Fine particle pollution during megafires contains potentially toxic elements.

Wildfires that raged across Australia during the 2019-2020 'Black Summer' produced an enormous quantity of particulate matter (PM) pollution, with plumes that cloaked many urban centres and ecosystems along the eastern seaboard. This has motivated a need to understand the magnitude and nature of PM exposure, so that its impact on both built and natural environments can be more accurately assessed. Here we present the potentially toxic fingerprint of PM captured by building heating, ventilation, and air conditioning filters in Sydney, Australia during the peak of the Wildfires, and from ambient urban emissions one year later (Reference period). Atmospheric PM and meteorological monitoring data were also assessed to determine the magnitude and source of high PM exposure. The wildfires were a major source of PM pollution in Sydney, exceeding the national standards on 19 % of days between November-February. Wildfire particles were finer and more spherical compared to Reference PM, with count median diameters of 892.1 ± 23.1 versus 1484.8 ± 96.7 nm (mean ± standard error). On an equal-mass basis, differences in potentially toxic elements were predominantly due to higher SO42--S (median 20.4 vs 4.7 mg g-1) and NO3--N (2.4 vs 1.2 mg g-1) in Wildfire PM, and higher PO43--P (10.4 vs 1.4 mg g-1) in Reference PM. Concentrations of remaining elements were similar or lower than Reference PM, except for enrichments to F-, Cl-, dissolved Mn, and particulate Mn, Co and Sb. Fractional solubilities of trace elements were similar or lower than Reference PM, except for enhanced Hg (12.1 vs 1.0 %) and greater variability in Cd, Hg and Mn solubility, which displayed upper quartiles exceeding that of Reference PM. These findings contribute to our understanding of human and ecosystem exposures to the toxic components of mixed smoke plumes, especially in regions downwind of the source.

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.

Preparation of matrix-matched standards for the analysis of teeth via laser ablation-inductively coupled plasma-mass spectrometry.

Mineralised tissue such as teeth can serve as a retrospective, chronological bioindicator of past exposure to toxic metals. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) can be used to determine the presence and spatial distribution of toxic metals in teeth, giving a record of when an exposure occurred. Concentrations of these metals are often determined by a one-point calibration against NIST glass using an equation that requires an internal standard factor that accounts for differences in ablation behaviour between the glass and the tooth. However, an ideal external calibration would contain multiple matrix-matched standards to obtain a calibration curve. Here, we investigated optimal procedures for preparing synthetic hydroxyapatite (HA) doped with elements of interest as a calibration material. The materials were examined for homogeneity of metal incorporation, matrix-matched ablation characteristics, linearity, and limits of detection. A homogenised and pelleted HA was the most suitable material, providing improved ablation characteristics over previous HA materials and NIST glass for the analysis of teeth. An ablation yield of 1.1 showed its suitability to analyse teeth, the metals were homogeneously incorporated, and it produced excellent linearity with limits of detection ranging from 0.1-2 µg kg-1 for magnesium, aluminium, nickel, copper, zinc, cadmium, barium and lead. A juvenile incisor from a remote indigenous community in Australia and an adult molar from Sri Lanka were assessed for toxic metal exposure. The molar showed evidence of exposure to cadmium and lead. The synthetic HA material was straightforward to prepare, and will improve confidence in the analysis of teeth and other biomineralised material when assessing toxic metal exposure.

Acetonitrile adduct analysis of underivatised amino acids offers improved sensitivity for hydrophilic interaction liquid chromatography tandem mass-spectrometry.

