Correlative imaging of trace elements and intact molecular species in a single-tissue sample at the 50 μm scale

Elemental and molecular imaging play a crucial role in understanding disease pathogenesis. To accurately correlate elemental and molecular markers, it is desirable to perform sequential elemental and molecular imaging on a single-tissue section. However, very little is known about the impact of performing these measurements in sequence. In this work, we highlight some of the challenges and successes associated with performing elemental mapping in sequence with mass spectrometry imaging. Specifically, the feasibility of molecular mapping using the mass spectrometry imaging (MSI) techniques matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) in sequence with the elemental mapping technique particle-induced X-ray emission (PIXE) is explored. Challenges for integration include substrate compatibility, as well as delocalization and spectral changes. We demonstrate that while sequential imaging comes with some compromises, sequential DESI-PIXE imaging is sufficient to correlate sulfur, iron, and lipid markers in a single tissue section at the 50 μm scale

Investigating the Feasibility of Multimodal Imaging using Ion Beam Analysis and Mass Spectrometry Imaging Techniques

This thesis investigates the possibility of using ion beam analysis (IBA) with mass spectrometry imaging (MSI) techniques for multimodal elemental and molecular imaging on single sample. This was found to be possible through the careful consideration of suitable substrates to mount the samples which would compensate for the different requirements with using IBA and MSI techniques. Appropriate workflows were also investigated to determine the effects of one analysis to the corresponding information obtained from the subsequent analysis.In this thesis, three techniques were used. The elemental imaging tool was particle induced X-ray emission (PIXE) which is a widely used IBA technique for mapping elements. The two MSI techniques tested here were matrix assisted laser desorption ionisation (MALDI) and desorption electrospray ionisation (DESI).Suitable substrates to mount tissue homogenates were investigated for the compatibility with IBA and MSI techniques. Investigated substrates included standard glass slides (used for MSI), polyethylene (PET) films, carbon film and highly ordered pyrolytic graphite (HOPG). For PIXE, the PET substrate was the most compatible due to the thin nature of the film (4 µm) and had low elemental impurities compared with the other investigated substrates. PET was also found to be compatible with DESI, where the intensity of molecular endogenous compounds and maps were comparable to standard glass slides. For MALDI analysis, the carbon foil and HOPG were more suited. PET was not compatible with the laser, causing holes to form in the substrate.Using the chosen candidate substrates for each technique, appropriate workflows for IBA with MSI on a single sample were investigated. Two workflows were adopted: ‘Workflow 1’ where the MSI technique (DESI or MALDI) was carried out prior to IBA, the other was ‘Workflow 2’ where IBA irradiations were carried out prior to the MSI technique. For Workflow 1, redistribution of elements in the tissue homogenate due to the MSI analysis was explored. It was found that whilst MALDI does not appear to have an adverse impact on trace element distribution, DESI redistributed Cl and K, but not Fe. This work explored whether prior PIXE analysis (involving 2.5 MeV proton beam irradiation (at low, medium and high fluence)) caused changes to the observed molecular compounds. The results presented here show irradiating the sample at medium and high fluence with the ion beam leads to some compounds (such as lipids) to change in intensity. It was found that beam damage can be reduced by application of a matrix prior to ion beam analysis. Finally, it was found that ion beam induced chemical changes are also less pronounced on the carbon foil substrate. This thesis concludes that this is due to the higher thermal conductivity of the substrate, and therefore that beam heating plays a role in the observed damage to tissue

Correlative Imaging of Trace Elements and Intact Molecular Species in a Single Tissue Sample at the 50 Micron Scale

Elemental and molecular imaging play a crucial role in understanding disease pathogenesis. To accurately correlate elemental and molecular markers, it is desirable to perform sequential elemental and molecular imaging on a single tissue section. However, very little is known about the impact of performing these measurements in sequence. In this work, we highlight some of the challenges and successes associated with performing elemental mapping in sequence with mass spectrometry imaging. Specifically, the feasibility of molecular mapping using the mass spectrometry imaging (MSI) techniques matrix assisted laser desorption ionisation (MALDI) and desorption electrospray ionisation (DESI) in sequence with the elemental mapping technique particle induced X-ray emission (PIXE) is explored. Challenges for integration include substrate compatibility, as well as delocalisation and spectral changes. We demonstrate that whilst sequential imaging comes with some compromises, sequential DESI-PIXE imaging is sufficient to correlate sulphur, iron and lipid markers in a single tissue section at the 50-micrometre scale