The chemical composition of animal cells and their intracellular compartments reconstructed from 3D mass spectrometry
The chemical composition of animal cells and their intracellular compartments reconstructed from 3D mass spectrometry
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DOI：
10.1002/anie.200604468
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Hagenhoff, Birgit
中科院分区：
文献类型：
--
作者：
Breitenstein, Daniel;Rommel, Christina E.;Hagenhoff, Birgit
分析动物细胞化学成分的空间分辨率的实验方法对于生物医学研究的许多领域是重要的。利用飞行时间二次离子质谱（TOF-SIMS）分析三维微结构是一种新兴的生物样品分子结构分析技术。在西姆斯中，样品表面被初级离子轰击。在所谓的碰撞级联中传输的一部分能量被引导回样品表面，并导致中性和带电化学物质（二次离子）从最上层分子层解吸。随后收集这些物质并对其质荷比进行分析。[1]今天，大多数用于有机应用的最先进的仪器使用TOF分析仪来测定解吸的二次离子的质量。[2]TOF-SIMS允许并行检测所有元素以及小有机分子，灵敏度低至ppm/飞摩尔范围。[3]用初级离子束扫描样品表面提供化学表面组成的2D图像。此外，长时间的离子轰击样品在一个恒定的位置导致溅射侵蚀。溅射材料的质量分析然后揭示样品的垂直组成。[1]有机材料的横向分布可以以约150-400 nm的分辨率成像，[4-6]而有机聚合物膜中的垂直分辨率被证明优于30 nm。[7]然而，迄今为止，从有机材料获得的有限信号强度以及碰撞级联破坏有机分子并因此破坏分子信息的事实阻碍了对生物样品如细胞和组织的应用。通过使用Au ~（3+）和Bi ~（3+）等多原子原离子，改善了表面分析中的低信号强度和溅射深度剖析中的分子信息损失。[3，8]此外，巴克敏斯特富勒烯已成为溅射腐蚀的新离子源。[9]发现C60+离子的影响对有机样品的破坏性小于普通溅射离子O2+和Cs+。[10]即使是完整的有机分子也能在溅射过程中幸存下来。[11]因此，本研究的目的是通过对样品表面进行重复的西姆斯分析，然后进行溅射腐蚀，将样品的更深层暴露于下一轮西姆斯分析（TOF-SIMS 3D微区分析），在三维中重建动物细胞的分子组成。在一个双束设置Bi 3+初级离子被用来确定表面的化学组成，和C60+离子被用于间歇溅射侵蚀。[12]正常大鼠肾（NRK）细胞，在普通细胞培养条件下的盖玻片上生长的6个汇合层，通过TOF-SIMS 3D微区分析后，细胞已被稳定的化学固定。化学固定是在高真空中保存生物样品结构的常规程序。[13]所有样品的TOF-SIMS结果都具有很好的重现性和相似性，因此这里的数据仅限于一个典型的实验。图1显示了TOF-SIMS微区分析的一个独特方面，它与光学图像采集非常不同。在实验开始时，培养基质上存在汇合的细胞层。西姆斯可接近的最上层分子层（图1a中的粗线）将由源自细胞表面的化学物质组成。在间歇溅射周期中，样品表面
Experimental approaches for analyzing the chemical composition of animal cells with spatial resolution are important for many fields of biomedical research. The analysis of threedimensional microstructures by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) is an emerging technique to make the molecular architecture of biological samples accessible. In SIMS the sample surface is bombarded by primary ions. A fraction of the energy transported in the socalled collision cascade is directed back to the sample surface and causes the desorption of neutral and charged chemical species (secondary ions) from the uppermost molecular layer. These are subsequently collected and analyzed with respect to their mass/charge ratio.[1] Today, most state-of-the-art instruments for organic applications use TOF analyzers for mass determination of the desorbed secondary ions.[2] TOF-SIMS allows the detection of all elements as well as small organic molecules in parallel and has a sensitivity down to the ppm/femtomole range.[3] Scanning the sample surface with the primary-ion beam provides a 2D image of the chemical surface composition. Moreover, prolonged ion bombardment of the sample at a constant position leads to sputter erosion. Mass analysis of the sputtered material then reveals the vertical composition of the sample.[1] The lateral distribution of organic material can be imaged with a resolution of about 150–400 nm,[4–6] whereas the vertical resolution in organic polymer films was shown to be better than 30 nm.[7] Application to biological samples like cells and tissues, however, has so far been hindered by the limited signal intensities obtained from organic materials and the fact that the collision cascade destroys organic molecules and, thus, molecular information. The low signal intensities in surface analysis and the loss of molecular information in sputter depth profiling have been improved by the use of polyatomic primary ions like Au3+ and Bi3+.[3, 8] Moreover, buckminsterfullerenes have become available as a new ion source for sputter erosion.[9] The impact of C60+ ions was found to be less destructive to organic samples than the common sputter ions O2+ and Cs+.[10] Even intact organic molecules survive the sputter process.[11] Thus, it was the objective of this study to reconstruct the molecular composition of animal cells in three dimensions by applying repeated cycles of SIMS analysis of the sample surface followed by sputter erosion that exposes a deeper layer of the sample to the next round of SIMS analysis (TOF-SIMS 3D microarea analysis). In a dual-beam setup Bi3+ primary ions were used to determine the chemical composition of the surface, and C60+ ions were used for intermittent sputter erosion.[12] Six confluent layers of normal rat kidney (NRK) cells, grown on cover slips under ordinary cell-culture conditions, were analyzed by TOF-SIMS 3D microarea analysis after the cells had been stabilized by chemical fixation. Chemical fixation is a routine procedure to preserve the structure of biological samples in high vacuum.[13] TOF-SIMS results were very reproducible and similar for all samples so that data presentation here is confined to one typical experiment. Figure 1 illustrates a unique aspect of TOF-SIMS microarea analysis that is very different from optical-image acquisition. At the beginning of the experiment a confluent cell layer is present on the culture substrate. The uppermost molecular layer (thick line in Figure 1a) accessible for SIMS will consist of chemical species originating from the cell surfaces. During intermittent sputter cycles the sample sur-