnitude. Consequently, we showed that SHeM micrographs of sub-monolayer films can exhibit strongly varying contrast, arising from the specific nature of the sub-resolution scattering, thereby enabling ångström-scale height characterisation of thin films across centimetre lateral length scales. Its unique ability to observe the effects of sub-nanoscale features upon scattered helium intensity enables the SHeM to evaluate nano-coatings and thin films across large areas. Looking ahead, once a material system is well-characterised with SHeM, such analysis could readily become a routine part of quality control; a valuable tool for improving production yields and throughput.4 and 7% Ar into the chamber (86% H2) while intermediate-diamond film morphology was achieved by interchanging Ar with 0.2% of N2 . To produce the nanodiamond films, a Si wafer  was seeded via ultrasonication in a nanoparticle solution comprised of positively charged nanodiamond particles (PlasmaChem) and DI water (0.005 wt%). After seeding, the synthesis was carried out using a microwave plasma-assisted chemical vapour deposition (CVD) system (Seki Diamond Microwave System AX6500X). During the main step of deposition, a gas mixture of 97% H2, 3% CH4 was used at a pressure of 40 Torr and a power of 3000 W. A deposition time of 90 min was selected to achieve the desired diamond growth coverage and quality.Micro- and intermediate-diamond films were grown onto <100> Si substrates in a home-built 3 kW 2.45 GHz Microwave Plasma Chemical Vapour Deposition (MPCVD) reactor. Deposition for both samples was conducted in hydrogen-rich plasma for 4 h at a pressure of 150 Torr, keeping the surface temperature at 840 °C. The micro-crystalline diamond film was grown by introducing 7% CHA microscope glass slide (Livingston 7105-PPA) with frosted end was additionally sandblasted in-house (Econoline PB600SS sandblaster) on the opposite end to the frosted area. After sandblasting, the glass slide was cut with a diamond cutter to provide a sample of ca. 15 × 7 mm size piece including all three surface finishes . The cut sample was cleaned by subsequent sonications in acetone then isopropyl alcohol (IPA), and attached to a SHeM sample slide with an adhesive carbon tab. For the purposes of HAS measurements, separate ca. 7 × 7 mm pieces of each surface treatment were cut with a diamond cutter from the same glass slide as used for the SHeM measurements. After sonication, each sample was mounted to a HAS sample holder via a metal clamp.A 2” diameter, 0.5 mm thick Si wafer from Sigma-Aldrich  was cleaved into ca. 7 × 5 mm, ca. 7 × 12 mm, and ca. 12 × 12 mm substrates. After rinsing with acetone and IPA, the samples were placed onto cellulose acetate  and a pinhole (5 μm diameter). During imaging, the sample chamber pressure was typically of the order 1 × 10−8 mbar. Image acquisition is specified in terms of a dwell time, consisting of a wait  and a read  component, in line with standard stagnation detector operation for neutral atom beams38. For all subsequent descriptions of SHeM micrographs, the dwell times are specified as wait/read. Although current recording times for the presented SHeM images can span up to several days, there are two important factors to be considered to put these times into perspective: (a) at this early stage of SHeM imaging technology development, the limiting factors in imaging times are the current neutral helium detector sensitivities and response times. Work is ongoing on improving neutral helium detectors41 as well as developing instruments utilising more than one detector43 decreasing imaging times by several orders of magnitude. (b) when considering the SHeM imaging time required to investigate sub-nanoscale features over areas of several mm2 it is important to note that other techniques  would require much longer imaging times (~weeks/months/years) to cover the same area at the resolution required to visualise sub-nanoscale features.Further details concerning the experimental apparatus used to collect the neutral helium micrographs in this manuscript can be found in the literatureThe micrographs of the masked gold layers were collected using a 600/3000 ms dwell. For micrograph ‘a’ this total dwell was achieved by summing two component images, each with 600/1500 ms dwell.SHeM data for the glass material system was collected in a single micrograph with 1000/1500 ms dwell. An image with a suitable background region for contrast calculations was conducted with identical dwell. The diamond material system used 5 component images, each with 1200/1800 ms dwell, which were then summed to produce the final micrograph. An image with a suitable background region was conducted with 1200/9000 ms dwell. Errors in the contrast are estimated using the standard deviation of the intensity in the regions used to calculate contrast and standard error propagation.To avoid potential detector drift, multiple SHeM micrographs were recorded for each gold layer thickness and then combined to generate the final image. In particular, for each SHeM micrograph shown, 15 separate SHeM scans were collected using 650/2000 ms dwell per pixel and then summed. An image with a suitable background region for contrast calculations was also conducted after each image set using a 1000/30,000 ms dwell. Errors in the contrast are estimated using the standard deviation of the intensity in the regions used to calculate contrast and standard error propagation.Micrograph ‘a’ used 1000/8500 ms dwell, while micrograph ‘c’ employed 500/1500 ms dwell.For the temperature studies in Supplementary Fig. To avoid potential detector drift, multiple SHeM micrographs were recorded and then combined to generate the final image. For the micrograph shown as the inset in Supplementary Fig. www.gwyddion.net) and are provided in Supplementary Discussion All AFM images were recorded on an Oxford Instrument Asylum Research Cypher atomic force microscope in Tapping mode (standard topography/AC Air top) with a Budget Sensor Tap300Al-G tip. A range of AFM images were recorded on the glass, diamond, and Au on Si samples. Note that the same samples were used for both AFM and HAS experiments. For the Au on Si samples, reference AFM images were recorded on the pure silicon surface for each of the different layer thicknesses to ensure consistency across the dataset. Detailed AFM analyses of the glass and diamond model surfaces  were calculated using the open-source microscopy suite Gwyddion (All SEM images were recorded on an eLine system from RAITH with the Everhart-Thornley detector (SE2) and a 10 keV probe beam.44. A sketch of the