Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.
J Struct Biol. 2014 Mar;185(3):278-84. doi: 10.1016/j.jsb.2013.11.008. Epub 2013 Dec 1.
Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce "keyframe imaging" as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a "target map" of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10(9) in a single automated experimental run.
在大视场中高效关联成像小目标是细胞生物学中的一个核心问题。在这里，我们展示了聚焦离子束扫描电子显微镜（FIB-SEM）在解决这个问题方面的一系列技术进展。我们报告了该过程在速度、稳健性和自动化方面的提高，并实现了一致的 z 切片厚度约为 3nm。我们引入了“关键帧成像”作为一种新方法，可以同时对大视场进行成像，并获得目标子体积的高分辨率 3D 图像。我们展示了这些进展在 HIV 核心的细胞质融合中间体成像中的应用。使用荧光标记的细胞膜、蛋白质和 HIV 核心，我们首先通过荧光显微镜生成 HIV 感染细胞的“目标图”。然后，我们生成整个细胞的相关 3D EM 体积以及单个 HIV 核心的高分辨率 3D 图像，在单个自动化实验运行中实现了跨 10^9 体积尺度的关联成像。
