Towards Establishing Best Practice in the Analysis of Hydrogen and Deuterium by Atom Probe Tomography.

As hydrogen is touted as a key player in the decarbonization of modern society, it is critical to enable quantitative hydrogen (H) analysis at high spatial resolution and, if possible, at the atomic scale. H has a known deleterious impact on the mechanical properties (strength, ductility, toughness) of most materials that can hinder their use as part of the infrastructure of a hydrogen-based economy. Enabling H mapping including local hydrogen concentration analyses at specific microstructural features is essential for understanding the multiple ways that H affect the properties of materials including embrittlement mechanisms and their synergies. In addition, spatial mapping and quantification of hydrogen isotopes is essential to accurately predict tritium inventory of future fusion power plants thus ensuring their safe and efficient operation. Atom probe tomography (APT) has the intrinsic capability to detect H and deuterium (D), and in principle the capacity for performing quantitative mapping of H within a material's microstructure. Yet, the accuracy and precision of H analysis by APT remain affected by complex field evaporation behavior and the influence of residual hydrogen from the ultrahigh vacuum chamber that can obscure the signal of H from within the material. The present article reports a summary of discussions at a focused workshop held at the Max-Planck Institute for Sustainable Materials in April 2024. The workshop was organized to pave the way to establishing best practices in reporting APT data for the analysis of H. We first summarize the key aspects of the intricacies of H analysis by APT and then propose a path for better reporting of the relevant data to support interpretation of APT-based H analysis in materials.

Subcellular dynamics studies of iron reveal how tissue-specific distribution patterns are established in developing wheat grains.

Understanding the mechanisms of iron trafficking in plants is key to enhancing the nutritional quality of crops. Because it is difficult to image iron in transit, we currently have an incomplete picture of the route(s) of iron translocation in developing seeds and how the tissue-specific distribution is established. We have used a novel approach, combining iron-57 (57 Fe) isotope labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), to visualize iron translocation between tissues and within cells in immature wheat grain, Triticum aestivum. This enabled us to track the main route of iron transport from maternal tissues to the embryo through the different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, with confirmation provided by histological staining and transmission electron microscopy energy dispersive X-ray spectroscopy (TEM-EDS). Almost all iron in both control and transgenic grains was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Furthermore, a new type of iron body, highly enriched in 57 Fe, was observed in aleurone cells and may represent iron being delivered to phytate globoids. Correlation of the 57 Fe enrichment profiles obtained by NanoSIMS with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification strategies.

Methods to Visualize Elements in Plants.

Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plant molecular biology, agronomy, plant physiology, plant nutrition, and ionomics. However, it is often challenging to evaluate the applicability of the wide range of techniques available, with each having its own strengths and limitations. Here, we compare scanning/transmission electron microscopy-based energy-dispersive x-ray spectroscopy, x-ray fluorescence microscopy, particle-induced x-ray emission, laser ablation inductively coupled plasma-mass spectrometry, nanoscale secondary ion mass spectroscopy, autoradiography, and confocal microscopy with fluorophores. For these various techniques, we compare their accessibility, their ability to analyze hydrated tissues (without sample preparation) and suitability for in vivo analyses, as well as examining their most important analytical merits, such as resolution, sensitivity, depth of analysis, and the range of elements that can be analyzed. We hope that this information will assist other researchers to select, access, and evaluate the approach that is most useful in their particular research program or application.

Microdistribution and quantification of the boron neutron capture therapy drug BPA in primary cell cultures of human glioblastoma tumour by NanoSIMS.

The ability of secondary ion mass spectrometry (SIMS) to provide high sensitivity imaging of elements and small-medium mass molecules in biological tissues and cells, makes it a very powerful tool for drug distribution studies. Here we report on the application of a high-resolution dynamic SIMS instrument for the quantification and localisation of therapeutic levels of the BNCT agent l-para-(dihydroxyboryl)-phenylalanine (BPA) in primary cell cultures from human patients exhibiting glioblastoma multiform tumours. Boron uptake and distribution was determined quantitatively as a function of cell-sampling location and different treatment regimes. Importantly, BPA was found to accumulate in cancer cells invading the 'brain around tumour' tissue in addition to the main tumour site. Pre-treatment of samples with l-tyrosine was found not to increase the uptake of BPA, nor change the intracellular drug distribution. In cultured cells from the tumour core and brain around tumour, with and without l-tyrosine pre-treatment, normalised boron-related signals were higher from cell nuclei than from cytoplasm. An efflux treatment was found to reduce BPA levels, but at a rate slower than the original uptake, and did not affect the intracellular drug distribution. To the best of our knowledge, these data represent the first published study of BPA uptake and l-amino acid pre-treatment in cultured primary human cells using dynamic SIMS, and the most detailed, subcellular distribution study of a BNCT agent in any cellular system.

