Pedal to the metal

It’s elemental, after all: metals are essential to the function of many biological systems and are thought to have some interaction with at least 30 per cent of all proteins, so taking a good look at those interactions has become a thriving field of research in the last couple of years. Metallomics has joined metabolomics and proteomics as part of the wider ‘omics’ families, and many are using its techniques to study certain diseases, particularly Alzheimer’s and Parkinson’s.

Allowing researchers a close look at those metal interactions has now become a little easier, with a team from the University of Technology, Sydney, led by analytical chemist Dr Philip Doble, in association with equipment multinational Agilent Technologies, developing a new technique called elemental bioimaging to map deposits of trace metals in biological tissues.

The technique has actually been adapted and refined from a technology used in forensic science, laser ablation combined with inductively coupled plasma mass spectrometry (ICP-MS), which has been used to analyse a variety of trace evidence. In fact, Doble and his team are shortly to publish a paper on using laser ablation on steel particles generated from the illicit removal of serial numbers on firearms (see p3).

Laser ablation/ICP-MS was developed to measure solid samples without the need for aggressive sample preparation and was swiftly adopted by geochemists to analyse rocks, but now it’s being used to analyse tissue samples. Doble and his team initially became interested in the biological application when they worked with Professors John Thompson and Richard Scolyer from the Melanoma and Skin Cancer Research Institute (MASCRI) at the University of Sydney.

In that project, the technique was used to image levels of the metalloid antimony in a lymph node biopsy. Surgeons use an antimony sulphide colloid injected into a tumour to track down the sentinel lymph node if the cancer has spread.

“It’s not always possible to determine whether you have the right lymph node after you’ve injected the material, so we image these cells for antimony,” Doble says. “And sure enough, when it was the sentinel lymph node the antimony went through the roof.

“You take a slice (of the biopsy) and you image it and you can see hot spots of antimony. We thought this was very cool so we talked to our friends from Agilent and over a period of years it has developed into this concept of elemental bioimaging.”

ICP-MS is an element analyser, designed to measure trace levels of the elements in the Periodic Table unlike other forms of “organic” MS that are used to identify and quantify molecular compounds. The laser ablation is a sample introduction system for ICP-MS that allows solid materials, including tissues, to be characterised.

Solid samples are ablated by a laser and vapourised, and then transported into the ICP-MS. Doble likens the end result to a dot matrix printer, with an image built up by multiple raster lines.

The vapourised sample is carried into a very hot argon plasma that operates at temperatures close to those at the centre of the sun. As the samples pass through the plasma they decompose to their atoms and these loose an electron to produce ions. The ions are transported into the mass spectrometer that separates the ions according to their mass to charge ratio.

“It’s a mass spectrometer so you can look at the isotopes of the elements; iron-56 and iron-57, for example, down to one atomic mass unit resolution,” Doble says.

“(The sample) ions in the plasma are directed into a quadrupole, which is essentially a mass filter; for a given set of control voltages, only one mass charge ratio will be allowed to pass through; change the settings and another mass will pass through. By sweeping the control settings, the quadrupole will sequentially pass each mass of interest and allow them to strike a detector.

The laser is directed at a position on the tissue, the laser fired for a short period and the analysis undertaken in the ICP-MS. The laser is then moved to a new position and the analysis repeated before moving to a new position. Gradually as the sampling takes place, an image showing the distribution of elements built up. It’s like a dot matrix printer.”

---PB--- Beer collaboration

Doble has been working on the technology for the last five years, first developing the idea in 2003 when he met Rod Minett, Agilent’s country manager for Australia, who then introduced him to Rudi Grimm, Agilent’s global business development manager for proteomics and metabolomics.

Agilent has since donated over a million dollars worth of equipment to UTS – including an ICP-MS, a triple quadrupole liquid chromatograph/mass spec, a gas chromatograph and a lab-on-a-chip bioanalyser – and enabled the university to open a new Elemental Bio-Imaging Facility at its inner-city Sydney campus in late June.