LC-MS/MS method development for native amino acid detection can be problematic due to low ionisation efficiencies, in source fragmentation, potential for cluster ion formation and incorrect application of chromatography techniques. This has led to the majority of the scientific community derivatising amino acids for more sensitive analysis. Derivatisation has several benefits including reduced signal-to-noise ratios, more efficient ionisation, and a change in polarity, allowing the use of reverse phase chromatography. However, derivatisation of amino acids can be expensive, requires additional sample preparation steps, is more time consuming and increases sample instability, due to the most derivatised amino acids only be stable for finite amount of time. While showing initial promise, development of reliable hydrophilic interaction liquid chromatography (HILIC) separation methods has presented difficulties for the analyst including irreproducible separation and poor sensitivity. This study aimed to find a means to improve the detection sensitivity of the 20 protein amino acids by HILIC-MS/MS. We describe the use of previously undescribed amino acid-acetonitrile (ACN) adducts to improve detection of 16 out of the 20 amino acids. While all amino acids examined did form an ACN adduct, 4 had low intensity adduct formation compared to their protonated state, 3 of which are classified as basic amino acids. For 15 of the 20 amino acids tested, we used the ACN adduct for both quantification and qualification ions and demonstrated a significant enhancement in signal-to-noise ratio, ranging from 23 to 1762% improvement. Lower LODs, LOQs and lower ranges of linearity were also achieved for these amino acids. The optimised method was applied to a human neuroblastoma cell line (SH-SY5Y) with the potential to be applied to other complex sample types. The improved sensitivity this method offers simplifies sample preparation and reduces the costs of amino acid analysis compared to those methods that rely on derivatisation for sensitivity.

ß-Methylamino-L-alanine-induced protein aggregation in vitro and protection by L-serine.

The cyanobacterial non-protein amino acid α-amino-ß-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system xc_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.

Pew2: Open-Source Imaging Software for Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry.

Open-sourced software is a key component of the mass spectrometry imaging field, where transparency in data processing is vital. Imaging of trace elements and immunohistochemically labeled biomolecules in tissue sections is typically performed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). However, efficient and facile processing of images is hampered by a lack of verifiable and user-friendly software that supports multiple LA-ICP-MS platforms. In this technical note, we introduce Pew2, a LA-ICP-MS specific and feature-rich open-source image processing software that is compatible with common ICP-MS vendors. Pew2 is designed to be fast and easy to use and adheres to modern visualization philosophies to maximize productivity and to minimize data interpretation errors and image anomalies.

Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging (iMSI) uses laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to determine the spatial expression of biomolecules in tissue sections following immunolabelling with antibodies conjugated to a metal reporter. As with all immunolabelling techniques, the binding efficiency of multiplexed staining can be affected by a number of factors including epitope blocking and other forms of steric hindrance. To date, the effects on the binding of metal-conjugated antibodies to their epitopes in a multiplexed analysis have yet to be quantitatively explored by iMSI. Here we describe a protocol to investigate the effects of multiplexing on reproducible binding using the muscle proteins, dystrophin, sarcospan, and myosin as a model, with antibodies conjugated with Maxpar® reagents before histological application to murine quadriceps sections using standard immunolabelling protocols and imaging with LA-ICP-MS. The antibodies were each individually applied to eight sections, and multiplexed to another eight sections. The average concentrations of the lanthanide analytes were determined, before statistical analyses found there was no significant difference between the individual and multiplexed application of the antibodies. These analyses provide a framework for ensuring reproducibility of antibody binding during multiplexed iMSI, which will allow quantitative exploration of protein-protein interactions and provide a greater understanding of fundamental biological processes during healthy and diseased states.

Bottom-Up Synthesis of Hexagonal Boron Nitride Nanoparticles with Intensity-Stabilized Quantum Emitters.

Fluorescent nanoparticles are widely utilized in a large range of nanoscale imaging and sensing applications. While ultra-small nanoparticles (size ≤10 nm) are highly desirable, at this size range, their photostability can be compromised due to effects such as intensity fluctuation and spectral diffusion caused by interaction with surface states. In this article, a facile, bottom-up technique for the fabrication of sub-10-nm hexagonal boron nitride (hBN) nanoparticles hosting photostable bright emitters via a catalyst-free hydrothermal reaction between boric acid and melamine is demonstrated. A simple stabilization protocol that significantly reduces intensity fluctuation by ≈85% and narrows the emission linewidth by ≈14% by employing a common sol-gel silica coating process is also implemented. This study advances a promising strategy for the scalable, bottom-up synthesis of high-quality quantum emitters in hBN nanoparticles.

Quantitative immuno-mass spectrometry imaging of skeletal muscle dystrophin.

