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Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

机译:二次离子MS成像可相对定量处理不同人群的单个细胞膜中的胆固醇

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Time-of-flight secondary ion mass spectrometry (TOF-SIMS)is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.
机译:飞行时间二次离子质谱(TOF-SIMS)是一种建立完善的生物分析方法,可直接对单细胞之间的化学分布进行成像。在这里,我们报告使用SIMS成像的协议,以比较量化两个细胞质膜之间胆固醇水平的相对差异。应该可以将此程序应用于其他选定脂质的研究。这种发展使得可以在单细胞水平上直接比较不同药物处理的化学效果和质膜中的孵育条件。相对定量TOF-SIMS成像已在此处用于比较处理后的胆固醇相对于对照细胞而言含量较高的巨噬细胞。在SIMS分析之前,使用具有两种不同膜染料的原位荧光显微镜技术来区分形态相似但差异处理的细胞。荧光鉴定的细胞的SIMS图像显示,这两个细胞群具有不同的外部小叶膜组成,其中胆固醇处理的巨噬细胞的膜中胆固醇含量是对照巨噬细胞的两倍以上。使用SIMS进行相对定量以比较单个细胞的化学组成可以提供有关正常生物学功能,疾病的病原体以及可能的疾病治疗方法的有价值的信息。

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