<p>Cholesterol is a neutral lipid widely implicated in neurological and other physiological processes. However, it is difficult to detect and precisely localise in situ using mass spectrometry imaging techniques due to low sensitivity and matrix suppression by co-localised lipids. Here, we present the application of large water gas cluster ion beams (GCIBs) for cholesterol analysis and localisation in murine brains using secondary ion mass spectrometry (SIMS). Optimal GCIB parameters were established through analysis of cholesterol standards. These clusters were then used to analyse whole brain sections with minimal sample preparation. Molecular images obtained with water clusters show that cholesterol is distributed across the whole brain, with variations in intensity. In addition, large water clusters were shown to simultaneously maximise secondary ion yields while reducing matrix effects for in situ cholesterol analysis. Depth profiling further revealed that different brain regions produced varying sputter yields. This study demonstrates that large water clusters are very well suited for cholesterol analysis in murine brains. Because this approach requires no derivatisation or matrix addition, it is likely to make analysis faster, simpler, and less prone to matrix effects.</p> Graphical Abstract <p></p>