Departments

Bilateral Project with partners from Ghent University, Belgium: ARRS-FWO

Project leader: J.T. van Elteren

PROJECT SYNOPSIS

Laser ablation-ICP-mass spectrometry (LA-ICP-MS) can be used for visualizing the distribution of (trace) elements across any solid material with high spatial resolution. The recent development of low-dispersion LA cells has provided gains in throughput and sensitivity of 2-3 orders of magnitude compared to conventional cells. The project focuses on development of methodologies for high-resolution 2D & 3D imaging of samples of a wide range of hardness, morphology, composition and complexity, taking full advantage of this next generation of LA cells. New algorithms and approaches to 2D & 3D mapping will be elaborated. Cross-validation with established molecular and elemental imaging microprobes will be performed to benchmark the new LA-ICP-MS protocols. Enhanced visualization of localized features in 2D and 3D elemental LA-ICP-MS images will be explored by merging these images with images obtained with complementary molecular and morphological techniques, potentially leading to better insight into bio- and geochemical processes on the (sub-) μm scale. Mathematical procedures for registration, data mining and data fusion of these multimodal images will be deployed and further developed, whilst fundamental issues regarding quantification and calibration will be addressed. Multimodal 2D and 3D imaging will be applied to biological tissues to monitor biochemical changes induced by drug admission or by external exposure to (heavy) metals in the context of environmental pollution. 

FUNDING BODIES

Slovenian Research Agency ARRS (Department of Analytical Chemistry, National Institute of Chemistry, Ljubljana) and the Research Foundation Flanders FWO (Department of Chemistry, Atomic & Mass Spectrometry, Ghent University, Belgium); funding period: 01.01.2017-31.12.2020; funded hours: 2049 (1.20 FTE).

SLOVENIAN GROUP MEMBERS (01.01.2017-31.12.2020)

J.T. van Elteren (head)
M. Šala (researcher)
V.S. Šelih (researcher)
A. Pavlišič (researcher)

PROJECT TARGETS

The project targets the following objectives: 

i)    Development and optimization of 2D and 3D LA-ICP-MS imaging;
ii)   Performance testing via cross-validation of 2D and 3D LA-ICP-MS imaging;
iii)  Multimodal imaging and data fusion for relevant samples;
iv)  Solving quantification issues in LA-ICP-MS imaging;
v)   Physical effects of cryogenic cooling on 2D and 3D LA-ICP-MS mapping. 

REALIZATION

Overcoming Instrumental Limitations
The major bottleneck in LA-ICP-MS imaging is the time it takes to generate a high-resolution map of a large(r) sample. The system response is directly responsible for the mapping time and is mostly determined by the washout of aerosol particles from the cell and their dispersion in the transfer line to the ICP, generally defined by the full width of a laser pulse at 1 % of the maximum (FW0.01M, in seconds). Ca. ten years ago the typical LA cell geometry restricted fast mapping as a result of a sluggish system response caused by turbulent flow conditions and large cell volumes, yielding FW0.01M values > 10 s. Recent developed LA cells by our FWO partner have small internal volumes and optimized tubing that transports the aerosol particles to the ICP in a much faster fashion. They can be regarded as rapid response cells with washout times as low as 1 ms. This development has led to a revolutionary advancement of elemental LA-ICP-MS imaging with imaging rates approaching 500 kpixels per hour, compared to ca. 10 kpixels per hour only several years ago.

Identifying LA-ICP-MS Artifacts
However, various imaging artefacts may be encountered when the LA-ICP-MS conditions such as beam size, acquisition and dwell times, repetition rate and scanning speed are not carefully selected. We pioneered strategies to optimize the LA-ICP-MS imaging parameters and eliminate or minimize artefacts such as pixilation, aliasing, blur and noise, guided by modelling strategies that accurately simulate the LA-ICP-MS imaging process and validate the image quality. Artefacts associated with aliasing in 2D laser ablation-ICP-MS elemental imaging, giving rise to interference patterns in images, were studied through simulation and eliminated experimentally by synchronization of the laser ablation repetition rate and the data acquisition time of ICP-MS [1,2]. The structural similarity (SSIM) index, an objective quantifier of differences between a distorted and a reference image, was used as a tool for optimizing the perceived visual image quality obtained by 2D LA-ICP-MS bioimaging [3]. As a result, new LA cell designs and improved interfaces with the ICP-MS were implemented to yield 20-50 times faster imaging, and at the same time produce high quality elemental images that convey detail, texture and shape.

