Applications of microscope mode imaging mass spectrometry

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Modern in vitro diagnostics lack the precision to rapidly identify the presence or absence of proteins at low concentrations. Improvements in their sensitivity and throughput will enable biomolecules to be quantified, and lead to more efficient protein recognition. The proposed research addresses these goals using a novel ion microscopy technique, microscope mass spectrometry imaging (MSI), to capture quantitative and mass-resolved chemical snapshots of complex surfaces. Microscope MSI differs from conventional microprobe MSI in the use of a defocused laser or ion beam to generate ions from large surface areas (~1 cm2) rather than from a single point. Using a specific electric field, the resulting ions are separated by their m/z, and electrostatically focused onto a two-dimensional detector array. The result is a series of ion images, one for each m/z. This multiplexed approach has the potential to: a) rapidly identify multiple biomarkers in clinical samples during a single experimental cycle; b) improve data acquisition rates by simultaneously analysing a large surface, and c) to improve sensitivity by using imaging sensors capable of recording single ion events. The proposed research explores this potential through three objectives: first, improve the sensitivity, mass range, and spatial and mass resolution of microscope MSI by incorporating reflectron mass spectrometry and pulsed ion extraction methods into an ion microscope; second, maximize sample throughput by demonstrating the simultaneous analysis of multiple biomarkers in sample arrays; and the third will apply these improvements to the analysis of complex biological samples by implementing machine learning for marker identification. Partnering with NPL will benefit each of these goals. NPL operates the National Centre of Excellence in Mass Spectrometry Imaging (NiCE-MSI), and contains a dedicated team of researchers, led by Professor Josephine Bunch, who have demonstrated expertise in mass spectrometry instrument development as well as the application of MSI to drug discovery. To better understand the performance and limitations of microscope MSI, it will be benchmarked against conventional microprobe MALDI methods where higher throughput is achieved by continuous movement of a sample under the laser beam, thus reducing the time between pixels where no data are acquired. This comparison will enable a fuller exploration of differences in the fundamental properties of these two imaging modes. Additionally, quantitation studies will further develop the use of microprobe MSI within clinically relevant contexts. Understanding the detection limits, sensitivity and dynamic range of microscope mode MSI will provide a solid metrological foundation for this new technology. Project objectives and Milestones: 1) Coupling microscope MSI with reflectron and pulsed extraction methods 2) Developing microscope ion optics for enhanced spatial resolution or mass range 3) Simultaneous microscope MSI of sample arrays 4) Microscope MSI of protein sites within tissues using mass tags with comparison to high-throughput microprobe MSI. Development of suitable machine learning algorithms 5) Use of microscope mode for quantitative MSI and comparison to microprobe mode. The proposed research will benefit from two patented technologies developed at Oxford and previously funded by the EPSRC. The first, Pixel Imaging Mass Spectrometry (PImMS), uses an event-triggered, time-stamping image sensor to record the position and arrival time for each detected ion to a precision of 12.5 ns, and effectively allows every resolved m/z to be imaged during one experimental MSI cycle. The second technology is a fast scintillator that enhances the time resolution of typical MSI detector arrays. This project falls within the EPSRC Physical Sciences research area, under the medical imaging, sensors and instrumentation, and analytical science portfolios and is a collaboration with NPL.