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Mass spectrometry imaging (MSI) is a powerful tool for mapping the spatial distribution and relative abundance of molecules across a sample surface. The distribution of proteins, metabolites, lipids and drugs can be determined and unlike other molecular imaging techniques, such as immunohistochemistry (IHC), MSI is completely label-free. Therefore, this technique does not require prior knowledge of the molecule to be imaged and thousands of molecules can be imaged at once. This is particularly useful for untargeted imaging, to discover molecules which are important for a certain condition. For example, a healthy tissue sample can be compared to a diseased sample in the same imaging run to help understand the molecules involved in the disease process. MSI has become a popular technique in fields such as neuroscience, drug distribution studies and as a biomarker discovery tool in cancer. However, the use of MSI in microbiology has to date been limited. In the present study MSI was used to investigate molecular host-microbe interactions of both beneficial and pathogenic bacteria of the gastrointestinal tract. In the initial part of this thesis, MSI was employed to discover molecular changes in the host, caused by Salmonella enterica serovar Typhimurium infection. S. Typhimurium is a Gram-negative facultative intracellular bacterium and is a leading cause of food-borne infection worldwide in humans. The symptoms include abdominal cramps and diarrhoea, and although this infection is usually self-limiting with individuals recovering without the requirement for treatment, it can be more serious in young, old, malnourished, or immunocompromised people. S. Typhimurium is transmitted by ingestion of contaminated food or water. The bacteria infect cells of the gastrointestinal tract, causing inflammation, and cross the epithelial layer of the gut to enter underlying specialised immune tissue, the Peyer’s patches. S. Typhimurium can be taken up by immune cells, but can survive within these cells and are transported to another specialised immune location, the mesenteric lymph nodes (MLNs). In the present study, an S. Typhimurium infected, gastroenteritis mouse model was used to investigate host-pathogen interaction. Various tissue types were collected from 72 h infected, 48 h infected and uninfected mice including colon, Peyer’s patches and MLN. IHC staining was used to locate tissue types and regions where S. Typhimurium were present and MSI was employed to find molecular changes caused by the infection. This preliminary analysis highlighted 73 molecules that differed in abundance or distribution between infected and uninfected samples, across all three tissue types. These molecules could be investigated further in future, however, subsequent analysis focused on one molecule, which was identified as palmitoylcarnitine, a molecule involved in fatty acid metabolism. This molecule was present at high abundance in areas of the MLNs 72 h post infection, where both bacteria and infection induced tissue damage were present. This molecule was also present in uninfected samples and areas of infected tissue where no bacteria were present at lower levels, therefore this molecule was deemed to be host derived. It was hypothesised that this molecule could either be; produced by the immune system to directly damage S. Typhimurium, be produced by the immune system to enhance the immune response, or be a by-product of tissue damage and could further damage host cells. Therefore, subsequent analysis focused on testing the effects of palmitoylcarnitine to investigate these hypotheses. No effects were found when testing this molecule on bacterial growth or virulence. Palmitoylcarnitine localised to areas of immune cell, T cell, B cell and macrophage, disruption in the MLNs. Cells from MLNs were isolated and cultured in the presence of palmitoylcarnitine to investigate any effects of this molecule on immune cell death or activation. Palmitoylcarnitine was found to cause cell death by apoptosis of a particular subset of immune cells, CD4+CD25+ T cells. These immune cells are mostly likely regulatory T cells, which protect the host against excess damage during an immune response. Therefore, the overall hypothesis was S. Typhimurium infection could be disrupting fatty acid metabolism, leading to accumulation of palmitoylcarnitine. This in turn causes death of CD4+CD25+ T cells, which could be responsible for causing the excess tissue damage found in the MLNs during infection. The second part of this thesis employed MSI to investigate the interaction between the beneficial bacteria of the gastrointestinal tract, the microbiota, and the host brain. An altered microbiota has recently been linked to diseases throughout the body and differences in the microbiota have been found in patients with neurological disorders, such as autism, Parkinson’s disease and depression. There is still little known about the links between the brain and microbiota and how the microbiota may be influencing the brain. In the present study the colons and brains of mice with absent or depleted microbiota were compared to conventionally colonized mice to investigate possible links between the gut and the brain. MSI was demonstrated to be an effective technique to image known molecules, as well as previously unknown molecules which changed between microbiota depleted and conventionally colonized mice. A previously unknown microbiota derived molecule was chosen for further identification and analysis. This study demonstrates the capabilities of MSI as a discovery tool to find molecules important for host-microbe interactions. This study also aids in advancing the use of this technique in the field of microbiology, which would be highly beneficial in future to help understand immune evasion strategies of pathogenic bacteria and how the microbiota is interacting and crucial to the host.