The idea for the new technique was actually germinated over a couple of beers, Grimm said at the facility opening. “I had been thinking about this for a while but everyone thought it was another of my crazy ideas,” he said. “This is a very emotional moment because it is a dream that has come true. This technology is going to provide completely new sets of biological data.”

Doble and his team are already working with a number of collaborators around the country, including a group at the Mental Health Research Institute in Victoria. “Essentially, they are in neuro diseases – Alzheimer’s and Parkinson’s,” he says.

“In Parkinson’s, the region of the brain of most interest is the substantia nigra, particularly the substantia nigra pars compacta. That area is known to have an accumulation of iron that is correlated with the destruction of doperminergic neurons.

“They don’t know whether it is cause or effect or if there’s some other mechanism, but it seems to be association with oxidative destruction of neurons. We are providing regional information about the distribution of metal ions. What they used to do before was to take a brain punch and analyse the bulk area, and indeed they found increases in iron in particular. But we can show it in situ – it is quite remarkable because you can see the needle tracks that the lesions have made.”

The team is also preparing for a project looking at regional distribution of metals in Alzheimer’s brains, with zinc one obvious candidate. And he is working with a team from Stanford University in the US looking at the formation of calcium phosphate crystals in osteoarthritic knees.

“We are looking at hearts as well,” Doble says. “We are imaging heart from an animal model and looking at certain LIM domain proteins that are associated with zinc. They are zinc-binding proteins that are suspected of an association with heart failure. But you can apply it to anything really: it’s not restricted to tissues. One example is imaging a leaf, looking at bioremediation of heavy metals. Where do those metals go?”

Also on the agenda is marrying the laser ablation-ICP-MS technology with matrix-assisted laser desorption/ionisation (MALDI), a soft ionisation technique for delicate tissues. “The idea is to do a MALDI image and then an elemental bioimaging image and put the maps together,” Doble says. “The full implications have not been realised yet.”

---PB--- Going gunbusters

Philip Doble started out as an analytical chemist, doing his undergraduate degree at UTS, where is now a senior lecturer in the Department of Chemistry, Materials and Forensic Science. In between, he has many years of experience in industry, working with Waters Corporation in the field of chromatography and then in capillary electrophoresis (CE), and worked in a number of roles from laboratory positions to customer support and then sales.

Feeling unfulfilled in the private sector, he approached Professor Paul Haddad from the chemistry school at the University of Tasmania and inquired about PhD scholarships, one of which he took up in CE. He did a post-doc in Tasmania and then returned to UTS as a lecturer in analytical chemistry, specialising in the analytical side of separation science, and then branching into forensic science.

It was the forensics department that first purchased an ICP-MS from Agilent, where it was used for trace evidence analysis. “A classic example is glass fragments,” he says. “You find glass fragments and then the question is: does that glass fragment come from a vehicle accident, or can you match two bits up? You fire a laser at a piece of trace evidence, whether it is a piece of glass or something else that has a unique elemental profile, then you match the profile and say these are consistent or these are quite different.

“One area that is working very well is looking at firearms. One of the common things people do is obliterate the serial numbers on firearms – they file it off or pinch it or drill it out. But if you file it off there are techniques that allow you to get it back.

“It’s like etching – you etch on the surface but you can see the impression underneath. Criminals are of course aware of this so they often drill out the number. The question was then asked: is it possible to discriminate a metal, a swarf for example that has been drilled out of a gun, and match it with the gun itself to make a model. And the answer is yes, we can.

“What we did was look at a series of elements, about 10 or so, which is quite a large number to think about. So we use a technique called principal component analysis and that finds hidden variables in the data. It reduces the dimensionality to something we can deal with.

“Then you plot those variables and the ones that are similar, cluster. So we can differentiate between the makes of guns and who the manufacturer is based on this. That’s really where all this started.”