Methodological developments for single-cell mass spectrometry imaging

Abstract

The work presented in this doctoral thesis addresses the development of mass spectrometry imaging (MSI) methods and focuses on their application to biological samples for analysis on the single-cell level. For this purpose, a home-built MSI ion source was developed and optimized for high lateral resolution in the low micrometer range. Sample preparation had to be optimized and multiple approaches were evaluated in order to fulfill the special requirements posed by high-lateral-resolution MSI at the single-cell level. In a first project, an approach alternative to the widely employed matrix-assisted laser desorption/ionization (MALDI) MSI was evaluated, involving a matrix-free sample preparation method using nanostructured membranes, so-called DIUTHAME (desorption ionization using through-hole alumina membrane). With this method, the MS image quality regarding contrast and signal homogeneity could be improved compared to MALDI MSI while employing a simpler and faster sample preparation workflow. DIUTHAME membranes were successfully demonstrated as useful ionization-assisting materials on a variety of tissue types. However, signal intensities were found to be reduced by about one order of magnitude compared to MALDI MSI and thereby the number of analytes detected with the DIUTHAME method was reduced. Sub-cellular resolution MSI was demonstrated to be possible with a lateral resolution down to 5 μm using DIUTHAME, but better lateral resolutions were impeded by low ionization efficiency. In a second project, the MALDI MSI sample preparation workflow was optimized for microglia single cells grown on glass slides for high-lateral-resolution MSI down to 1.5 μm. Separate workflows have been developed for bulk lipidomics analysis and for high-lateral-resolution MSI to extend the usability of the method in general. It was possible to statistically differentiate multiple microglia cell lines from each other and find molecular markers for inflammatory stimulation. By employing an imaging approach, the cell morphology could be resolved in high detail while simultaneously pinpointing cellular heterogeneity on the level of distinct molecules. Intra-cellular heterogeneity involving the formation and varying composition of lipid droplets was observed and the influence of inflammatory stimulation was investigated. Further, inter-cellular phospholipid heterogeneity was described and visualized for the first time in microglia cells grown under identical conditions. In summary, the methods and home-built instrumentation described in this doctoral thesis successfully enabled single-cell mass spectrometry imaging at sub-cellular lateral resolution and fundamentally improved its application to biological samples. In an outlook, further possible applications of single-cell MSI are showcased. Lipidomic differences in rested and sleep-deprived Drosophila melanogaster brains were identified and a potential localization to sleep-related neurons was investigated in a multimodal approach involving fluorescence microscopy.