Spatial distribution of trace metals and associated transport proteins during bacterial infection.

Innate immune systems alter the concentrations of trace elements in host niches in response to invading pathogens during infection. This work reports the interplay between d-block metal ions and their associated biomolecules using hyphenated elemental techniques to spatially quantify both elemental distributions and the abundance of specific transport proteins. Here, lung tissues were collected for analyses from naïve and Streptococcus pneumoniae-infected mice fed on a zinc-restricted or zinc-supplemented diet. Spatiotemporal distributions of manganese (55Mn), iron (56Fe), copper (63Cu), and zinc (66Zn) were determined by quantitative laser ablation-inductively coupled plasma-mass spectrometry. The murine transport proteins ZIP8 and ZIP14, which are associated with zinc transport, were also imaged by incorporation of immunohistochemistry techniques into the analytical workflow. Collectively, this work demonstrates the potential of a single instrumental platform suitable for multiplex analyses of tissues and labelled antibodies to investigate complex elemental interactions at the host-pathogen interface. Further, these methods have the potential for broad application to investigations of biological pathways where concomitant measurement of elements and biomolecules is crucial to understand the basis of disease and aid in development of new therapeutic approaches.

Immunoaffinity extraction followed by enzymatic digestion for the isolation and identification of proteins employing automated µSPE reactors and mass spectrometry.

This work describes a novel automated and rapid method for bottom-up proteomics combining protein isolation with a micro-immobilised enzyme reactor (IMER). Crosslinking chemistry based on 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling was exploited to immobilise trypsin and antibodies onto customisable silica particles coated with carboxymethylated dextran (CMD). This novel silica-CMD solid-phase extraction material was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), conductometric titrations and enzymatic colorimetric assays. Micro-solid-phase extraction (µSPE) cartridges equipped with the modified CMD material were employed and integrated into an automated and repeatable workflow using a sample preparation workstation to achieve rapid and repeatable protein isolation and pre-concentration, followed by tryptic digestion producing peptide fragments that were identified by liquid chromatography mass spectrometry (LC-MS).

Host-Mediated Copper Stress Is Not Protective against Streptococcus pneumoniae D39 Infection.

Metal ions are required by all organisms for the chemical processes that support life. However, in excess they can also exert toxicity within biological systems. During infection, bacterial pathogens such as Streptococcus pneumoniae are exposed to host-imposed metal intoxication, where the toxic properties of metals, such as copper, are exploited to aid in microbial clearance. However, previous studies investigating the antimicrobial efficacy of copper in vivo have reported variable findings. Here, we use a highly copper-sensitive strain of S. pneumoniae, lacking both copper efflux and intracellular copper buffering by glutathione, to investigate how copper stress is managed and where it is encountered during infection. We show that this strain exhibits highly dysregulated copper homeostasis, leading to the attenuation of growth and hyperaccumulation of copper in vitro. In a murine infection model, whole-tissue copper quantitation and elemental bioimaging of the murine lung revealed that infection with S. pneumoniae resulted in increased copper abundance in specific tissues, with the formation of spatially discrete copper hot spots throughout the lung. While the increased copper was able to reduce the viability of the highly copper-sensitive strain in a pneumonia model, copper levels in professional phagocytes and in a bacteremic model were insufficient to prosecute bacterial clearance. Collectively, this study reveals that host copper is redistributed to sites of infection and can impact bacterial viability in a hypersusceptible strain. However, in wild-type S. pneumoniae, the concerted actions of the copper homeostatic mechanisms are sufficient to facilitate continued viability and virulence of the pathogen. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is one of the world's foremost bacterial pathogens. Treatment of both localized and systemic pneumococcal infection is becoming complicated by increasing rates of multidrug resistance globally. Copper is a potent antimicrobial agent used by the mammalian immune system in the defense against bacterial pathogens. However, unlike other bacterial species, this copper stress is unable to prosecute pneumococcal clearance. This study determines how the mammalian host inflicts copper stress on S. pneumoniae and the bacterial copper tolerance mechanisms that contribute to maintenance of viability and virulence in vitro and in vivo. This work has provided insight into the chemical biology of the host-pneumococcal interaction and identified a potential avenue for novel antimicrobial development.

Quantitative multiplexed analysis of MMP-11 and CD45 in metastatic breast cancer tissues by immunohistochemistry-assisted LA-ICP-MS.