The Nodulin 26-like intrinsic membrane protein OsNIP3;2 is involved in arsenite uptake by lateral roots in rice.

Previous studies have shown that the Nodulin 26-like intrinsic membrane protein (NIP) Lsi1 (OsNIP2;1) is involved in arsenite [As(III)] uptake in rice (Oryza sativa). However, the role of other rice NIPs in As(III) accumulation in planta remains unknown. In the present study, we investigated the role OsNIP3;2 in As(III) uptake in rice. When expressed in Xenopus laevis oocytes, OsNIP3;2 showed a high transport activity for As(III). Quantitative real-time RT-PCR showed that the expression of OsNIP3;2 was suppressed by 5 µM As(III), but enhanced by 20 and 100 µM As(III). Transgenic rice plants expressing OsNIP3;2pro-GUS showed that the gene was predominantly expressed in the lateral roots and the stele region of the primary roots. Transient expression of OsNIP3;2:GFP fusion protein in rice protoplasts showed that the protein was localized in the plasma membrane. Knockout of OsNIP3;2 significantly decreased As concentration in the roots, but had little effect on shoot As concentration. Synchrotron microfocus X-ray fluorescence showed decreased As accumulation in the stele of the lateral roots in the mutants compared with wild-type. Our results indicate that OsNIP3;2 is involved in As(III) uptake by lateral roots, but its contribution to As accumulation in the shoots is limited.

Submicron Patterning of Polymer Brushes: An Unexpected Discovery from Inkjet Printing of Polyelectrolyte Macroinitiators.

Using an electrostatic-based super inkjet printer we report the high-resolution deposition of polyelectrolyte macroinitiators and subsequent polymer brush growth using SI-ARGET-ATRP. We go on to demonstrate for the first time a submicron patterning phenomenon through the addition of either a like charged polyelectrolyte homopolymer or through careful control of ionic strength. As a result patterning of polymer brushes down to ca. 300 nm is reported. We present a possible mechanistic model and consider how this may be applied to other polyelectrolyte-based systems as a general method for submicron patterning.

The dynamics of protein body formation in developing wheat grain.

Wheat is a major source of protein in the diets of humans and livestock but we know little about the mechanisms that determine the patterns of protein synthesis in the developing endosperm. We have used a combination of enrichment with (15) N glutamine and NanoSIMS imaging to establish that the substrate required for protein synthesis is transported radially from its point of entrance in the endosperm cavity across the starchy endosperm tissues, before becoming concentrated in the cells immediately below the aleurone layer. This transport occurs continuously during grain development but may be slower in the later stages. Although older starchy endosperm cells tend to contain larger protein deposits formed by the fusion of small protein bodies, small highly enriched protein bodies may also be present in the same cells. This shows a continuous process of protein body initiation, in both older and younger starchy endosperm cells and in all regions of the tissue. Immunolabeling with specific antibodies shows that the patterns of enrichment are not related to the contents of gluten proteins in the protein bodies. In addition to providing new information on the dynamics of protein deposition, the study demonstrates the wider utility of NanoSIMS and isotope labelling for studying complex developmental processes in plant tissues.

Identification of the primary lesion of toxic aluminum in plant roots.

Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µm Al reduced root growth after only 5 min (or 30 min at 30 µm Al), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.

The role of nodes in arsenic storage and distribution in rice.

Knowledge of arsenic (As) accumulation in rice (Oryza sativa L.) is important for minimizing As transfer to the food chain. The aim of this study was to investigate the role of rice nodes in As storage and distribution. Synchrotron µX-ray fluorescence (µ-XRF) was used to map As distribution in the top node and internode of a lsi2 mutant defective in silicon/arsenite efflux carrier and its wild-type (WT) grown in soil. Lsi2 expression in different tissues during grain filling was investigated by quantitative RT-PCR. Arsenite or dimethylarsinic acid (DMA) was supplied to excised panicles to investigate the roles of Lsi2 and phytochelatins (PC) in As distribution. µ-XRF mapping revealed As storage in the phloem of different vascular bundles in the top node and internode. Soil-grown plants of lsi2 had markedly decreased As accumulation in the phloem compared with the WT. Lsi2 was strongly expressed, not only in the roots but also in the nodes. When excised panicles were exposed to As(III), the lsi2 mutant distributed more As to the node and flag leaf but less As to the grain compared with the WT, while there was no significant difference in DMA distribution. Inhibition of PC synthesis by l-buthionine-sulphoximine decreased As(III) deposition in the top node but increased As accumulation in the grain and flag leaf. The results suggest that rice nodes serve as a filter restricting As(III) distribution to the grain. Furthermore, Lsi2 plays a role in As(III) distribution in rice nodes and phytochelatins are important compounds for As(III) storage in the nodes.

