Laser Ablation Inductively Coupled Plasma Mass Spectrometry: An Emerging Technology for Multiparametric Analysis of Tissue Antigens.
ABSTRACT: New analytical techniques for multiparametric characterisation of individual cells are likely to reveal important information about the heterogeneity of immunological responses at the single-cell level. In this proof-of-principle study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was applied to the problem of concurrently detecting 24 lineage and activation markers expressed by human leucocytes. This approach was sufficiently sensitive and specific to identify subpopulations of isolated T, B, and natural killer cells. Leucocyte subsets were also accurately detected within unfractionated peripheral blood mononuclear cells preparations. Accordingly, we judge LA-ICP-MS to be a suitable method for assessing expression of multiple tissue antigens in solid-phase biological specimens, such as tissue sections, cytospins, or cells grown on slides. These results augur well for future development of LA-ICP-MS-based bioimaging instruments for general users.
Project description:Imaging the size distribution of metal nanoparticles (NPs) in a tissue has important implications in terms of evaluating NP toxicity. Microscopy techniques used to image tissue NPs are limited by complicated sample preparation or poor resolution. In this study, we developed a laser ablation (LA) system coupled to single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) for quantitative imaging of gold (G)NPs in tissue samples. In this system, GNPs were ablated but did not disintegrate and integrate under optimised operation conditions, which were verified by characterising LA particles by scanning electron microscopy. The feasibility of imaging size distributions in tissue was validated using reference GNPs 60 and 80?nm in size on matrix-matched kidney. A transport efficiency of 6.07% was obtained by LA-SP-ICP-MS under optimal conditions. We used this system to image 80-nm GNPs in mouse liver and the size distribution thus obtained was in accordance with that determined by nebuliser SP-ICP-MS. The images revealed that 80-nm GNPs mainly accumulate in the liver and did not obviously aggregate. Our results demonstrate that LA-SP-ICP-MS is an effective tool for evaluating the size distribution of metal NPs in tissue.
Project description:Functionalized gold nanoparticles (AuNPs) have unique properties that make them important biomedical materials. Optimal use of these materials, though, requires an understanding of their fate in vivo. Here we describe the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to image the biodistributions of AuNPs in tissues from mice intravenously injected with AuNPs. We demonstrate for the first time that the distributions of very small (?2 nm core) monolayer-protected AuNPs can be imaged in animal tissues at concentrations in the low parts-per-billion range. Moreover, the LA-ICP-MS images reveal that the monolayer coatings on the injected AuNPs influence their distributions, suggesting that the AuNPs remain intact in vivo and their surface chemistry influences how they interact with different organs. We also demonstrate that quantitative images of the AuNPs can be generated when the appropriate tissue homogenates are chosen for matrix matching. Overall, these results demonstrate the utility of LA-ICP-MS for tracking the fate of biomedically-relevant AuNPs in vivo, facilitating the design of improved AuNP-based therapeutics.
Project description:In order to quantify and compare the uptake of aluminum oxide nanoparticles of three different sizes into two human cell lines (skin keratinocytes (HaCaT) and lung epithelial cells (A549)), three analytical methods were applied: digestion followed by nebulization inductively coupled plasma mass spectrometry (neb-ICP-MS), direct laser ablation ICP-MS (LA-ICP-MS), and flow cytometry. Light and electron microscopy revealed an accumulation and agglomeration of all particle types within the cell cytoplasm, whereas no particles were detected in the cell nuclei. The internalized Al2O3 particles exerted no toxicity in the two cell lines after 24 h of exposure. The smallest particles with a primary particle size (x BET) of 14 nm (Alu1) showed the lowest sedimentation velocity within the cell culture media, but were calculated to have settled completely after 20 h. Alu2 (x BET = 111 nm) and Alu3 (x BET = 750 nm) were calculated to reach the cell surface after 7 h and 3 min, respectively. The internal concentrations determined with the different methods lay in a comparable range of 2-8 µg Al2O3/cm(2) cell layer, indicating the suitability of all methods to quantify the nanoparticle uptake. Nevertheless, particle size limitations of analytical methods using optical devices were demonstrated for LA-ICP-MS and flow cytometry. Furthermore, the consideration and comparison of particle properties as parameters for particle internalization revealed the particle size and the exposure concentration as determining factors for particle uptake.
Project description:Metals have a number of important roles within the brain. We used laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to map the three-dimensional concentrations and distributions of transition metals, in particular iron (Fe), copper (Cu) and zinc (Zn) within the murine brain. LA-ICP-MS is one of the leading analytical tools for measuring metals in tissue samples. Here, we present a complete data reduction protocol for measuring metals in biological samples, including the application of a pyramidal voxel registration technique to reproducibly align tissue sections. We used gold (Au) nanoparticle and ytterbium (Yb)-tagged tyrosine hydroxylase antibodies to assess the co-localisation of Fe and dopamine throughout the entire mouse brain. We also examined the natural clustering of metal concentrations within the murine brain to elucidate areas of similar composition. This clustering technique uses a mathematical approach to identify multiple 'elemental clusters', avoiding user bias and showing that metal composition follows a hierarchical organisation of neuroanatomical structures. This work provides new insight into the distinct compartmentalisation of metals in the brain, and presents new avenues of exploration with regard to region-specific, metal-associated neurodegeneration observed in several chronic neurodegenerative diseases.