Breast cancer is the leading cause of cancer death in woman and tremendous efforts are undertaken to limit its dissemination and to provide effective treatment. Various histopathological parameters are routinely assessed in breast cancer biopsies to provide valuable diagnostic and prognostic information. MMP-11 and CD45 are tumor-associated antigens and potentially valuable biomarkers for grading aggressiveness and metastatic probability. This paper presents methods for quantitative and multiplexed imaging of MMP-11 and CD45 in breast cancer tissues and investigates their potential for improved cancer characterization and patient stratification. An immunohistochemistry-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method was successfully developed and optimized using lanthanide-tagged monoclonal antibodies as proxies to determine spatial distributions and concentrations of the two breast cancer biomarkers. The labeling degree of antibodies was determined via size exclusion-ICP-tandem mass spectrometry (SEC-ICP-MS/MS) employing online calibration via post-column isotope dilution analysis (IDA). The calibration of spatial distributions of labeled lanthanides in tissues was performed by ablating mold-prepared gelatin standards spiked with element standards. Knowledge of labeling degrees enabled the translation of lanthanide concentrations into biomarkers concentrations. The k-means clustering was used to select tissue areas for statistical analysis and mean concentrations were compared for sets of metastatic, non-metastatic and healthy samples. MMP-11 was expressed in stroma surrounding tumor areas, while CD45 was predominantly found inside tumor areas with high cell density. There was no significant correlation between CD45 and metastasis (P = 0.70); however, MMP-11 was significantly up-regulated (202%) in metastatic samples compared to non-metastatic (P = 0.0077) and healthy tissues (P = 0.0087).

Separation of intact proteins by capillary electrophoresis.

This work introduces novel and universal workflows for the analysis of intact proteins by capillary electrophoresis and presents guidelines for the targeted selection of appropriate background electrolytes (BGEs) by consideration of the target proteins' isoelectric point (pI). The suitability of neutral dimethyl polysiloxane (PDMS) capillaries with dynamic coatings of cationic cetyltrimethylammonium bromide (CTAB) or anionic sodium dodecyl sulfate (SDS), and bare fused silica (BFS) capillaries were systematically evaluated for the analysis of histidine and seven model proteins in six BGEs with pH values between 3.0 and 9.6. Multiple capillary and BGE combinations were suitable for the analysis of all proteins with molecular weights ranging from 13.7-150 kDa, and pIs between 4.7 and 9.6. The CTAB-PDMS capillary was best suited for low pH BGEs, while the SDS-PDMS and BFS capillary were superior for high pH BGEs. These combinations consistently resulted in sharp peak shapes and rapid migration times. pH values of BGEs closer to the proteins' pI produced poorer peak shapes and decreased effective mobilities due to suppressed ionisation. Plots of mobility vs. pH crossed at approximately the pI of the protein in most cases. The workflow was applied to the analysis of caseins and whey proteins in milk for the separation of the seven most abundant proteins, including the isoforms of A1 and A2 ß-casein and ß-lactoglobulin A and B.

Analysis of Ti- and Pb-based particles in the aqueous environment of Melbourne (Australia) via single particle ICP-MS.

The analysis of natural and anthropogenic nanomaterials (NMs) in the environment is challenging and requires methods capable to identify and characterise structures on the nanoscale regarding particle number concentrations (PNCs), elemental composition, size, and mass distributions. In this study, we employed single particle inductively coupled plasma-mass spectrometry (SP ICP-MS) to investigate the occurrence of NMs in the Melbourne area (Australia) across 63 locations. Poisson statistics were used to discriminate between signals from nanoparticulate matter and ionic background. TiO2-based NMs were frequently detected and corresponding NM signals were calibated with an automated data processing platform. Additionally, a method utilising a larger mass bandpass was developed to screen for particulate high-mass elements. This procedure identified Pb-based NMs in various samples. The effects of different environmental matrices consisting of fresh, brackish, or seawater were mitigated with an aerosol dilution method reducing the introduction of salt into the plasma and avoiding signal drift. Signals from TiO2- and Pb-based NMs were counted, integrated, and subsequently calibrated to determine PNCs as well as mass and size distributions. PNCs, mean sizes, particulate masses, and ionic background levels were compared across different locations and environments.

An integrated mass spectrometry imaging and digital pathology workflow for objective detection of colorectal tumours by unique atomic signatures.

Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells.

"Simultaneous targeted and non-targeted analysis of per- and polyfluoroalkyl substances in environmental samples by liquid chromatography-ion mobility-quadrupole time of flight-mass spectrometry and mass defect analysis".

