An integrated mass spectrometry imaging and digital pathology workflow for objective detection of colorectal tumours by unique atomic signatures.

Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS).

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Determination of selenium in serum in the presence of gadolinium with ICP-QQQ-MS.

Gadolinium (Gd)-based magnetic resonance imaging (MRI) contrasting agents interfere with the determination of selenium (Se) when analysed by single quadrupole inductively coupled plasma-mass spectrometry (ICP-MS). This paper demonstrates that an ICP-triple quadrupole-MS (ICP-QQQ-MS) with oxygen mass shift overcomes Gd(++) interference on Se(+) and mitigates typically encountered matrix and spectral based interferences. Normal human serum was diluted in a solution containing isopropanol, EDTA, NH4OH and Triton X-100. Samples were unspiked (control) serum; serum spiked with 0.127 µmol L(-1) Se or 127 µmol L(-1) Gd; and serum spiked with both 0.127 µmol L(-1) Se and 127 µmol L(-1) Gd. Consideration of collision/reaction gases and conditions for interference mitigation included helium (He); a 'low' and 'high' hydrogen (H2) flow, and oxygen (O2). The instrument tune for O2 was optimised for effective elimination of interferences via a mass shift reaction of Se(+) to SeO(+). The ICP-QQQ-MS was capable of detecting trace (>9.34 nmol L(-1)) levels of Se in serum in the presence of Gd in our simulated post-MRI serum sample. The multi-tune capabilities of the ICP-QQQ-MS may be adapted to eliminate other specific isobaric interferences that cause false positive results in other analyses where the analyte is confounded by doubly charged and/or polyatomic species.

High-resolution elemental bioimaging of Ca, Mn, Fe, Co, Cu, and Zn employing LA-ICP-MS and hydrogen reaction gas.

Imaging of trace metal distribution in tissue sections by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is typically performed using spatial resolutions of 30 µm(2) and above. Higher resolution imaging is desirable for many biological applications in order to approach the dimensions of a single cell. The limiting factor for increasing resolution is sensitivity, where signal-to-noise ratios are poor due to inherent background spectral interferences and reduced sample volume with decreasing laser beam diameter. Several prominent spectral interferences are present for a number of biologically relevant isotopes, including the (40)Ar(16)O(+) spectral interference on (56)Fe(+). We examined if H(2) as a reaction gas could improve the analytical performance of imaging experiments for a range of masses with spectral interferences. At low (<1 mL min(-1)) H(2) flow rates, greater spectral interference due to H(+) adducts was observed for (55)Mn, (57)Fe, and (59)Co. At higher flow rates of up to 3 mL H(2) per minute, the spectral interferences were reduced leading to improvement in limits of analysis for masses with O- and N-based polyatomic interferences. Enhanced sensitivity with the reaction cell allowed construction of high resolution (6 µm(2)) imaging of (56)Fe in the mouse brain that approached the dimensions of single cells.

Elemental bio-imaging of thorium, uranium, and plutonium in tissues from occupationally exposed former nuclear workers.

Internal exposure from naturally occurring radionuclides (including the inhaled long-lived actinides (232)Th and (238)U) is a component of the ubiquitous background radiation dose (National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States; NCRP Report No. 160; NCRP: Bethesda, MD, 2009). It is of interest to compare the concentration distribution of these natural alpha-emitters in the lungs and respiratory lymph nodes with those resulting from occupational exposure, including exposure to anthropogenic plutonium and depleted and enriched uranium. This study examines the application of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) to quantifying and visualizing the mass distribution of uranium and thorium isotopes from both occupational and natural background exposure in human respiratory tissues and, for the first time, extends this application to the direct imaging of plutonium isotopes. Sections of lymphatic and lung tissues taken from deceased former nuclear workers with a known history of occupational exposure to specific actinide elements (uranium, plutonium, or americium) were analyzed by LA-ICPMS. Using a previously developed LA-ICPMS protocol for elemental bio-imaging of trace elements in human tissue and a new software tool, we generated images of thorium ((232)Th), uranium ((235)U and (238)U), and plutonium ((239)Pu and (240)Pu) mass distributions in sections of tissue. We used a laboratory-produced matrix-matched standard to quantify the (232)Th, (235)U, and (238)U concentrations. The plutonium isotopes (239)Pu and (240)Pu were detected by LA-ICPMS in 65 mum diameter localized regions of both a paratracheal lymph node and a sample of lung tissue from a person who was occupationally exposed to refractory plutonium (plutonium dioxide). The average (overall) (239)Pu concentration in the lymph node was 39.2 ng/g, measured by high purity germanium (HPGe) gamma-spectrometry (Lynch, T. P.; Tolmachev, S. Y.; James, A. C. Radiat. Prot. Dosim. 2009, 134, 94-101). Localized mass concentrations of thorium ((232)Th) and uranium ((238)U) in lymph node tissue from a person not occupationally exposed to these elements (chronic natural background inhalation exposure) ranged up to 400 and 375 ng/g, respectively. In lung samples of occupationally nonexposed to thorium and uranium workers, (232)Th and (238)U concentrations ranged up to 200 and 170 ng/g, respectively. In a person occupationally exposed to air-oxidized uranium metal (Adley, F. E.; Gill, W. E.; Scott, R. H. Study of atmospheric contaminiation in the melt plant buiding. HW-23352(Rev.); United States Atomic Energy Commission: Oakridge, TN, 1952, p 1-97), the maximum (235)U and (238)U isotopic mass concentrations in a lymph node, measured at higher resolution (with a 30 mum laser spot diameter), were 70 and 8500 ng/g, respectively. The ratio of these simultaneously measured mass concentrations signifies natural uranium. The current technique was not sufficiently sensitive, even with a 65 mum laser spot diameter, to detect (241)Am (at an overall tissue concentration of 0.024 ng/g, i.e., 3 Bq/g).

Elemental bio-imaging of melanoma in lymph node biopsies.

The spatial distribution of trace elements in human lymph nodes partially infiltrated by melanoma cells was determined by elemental bio-imaging. Imaging of (31)P within the nodal capsule and normal lymph node tissue showed a clear demarcation of the tumour boundary, with a significant decrease in relative (31)P concentration within the tumour. The location of the tumour boundary was confirmed by haematoxylin and eosin staining of serial sections and observation by light microscopy. Further enhancement of the tumour boundary was achieved by imaging the (31)P/(34)S ratio. (31)P/(66)Zn ratio images showed a decreasing ratio beyond the tumour boundary that extended into peritumour normal lymph node tissue.