胆固醇是中枢神经系统和组织的重要组成部分，了解其空间分布对于生物学、病理生理学和诊断至关重要。然而，由于难以电离甾醇分子，使用基质辅助激光解吸/电离质谱成像（MALDI-MSI）对胆固醇进行直接成像仍然具有挑战性且耗时。为了解决这个问题，建立了MALDI-MSI方法，通过MALDI-MSI直接快速分析阿尔茨海默病（AD）、不同癌症组织和器官中胆固醇的空间分布。这种优异的成像性能取决于对影响MALDI-MSI成像的各种条件的研究和系统优化。在本例中，我们报告了 AD 小鼠大脑特定结构以及不同肿瘤组织和器官中胆固醇的分布和水平。结果显示，AD小鼠小脑，特别是侧柏中的胆固醇含量明显高于野生型（WT）模型。此外，我们使用 MALDI-MSI 成功地可视化胆固醇在其他器官中的分布，如心脏、肝脏、脾脏、肾脏、胰腺以及肿瘤组织实质和间质。值得注意的是，系统地研究了胆固醇 MS/MS 碳氢化合物片段的归属。我们提出的优化策略和建立的 MALDI-MSI 方法可以很容易地推广到不同的动物组织或活体样本，从而促进 MALDI-MSI 在临床、医学和生物学研究中的应用潜力。

Cholesterol is a vital component of the central nervous system and tissues, and understanding its spatial distribution is crucial for biology, pathophysiology, and diagnostics. However, direct imaging of cholesterol using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) remains challenging and time-consuming due to the difficulty in ionizing the sterol molecule. To tackle this issue, a MALDI-MSI method is established for direct and rapid analysis of the spatial distribution of cholesterol in Alzheimer's disease (AD), different cancer tissues and organs via MALDI-MSI. This excellent imaging performance depends on the study and systemic optimization of various conditions that affect the imaging of MALDI-MSI. In this case, we report the distribution and levels of cholesterol across specific structures of the AD mouse brain and different tumor tissue and organs. According to the results, the content of cholesterol in the AD mouse cerebellum, especially in the arborvitae, was significantly higher than that in the wild type (WT) model. Furthermore, we successfully visualize the distribution of cholesterol in other organs, such as the heart, liver, spleen, kidney, pancreas, as well as tumor tissues parenchyma and interstitium using MALDI-MSI. Notably, the attribution of cholesterol MS/MS hydrocarbon fragments was systematically investigated. Our presented optimization strategy and established MALDI-MSI method can be easily generalized for different animal tissues or live samples, thereby facilitating the potential for applications of MALDI-MSI in clinical, medical and biological research.