Chen Yanyan, Shi Rui, Wang Jianing, Xie Chengyi, Song Yuanyuan, Li Ruxin, Wen Luyao, Lam Thomas Ka-Yam, Yang Zhu, Cai Zongwei
State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Hong Kong SAR 315200, China.
Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR 315200, China.
Anal Chem. 2025 Jul 29. doi: 10.1021/acs.analchem.5c03289.
High-resolution mass spectrometry imaging (MSI) plays a vital role in lipidomics, yet challenges persist in analyzing lipids at the single-cell level due to limitations in spatial resolution and lipid coverage. While existing strategies based on a single matrix application step for dual-polarity provide high lipid coverage from the same sample and enable easy sample preparation, matrix depletion limits their spatial resolution to 10 μm, preventing their application to single-cell imaging. Here, we present a single-cell/subcellular resolution strategy for dual-polarity matrix-assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) that eliminates the need for matrix reapplication. This approach achieves 5 μm spatial resolution while maintaining lipid coverage comparable to multistep single-cell imaging methods. This is enabled by a fine-tuned matrix deposition technique that fully utilizes the high sensitivity of -(1-naphthyl)-ethylenediamine dihydrochloride (NEDC) in dual polarities and optimized acquisition conditions, allowing single-deposition workflows without the need for washing, repreparation, or image recalibration. This single-cell resolution MALDI-MSI strategy successfully imaged a broader range of lipid species with distinctive spatial detail in mouse kidney tissue and lung carcinoma cells (A549). Using spatial probabilistic latent semantic analysis (PLSA), we identified three distinct lipid distribution patterns within a single-cell population in both polarities, and histogram analysis revealed substantial cell-to-cell lipidomic heterogeneity. This strategy overcomes limitations of traditional dual-polarity MSI and provides a powerful tool for advancing cellular lipidomics, elucidating disease mechanisms, and investigating environmental toxicology.
高分辨率质谱成像（MSI）在脂质组学中发挥着至关重要的作用，但由于空间分辨率和脂质覆盖范围的限制，在单细胞水平分析脂质仍存在挑战。虽然现有的基于单步基质应用的双极性策略可从同一样本中获得高脂质覆盖范围并简化样本制备，但基质耗尽将其空间分辨率限制在10μm，阻碍了其在单细胞成像中的应用。在此，我们提出了一种用于双极性基质辅助激光解吸电离质谱成像（MALDI-MSI）的单细胞/亚细胞分辨率策略，该策略无需重新应用基质。这种方法实现了5μm的空间分辨率，同时保持了与多步单细胞成像方法相当的脂质覆盖范围。这得益于一种经过微调的基质沉积技术，该技术充分利用了盐酸-（1-萘基）乙二胺（NEDC）在双极性中的高灵敏度以及优化的采集条件，允许进行单步沉积工作流程，无需洗涤、重新制备或图像重新校准。这种单细胞分辨率的MALDI-MSI策略成功地在小鼠肾脏组织和肺癌细胞（A549）中对更广泛的脂质种类进行了具有独特空间细节的成像。使用空间概率潜在语义分析（PLSA），我们在双极性的单细胞群体中识别出三种不同的脂质分布模式，直方图分析揭示了细胞间脂质组的显著异质性。该策略克服了传统双极性MSI的局限性，为推进细胞脂质组学、阐明疾病机制和研究环境毒理学提供了一个强大的工具。
