Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography.
Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography.
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在纳米级3D中看到分枝杆菌感染的细胞：通过光学显微镜和FIB/SEM层析成像进行相关成像。
DOI：
10.1371/journal.pone.0134644
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Halaas Ø
中科院分区：
文献类型：
--
作者：
Beckwith MS;Beckwith KS;Sikorski P;Skogaker NT;Flo TH;Halaas Ø
分枝杆菌对当今世界的健康构成威胁，致病性和机会性细菌在大部分人口中引起结核病和非结核病。关于感染和疾病期间细菌与宿主之间的相互作用，仍有许多未知之处，需要进行更多的研究以应对耐药性和低效疫苗的挑战。这项工作建立了一个可靠的和可重复的方法进行相关成像的人巨噬细胞感染分枝杆菌在超高分辨率和3D。应用聚焦离子束/扫描电子显微镜（FIB/SEM）断层扫描，连同共聚焦荧光显微镜用于适当感染细胞的定位。该方法是基于Aclar聚（氯-三氟）乙烯基板，通过简单的热成型工艺微图案化成有利的几何形状。该平台提高了FIB/SEM断层扫描分析的吞吐量和质量，并成功应用于详细描述3D中整个分枝杆菌感染的巨噬细胞的细胞内环境。
Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.