Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging
Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging
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DOI：
10.1016/j.ymeth.2015.03.011
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发表时间：
2015-10-15
期刊：
影响因子：
4.8
通讯作者：
Soeller, Christian
中科院分区：
文献类型：
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作者：
Crossman, David J.;Hou, Yufeng;Soeller, Christian
许多生物学问题需要不同空间尺度的信息，包括分子、细胞器、细胞和组织尺度。在这里，我们详细介绍了一种通过将直接随机光学重建显微镜（dSTORM）的纳米级数据与共聚焦显微镜提供的细胞和组织水平数据相关联，对人类心脏组织进行多尺度成像的方法。通过利用传统的荧光染料，可以用两种方式成像相同的细胞结构。人类心脏组织首次在纳米尺度上成像，以鉴定含有心肌蛋白结膜亲蛋白（JPH）和ryanodine受体（RyR）的大分子膜复合物。随后用共聚焦显微镜对这些蛋白和另一个细胞膜标记物（小麦胚芽凝集素，WGA）的分布进行成像。通过将dSTORM数据分割为膜和非膜成分，我们发现与细胞内区室相比，RyR与JPH在质膜上的共定位增加。描述了抗体标记，质量控制，定位和对齐模式之间的结构以及组合的多尺度数据集分析的策略。(C) 2015出版的爱思唯尔公司。
Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described. (C) 2015 Published by Elsevier Inc.