A targeted 3D EM and correlative microscopy method using SEM array tomography
A targeted 3D EM and correlative microscopy method using SEM array tomography
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DOI：
10.1242/dev.160879
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发表时间：
2018-06-01
期刊：
影响因子：
4.6
通讯作者：
Kolotuev, Irina
中科院分区：
文献类型：
--
作者：
Burel, Agnes;Lavault, Marie-Theprimerese;Kolotuev, Irina
使用电子显微镜定位复杂组织中罕见的细胞事件或结构具有挑战性。相关的光学和电子显微镜程序已开发链接荧光蛋白表达与超微结构分辨率。在这里，我们提出了一个优化的扫描电子显微镜（SEM）工作流程的体积阵列断层扫描的不对称样品和模式生物（秀丽隐杆线虫，果蝇，斑马鱼）。我们修改了金刚石刀，以简化连续切片阵列采集，最大限度地减少伪影。阵列采集后，将阵列转移到玻璃盖玻片或硅晶片载体上。使用光学显微镜，快速筛选阵列，以初步识别全局解剖特征（器官或身体特征）。然后，使用SEM，对感兴趣的细胞和/或器官进行深入研究。我们的手动和自动数据采集策略使3D数据采集和关联比其他方法更简单、更精确。该方法可用于解决细胞和发育生物学中需要有效识别标记细胞或细胞器的问题。
Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.