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg-1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

Controlled Doping of GeV and SnV Color Centers in Diamond Using Chemical Vapor Deposition.

Group IV color centers in diamond (Si, Ge, Sn, and Pb) have recently emerged as promising candidates for realization of scalable quantum photonics. However, their synthesis in nanoscale diamond is still in its infancy. In this work we demonstrate controlled synthesis of selected group IV defects (Ge and Sn) into nanodiamonds and nanoscale single crystal diamond membranes by microwave plasma chemical vapor deposition. We take advantage of inorganic salts to prepare the chemical precursors that contain the required ions that are then incorporated into the growing diamond. Photoluminescence measurements confirm that the selected group IV emitters are present in the diamond without degrading its structural quality. Our results are important to expand the versatile synthesis of color centers in diamond.

Low background mould-prepared gelatine standards for reproducible quantification in elemental bio-imaging.

Standard preparation for elemental bio-imaging by laser ablation-inductively coupled plasma-mass spectrometry is confounded by the chemical and physical differences between standard and sample matrices. These differences lead to variable ablation, aerosol generation and transportation characteristics and must be considered when designing matrix-matched standards for reliable calibration and quantification. The ability to precisely mimic sample matrices is hampered due to the complexity and heterogeneity of biological tissue and small variabilities in standard matrices and sample composition often negatively impact accuracy, precision and robustness. Furthermore, cumbersome preparation protocols may limit reproducibility and traceability. This work presents novel facile methods for the preparation of gelatine standards using both commercial and laboratory-made moulds. Surface roughness, thickness and robustness of the mould-prepared standards were compared against cryo-sectioned gelatine and homogenised brain tissue standards. The mould-prepared standards had excellent thickness accuracy and signal precision which allowed robust quantification, were easier to prepare and therefore easier to reproduce. We also compared gelatine standards prepared from a variety of animal sources and discuss their suitability to calibrate low level elemental concentrations. Finally, we present a simple method to remove background metals in gelatine using various chelating resins to increase the dynamic calibration range and to improve limits of analysis.

Super-Resolution Reconstruction for Two- and Three-Dimensional LA-ICP-MS Bioimaging.

The resolution of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) elemental bioimaging is usually constrained by the diameter of the laser spot size and is often not adequate to explore in situ subcellular distributions of elements and proteins in biological tissue sections. Super-resolution reconstruction is a method typically used for many imaging modalities and combines multiple lower resolution images to create a higher resolution image. Here, we present a super-resolution reconstruction method for LA-ICP-MS imaging by ablating consecutive layers of a biological specimen with offset orthogonal scans, resulting in a 10× improvement in resolution for quantitative measurement of dystrophin in murine muscle fibers. Layer-by-layer image reconstruction was also extended to the third dimension without the requirement of image registration across multiple thin section specimens. Quantitative super-resolution reconstruction, combined with Gaussian filtering and application of the Richardson-Lucy total variation algorithm, provided superior image clarity and fidelity in two- and three-dimensions.

From Lead(II) Dithiocarbamate Precursors to a Fast Response PbS Positive Temperature Coefficient Thermistor.

PbS submicron crystals were formed by thermolysis of two different lead dithiocarbamate complexes. These precursors were readily synthesized and fully characterized, and in situ synchrotron powder diffraction experiments were performed to characterize their decomposition. The structure and purity of resultant PbS was examined using scanning electron and transmission electron microscopies, powder X-ray diffraction, and infrared spectroscopy. Submicron crystalline PbS was used to create a new PbS thermistor with excellent sensitivity and an ultrarapid thermal response time.

Remarkable thermal stability of gold nanoparticles functionalised with ruthenium phthalocyanine complexes.

A gold nanoparticle (AuNP) ruthenium phthalocyanine (RuPc) nanocomposite has been synthesised that exhibits high thermal stability. Electrical resistance measurements revealed that the nanocomposite is stable up to ∼320 °C. Examination of the nanocomposite and the RuPc stabiliser complex using thermogravimetric analysis and differential scanning calorimetry show that the remarkable thermal stability is due to the RuPc molecules, which provide an effective barrier to sintering of the AuNPs.