Imaging mass spectrometry for in situ lipidomics: from cell structures to cardiac tissue

Doctoral thesis, 2018

Imaging of cells and tissues is important for studying different processes within biological systems due to the spatial information provided for different molecular species during imaging. One powerful imaging technique is mass spectrometry imaging (MSI). It is a label free technique that provides chemical information of a sample at the same time as it allows for imaging at high spatial resolution. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) uses a focused primary ion beam to ablate and ionise molecules from the top layers of the sample surface which makes it a very surface sensitive technique. Recent developments in high energy gas cluster ion beam (GCIB) technology for ToF-SIMS has greatly improved the imaging of higher mass species, such as intact lipids. Lipids are important molecules found in all living organisms. They are used as building blocks for cells and are involved in a variety of important cellular processes such as energy storage and acting as important mediators in many signalling pathways, making them an interesting target for imaging studies. In this thesis, the ToF-SIMS imaging technique has been applied to both tissues and cells in order to perform in situ lipidomics analysis of various samples. Development of sample treatment methods that provides easier data interpretation and other method development for improving secondary ion yields have also been implemented in this work. In paper I, enhancement of negative secondary ion yields was induced by a combination of ion bombardment using a GCIB with simultaneous caesium flooding, for both inorganic and organic substrates. In paper II, ToF-SIMS imaging with a GCIB was used together with LC-MS to elucidate changes in lipid composition 6 hours after an induced myocardial infarction in mouse heart. The spatial information from the MSI allowed correlation of specific lipid species to infarcted and non-infarcted regions of the heart. Localised lipid accumulation was discovered in specific regions of the heart. In paper IV, these lipid changes were tracked over longer periods of time, 24 hours and 48 hours after infarction, and progression of the infarcted area was observed. In paper III, a simple method was developed in order to aid interpretation of the complex mass spectra collected from ToF-SIMS experiments of complex tissue sample such as heart tissue. Salt adduct formation was demonstrated as a means to discriminate between diacylglyceride and triacylglyceride, which are usually impossible to distinguish during ToF-SIMS analysis. In paper V, lipid changes in PC12 cell membranes were analysed after incubation with the essential fatty acids, omega-3 and omega-6. Using deuterium labelled fatty acids made it possible to track incorporation into phospholipids as well as the relative amount of each.