Streamlining LA-ICP-MS Imaging
Having identified the LA-ICP-MS bottlenecks we set out to streamline the 2D and 3D imaging procedures even further. An all-inclusive “artefacts paper” discusses and offers solutions for best practices in LA-ICP-MS imaging [4], paving the way for introducing an online app for optimizing the LA-ICP-MS imaging conditions for fastest analysis with best possible image quality [5]. Several papers have been published to promote our LA-ICP-MS imaging approach which encourages the use of a high laser dosage, i.e., the number of laser pulses per pixel. The analytical performance of a LA unit with an experimental 500 Hz laser head for ultrafast mapping showed no problems related to particle saturation, elemental fractionation, ionisation efficiency, etc. [6] and it was shown that superior signal-to-noise ratios were obtained using a dosage of 10 for low-concentration mapping of elements in a biological (murine brain tissue) and a mineralogical (asbestos fibers) sample [7]. Teledyne Cetac Technologies, the largest laser ablation equipment supplier in the world, has set out to include our optimization approach as a frontend plugin in their operating software.

Calibration Issues
For quantification purposes in bioimaging, commonly in-house prepared calibration standards are used based on matrix-matching via spiking of materials such as cellulose paper, filter paper, agarose, gelatin, etc. or the use of pressed pellets based on (doping of) certified reference biomaterials (CRMs). The Achilles' heel in all these calibration approaches is that the sample and standard matrices need to be exactly the same to circumvent elemental fractionation and differences in ablation rate. We fabricated highly homogeneous multi-element gelatin calibration standards for quantitative LA-ICP-MS bioimaging. Heterogeneity issues caused by the so-called “coffee-stain” and/or “Marangoni” effects were found to be element-dependent but could be circumvented by careful selection of drying/setting conditions. A micro-homogeneity test was developed for certification of the standards [8].

Regular and Complementary Imaging Applications
The imaging strategies have been applied to a variety of topical questions in several interdisciplinary fields. Some of the most noteworthy research carried out is related to the following work: i) a study on mercury (Hg) localization, speciation and ligand environment in edible mushrooms: Boletus edulis, B. aereus and Scutiger pescaprae by LA-ICP-MS, SR-μ-XRF and Hg L3-edge XANES and EXAFS [9] and ii) development of a cheap ion chromatography methodology to study the stability of historic glass, after extraction of ions from the glass, through comparison with LA-ICP-MS depth profiling [10]. A book chapter was co-authored to highlight the complementarity of elemental imaging techniques, including LA-ICP-MS, µPIXE, µXRF, SIMS, µXANES, etc., for the analysis of plant tissues [11].

LA-Single Particle-ICPMS as a Multimodal Imaging Tool
All of the above LA-ICP-MS imaging is related to measurement of the elemental distribution, but the next big step is the inclusion of nanoparticles in the imaging protocols due to concerns about their safety, especially as uptake mechanisms for metal nanoparticles are different from these for dissolved metal species. Although in some cases the spatial distribution of nanoparticles in biological tissues has been measured by LA-ICP-MS, they were not measured as single particles but as an elemental distribution profile, thus not identifying individual particles. We pioneered the analysis of nanoparticles by LA-single particle-ICP-MS to measure individual nanoparticle events, and through a custom data processing protocol we were able to retrieve the nanoparticle number concentration and spherical-equivalent size [12]. This novel technique gives a new dimension to LA-ICP-MS imaging and connects directly to the field of nanometallomics and nanotoxicology. 

Advanced data processing and visualization techniques were applied to LA-single particle-ICPMS data obtained from imaging of roots of sunflower (Helianthus annuus L.) which were exposed to ionic silver (Ag+) [13]. Highly multiplexed images with tissue level resolution were generated, showing the uptake and transformation of Ag+ to silver nanoparticles (AgNPs) in whole root cross-sections, and revealing that the size of biosynthesized AgNPs is predisposed by the reducing power of particular root compartments. Various Ag+ and AgNP visualization strategies were shown with special emphasis on spatially resolved information associated with the number and spherical-equivalent size of individual AgNPs in user-selected root cross-sectional regions, including statistical analysis of the AgNP distribution. Results compared favourably to localized speciation results obtained by synchrotron techniques such as XANES. Fundamentals of this novel technique were addressed in a separate paper for correctly setting up the LA-ICP-MS instrument and data processing [14]. Normally, this multimodality information can only be obtained by combining the results of techniques from several complementary platforms.

REFERENCES

[1]    J.T. van Elteren, V.S. Šelih, M. Šala, S.J.M. Van Malderen and F. Vanhaecke, Imaging artifacts in continuous scanning 2D LA-ICP-MS Imaging due to non-synchronization issues, Anal. Chem. 90 (2018) 2896-2901. doi.org/10.1021/acs.analchem.7b05134.