Per- and polyfluoroalkyl substances (PFAS) represent a large group of synthetic organic compounds which exhibit unique properties and have been extensively used for consumer and industrial products, resulting in a widespread presence in the environment. Regulation requiring PFAS monitoring has been implemented worldwide due to their potential health and eco-toxicological effects. Targeted methods are commonly used to monitor between twenty to forty PFAS compounds, representing only a small fraction of the number of compounds that may be present. Consequently, there is an increasing interest in complementary non-targeted methods to screen and identify unknown PFAS compounds with the aim to improve knowledge and to generate more accurate models regarding their environmental mobility and persistence. This work details the development of a method that simultaneously provided targeted and non-targeted PFAS analysis. Ultra-high performance liquid chromatography (UHPLC) was coupled to ion mobility-quadrupole time of flight-mass spectrometry (IMS-QTOF-MS) and used to quantify known and screen unknown PFAS in environmental samples collected within the greater Sydney basin (Australia). The method was validated for the quantification of 14 sulfonate-based PFAS, and a non-targeted data analysis workflow was developed using a combination of mass defect analysis with common fragment and neutral loss filtering to identify fluorine-containing species. The optimised method was applied to the environmental samples and enabled the determination of 3-7 compounds from the targeted list and the detection of a further 56-107 untargeted PFAS. This simultaneous analysis reduces the complexity of multiple analyses, and allows for greater interrogation of the full PFAS load in environmental samples.

Characterisation of microplastics and unicellular algae in seawater by targeting carbon via single particle and single cell ICP-MS.

The discharge of plastic waste and subsequent formation and global distribution of microplastics (MPs) has caused great concern and highlighted the need for dedicated methods to characterise MPs in complex environmental matrices like seawater. Single particle inductively coupled plasma - mass spectrometry (SP ICP-MS) is an elegant method for the rapid analysis of nano- and microparticles and to characterise number concentrations, mass, and size distributions. However, the analysis of carbon (C)-based microstructures such as MPs by SP ICP-MS is at an early stage. This paper investigates various strategies to improve figures of merit to detect and characterise MPs in complex matrices, such as seawater. Ten methods operating distinct acquisition modes with various collision/reaction gases, tandem MS (ICP-MS/MS) and targeting 12C or 13C were developed and compared for the analysis of polystyrene-based MPs standards in ultra-pure water and seawater. The robust analysis of MPs in seawater was accomplished by on-line aerosol dilution enabling repeatable size calibration while minimising drift effects. However, the direct analysis of seawater decreased ion transmission and required matrix-matching for accurate size calibration. Analysis of the 12C isotope instead of 13C improved the size detection limits (sDL) to 0.62 µm in ultra-pure water and to 0.96 µm in seawater. ICP-MS/MS methods decreased ion transmission but also reduced background signal and increased selectivity, particularly in the presence of spectral interferences. In the second part of this study, it was demonstrated that the developed methods were applicable for the analysis of C in unicellular organisms and allowed calibration of physical dimensions. This is relevant for the investigation and understanding of phenotypical traits associated, for example, with climate change resilience as well as oceanic C storage. SP/SC ICP-MS was employed to target five different intact Symbiodiniaceae algae strains with diverse life-histories in seawater and polystyrene-based MPs were used to calibrate cellular C masses, which were between 51 and 83 pg. The C mass distribution across the analysed unicellular cells was used for modelling cell sizes, which were in the range of 7.6 and 10.1 µm. Determined values were in line with values obtained with complementary techniques (Coulter-counting, total organic C analysis and microscopic analysis).

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.

Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry Imaging in Biology.

Elemental imaging gives insight into the fundamental chemical makeup of living organisms. Every cell on Earth is comprised of a complex and dynamic mixture of the chemical elements that define structure and function. Many disease states feature a disturbance in elemental homeostasis, and understanding how, and most importantly where, has driven the development of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) as the principal elemental imaging technique for biologists. This review provides an outline of ICP-MS technology, laser ablation cell designs, imaging workflows, and methods of quantification. Detailed examples of imaging applications including analyses of cancers, elemental uptake and accumulation, plant bioimaging, nanomaterials in the environment, and exposure science and neuroscience are presented and discussed. Recent incorporation of immunohistochemical workflows for imaging biomolecules, complementary and multimodal imaging techniques, and image processing methods is also reviewed.

The Prevalence of Inorganic Mercury in Human Kidneys Suggests a Role for Toxic Metals in Essential Hypertension.

