Maarten De Rijcke

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.

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.

High resolution mass spectrometry-based screening reveals lipophilic toxins in multiple trophic levels from the North Sea

Lipophilic marine biotoxins, which are mainly produced by small dinoflagellates, are increasingly detected in coastal waters across the globe. As these producers are consumed by zooplankton and shellfish, the toxins are introduced, bioaccumulated and possibly biomagnified throughout marine food chains. Recent research has demonstrated that ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is an excellent tool to detect marine toxins in algae and seafood. In this study, UHPLC-HRMS was used to screen lipophilic marine biotoxins in organisms from different trophic levels of the Belgian coastal zone ecosystem. A total of 20 tentatively identified lipophilic compounds was detected. Hereby, the trophic transfer of lipophilic marine biotoxins to the upper trophic level was considered to be rather limited. Furthermore, 36% of the compounds was clearly transferred between different organisms. A significant biotransformation of compounds from the okadaic acid and spirolide toxin groups was observed (64%), mainly in filter feeders. Through a multi-targeted approach, this study showed that marine organisms in the Belgian coastal zone are exposed to a multi-toxin mixture. Further research on both single compound and interactive toxic effects of the frequently detected lipophilic marine toxin ester metabolites throughout the food chain is therefore needed. As a future perspective, confirmatory identification of potential toxins by studying their fragmentation spectra (using new tools such as hybrid quadrupole Q-Exactive™ Orbitrap-MS) is designated.

Three-Dimensional Reconstruction of the Tissue-Specific Multielemental Distribution within Ceriodaphnia dubia via Multimodal Registration Using Laser Ablation ICP-Mass Spectrometry and X-ray Spectroscopic Techniques

In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 μm via a data fusion approach employing state-of-the-art laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) and laboratory-based absorption microcomputed tomography (μ-CT). C. dubia was exposed to elevated Cu, Ni, and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to μ-CT analysis. Subsequently, the sample was cut into 5 μm thin sections that were subjected to LA-ICP-TOFMS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOFMS images relative to the corresponding slices of the 3D μ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOFMS data were projected onto the μ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal μ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.

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.