experimental setup used in MAGIE is shown in Fig. 13. The neutral helium beam was generated by a free-jet expansion from a (10 ± 1) μm nozzle  at a stagnation pressure of (80 ± 1) bar. Note, this stagnation pressure was lower than that for the SHeM (200 bar), since MAGIE is not equipped for long term measurements at such high pressures. The stagnation temperatures for the Au on Si, diamond, and glass measurements were 300 K, 300 K and 305 K, respectively. The elastic energy of the helium beam, Ei was evaluated experimentally from a TOF measurement on the direct beam. It was found to be Ei = 67.20 ± 0.25 meV and Ei = 68.25 ± 0.25 meV for the 300 K and the 305 K beam respectively. All samples were kept at room temperature (Ts = 294.5 ± 1 K) during the measurements. To mimic the SHeM conditions as closely as possible, no sample cleaning procedures were performed. The base pressure in the sample chamber during operation was around 3 × 10−8 mbar. The helium beam spot size on the sample was ca. 4 mm for all experiments. The intensity was recorded using a home-built ionisation detector44 with an opening of 4.6 × 6.6 mm. Detector sensitivity changes between the measurements were accounted for by calibration to the same background signal for each measurement. No data were excluded from the analysis. All HAS measurements were performed on one set of samples.All HAS measurements were performed using the molecular beam scattering apparatus located at the University of Bergen, henceforth referred to as MAGIEαD) was fixed at 90°, and the chopper disc was removed from the beam line. Rocking curves were then recorded by varying the incoming beam angle θi  relative to the sample surface normal and measuring the total reflected intensity. Reproducibility of the rocking curves was ensured by recording measurements at the same parameter set repeatedly at different times for the glass and the Au on Si sample sets. For the Au on Si samples each layer thickness had its own Si reference surface on the same sample providing for an additional reproducibility check on the Si results. For the diamond sample set just one set of measurements was performed. The total number of recorded rocking curves for the various samples were as follows: 5, 6, and 3 for smooth, frosted, and sandblasted glass (respectively); 1 for each of the micro and intermediate-diamond samples; 2, 3, and 2 for Au on Si samples of 9 Å, 26 Å, and 235 Å thicknesses (respectively).For rocking curve measurements, the source-to-detector angle (45. The chopper disc to sample distance was (1003 ± 3) mm and the sample to detector distance was (902 ± 3) mm. Knowing the initial energy of the atoms, the path length they travel from the chopper to the sample, and the path length from the sample to the detector, the arrival time can be converted into a velocity and thus an energy. Specular measurements (αM = αD/2) were recorded at αD = 90° and αM = 45°, where again for off-specular measurement conditions (αM ≠ αD/2) either αD was slightly increased or decreased (with αM = 45°) or αD was kept at 90° and αM was decreased or increased. TOF measurement spectra where then transformed into energy-resolved spectra. The final energy spectra are presented as an energy change of the reflected atoms relative to the initial energy of the atoms in the beam. All TOF measurements were recorded over 511 channels with a channel width of 4.35 µs, a delay time of dt = 12 µs and a measurement time of tm = 3000 s. Based on these settings, each TOF channel (time channel) is addressed 1.35 × 106 times per measurement. To ensure reproducibility, several TOF curves were recorded for the glass and the Au on Si sample sets at the same detector and manipulator positions at different times. For the Au on Si samples each layer thickness had its own Si reference surface on the same sample providing for an additional reproducibility check on the Si results. For the diamond sample set just one set of measurements was performed. The total amount of recorded TOF files for the various sample sets were as follows: 10, 12 and 10 for smooth, frosted and sandblasted glass (respectively); 4 for each of the micro and intermediate-diamond samples; 8, 15 and 6 for Au on Si samples of 9 Å, 26 Å, and 235 Å thicknesses (respectively). After accounting for detector sensitivity changes, the redundancy measurements at the same detector and manipulator position concurred well with each other. Note that for clarity, representative TOF measurements are presented in Supplementary Discussion Energy-resolved spectra were obtained using TOF; recording the time-resolved intensity of reflected beam packages generated by mechanical chopping using a pseudorandom chopper (chopper rotation frequency 450 Hz)ance was 003 ± 3 mThe facet-scattering model (MATLAB) estimates experimental SHeM micrograph intensities using the height maps collected or estimated from AFM or SEM (respectively). The following section describes the logical process by which the model simulates SHeM intensities.6, the imported height map is rastered line-by-line underneath a fixed ‘beam’, with the facets that are struck by the incoming rays stored in a ‘hit matrix’ .The SHeM scattering geometry is used to generate a lookup table that encodes the probability for a ray to scatter specularly from a single, isolated facet into the detector  via ray tracing. Next, an appropriate height map is imported and scaled in all three Cartesian axes. Using nearest neighbours interpolation, the local gradient—and thus tilt angle at each facet—is obtained. To model the effects of shadowing6). If no such features block the path of an outgoing ray, only the local facet angle will determine whether the ray enters the detector and is marked as a ‘hit’; however, if masking occurs the hit is nullified. The final intensity is then calculated by counting the relative fraction of incoming rays that strike the detector aperture after interacting with the sample. For each imported height map, the simulation is run for the map rotated at angles of 0, 90, 180, and 270 degrees. The mean and standard deviation of the relative intensities for all rotations are recorded, with standard error propagation then utilised to compute the datapoints and error bars given in Figs. The hit matrix, local facet angle, and the lookup table determine which particular facets lead to outgoing rays striking the detector. The outgoing, specularly scattered ray is tracked through the height map (with periodic boundary conditions) to see if a tall feature intersects it prior to entering the detector (maskingSupplementary Information FilePeer Review File