3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.
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DOI：
10.1242/jcs.188433
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发表时间：
2017-01-01
影响因子：
4
通讯作者：
Collinson LM
中科院分区：
文献类型：
--
作者：
Russell MR;Lerner TR;Burden JJ;Nkwe DO;Pelchen-Matthews A;Domart MC;Durgan J;Weston A;Jones ML;Peddie CJ;Carzaniga R;Florey O;Marsh M;Gutierrez MG;Collinson LM
生命的过程发生在多个维度上，但即使在三维空间中想象这些过程也是具有挑战性的。在这里，我们描述了一个工作流程，三维相关的光和电子显微镜（CLEM）的细胞单层，使用荧光显微镜来识别和跟踪生物事件，结合系列块面扫描电子显微镜分析底层的超微结构。工作流程包括从细胞培养到样品处理、成像策略以及3D图像处理和分析的所有步骤。我们展示了工作流程在三项研究中的成功应用，每项研究都旨在更好地了解复杂和动态的生物学过程，包括培养细胞的细菌和病毒感染以及肿瘤中常见的细胞内细胞结构的形成。我们的工作流程揭示了对结核分枝杆菌在原代人淋巴管内皮细胞中的复制生态位，人类单核细胞衍生的巨噬细胞中的HIV-1以及肠空泡的组成的新见解。这种3D CLEM技术的广泛应用将使其成为生物医学研究相关成像工具箱的有益补充。总结：细胞单层的3D相关光学和电子显微镜的新工作流程，适用于M.-结核病感染的细胞、HIV-1感染的细胞和细胞内细胞结构。
The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. Summary: A new workflow for 3D correlative light and electron microscopy of cell monolayers, applied to studies of M.-tuberculosis-infected cells, HIV-1-infected cells and entotic cell-in-cell structures.