The kidney plays a dominant role in the pathogenesis of essential hypertension, but the initial pathogenic events in the kidney leading to hypertension are not known. Exposure to mercury has been linked to many diseases including hypertension in epidemiological and experimental studies, so we studied the distribution and prevalence of mercury in the human kidney. Paraffin sections of kidneys were available from 129 people ranging in age from 1 to 104 years who had forensic/coronial autopsies. One individual had injected himself with metallic mercury, the other 128 were from varied clinicopathological backgrounds without known exposure to mercury. Sections were stained for inorganic mercury using autometallography. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used on six samples to confirm the presence of autometallography-detected mercury and to look for other toxic metals. In the 128 people without known mercury exposure, mercury was found in: (1) proximal tubules of the cortex and Henle thin loops of the medulla, in 25% of kidneys (and also in the man who injected himself with mercury), (2) proximal tubules only in 16% of kidneys, and (3) Henle thin loops only in 23% of kidneys. The age-related proportion of people who had any mercury in their kidney was 0% at 1-20 years, 66% at 21-40 years, 77% at 41-60 years, 84% at 61-80 years, and 64% at 81-104 years. LA-ICP-MS confirmed the presence of mercury in samples staining with autometallography and showed cadmium, lead, iron, nickel, and silver in some kidneys. In conclusion, mercury is found commonly in the adult human kidney, where it appears to accumulate in proximal tubules and Henle thin loops until an advanced age. Dysfunctions of both these cortical and medullary regions have been implicated in the pathogenesis of essential hypertension, so these findings suggest that further studies of the effects of mercury on blood pressure are warranted.

Mercury in the human adrenal medulla could contribute to increased plasma noradrenaline in aging.

Plasma noradrenaline levels increase with aging, and this could contribute to the sympathetic overactivity that is associated with essential hypertension and the metabolic syndrome. The underlying cause of this rise in noradrenaline is unknown, but a clue may be that mercury increases noradrenaline output from the adrenal medulla of experimental animals. We therefore determined the proportion of people from 2 to 104 years of age who had mercury in their adrenal medulla. Mercury was detected in paraffin sections of autopsied adrenal glands using two methods of elemental bioimaging, autometallography and laser ablation-inductively coupled plasma-mass spectrometry. Mercury first appeared in cells of the adrenal medulla in the 21-40 years group, where it was present in 52% of samples, and increased progressively in frequency in older age groups, until it was detected in 90% of samples from people aged over 80 years. In conclusion, the proportion of people having mercury in their adrenal medulla increases with aging. Mercury could alter the metabolism of catecholamines in the adrenal medulla that leads to the raised levels of plasma noradrenaline in aging. This retrospective autopsy study was not able to provide a definitive link between adrenal mercury, noradrenaline levels and hypertension, but future functional human and experimental studies could provide further evidence for these associations.

Mercury in the human thyroid gland: Potential implications for thyroid cancer, autoimmune thyroiditis, and hypothyroidism.

OBJECTIVE: Mercury and other toxic metals have been suggested to be involved in thyroid disorders, but the distribution and prevalence of mercury in the human thyroid gland is not known. We therefore used two elemental bio-imaging techniques to look at the distribution of mercury and other toxic metals in the thyroid glands of people over a wide range of ages. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded thyroid tissue blocks were obtained from 115 people aged 1-104 years old, with varied clinicopathological conditions, who had thyroid samples removed during forensic/coronial autopsies. Seven-micron sections from these tissue blocks were used to detect intracellular inorganic mercury using autometallography. The presence of mercury was confirmed using laser ablation-inductively coupled plasma-mass spectrometry which can detect multiple elements. RESULTS: Mercury was found on autometallography in the thyroid follicular cells of 4% of people aged 1-29 years, 9% aged 30-59 years, and 38% aged 60-104 years. Laser ablation-inductively coupled plasma-mass spectrometry confirmed the presence of mercury in samples staining with autometallography, and detected cadmium, lead, iron, nickel and silver in selected samples. CONCLUSIONS: The proportion of people with mercury in their thyroid follicular cells increases with age, until it is present in over one-third of people aged 60 years and over. Other toxic metals in thyroid cells could enhance mercury toxicity. Mercury can trigger genotoxicity, autoimmune reactions, and oxidative damage, which raises the possibility that mercury could play a role in the pathogenesis of thyroid cancers, autoimmune thyroiditis, and hypothyroidism.

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.

Mercury in Pancreatic Cells of People with and without Pancreatic Cancer.

