Russell Matthew R G, Lerner Thomas R, Burden Jemima J, Nkwe David O, Pelchen-Matthews Annegret, Domart Marie-Charlotte, Durgan Joanne, Weston Anne, Jones Martin L, Peddie Christopher J, Carzaniga Raffaella, Florey Oliver, Marsh Mark, Gutierrez Maximiliano G, Collinson Lucy M
Electron Microscopy Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Host-pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK.
J Cell Sci. 2017 Jan 1;130(1):278-291. doi: 10.1242/jcs.188433. Epub 2016 Jul 21.
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.
生命过程发生在多个维度中，但即使对这些过程进行三维成像也具有挑战性。在此，我们描述了一种用于细胞单层三维关联光电子显微镜（CLEM）的工作流程，该流程使用荧光显微镜来识别和追踪生物事件，并结合连续块面扫描电子显微镜来分析潜在的超微结构。该工作流程涵盖了从细胞培养到样品处理、成像策略以及三维图像处理与分析的所有步骤。我们展示了该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理解复杂且动态的生物过程，包括培养细胞的细菌和病毒感染以及肿瘤中常见的内吞细胞内结构的形成。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微环境、HIV - 1在人单核细胞衍生巨噬细胞中的复制微环境以及内吞液泡组成的新见解。这种三维CLEM技术的广泛应用将使其成为生物医学研究关联成像工具箱中的一个有用补充。
