Eva Vergucht

A novel calibration strategy for the quantitative imaging of iron in biological tissues by LA-ICP-MS using matrix-matched standards and internal standardisation

The development of a novel and straightforward procedure for the preparation of matrix-matched calibration standards for the quantitative imaging of iron (Fe) in biological tissues by laser ablation (LA)-ICP-MS with on-tissue internal standard addition is described. This simple approach enabled on-tissue addition of Rh as internal standard to samples (with heterogeneous Fe distribution) and calibrants (with homogeneous Fe distribution). This is achieved without altering the original Fe distribution of the sample. Calibration standards were prepared by full horizontal immersion of slides with mounted homogenised sheep brain tissue section into the corresponding solution containing 0.5, 0.75, 1, 5, 10 and 20 mg kg−1 Fe (each also containing 250 µg kg−1 Rh as IS) in pure methanol for 30 minutes (6 immersions, each for 5 minutes). Subsequent air-drying (bench drying at room temperature) for approximately 5 minutes was undertaken in between consecutive immersions, to prevent long-term exposure of the tissue to lipid degradation. Tissue-matched standards were characterised in-house for Fe composition, homogeneity and stability (at storage temperatures of −80 °C, −20 °C, 4 °C and 25 °C for up to 2 months) in order to investigate their suitability as calibrants for quantitative LA-ICP-MS. The homogeneity data suggested that the materials are homogeneous in terms of Fe and Rh distribution with RSDs (n = 30) of 8.3% and 4.7%, respectively. The Fe measurement precision was improved by approximately a factor of 2 when normalising 56Fe intensities to 103Rh intensities; the RSD (n = 30) for 56Fe/103Rh was 3.6%. The produced calibration standards were found to be stable when stored at room temperature for approximately 50 days, suggesting that they can be reused for multiple batches. Using LA coupled to double-focusing sector field ICP-MS in medium resolution mode (m/Δm = 4000), linear calibration over a range of 107 to 1519 mg kg−1 Fe (R2 = 0.99) was achieved with a limit of detection of 1.84 mg kg−1 Fe. Assessment of the accuracy of the method for the quantitative imaging of Fe in tissues was undertaken by comparison of the LA-ICP-MS data with that obtained by micro-XRF; the average Fe concentrations in selected tissue regions obtained by using XRF fell within the window defined by the LA-ICP-MS values and their associated standard deviations.

The combined effect of dissolved organic carbon and salinity on the bioaccumulation of copper in marine mussel larvae.

Larvae of Mytilus spp. are among the most Cu sensitive marine species. In this study we assessed the combined effect of salinity and dissolved organic carbon (DOC) on Cu accumulation on mussel larvae. Larvae were exposed for 48 h to three Cu concentrations in each of nine salinity/DOC treatments. Synchrotron radiation X-ray fluorescence was used to determine the Cu concentration in 36 individual larvae with a spatial resolution of 10 × 10 μm. Cu body burden concentrations varied between 1.1 and 27.6 μg/g DW larvae across all treatments and Cu was homogeneously distributed at this spatial resolution level. Our results indicate decreasing Cu accumulation with increasing DOC concentrations which can be explained by an increase in Cu complexation. In contrast, salinity had a nonlinear effect on Cu. This cannot be explained by copper speciation or competition processes and suggests a salinity-induced alteration in physiology.

Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards

This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 μg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions.

Salinity and dissolved organic carbon both affect copper toxicity in mussel larvae: Copper speciation or competition cannot explain everything

Predicting copper (Cu) toxicity in marine and estuarine environments is challenging because of the influence of anions on Cu speciation, competition between Cu(2+) and other cations at the biotic ligand and the effect of salinity on the physiology of the organism. In the present study the combined effect of salinity and dissolved organic carbon (DOC) on Cu toxicity to larvae of Mytilus galloprovincialis was assessed. Two statistical models were developed and used to elucidate the relationship between Cu toxicity, salinity, and DOC. All models based on dissolved Cu indicate a decrease in Cu toxicity with increasing DOC concentrations, which can partly be explained by complexation of Cu(2+) ions with DOC. These models also indicate an increase in Cu toxicity (modeled with dissolved Cu or Cu(2+) activity) with increasing salinity, suggesting a salinity-induced alteration in the physiology of the mussel larvae. When based on Cu body burdens, neither of the models indicates an effect of salinity or DOC. This shows that the Cu body burden is a more constant predictor of Cu toxicity, regardless of the water chemistry influencing Cu speciation or competition and possible physiological alterations or changes in Cu speciation or competition.

In vivo X-ray elemental imaging of single cell model organisms manipulated by laser-based optical tweezers

We report on a radically new elemental imaging approach for the analysis of biological model organisms and single cells in their natural, in vivo state. The methodology combines optical tweezers (OT) technology for non-contact, laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time. The main objective of this work is to establish a new method for in vivo elemental imaging in a two-dimensional (2D) projection mode in free-standing biological microorganisms or single cells, present in their aqueous environment. Using the model organism Scrippsiella trochoidea, a first proof of principle experiment at beamline ID13 of the European Synchrotron Radiation Facility (ESRF) demonstrates the feasibility of the OT XRF methodology, which is applied to study mixture toxicity of Cu-Ni and Cu-Zn as a result of elevated exposure. We expect that the new OT XRF methodology will significantly contribute to the new trend of investigating microorganisms at the cellular level with added in vivo capability.

Methodological challenges of optical tweezers- based X-ray fluorescence imaging of biological model organisms at synchrotron facilities

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence

High pressure frozen (HPF), cryo-substituted microtome sections of 2 μm thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1–2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis.