J. Sabine Becker

Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS)

The distribution analysis of (essential, beneficial, or toxic) metals (e.g., Cu, Fe, Zn, Pb, and others), metalloids, and non-metals in biological tissues is of key interest in life science. Over the past few years, the development and application of several imaging mass spectrometric techniques has been rapidly growing in biology and medicine. Especially, in brain research metalloproteins are in the focus of targeted therapy approaches of neurodegenerative diseases such as Alzheimers and Parkinsons disease, or stroke, or tumor growth. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using double-focusing sector field (LA-ICP-SFMS) or quadrupole-based mass spectrometers (LA-ICP-QMS) has been successfully applied as a powerful imaging (mapping) technique to produce quantitative images of detailed regionally specific element distributions in thin tissue sections of human or rodent brain. Imaging LA-ICP-QMS was also applied to investigate metal distributions in plant and animal sections to study, for example, the uptake and transport of nutrient and toxic elements or environmental contamination. The combination of imaging LA-ICP-MS of metals with proteomic studies using biomolecular mass spectrometry identifies metal-containing proteins and also phosphoproteins. Metal-containing proteins were imaged in a two-dimensional gel after electrophoretic separation of proteins (SDS or Blue Native PAGE). Recent progress in LA-ICP-MS imaging as a stand-alone technique and in combination with MALDI/ESI-MS for selected life science applications is summarized.

Cerebral bioimaging of Cu, Fe, Zn, and Mn in the MPTP mouse model of Parkinson’s disease using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been established as a powerful technique for the determination of metal and nonmetal distributions within biological systems with high sensitivity. An imaging LA-ICP-MS technique for Fe, Cu, Zn, and Mn was developed to produce large series of quantitative element maps in native brain sections of mice subchronically intoxicated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) as a model of Parkinson’s disease. Images were calibrated using matrix-matched laboratory standards. A software solution allowing a precise delineation of anatomical structures was implemented. Coronal brain sections were analyzed crossing the striatum and the substantia nigra, respectively. Animals sacrificed 2 h, 7 d, or 28 d after the last MPTP injection and controls were investigated.We observed significant decreases of Cu concentrations in the periventricular zone and the fascia dentata at 2 h and 7d and a recovery or overcompensation at 28 d, most pronounced in the rostral periventricular zone (+40%). In the cortex Cu decreased slightly to −10%. Fe increased in the interpeduncular nucleus (+40%) but not in the substantia nigra. This pattern is in line with a differential regulation of periventricular and parenchymal Cu, and with the histochemical localization of Fe, and congruent to regions of preferential MPTP binding described in the rodent brain.The LA-ICP-MS technique yielded valid and statistically robust results in the present study on 39 slices from 19 animals. Our findings underline the value of routine micro-local analytical techniques in the life sciences and affirm a role of Cu availability in Parkinson’s disease.

The synergy of elemental and biomolecular mass spectrometry: new analytical strategies in life sciences

The application of mass spectrometry with soft ionization techniques (ESI, electrospray ionization, and MALDI, matrix-assisted laser desorption ionization) in the life sciences for the detection and identification of biomolecules is already well established, whereas the application of elemental mass spectrometry and in particular inductively coupled plasma mass spectrometry (ICP-MS) for the determination of metals, metalloids and non-metals in biomolecules is rather new and there is some hesitation in accepting this analytical method, although it offers many advantages. Therefore, it is the aim of this tutorial review to highlight new analytical strategies consisting of the combined applications of elemental and molecular mass spectrometric techniques. In fact, elemental and biomolecular mass spectrometric methods are highly complementary: elemental mass spectrometry methods, such as ICP-MS, offer very sensitive element analysis in the trace and ultra-trace concentration range with multielement capability and the excellent and uniform sensitivity is structure-independent and can be used analytically for accurate quantification as well as for fast screening of specific elements even in complex samples. Laser ablation (LA) ICP-MS, as a solid state mass spectrometric technique, allows the direct determination of trace elements in biological and environmental samples and is applied for microlocal analysis with spatial resolution in the mum range. In contrast, molecular weight determination and structural information is completely lost during the ionization step so that these features have to be provided by biomolecular mass spectrometry and in particular by ESI- and MALDI-MS. On the basis of selected examples, it will be shown that only the combination of different elemental and biomolecular mass spectrometric techniques can solve analytical problems in the life sciences and environmental research in a synergistic way where neither technique alone would be successful. This synergy will be demonstrated by selected applications from various areas: food and nutrition, toxicology, clinical and pharmaceutical research, biochemistry and in particular proteomics. Future developments and trends will be discussed concerning instrumental developments of new mass spectrometric techniques providing high sensitivity with lower detection limits for many elements measured quasi-simultaneously so that new analytical information about biological systems can be drawn from isotopic information and the application of stable non-radioactive isotopic tracers. In addition, elemental labels enable the development of new high-throughput screening techniques based on multiplexed biomarkers. Advanced powerful surface mass spectrometric techniques are required for the imaging of elemental and molecular information in order to analyse tissue samples and to develop novel array-based biochips.

