Metals in biological systems
Living organisms use fluxes in chemical elements to control fundamental processes such as metabolism, homeostatic regulation, the cell cycle and fertilization. Up to 60 chemical elements can be detected in the human body, about 25 of which are considered to be essential. Dysregulation of essential elements underpins neurological disorders such as Alzheimer’s disease (copper, zinc, iron and aluminum), angiogenesis and tumor formation in cancer (copper), immune response during infectious disease (iron) and specific diseases such as Wilson’s and Menkes disease that are linked to copper dysregulation. Both essential and non-essential elements are potentially toxic when dysregulated. Platinum (Pt), gadolinium (Gd), ruthenium (Ru) and arsenic are used in medicine as therapeutics or imaging contrast agents.
Studying metals
Understanding fundamental processes in human health and disease relies on the ability to measure elemental concentration, distribution, and chemical binding form. Only the first of these measures is widely accessible to researchers. Measuring the concentration of an element in a biological system involves sample dehydration, homogenization, and dissolution, which provides a dry, volume-averaged value removed from a biological context. While this metric is undoubtedly useful, a deeper understanding of biological systems requires knowledge of the other parameters: where elements are within the tissue or cell and what they are bound to. Elemental distribution is arguably equally as – if not more – important than volume-averaged concentration because it can provide direct information about biological processes.
Additionally, rare elements, such as the lanthanide series, are now used as specific metal tags for antibodies that react with antigens. This technique, termed mass cytometry (21, 22), is essentially equivalent to immunohistochemistry, except that it uses mass-specific probes rather than fluorescent specific probes. The possibility of using mass cytometry in tandem with elemental imaging opens up new possibilities in the study of human diseases linked to metal dysregulation.
Elemental Imaging
Elemental imaging techniques collect quantitative and spatially resolved information on the micron to sub-micron scale from suites of approximately 10 – 20 elements, making them preferable to metal-specific stains or fluorophores, which are subject to artefacts from chemical binding form, stain permeability and overlapping fluorescence. Elemental imaging frequently provides hypothesis-generating information, but the complexity of sample preparation and quantitation have made it somewhat of a niche technique (23). Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a laboratory-based elemental imaging technique which interfaces two bench top instruments. The laser ablation system uses a neodymium yttrium-aluminum-garnet (Nd-YAG) or argon fluoride (ArF) laser to generate UV laser energy of wavelengths 266nm, 213nm or 193 nm. All of these wavelengths are suitable for elemental imaging because they effectively couple with the biological surface to generate a plume of sample particulates that are a carried in a stream of helium gas to the ICP-MS. In the ICP-MS the particles are atomized and ionized by the plasma (ICP) and the ions are extracted into the mass spectrometer (MS) where the selected analyte ions are counted. LA-ICP-MS generally achieves equivalent or better (lower) elemental detection than synchrotron X-ray fluorescence (SXRF), a powerful elemental imaging technique available at national user facilities. LA-ICP-MS operates within a spatial resolution of 4-100 µm, making it ideal for biological work.
LA-ICP-MS instrumentation
BNEIR operates an Elemental Scientific lasers NWR213 laser ablation system (Elemental Scientific Lasers, Bozeman, MT). The NWR laser is a Nd-YAG frequency quintupled with a wavelength of 213 nm. The laser can be focused with either an infinitely variable aperture (IVA) resulting in circular spot sizes from 4 µm – 110 µm in 1 µm steps or X and Y rotation aperture (XYR) which allows for square or rectangular ablation, adjustable at 1 µm in X and Y dimensions and adjustable in rotation in 1◦ increments. It has a two-volume sample cell (TV2), which has a total cell area of 10 cm X 10 cm and allows for loading four standard biological slides into the sample chamber. A small sample cup tracks the stage motion with the ablation plume being confined to this low volume sample cup. This greatly increases the signal to noise ratio of the ablation plume and decreases washout time. The NWR laser has a repetition rate of 1 – 20 Hz and would typically be operated at 10 or 20Hz (i.e. 10 – 20 laser shots per second). The NWR system is controlled by ActiveView2 software which allows scalable viewing of the sample chamber at 1 mm resolution down the low micron range at the sample level. The TEAC has also acquired a dual concentric injector (DCI) optional accessory that can be used, along with the TV2 sample chamber design, to rapidly transport the laser sample plume such that it arrives at the ICP-MS in discrete ’packets’ resulting from a single laser shot. Operating in this mode with the DCI allows for the fastest image generation moving the sampling stage 20 times a second and collecting ICP-MS data on each laser shot. The drawback to the DCI, when using a quadrupole ICP-MS, is that the user can only measure ≤ 3 isotopes in the limited time window (< 50 msec) of each laser pulse.