Project description:This paper reports LA-ICP-MS 87Sr/86Sr isotopic data collected from archaeological human remains uncovered in Manzherok region, Altai Republic, Russian Federation ("Mobility of nomads in central Asia: chronology and 87Sr/86Sr isotope evidence from the Pazyryk barrows of northern Altai, Russia" . The skeletal remains derive from Scythian barrows dated to 6th - 3rd century BC located at Chultukov Log 1 cemetery. The Chultukov Log cemetery, located approximately 470km south of Novosibirsk, is considered the biggest nomadic burial ground in the Upper Altai and the Sayan Mountains. To enrich the information on prehistoric mobility of ancient nomadic populations in Central Asia, strontium isotopic data were collected using a Nu plasma (II) MC-ICP-MS equipped with ESI NWR193-based laser ablation system from premolar teeth of 8 adult individuals (4 males and 4 females), associated mainly with the Pazyryk culture. Additionally, we report bioavailable strontium data from single Equus caballus specimen (found at Chultukov Log 9 settlement) from Manzherok territory. In this study we have successfully applied and tested new in-depth decontamination protocol for total (<95%) removal of contaminants, necrotic tissue and dental calculus in archaeological materials based on a clinical irrigation procedure with NaOCl and EDTA. Strontium LA-ICP-MS 87Sr/86Sr isotopic data presented in this paper were obtained from prehistoric human teeth previously decontaminated according to this method. These data will provide valuable resources for isotopic analyses of prehistoric transportation systems in Central Asia, including residential mobility of ancient nomads inhabiting steppe zone, Mongolia and NW China.
Project description:The selection of drug candidates for entering clinical development relies on in vivo testing in (solid) tumor animal models. However, the heterogeneity of tumor tissue (e.g. in terms of drug uptake or tissue composition) is rarely considered when testing novel drug candidates. Therefore, we used the murine colon cancer CT-26 tumor model to study the spatially-resolved drug distribution in tumor tissue upon repetitive treatment of animals over two weeks with three investigational platinum(IV)-based anticancer agents, oxaliplatin or satraplatin. A quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method revealed a heterogeneous platinum distribution, which correlated well with the histologic features of the tumor and surrounding tissue at the microscopic level. In most of the cases, higher amounts of intratumoral platinum were found in the surrounding tissue than in the malignant parts of the sample. This indicates that determination of average platinum amounts (e.g. by microwave-assisted digestion of the sample followed by analysis with ICP-MS) might overestimate the drug uptake in tumor tissue causing misleading conclusions. In addition, we studied the platinum distribution in the kidneys of treated animals to probe if accumulation in the cortex and medulla predict potential nephrotoxicity. A 10-fold increase of platinum in the cortex of the kidney over the medulla was observed for oxaliplatin and satraplatin. Although these findings are similar to those in the platinum distribution of the nephrotoxic anticancer drug cisplatin, treatment with the compounds of our study did not show signs of nephrotoxicity in clinical use or clinical trials (oxaliplatin, satraplatin) and did not result in the alteration of renal structures. Thus, predicting the side effects based on bioimaging data by LA-ICP-MS should be considered with caution. To the best of our knowledge, this is the first LA-ICP-MS study on spatially-resolved platinum accumulation in tissues after repetitive platinum-based anticancer drug treatment of mice bearing a preclinical tumor model.
Project description:Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics.A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5).Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging.Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods.
Project description:Wilson disease is a rare inherited autosomal recessive disorder. As a consequence of genetic alterations in the ATP7B gene, copper begins to accumulate in the body, particularly in the liver and brain. Affected persons are prone to develop liver cancer and severe psychiatric and neurological symptoms. Clinically, the development of corneal Kayser-Fleischer rings and low ceruloplasmin concentrations (<20 mg/dL) are indicative of Wilson disease. However, the detection of elevated hepatic copper content (>250 µg/g dry weight) alone is still considered as the best but not exclusive diagnostic test for Wilson disease. Presently, specific copper stains (e.g., rhodanine) or indirect staining for copper-associated proteins (e.g., orcein) are widely used to histochemically visualize hepatic copper deposits. However, these procedures only detect lysosomal copper, while cytosolic copper is not detectable. Similarly, elemental analysis in scanning electron microscope with energy dispersive X-ray analysis (EDX) often leads to false negative results and inconsistencies. Here, we tested the diagnostic potential of laser ablation inductively-coupled mass spectrometry (LA-ICP-MS) that allows quantitative analysis of multiple elements. Comparative studies were performed in wild type and the Atp7b null mouse model. We propose LA-ICP-MS as a versatile and powerful method for the accurate determination of hepatic copper in people with Wilson disease with high spatial resolution.
Project description:In this study, for a first time (according to our knowledge), we couple the methodologies of chlorophyll fluorescence imaging analysis (CF-IA) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), in order to investigate the effects of cadmium (Cd) accumulation on photosystem II (PSII) photochemistry. We used as plant material Salvia sclarea that grew hydroponically with or without (control) 100 μM Cd for five days. The spatial heterogeneity of a decreased effective quantum yield of electron transport (ΦPSΙΙ) that was observed after exposure to Cd was linked to the spatial pattern of high Cd accumulation. However, the high increase of non-photochemical quenching (NPQ), at the leaf part with the high Cd accumulation, resulted in the decrease of the quantum yield of non-regulated energy loss (ΦNO) even more than that of control leaves. Thus, S. sclarea leaves exposed to 100 μM Cd exhibited lower reactive oxygen species (ROS) production as singlet oxygen (1O2). In addition, the increased photoprotective heat dissipation (NPQ) in the whole leaf under Cd exposure was sufficient enough to retain the same fraction of open reaction centers (qp) with control leaves. Our results demonstrated that CF-IA and LA-ICP-MS could be successfully combined to monitor heavy metal effects and plant tolerance mechanisms.
Project description:BACKGROUND AND AIMS: Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult. METHODS: In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers. RESULTS: Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples. CONCLUSIONS: Biometal imaging via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.