Trace metal imaging with high spatial resolution: Applications in biomedicine

New generations of analytical techniques for imaging of metals are pushing hitherto boundaries of spatial resolution and quantitative analysis in biology. Because of this, the application of these imaging techniques described herein to the study of the organization and dynamics of metal cations and metal-containing biomolecules in biological cell and tissue is becoming an important issue in biomedical research. In the current review, three common metal imaging techniques in biomedical research are introduced, including synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). These are exemplified by a demonstration of the dopamine-Fe complexes, by assessment of boron distribution in a boron neutron capture therapy cell model, by mapping Cu and Zn in human brain cancer and a rat brain tumor model, and by the analysis of metal topography within neuromelanin. These studies have provided solid evidence that demonstrates that the sensitivity, spatial resolution, specificity, and quantification ability of metal imaging techniques is suitable and highly desirable for biomedical research. Moreover, these novel studies on the nanometre scale (e.g., of individual single cells or cell organelles) will lead to a better understanding of metal processes in cells and tissues.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in elemental imaging of biological tissues and in proteomics

Of all the inorganic mass spectrometric techniques, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) plays a key role as a powerful and sensitive microanalytical technique, enabling multi-element trace analysis and isotope ratio measurements at the trace and ultra-trace level in the life sciences. LA-ICP-MS was used to produce images of detailed regionally specific element distribution in thin sections of tissue from different parts of the human brain. The quantitative determination of copper, zinc and other elements distributed in thin slices of human brain samples was performed using matrix-matched laboratory standards. Imaging mass spectrometry provides new information on the spatially inhomogeneous element distribution in thin sections of human tissue, for example of different brain regions (e.g., insular region) or brain tumour tissue. The detection limits obtained for Cu and Zn determination in tissue sections were in the sub-µg g–1 range. Possible strategies will be discussed for applying LA-ICP-MS in brain research and the life sciences, including the imaging of thin slices of brain tissue in order to obtain element distributions or applications in proteome analysis in combination with MALDI-MS to study phospho- and metal-containing proteins.

Application of double-focusing sector field ICP mass spectrometry with shielded torch using different nebulizers for ultratrace and precise isotope analysis of long-lived radionuclides

The capability of double-focusing sector field ICP-MS with a plasma-shielded torch using different nebulizers (a Meinhard nebulizer with a Scott-type spray chamber with a solution uptake rate of 1 ml min –1 ; a MicroMist microconcentric nebulizer used with a minicyclonic spray chamber with a solution uptake rate of 0.085 ml min –1 ; an ultrasonic nebulizer with a solution uptake rate of 2 ml min –1 ; and a direct injection high-efficiency nebulizer with a solution uptake rate of 0.085 ml min –1 ) for the introduction of radioactive sample solutions into the ICP was investigated. The total amount of analyte for each long-lived radionuclide ( 226 Ra, 230 Th, 237 Np, 238 U, 239 Pu and 241 Am; concentration of each was 1 ng l –1 in the aqueous solution) using different nebulizers was 5 pg for the Meinhard nebulizer, 0.4 pg for the MicroMist microconcentric nebulizer and 10 pg for the ultrasonic nebulizer. The application of the shielded torch yielded an increase in sensitivity for all these nebulizers of up to a factor of 5 compared with the original configuration without a shielded torch. Sensitivities of about 2000 MHz ppm –1 were measured for the radionuclides investigated (except for 226 Ra) using the MicroMist microconcentric nebulizer with a shielded torch. The detection limits were in the sub-pg l –1 range and the precision ranged from 1 to 2% RSD (n=5) for the 1 ng l –1 concentration level (0.4 pg sample size). A further increase in sensitivity for long-lived radionuclides of nearly one order of magnitude in comparison with the MicroMist microconcentric nebulizer was observed using ultrasonic nebulization, but the amount of analyte required was significantly higher (by a factor of 25). In contrast, the direct injection high-efficiency nebulizer (DIHEN) in double-focusing sector field ICP-MS (DF-ICP-MS) with a shielded torch resulted in a decrease in sensitivity in comparison with the unshielded torch because of a higher water load due to the direct injection of aqueous solution into the plasma. At low solution uptake rates (down to several µl min –1 ), the uranium solutions were analyzed by DIHEN-ICP-MS using a double-focusing sector field instrument with higher sensitivity than quadrupole-based ICP-MS. Flow injection was used for sample introduction to measure small sample volumes of radioactive waste solutions (20 µl). The determination of 237 Np at a concentration of 10 ng l –1 by flow injection DF-ICP-MS was possible with a precision of 2.0% (RSD, n=5). In order to avoid mass spectral interferences and matrix effects long-lived radionuclides (e.g., of U, Th and 99 Tc) were separated from the radioactive waste matrix by liquid-liquid extraction or ion exchange. The methods developed for the precise determination of the concentration and isotopic ratios of long-lived radionuclides were applied to aqueous standard solutions and radioactive wastes by double-focusing sector field ICP-MS. The precision of Pu isotopic analysis by double-focusing ICP-MS with a shielded torch was 0.2, 2 and 14% for 1000, 100 and 10 pg l –1 (amount of analyte: 500, 50 and 5 fg), respectively.