Combined NanoSIMS and synchrotron X-ray fluorescence reveal distinct cellular and subcellular distribution patterns of trace elements in rice tissues.

The cellular and subcellular distributions of trace elements can provide important clues to understanding how the elements are transported and stored in plant cells, but mapping their distributions is a challenging task. The distributions of arsenic, iron, zinc, manganese and copper, as well as physiologically related macro-elements, were mapped in the node, internode and leaf sheath of rice (Oryza sativa) using synchrotron X-ray fluorescence (S-XRF) and high-resolution secondary ion mass spectrometry (NanoSIMS). Although copper and silicon generally showed cell wall localization, arsenic, iron and zinc were strongly localized in the vacuoles of specific cell types. Arsenic was highly localized in the companion cell vacuoles of the phloem in all vascular bundles, showing a strong co-localization with sulfur, consistent with As(III)-thiol complexation. Within the node, zinc was localized in the vacuoles of the parenchyma cell bridge bordering the enlarged and diffuse vascular bundles, whereas iron and manganese were localized in the fundamental parenchyma cells, with iron being strongly co-localized with phosphorus in the vacuoles. The highly heterogeneous and contrasting distribution patterns of these elements imply different transport activities and/or storage capacities among different cell types. Sequestration of arsenic in companion cell vacuoles may explain the limited phloem mobility of arsenite.

Multi-scale correlative microscopy investigation of both structure and chemistry of deformation twin bundles in Fe-Mn-C steel.

A multi-scale investigation of twin bundles in Fe-22Mn-0.6C (wt%) twinning-induced plasticity steel after tensile deformation has been carried out by truly correlative means; using electron channelling contrast imaging combined with electron backscatter diffraction, high-resolution secondary ion mass spectrometry, scanning transmission electron microscopy, and atom probe tomography on the exact same region of interest in the sample. It was revealed that there was no significant segregation of Mn or C to the twin boundary interfaces.

High-resolution secondary ion mass spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots.

Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.

Elemental imaging at the nanoscale: NanoSIMS and complementary techniques for element localisation in plants.

The ability to locate and quantify elemental distributions in plants is crucial to understanding plant metabolisms, the mechanisms of uptake and transport of minerals and how plants cope with toxic elements or elemental deficiencies. High-resolution secondary ion mass spectrometry (SIMS) is emerging as an important technique for the analysis of biological material at the subcellular scale. This article reviews recent work using the CAMECA NanoSIMS to determine elemental distributions in plants. The NanoSIMS is able to map elemental distributions at high resolution, down to 50 nm, and can detect very low concentrations (milligrams per kilogram) for some elements. It is also capable of mapping almost all elements in the periodic table (from hydrogen to uranium) and can distinguish between stable isotopes, which allows the design of tracer experiments. In this review, particular focus is placed upon studying the same or similar specimens with both the NanoSIMS and a wide range of complementary techniques, showing how the advantages of each technique can be combined to provide a fuller data set to address complex scientific questions. Techniques covered include optical microscopy, synchrotron techniques, including X-ray fluorescence and X-ray absorption spectroscopy, transmission electron microscopy, electron probe microanalysis, particle-induced X-ray emission and inductively coupled plasma mass spectrometry. Some of the challenges associated with sample preparation of plant material for SIMS analysis, the artefacts and limitations of the technique and future trends are also discussed.

Elucidating heterogeneous iron biomineralization patterns in a denitrifying As(iii)-oxidizing bacterium: implications for arsenic immobilization.

