Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles
Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles
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DOI：
10.1016/j.jsb.2005.09.013
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发表时间：
2006-07-01
影响因子：
3
通讯作者：
Fahrni, Christoph J.
中科院分区：
文献类型：
--
作者：
McRae, Reagan;Lai, Barry;Fahrni, Christoph J.
基于同步加速器的 X 射线荧光显微镜 (microXRF) 是研究组织和细胞中各种生物相关元素的二维分布的强大工具。通过直接在聚甲醛碳涂层的电子显微镜网格上培养小鼠成纤维细胞，获得了具有明确亚细胞分辨率的 microXRF 元素图。为了将元素分布与特定细胞结构和细胞器的位置共定位，我们探索了与 FluoroNanogold 缀合的市售二抗的应用，FluoroNanogold 是一种双标记，将常规有机荧光团与 1.4 nm Au 簇结合在一起作为 microXRF 成像的异生标记。贴壁小鼠成纤维细胞在用作生物相容性 XRF 支持基底的氮化硅窗上生长，并分别用 FluoroNanogold 与线粒体或高尔基体特异性一抗组合进行标记。使用 11.95 keV 的入射 X 射线能量对空气中干燥的细胞进行光栅扫描，足以确保 Au L α 线的激发，为 An 以及大多数生物相关元素提供了亚微米分辨率的二维图。 MicroXRF 被证明具有足够的灵敏度，能够对 Au 标记细胞器的位置和结构细节进行成像，这与通过光学荧光显微镜观察到的亚细胞分布密切相关。 (c) 2006 Elsevier Inc. 保留所有权利。
Synchrotron-based X-ray fluorescence microscopy (microXRF) is a powerful tool to study the two-dimensional distribution of a wide range of biologically relevant elements in tissues and cells. By growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids, microXRF elemental maps with well-defined subcellular resolution were obtained. In order to colocalize the elemental distribution with the location of specific cellular structures and organelles, we explored the application of a commercially available secondary antibody conjugated to FluoroNanogold, a dual-label that combines a regular organic fluorophore with a 1.4 nm Au-cluster as xenobiotic label for microXRF imaging. Adherent mouse fibroblast cells were grown on silicon nitride windows serving as biocompatible XRF support substrate, and labeled with FluoroNanogold in combination with primary antibodies specific for mitochondria or the Golgi apparatus, respectively. Raster scanning of the in-air dried cells with an incident X-ray energy of 11.95 keV, sufficient to ensure excitation of the Au L alpha line, provided two-dimensional maps with submicron resolution for An as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the Au-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy. (c) 2006 Elsevier Inc. All rights reserved.