Toxic metals have been implicated in the pathogenesis of pancreatic cancer. Human exposure to mercury is widespread, but it is not known how often mercury is present in the human pancreas and which cells might contain mercury. We therefore aimed to determine, in people with and without pancreatic cancer, the distribution and prevalence of mercury in pancreatic cells. Paraffin-embedded sections of normal pancreatic tissue were obtained from pancreatectomy samples of 45 people who had pancreatic adenocarcinoma, and from autopsy samples of 38 people without pancreatic cancer. Mercury was identified using two methods of elemental bio-imaging: (1) With autometallography, inorganic mercury was seen in islet cells in 14 of 30 males (47%) with pancreatic cancer compared to two of 17 males (12%) without pancreatic cancer (p = 0.024), and in 10 of 15 females (67%) with pancreatic cancer compared to four of 22 females (19%) without pancreatic cancer (p = 0.006). Autometallographic mercury was present in acinar cells in 24% and in periductal cells in 11% of people with pancreatic cancer, but not in those without pancreatic cancer. (2) Laser ablation-inductively coupled plasma-mass spectrometry confirmed the presence of mercury in islets that stained with autometallography and detected cadmium, lead, chromium, iron, nickel and aluminium in some samples. In conclusion, the genotoxic metal mercury is found in normal pancreatic cells in more people with, than without, pancreatic cancer. These findings support the hypothesis that toxic metals such as mercury contribute to the pathogenesis of pancreatic cancer.

Characterization of Upconversion Nanoparticles by Single-Particle ICP-MS Employing a Quadrupole Mass Filter with Increased Bandpass.

This work introduces new methods to characterize dispersions of small-diameter or low-mass-fraction nanoparticles (NPs) by single-particle inductively coupled plasma-mass spectrometry (SP ICP-MS). The optimization of ion extraction, ion transport, and the operation of the quadrupole with increased mass bandwidth improved the signal-to-noise ratios significantly and decreased the size detection limits for all NP dispersions investigated. As a model system, 10.9 ± 1.0 nm Au NPs were analyzed to demonstrate the effects of increasing ion transmission. Specifically, increasing the mass bandwidth of the quadrupole improved the size detection limit to 4.2 nm and enabled the resolution of NP signals from ionic background and noise. Subsequently, the methods were applied to the characterization of lanthanide-doped upconversion nanoparticles (UCNPs) by SP ICP-MS. Three different types of UCNPs (90 nm NaYF4: 20% Yb, 2% Er; 20 nm NaGdF4: 20% Yb, 1% Er; 15 nm NaYF4: 20% Yb, 2% Er) were investigated. Y showed the best signal-to-noise ratios with optimized ion extraction and transport parameters only, whereas the signal-to-noise ratios of Gd, Er, and Yb were further improved by increasing the mass bandwidth of a quadrupole mass filter. The novel methods were suitable for detailed characterization of diluted UCNP dispersions including particle stoichiometries and size distributions. A Poisson model was further applied to assess particle-particle interactions in the aqueous dispersions. The methods have considerable potential for the characterization of small-diameter and/or low-mass-fraction nanoparticles.

The distribution of toxic metals in the human retina and optic nerve head: Implications for age-related macular degeneration.

OBJECTIVE: Toxic metals are suspected to play a role in the pathogenesis of age-related macular degeneration. However, difficulties in detecting the presence of multiple toxic metals within the intact human retina, and in separating primary metal toxicity from the secondary uptake of metals in damaged tissue, have hindered progress in this field. We therefore looked for the presence of several toxic metals in the posterior segment of normal adult eyes using elemental bioimaging. METHODS: Paraffin sections of the posterior segment of the eye from seven tissue donors (age range 54-74 years) to an eye bank were examined for toxic metals in situ using laser ablation-inductively coupled plasma-mass spectrometry, a technique that detects multiple elements in tissues, as well as the histochemical technique of autometallography that demonstrates inorganic mercury, silver, and bismuth. No donor had a visual impairment, and no significant retinal abnormalities were seen on post mortem fundoscopy and histology. RESULTS: Metals found by laser ablation-inductively coupled plasma-mass spectrometry in the retinal pigment epithelium and choriocapillaris were lead (n = 7), nickel (n = 7), iron (n = 7), cadmium (n = 6), mercury (n = 6), bismuth (n = 5), aluminium (n = 3), and silver (n = 1). In the neural retina, mercury was present in six samples, and iron in one. Metals detected in the optic nerve head were iron (N = 7), mercury (N = 7), nickel (N = 4), and aluminium (N = 1). No gold or chromium was seen. Autometallography demonstrated probable inorganic mercury in the retinal pigment epithelium of one donor. CONCLUSION: Several toxic metals are taken up by the human retina and optic nerve head. Injury to the retinal pigment epithelium from toxic metals could damage the neuroprotective functions of the retinal pigment epithelium and allow toxic metals to enter the outer neural retina. These findings support the hypothesis that accumulations of toxic metals in the retina could contribute to the pathogenesis of age-related macular degeneration.