VIDEO LINKS about ICP-MS
https://www.labtube.tv/video/MTA3NjMz
The TEAC possesses two quadrupole ICP-MS systems, an Agilent 7900 and an Agilent 8900 (Agilent Technologies, Wilmington, DE). The Agilent 7900 can be operated in normal, helium and hydrogen modes. Normal mode provides the highest sensitivity but lowest protection from mass spectral (polyatomic) interferences. Helium mode provides general (across the mass range) protection from interferences through collisional kinetic energy discrimination (KED) of analyte from polyatomics but causes some analyte signal suppression. Hydrogen mode can be useful because hydrogen can react specifically with some argon-based polyatomics (ArO on 56Fe, ArAr on 78Se and 80Se) without causing as much signal suppression of the analyte ion as He mode. The 8900 ICP-MS is a triple quadrupole instrument with the cell placed between the two quadrupoles. The control of analyte masses into the cell through quadrupole 1 significantly enhances the utility of the reaction cell approach because, by eliminating all masses except the analyte mass into the cell, it eliminates the possibility of forming new interfering species with very reactive gases used in the cell (O2 and NH3). The 8900 ICP-MS has excellent sensitivity for phosphorus (P) (m/z 31) and sulfur (S) (m/z 32 and 34), which by single quadrupole ICP-MS have major polyatomic interferences of NO (m/z 31) and O2 (m/z 32 and 34). By using O2 as a reaction gas, both P and S are quantitatively mass shifted in the cell to PO and SO species, and by setting quadrupole 2 to these new m/z values (47, 48, 50) then P and S can be measured with excellent sensitivity due to the much reduced background at their mass shifted m/z. In most cases the combination of the LA unit interfaced with the 7900 ICP-MS is perfectly sufficient for elemental imaging and this is the default LA-ICP-MS set up at the TEAC. On the rare occasions where the required analyte has a significant polyatomic interferent which is better suited by using the 8900 ICP-MS, this can be easily achieved as the LA system is on a laboratory cart and can be moved between the two ICP-MS instruments. Both ICP-MS instruments utilize the same Mass Hunter software so the resulting data output will be essentially the same whether a user is using the 7900 or 8900 ICP-MS. It should be noted that, because of the time constraints of LA-ICP-MS imaging, the ICP-MS can only be operated in one mode, i.e. either no gas or He or a reaction gas. It takes too much time to switch between gas modes for it to be a useful strategy for time resolved analysis such as laser ablation. Selection of which gas mode to use and which isotopes to select is just one area where working with an experienced team in a user facility can help a novice user get the correct data in their limited user time.
Quadrupole ICP-MS instruments (by far the most common commercial systems) are very robust and modern quadrupoles have excellent sensitivity. The market dominance of quadrupole ICP-MS instruments also ensures comprehensive industry support and software for the coupling of LA systems to quadrupole ICP-MS systems. The advent of reaction cell technology has enabled quadrupole instruments to achieve excellent (µg/kg) sensitivity even for ‘problem’ analytes with common polyatomic interferents. For all these reasons Q-ICP-MS systems are an excellent choice as the detection system for LA-ICP-MS, however, the effect of using a quadrupole as the mass filter and its limitation on the number of analytes that can be measured, and speed of mapping should be noted. Quadrupole mass spectrometers measure each analyte mass sequentially, albeit with very quick scanning, rather than truly simultaneously, and this places some restrictions on the number of analytes that can be included in any elemental imaging experiment and limits the speed of image generation. For most elemental imaging of multiple analytes, data collection is limited to 2– 5 pixels per second unless ≤3 analytes are requested in which case faster scanning is possible using short dwell times per analyte and collecting one pixel per laser shot. With our current laser system, the maximum frequency is 20 laser shots per second and so 20 pixels per second. Even at this relatively modest laser firing frequency, each laser pulse peak is ca. 30 milliseconds wide and all analyte data needs to be collected across that peak at multiple points, with ca 5 data points per analyte per peak to fully integrate the peak for each analyte. Either undersampling or oversampling can cause image aberrations, especially with fast washout cells (24-26) and this is another rationale for working with an experienced team in a user facility so that the user can be advised on the optimum settings to avoid these issues.