Anaerobic nitrate-dependent iron(ii) oxidation is a process common to many bacterial species, which promotes the formation of Fe(iii) minerals that can influence the fate of soil and groundwater pollutants, such as arsenic. Herein, we investigated simultaneous nitrate-dependent Fe(ii) and As(iii) oxidation by Acidovorax sp. strain ST3 with the aim of studying the Fe biominerals formed, their As immobilization capabilities and the metabolic effect on cells. X-ray powder diffraction (XRD) and scanning transmission electron microscopy (STEM) nanodiffraction were applied for biomineral characterization in bulk and at the nanoscale, respectively. NanoSIMS (nanoscale secondary ion mass spectrometry) was used to map the intra and extracellular As and Fe distribution at the single-cell level and to trace metabolically active cells, by incorporation of a 13C-labeled substrate (acetate). Metabolic heterogeneity among bacterial cells was detected, with periplasmic Fe mineral encrustation deleterious to cell metabolism. Interestingly, Fe and As were not co-localized in all cells, indicating delocalized sites of As(iii) and Fe(ii) oxidation. The Fe(iii) minerals lepidocrocite and goethite were identified in XRD, although only lepidocrocite was identified via STEM nanodiffraction. Extracellular amorphous nanoparticles were formed earlier and retained more As(iii/v) than crystalline "flakes" of lepidocrocite, indicating that longer incubation periods promote the formation of more crystalline minerals with lower As retention capabilities. Thus, the addition of nitrate promotes Fe(ii) oxidation and formation of Fe(iii) biominerals by ST3 cells which retain As(iii/v), and although this process was metabolically detrimental to some cells, it warrants further examination as a viable mechanism for As removal in anoxic environments by biostimulation with nitrate.

Dissimilatory Fe(III) Reduction Controls on Arsenic Mobilization: A Combined Biogeochemical and NanoSIMS Imaging Approach.

Microbial metabolism plays a key role in controlling the fate of toxic groundwater contaminants, such as arsenic. Dissimilatory metal reduction catalyzed by subsurface bacteria can facilitate the mobilization of arsenic via the reductive dissolution of As(V)-bearing Fe(III) mineral assemblages. The mobility of liberated As(V) can then be amplified via reduction to the more soluble As(III) by As(V)-respiring bacteria. This investigation focused on the reductive dissolution of As(V) sorbed onto Fe(III)-(oxyhydr)oxide by model Fe(III)- and As(V)-reducing bacteria, to elucidate the mechanisms underpinning these processes at the single-cell scale. Axenic cultures of Shewanella sp. ANA-3 wild-type (WT) cells [able to respire both Fe(III) and As(V)] were grown using 13C-labeled lactate on an arsenical Fe(III)-(oxyhydr)oxide thin film, and after colonization, the distribution of Fe and As in the solid phase was assessed using nanoscale secondary ion mass spectrometry (NanoSIMS), complemented with aqueous geochemistry analyses. Parallel experiments were conducted using an arrA mutant, able to respire Fe(III) but not As(V). NanoSIMS imaging showed that most metabolically active cells were not in direct contact with the Fe(III) mineral. Flavins were released by both strains, suggesting that these cell-secreted electron shuttles mediated extracellular Fe(III)-(oxyhydr)oxide reduction, but did not facilitate extracellular As(V) reduction, demonstrated by the presence of flavins yet lack of As(III) in the supernatants of the arrA deletion mutant strain. 3D reconstructions of NanoSIMS depth-profiled single cells revealed that As and Fe were associated with the cell surface in the WT cells, whereas for the arrA mutant, only Fe was associated with the biomass. These data were consistent with Shewanella sp. ANA-3 respiring As(V) in a multistep process; first, the reductive dissolution of the Fe(III) mineral released As(V), and once in solution, As(V) was respired by the cells to As(III). As well as highlighting Fe(III) reduction as the primary release mechanism for arsenic, our data also identified unexpected cellular As(III) retention mechanisms that require further investigation.

Evidence of hydrogen trapping at second phase particles in zirconium alloys.

Zirconium alloys are used in safety-critical roles in the nuclear industry and their degradation due to ingress of hydrogen in service is a concern. In this work experimental evidence, supported by density functional theory modelling, shows that the α-Zr matrix surrounding second phase particles acts as a trapping site for hydrogen, which has not been previously reported in zirconium. This is unaccounted for in current models of hydrogen behaviour in Zr alloys and as such could impact development of these models. Zircaloy-2 and Zircaloy-4 samples were corroded at 350 °C in simulated pressurised water reactor coolant before being isotopically spiked with 2H2O in a second autoclave step. The distribution of 2H, Fe and Cr was characterised using nanoscale secondary ion mass spectrometry (NanoSIMS) and high-resolution energy dispersive X-ray spectroscopy. 2H- was found to be concentrated around second phase particles in the α-Zr lattice with peak hydrogen isotope ratios of 2H/1H = 0.018-0.082. DFT modelling confirms that the hydrogen thermodynamically favours sitting in the surrounding zirconium matrix rather than within the second phase particles. Knowledge of this trapping mechanism will inform the development of current understanding of zirconium alloy degradation through-life.