[2]    S.J.M. Van Malderen, J.T. van Elteren, V.S. Šelih and F. Vanhaecke, Considerations on data acquisition with scanning mass spectrometers in laser ablation-inductively coupled plasma-mass spectrometry with low-dispersion interfaces, Spectrochim. Acta Part B At. Spectrosc. 140 (2017) 29-34. doi.org/10.1016/j.sab.2017.11.007.

[3]     J.T. van Elteren, M. Šala and V.S. Šelih, Perceptual image quality metrics concept in continuous scanning 2D laser ablation-inductively coupled plasma mass spectrometry, Anal. Chem. 90 (2018) 5916-5922. pubs.acs.org/doi/10.1021/acs.analchem.8b00751.

[4]     J.T. van Elteren, M. Šala and V.S. Šelih, Insights into the selection of 2D LA-ICP-MS (multi) elemental mapping conditions, J. Anal. At. Spectrom. 34 (2019) 1919-1931. doi.org/10.1039/c9ja00166b.

[5]    J.T. van Elteren, D. Metarapi, M. Šala, V.S. Šelih and C.C. Stremtan, Fine-tuning of LA-ICP-QMS conditions for elemental mapping, J. Anal. At. Spectrom. 35 (2020) 2494-2497. doi.org/10.1039/d0ja00322k.

[6]    M. Šala, V.S. Šelih, C.C. Stremtan and J.T. Van Elteren, Analytical performance of a high-repetition rate laser head (500 Hz) for HR LA-ICP-QMS imaging, J. Anal. At. Spectrom. 35 (2020) 1827-1831. doi.org/10.1039/c9ja00421a.

[7]    M. Šala, V.S. Šelih, C.C. Stremtan, T. Ťamaş and J.T. van Elteren, Implications of laser shot dosage on image quality in LA-ICP-QMS imaging, J. Anal. At. Spectrom. 36 (2021) 75-79. doi.org/10.1039/d0ja00381f.

[8]    M. Šala, V.S. Šelih and J.T. van Elteren, Gelatin gels as multi-element calibration standards in LA-ICP-MS bioimaging: fabrication of homogeneous standards and micro-homogeneity testing, Analyst 142 (2017) 3356-3359. doi.org/10.1039/C7AN01361B.

[9]    A. Kavčič, K. Mikuš, M. Debeljak,  J.T. van Elteren, I. Arčon, A. Kodre, P- Kump, A.-G. Karydas, A. Migliori,  M. Czyzycki and K. Vogel-Mikuš, Localization, ligand environment, bioavailability and toxicity of mercury in Boletus spp. and Scutiger pes-caprae mushrooms, Ecotoxicol. Environ. Saf. 184 (2019) 109623. doi.org/10.1016/j.ecoenv.2019.109623.

[10]    G. Verhaar, N.H. Tennent, J.T. van Elteren, V.S. Šelih, and M. Šala, Investigating ion depletion in unstable historic glass samples using laser ablation-inductively coupled plasma-mass spectrometry and ion chromatography, Recent Advances in Glass and Ceramics Conservation 2019: Interim Meeting of the ICOM-CC Glass and Ceramics Working Group and Icon Ceramics and Glass Group Conference, 5-7 September 2019, London, United Kingdom. ISBN: 9789290124702.

[11]    K. Vogel-Mikuš, J.T. van Elteren, M. Regvar, J. Chaiprapa, B. Jenčič, I. Arčon, A. Kodre, P. Kump, A. Kavčič, M. Kelemen, D. Metarapi, M. Nećemer, P. Vavpetić, P. Pelicon and P. Pongrac. Recent advances in 2D imaging of element distribution in Plants by focused beam techniques (in Plant metallomics and functional omics : a system-wide perspective). G. Sablok (ed.). Springer, 2019. p. 169-207. ISBN: 978-3-030-19102-3.

[12]    D. Metarapi, M. Šala, K. Vogel-Mikuš, V.S. Šelih and J.T. van Elteren, Nanoparticle Analysis in Biomaterials Using Laser Ablation−Single Particle−Inductively Coupled Plasma Mass Spectrometry, Anal. Chem. 91 (2019) 6200–6205. doi.org/10.1021/acs.analchem.9b00853.

[13]    D. Metarapi, J.T. van Elteren, M. Šala, K. Vogel-Mikuš, I. Arčon, V.S. Šelih, M. Kolar and S. Hočevar, Laser ablation-single-particle-inductively coupled plasma mass spectrometry as a multimodality bioimaging tool in nano-based omics, Environ. Sci. Nano. 8 (2021) 647-656. doi.org/10.1039/d0en01134g.

[14]    D. Metarapi and J.T. van Elteren, Fundamentals of single particle analysis in biomatrices by laser ablation-inductively coupled plasma mass spectrometry, J. Anal. At. Spectrom. 35 (2020) 784-793. doi.org/10.1039/D0JA00003E.