Abstract
Current histological and anatomical analysis techniques, including fluorescence in situ hybridisation, immunohistochemistry, immunofluorescence, immunoelectron microscopy and fluorescent fusion protein, have revealed great distribution diversity of mRNA and proteins in the brain. However, the distributional pattern of small biomolecules, such as lipids, remains unclear. To this end, we have developed and optimised imaging mass spectrometry (IMS), a combined technique incorporating mass spectrometry and microscopy, which is capable of comprehensively visualising biomolecule distribution. We demonstrated the differential distribution of phospholipids throughout the cell body and axon of neuronal cells using IMS analysis. In this study, we used solarix XR, a high mass resolution and highly sensitive MALDI-FT-ICR-MS capable of detecting higher number of molecules than conventional MALDI-TOF-MS instruments, to create a molecular distribution dataset. We examined the diversity of biomolecule distribution in rat brains using IMS and hypothesised that unsupervised machine learning reconstructs brain structures such as the grey and white matters. We have demonstrated that principal component analysis (PCA) can reassemble the grey and white matters without assigning brain anatomical regions. Hierarchical clustering allowed us to classify the 10 groups of observed molecules according to their distributions. Furthermore, the group of molecules specifically localised in the cerebellar cortex was estimated to be composed of phospholipids.