Spatial analysis of lipids in tissue samples applying mass spectrometry imaging

Lipids are important naturally occurring components in all living cellular organisms. They serve as the main building blocks of cellular membranes, participate in many signaling pathways and are also stored as an energy source. Due to the extreme complex cellular chemistry and structure of lipids, there is a real need to have a label-free technique with high chemical specificity, high accuracy and high sensitivity for study of lipids within the cell membrane. Mass spectrometry imaging (MSI) is capable of providing information on the chemical composition and spatial distribution of complex biological molecules. MSI is a powerful label-free tool for lipid analysis across biological materials. Both matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS), the two most common MSI techniques, have recently undergone many developments to improve spatial resolution and provide high sensitivity, mainly for higher mass species. These two techniques offer different capabilities in the analysis of a biological system. The main differences are that larger molecules can be ionized and detected using MALDI, whereas SIMS is capable of detecting mainly small molecules but at higher spatial resolution compared to MALDI. This thesis mainly focuses on two scopes of investigation with different sample modifications and also on the overall applicability of MSI for analysis of tissue samples. In recent years, some surface modifications have been developed to enhance the yield of intact molecular species in SIMS. One of them is matrix enhancement secondary ion mass spectrometry (ME-SIMS), which is the combination of the protocol for MALDI sample preparation and normal SIMS. In paper I, the possible mechanism of the signal enhancement in ME-SIMS was studied. Here, sublimation was used to deposit a thin layer of an organic matrix on the surface of a brain tissue slice analyzed with SIMS. In this work, I showed that sublimation could successfully provide enhancement in ion yields for a multitude of lipid species in SIMS. The mechanism of this enhancement could be due to a lower ion suppression followed by removal of the cholesterol crystals from the surface of sample allowing detection for less abundant species. It is also possible that the extraction of some specific lipids into the deposited matrix directly leads to an increase of higher mass lipid ion yield. In paper II, two different surface modifications, including matrix sublimation and nanoparticle deposition were applied on Drosophila brain samples and lipid information obtained from MALDI analysis were compared. Here, it was shown that each technique can be used in a complementary approach to detect a variety of lipid species. In paper III, SIMS imaging was employed to investigate the effect of specially processed cereals, as a specific diet on the alteration of lipid composition across the rodent intestine tissue. In paper IV, I continued the study of changes in lipid content, this time on brain samples of animals exposed to the same diet. Intake of such cereals increases active antisecretory factor (AF) in plasma, an endogenous protein with proven regulatory function on inflammation and fluid secretion. Although, the exact mechanism for the activation process of AF at the cellular level remains unclear. The results show changes in lipid content of cell membrane in response to this cereals intake suggesting a relation to activating AF. In paper V, the techniques for developing of sample preparation in SIMS imaging were investigated to improve the signal intensity of intact molecules at higher resolution