Approaches to increasing analytical throughput of human samples with multi-isotope imaging mass spectrometry.
Approaches to increasing analytical throughput of human samples with multi-isotope imaging mass spectrometry.
复制标题
利用多同位素成像质谱提高人体样本分析通量的方法。
DOI：
10.1002/sia.5580
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Lechene,ClaudeP
中科院分区：
文献类型：
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作者：
Steinhauser,MatthewL;Guillermier,Christelle;Wang,Mei;Lechene,ClaudeP
多同位素成像质谱（MIMS）将稳定同位素示踪剂与NanoSIMS离子显微镜的定量成像相结合。由于有广泛的安全先例，在MIMS应用中使用稳定同位素开辟了研究人类各种生物学问题的可能性。在这里，我们描述了一系列的方法，以提高有效的分析通量检测罕见的核标记事件与MIMS。在样品制备水平，将悬浮液中的细胞以高密度涂抹或将沉淀的细胞包埋并切片以达到核深度。对每种细胞类型的预溅射条件进行了优化，以确保细胞核的可重复采样。脂肪组织提出了不同的挑战，因为脂肪细胞的大体积导致在任何给定平面中的细胞核的强制性低密度。在将样品引入NanoSIMS仪器之前，对所有细胞核进行荧光染色和成像，并记录它们的坐标，从而允许对包含至少一个细胞核的区域进行自动分析，从而最大限度地减少死腔分析。这些数据强调了人类研究所面临的独特挑战，其中伦理和实际问题可能会限制长期使用稳定同位素标记，因为这可能是在分裂不频繁的细胞中实现高标记频率所必需的。版权所有© 2014约翰威利父子有限公司.
Multi‐isotope imaging mass spectrometry (MIMS) combines stable isotope tracers with the quantitative imaging of NanoSIMS ion microscopy. With extensive safety precedent, use of stable isotopes in MIMS applications opens the possibility of studying a wide array of biological questions in humans. Here we describe a series of approaches to increase the effective analytical throughput for detecting rare nuclear labeling events with MIMS. At the level of sample preparation, cells in suspension were smeared at high density or pelleted cells were embedded and sectioned to reach nuclear depth. Presputtering conditions were optimized for each cell type to ensure the reproducible sampling of nuclei. Adipose tissue posed a different challenge as the large volume of adipocytes results in an obligatorily low density of nuclei in any given plane. Before introducing samples to the NanoSIMS instrument, all nuclei were fluorescently stained and imaged, and their coordinates were recorded, allowing automated analysis of fields that contained at least one nucleus and therefore minimizing analysis of dead space. These data emphasize unique challenges posed by human studies, where both ethical and practical issues may limit the administration of stable isotope labels for prolonged periods of time as may be necessary to achieve high labeling frequencies in cells that divide infrequently. Copyright © 2014 John Wiley & Sons, Ltd.