Cholesterol is one of the most abundant lipids in the brain. It is heavily enriched in myelin sheaths which wrap around neurons and also found in plasma membranes of all cell types of the brain. Cholesterol is linked to diseases of aging, particularly neurodegenerative diseases, hence there is considerable interest in the use of mass spectrometry imaging of brain to see if cholesterol levels are disturbed in regions of pathology. Being a neutral molecule cholesterol is less amenable to ionisation than other membrane lipids e.g. phospholipids, and is thus discriminated against in mass spectrometry imaging studies. Here we report the use of two ambient ionisation technologies AP/MALDI (MassTech) and LESA Plus (Advion) combined with Orbitrap (Thermo Scientific) mass spectrometry to image cholesterol and its metabolites in brain tissue sections (10 μm) at high spatial resolution (10 μm pixel) or at high sensitivity (pg/mg). By application of on-tissue derivatisation we have imaged cholesterol at high spatial resolution by AP/MALDI-MS and MS/MS, while for less abundant metabolites, where it is necessary to perform analysis at much lower resolution (800 μm) to achieve sufficient sensitivity, we have utilised the TriVersa NanoMate with LESA Plus. The advantage of the TriVersa NanoMate is that it allows chromatographic separations after extraction and before ionisation. This is important as many cholesterol metabolites exist as isomers. We demonstrate the application of these technologies on mouse and human brain [1, 2]. 胆固醇是脑组织中最丰富的脂质。他们富集在包裹着神经元的髓鞘，也存在于脑组织中所有细胞的细胞质膜中。胆固醇与衰老，特别是神经退行性疾病相关，因此在病理学中，运用质谱成像来分析胆固醇水平在脑组织中的分布引起了广泛的兴趣。胆固醇作为一种中性分子，比其他的细胞膜脂质（如磷脂）更难电离，因此质谱成像研究很少涉及这类分子。 在此，我们将报道两种原位电离技术 AP/MALDI （MassTech公司）和 LESA Plus （Advion公司）与Orbitrap 质谱（Thermo公司）来对脑组织切片（10 μm厚）中的胆固醇及其代谢物进行高空间分辨率（10 μm 分辨率）或高灵敏度（pg/mg）成像。我们使用了组织原位衍生化法对胆固醇进行了 AP/MALDI-MS 和 MS/MS 成像，而对于低丰度的代谢物，我们使用了低分辨率（800 μm）的LESA Plus 来获取足够的灵敏度进行分析。许多胆固醇代谢物都有同分异构体，因此运用LESA Plus技术非常重要，它能够实现在原位萃取后的色谱分离，然后再进行离子化。我们将展示这些技术在鼠脑和人脑中的应用成果。[1, 2]