The human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitor resistance mutation I132M confers hypersensitivity to nucleoside analogs.

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.

NanoSIMS analysis of arsenic and selenium in cereal grain.

*Cereals are an important source of selenium (Se) to humans and many people have inadequate intakes of this essential trace element. Conversely, arsenic (As) is toxic and may accumulate in rice grain at levels that pose a health risk. Knowledge of the localization of selenium and arsenic within the cereal grain will aid understanding of their deposition patterns and the impact of processes such as milling. *High-resolution secondary ion mass spectrometry (NanoSIMS) was used to determine the localization of Se in wheat (Triticum aestivum) and As in rice (Oryza sativa). Combined synchrotron X-ray fluorescence (S-XRF) and NanoSIMS analysis utilized the strengths of both techniques. *Selenium was concentrated in the protein surrounding the starch granules in the starchy endosperm cells and more homogeneously distributed in the aleurone cells but with Se-rich hotspots. Arsenic was concentrated in the subaleurone endosperm cells in association with the protein matrix rather than in the aleurone cells. NanoSIMS indicated that the high intensity of As identified in the S-XRF image was localized in micron-sized hotspots near the ovular vascular trace and nucellar projection. *This is the first study showing subcellular localization in grain samples containing parts per million concentrations of Se and As. There is good quantitative agreement between NanoSIMS and S-XRF.

NanoSIMS Imaging and Analysis in Materials Science.

High-resolution SIMS analysis can be used to explore a wide range of problems in material science and engineering materials, especially when chemical imaging with good spatial resolution (50-100 nm) can be combined with efficient detection of light elements and precise separation of isotopes and isobaric species. Here, applications of the NanoSIMS instrument in the analysis of inorganic materials are reviewed, focusing on areas of current interest in the development of new materials and degradation mechanisms under service conditions. We have chosen examples illustrating NanoSIMS analysis of grain boundary segregation, chemical processes in cracking, and corrosion of nuclear components. An area where NanoSIMS analysis shows potential is in the localization of light elements, in particular, hydrogen and deuterium. Hydrogen embrittlement is a serious problem for industries where safety is critical, including aerospace, nuclear, and oil/gas, so it is imperative to know where in the microstructure hydrogen is located. By charging the metal with deuterium, to avoid uncertainty in the origin of the hydrogen, the microstructural features that can trap hydrogenic species, such as precipitates and grain and phase boundaries, can be determined by NanoSIMS analysis on a microstructurally relevant scale.

Pb nanospheres in ancient zircon yield model ages for zircon formation and Pb mobilization.

Nanospheres of lead (Pb) have recently been identified in zircon (ZrSiO4) with the potential to compromise the veracity of U-Pb age determinations. The key assumption that the determined age is robust against the effects of Pb mobility, as long as Pb is not lost from the zircon during subsequent geological events, is now in question. To determine the effect of nanosphere formation on age determination, and whether analysis of nanospheres can yield additional information about the timing of both zircon growth and nanosphere formation, zircons from the Napier Complex in Enderby Land, East Antarctica, were investigated by high-spatial resolution NanoSIMS (Secondary Ion Mass Spectrometry) mapping. Conventional SIMS analyses with >µm resolution potentially mixes Pb from multiple nanospheres with the zircon host, yielding variable average values and therefore unreliable ages. NanoSIMS analyses were obtained of 207Pb/206Pb in nanospheres a few nanometres in diameter that were resolved from 207Pb/206Pb measurements in the zircon host. We demonstrate that analysis for 207Pb/206Pb in multiple individual Pb nanospheres, along with separate analysis of 207Pb/206Pb in the zircon host, can not only accurately yield the age of zircon crystallization, but also the time of nanosphere formation resulting from Pb mobilization during metamorphism. Model ages for both events can be derived that are correlated due to the limited range of possible solutions that can be satisfied by the measured 207Pb/206Pb ratios of nanospheres and zircon host. For the Napier Complex zircons, this yields a model age of ca 3110 Ma for zircon formation and a late Archean model age of 2610 Ma for the metamorphism that produced the nanospheres. The Nanosphere Model Age (NMA) method constrains both the crystallization age and age of the metamorphism to ~±135 Ma, a significant improvement on errors derived from counting statistics.