Bioimaging mass spectrometry of trace elements - recent advance and applications of LA-ICP-MS: A review.

Bioimaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) offers the capability to quantify trace elements and isotopes within tissue sections with a spatial resolution ranging about 10-100 μm. Distribution analysis adds to clarifying basic questions of biomedical research and enables bioaccumulation and bioavailability studies for ecological and toxicological risk assessment in humans, animals and plants. Major application fields of mass spectrometry imaging (MSI) and metallomics have been in brain and cancer research, animal model validation, drug development and plant science. Here we give an overview of latest achievements in methods and applications. Recent improvements in ablation systems, operation and cell design enabled progressively better spatial resolutions down to 1 μm. Meanwhile, a body of research has accumulated covering basic principles of the element architecture in animals and plants that could consistently be reproduced by several laboratories such as the distribution of Fe, Cu, Zn in rodent brain. Several studies investigated the distribution and delivery of metallo-drugs in animals. Hyper-accumulating plants and pollution indicator organisms have been the key topics in environmental science. Increasingly, larger series of samples are analyzed, may it be in the frame of comparisons between intervention and control groups, of time kinetics or of three-dimensional atlas approaches.

Inorganic mass spectrometric methods for trace, ultratrace, isotope, and surface analysis

Abstract Inorganic mass spectrometric methods are widely used for multielemental determination at the trace and ultratrace level for isotope ratio measurements and surface analysis (depth profiling, imaging) in quite different materials (e.g. conducting, semiconducting, and nonconducting solid samples; technical, environmental, biological, geological, and water samples). The capability of spark source mass spectrometry (SSMS), laser ionization mass spectrometry (LIMS), glow discharge mass spectrometry (GDMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), secondary ion mass spectrometry (SIMS), sputtered neutral mass spectrometry (SNMS), and inductively coupled plasma mass spectrometry (ICP-MS) have been applied as the most important mass spectrometric techniques with their multielemental capability for the characterization of solid and aqueous samples. The detection limits for the direct analysis of solid samples by inorganic solid mass spectrometry were determined up to sub-ng g−1 and for aqueous solutions by ICP-MS up to sub-pg L−1. This article discusses the most important inorganic mass spectrometric techniques and their application for quantitative determination of trace element, isotope ratio measurements, and in-surface analysis.

Imaging of nutrient elements in the leaves of Elsholtzia splendens by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of nutrient elements (such as K, Mg, Mn, Cu, P, S and B) in the leaves of Elsholtzia splendens. The plant leaves were scanned directly with a focused Nd:YAG laser in the laser ablation chamber. The ablated material was transported with argon as carrier gas to a quadrupole-based ICP-MS (ICP-QMS), and the ion intensities of (39)K(+), (24)Mg(+), (55)Mn(+), (63)Cu(+), (31)P(+), (34)S(+) and (11)B(+) were measured by ICP-QMS to study the distribution of the elements of interest. The imaging technique using LA-ICP-MS on plant leaves does not require any sample preparation. Carbon ((13)C(+)) was used as an internal standard element to compensate for the difference in the amount of material ablated. Additional experiments were performed in order to study the influence of the water content of the analyzed leaves on the intensity signal of the analyte. For quantification purposes, standard reference material (NIST SRM 1515 Apple Leaves) was selected and doped with standard solutions of the analytes within the concentration range of 0.1-2000 mg L(-1). The synthetic laboratory standards together with the samples were measured by LA-ICP-MS. The shape and structure of the leaves was clearly given by LA-ICP-MS imaging of all the elements measured. The elemental distribution varied according to the element, but with a high content in the veins for all the elements investigated. Specifically, Cu was located uniformly in the mesophyll with a slightly higher concentration in the main vein. High ion intensity was measured for S with a high amount of this element in the veins similar to the images of the metals, whereas most of the B was detected at the tip of the leaf. With synthetic laboratory standard calibration, the concentrations of elements in the leaves measured by LA-ICP-MS were between 20 microg g(-1) for Cu and 14,000 microg g(-1) for K.

Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinsons disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.