A modular platform for automated cryo-FIB workflows.
A modular platform for automated cryo-FIB workflows.
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DOI：
10.7554/elife.70506
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发表时间：
2021-12-24
期刊：
影响因子：
7.7
通讯作者：
Mahamid J
中科院分区：
文献类型：
--
作者：
Klumpe S;Fung HK;Goetz SK;Zagoriy I;Hampoelz B;Zhang X;Erdmann PS;Baumbach J;Müller CW;Beck M;Plitzko JM;Mahamid J
通过低温聚焦离子束铣削（cryo-FIB）进行的薄片微加工已成熟为广泛用于细胞冷冻电子断层扫描的制备方法。由于维持玻璃体标本结构完整性所需的低 Ga+ 离子束电流的烧蚀率有限，因此常见的制备方案既耗时又费力。新一代冷冻 FIB 仪器的稳定性得到提高，现在可以实现自动化操作。在这里，我们提出了一个开源软件工具 SerialFIB，用于创建自动化和可定制的冷冻 FIB 准备协议。该软件包含一个用于轻松执行常规片层制备的图形用户界面、一个与可用 Python 包兼容的脚本模块以及与三维关联光学和电子显微镜 (CLEM) 工具的接口。 SerialFIB 能够简化先进的冷冻 FIB 方案，例如多模态成像、CLEM 引导的薄片制备和原位薄片提取程序。因此，我们的软件为进一步开发先进的低温成像和样品制备方案奠定了基础。
Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.