Macrophages are important immune cells that respond to environmental cues acquiring a range of activation statuses represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in vitro differentiation of macrophages into naïve, M1, and M2 phenotypes using different cytokines. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches but also allows the study of cell-to-cell variance. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data-driven multivariate approach highlighting amino acids and other small molecules. The diamino acids alanylasparagine and lipid sphingomyelin SM(d18/16:0) are uniquely found in M1 macrophages, while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. The first demonstration of this capability illustrates the great potential of direct cell analysis for in situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single-cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases.

中文翻译：

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使用 3D OrbiSIMS 对巨噬细胞进行单细胞代谢分析：与表型的相关性

巨噬细胞是重要的免疫细胞，它们对环境线索作出反应，在其光谱的每一端获得一系列以促炎 (M1) 和抗炎 (M2) 表型为代表的激活状态。表征不同巨噬细胞亚群的代谢特征（代谢谱）是了解人体免疫系统对不同刺激反应的有力工具。在这里，应用最近开发的 3D OrbiSIMS 仪器，在使用不同的细胞因子将巨噬细胞体外分化为幼稚、M1 和 M2 表型后，对单个细胞的代谢组进行有用的洞察。这种分析策略不仅需要比传统质谱方法少 6 个数量级以上的样本，而且还允许研究细胞间差异。巨噬细胞亚群中的特征性代谢物使用靶向脂质和数据驱动的多变量方法来识别，突出氨基酸和其他小分子。二氨基酸丙氨酰天冬酰胺和脂质鞘磷脂 SM(d18/16:0) 是在 M1 巨噬细胞中唯一发现的，而在 M2 巨噬细胞中观察到吡啶和嘧啶的强度增加，这些发现与已知的生物途径有关。这种能力的首次展示说明了使用 3D OrbiSIMS 进行原位代谢物分析的直接细胞分析的巨大潜力，可以利用分子特征在单细胞水平上探测功能表型，并了解人体对植入设备和免疫系统的反应疾病。