High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.
High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.
复制标题
DOI：
10.1186/jbiol42
复制
发表时间：
2006
影响因子：
--
通讯作者：
Slodzian G
中科院分区：
文献类型：
--
作者：
Lechene C;Hillion F;McMahon G;Benson D;Kleinfeld AM;Kampf JP;Distel D;Luyten Y;Bonventre J;Hentschel D;Park KM;Ito S;Schwartz M;Benichou G;Slodzian G
二次离子质谱（SIMS）是化学和材料科学中研究同位素组成的重要工具，但其在生物学中的应用受到技术因素的限制。多同位素成像质谱（Multi-Isotope Imaging Mass Spectrometry，MIMS）是将新一代的SIMS仪器与先进的离子光学、稳定同位素标记和定量图像分析软件相结合，用于生物材料的研究。新仪器可以产生高横向分辨率（低至33纳米）的质量图像，以及同时对几种同位素进行计数或成像。由于MIMS可以区分质量非常相似的离子，例如12C15N-和13C14N-，因此它能够精确和可重复地测量同位素比，从而在小于立方微米的体积内测量特定同位素标记的富集水平。MIMS的灵敏度至少是14C放射自显影的1,000倍。深度分辨率可以小于1 nm，因为仅需要几个原子层来创建原子质量图像。我们举例说明了使用MIMS成像未标记的哺乳动物培养细胞和组织切片;使用13 C-油酸分析脂肪细胞脂滴中的脂肪酸转运;使用15 N气态氮检查细菌中的固氮作用;使用15 N-亮氨酸测量耳蜗和缺血后肾细胞中的蛋白质更新水平;用15 N-尿苷和溴脱氧尿苷或14 C-胸苷的81 Br研究细胞核中DNA和RNA的共分布和尿苷掺入，用天然同位素12 C、16 O、14 N和31 P揭示培养的内皮细胞中的结构域;并追踪宿主小鼠淋巴结中的一些15N标记的供体脾细胞。MIMS首次使亚细胞区室中用稳定或放射性同位素标记的分子的成像和定量成为可能。
Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.