Bacquart Thomas, Devès Guillaume, Carmona Asunción, Tucoulou Rémi, Bohic Sylvain, Ortega Richard
Cellular Chemical Imaging and Speciation Group, CNAB UMR 5084, CNRS/Université de Bordeaux 1, BP 120 Le Haut Vigneau, 33175 Gradignan cedex, France.
Anal Chem. 2007 Oct 1;79(19):7353-9. doi: 10.1021/ac0711135. Epub 2007 Sep 7.
Identification of chemical species at a subcellular level is a key to understand the mechanisms involved in the biology of chemical elements. When performed with a microbeam, X-ray absorption near-edge structure (micro-XANES) enables the direct speciation analysis of oxidation states in subcellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. Here we report the principal characteristics in terms of spatial resolution, detection limit, reproducibility, and repeatability of a micro-XANES experimental setup based on Kirkpatrick-Baez X-ray focusing optics that maintains high flux of incoming radiation (>10(11) photons/s) at micrometric spatial resolution (1.5 x 4.0 microm2). Applications and limitations of this setup are illustrated by examples of iron and arsenic absorption spectra obtained from the cytosol, nucleus, and mitochondrial network of cultured cells. A better repeatability and sensitivity with no oxidation state modification and minimal beam damage is achieved when cells are analyzed in a frozen hydrated state, as compared to freeze-dried cells. This original experimental setup can now be applied for the direct speciation analysis of most trace elements at the subcellular level.
在亚细胞水平上识别化学物种是理解化学元素生物学所涉及机制的关键。当使用微束进行时，X射线吸收近边结构（微XANES）能够对亚细胞区室中的氧化态进行直接形态分析，避免了可能改变化学物种的细胞分级分离和其他制备步骤。在此，我们报告了基于柯克帕特里克-贝兹X射线聚焦光学器件的微XANES实验装置在空间分辨率、检测限、重现性和可重复性方面的主要特征，该装置在微米级空间分辨率（1.5×4.0微米²）下保持高入射辐射通量（>10¹¹光子/秒）。通过从培养细胞的细胞质、细胞核和线粒体网络获得的铁和砷吸收光谱示例说明了该装置的应用和局限性。与冻干细胞相比，当在冷冻水合状态下分析细胞时，可实现更好的可重复性和灵敏度，且无氧化态改变且束损伤最小。这种原始实验装置现在可用于亚细胞水平上大多数微量元素的直接形态分析。
