Developing methods for mass spectral imaging of environmental samples

The ability to create an image is fundamental to advancing scientific understanding. This is true at all scales - for instance, it is immediately obvious how a pollution map could be used to visualize and understand environmental threats. At the small scale as well, the ability to image individual cells and parts of cells is an essential part of modern science. There are well-established techniques to image large molecules (genes and proteins) in a biological sample, but this is much less routine for smaller things such as metabolites, metal ions, or molecules of chemical pollutants. One technique that can do this is secondary ion mass spectrometry (SIMS), in which a beam of ions (charged particles) is played across a sample. This produces secondary ions from its surface, which are collected and fed into a mass spectrometer, which sorts and counts them. The signal can then be interpreted to give you not only a detailed image of the sample, but also information about its chemical composition.

There are a number of different kinds of SIMS instrument, and one in particular, the Ion-Tof TOF-SIMS (standing for time-of-flight SIMS) has very high resolution in two different ways. Firstly, the detector (mass spectrometer) is high resolution, so that it can distinguish two different signals that a traditional instrument would lump together. Secondly, it has very high spatial resolution: it can easily create detailed images of something the size of a single cell, and resolve sub-cellular structures. This means that it has immense potential for studying the biochemistry of surfaces - for instance, a slice through a cell or tissue. It can also work just as well with non-model ecologically relevant organisms, particularly important for environmental research. However, there are many factors that could affect the potential quality of images produced by TOF-SIMS. In particular, how you prepare a sample to go into the instrument is likely to be crucial.

We propose to demonstrate the versatility and utility of TOF-SIMS for environmental research by applying it to three contrasting exemplar projects:

1. Imaging metal ions at a sub-cellular distribution in samples from woodlice. Woodlice are common terrestrial invertebrates, and their responses to pollution can tell us about the ecological health of a contaminated site. Two different woodlouse species handle the toxic metals lead and zinc very differently, and we will generate detailed maps of exactly where these metals are found within their cells. We may also be able to determine what kind of complexes these metal ions form.

2. Imaging nanoparticles in earthworms. The potential toxicity of very small particles - nanoparticles - is not at all well understood, so is a current hot research topic. It's obviously vital to be able to identify where they end up in a biological sample, but this is challenging. We will use TOF-SIMS as a complementary analysis for samples of nanoparticles in earthworms, generated from a separate project.

3. Imaging a unique earthworm metabolite. Earthworms are a common soil animal, and play a key role in maintaining soil quality. They produce a unique chemical, and no-one knows exactly what for. It's present in all earthworm species, so must play an important biological role: our best guess is that it helps them to survive desiccation when soil dries out, by helping stabilize membranes. We will produce a detailed map of where this chemical is found in earthworm cells, the first crucial step in understanding just what it does.

Thus, our project aims to provide a proof-of-principle for using TOF-SIMS in environmental research. For projects 1 and 2 above, we will be able to make a direct comparison to imaging data acquired (by a collaborating lab) at the Diamond synchrotron (a huge, complex, specialist facility): we expect the TOF-SIMS will have many advantages, such as higher-resolution images, at a fraction of the cost and complexity.

1. Future academic impact. This is a technology-led grant for optimizing and applying methods within the environmental sciences, and so much of its impact is likely to come from the future use of methods in projects in the environmental (and other) sciences. It could be used to help increase fundamental understanding in a wide range of possible scientific questions, and indeed our three exemplar projects are chosen to cover a wide range of different applications: (i) imaging metal ions, (ii) imaging nanoparticles, and (iii) imaging a small organic molecule. Hence it could be an enabling technology in many possible future NERC projects, giving it potentially very wide impact.

2. Timeliness. Imaging mass spectrometry is a rapidly developing field. SIMS is still relatively underexplored compared to MALDI for biological applications, even though it has some inherent advantages. Our application will make use of a new and highly specialized piece of equipment (TOF-SIMS/LEIS) together with an equally specialized FIB-SIMS. Both of these are found in only a small handful of university labs, and both together is even more uncommon. Funding this project would help maintain UK competitiveness in this rapidly advancing area.

3. Cost-effectiveness. One of the exciting aspects of our proposal is that we will be able directly to compare results to data obtained from the Diamond synchrotron facility. Our current application has the advantage that we will not be charged for use of the TOF-SIMS instrument (as the costs are still covered by the original grant application which secured the instrument). However, when it is eventually installed as a charge-out university facility, the planned facility charge will be £640/day. This is, of course, dramatically less than for Diamond, which is a massive national facility. The precise full economic cost (FEC) for Diamond time is difficult to estimate, but it is probably at least twenty times more expensive per day for an individual researcher than the TOF-SIMS FEC. We think it is very likely that the TOF-SIMS data will be at least as valuable as the synchrotron data (and may even be better in some respects, as they will have a higher spatial resolution). It thus offers a potential huge cost saving for UK science, if some future experiments could be carried out at a vastly reduced cost.