Cryo-plasma FIB/SEM volume imaging of biological specimens.
Cryo-plasma FIB/SEM volume imaging of biological specimens.
复制标题
生物标本的冷冻血浆FIB/SEM体积成像。
DOI：
10.7554/elife.83623
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发表时间：
2023-02-21
期刊：
影响因子：
7.7
通讯作者：
Grange M
中科院分区：
文献类型：
--
作者：
Dumoux M;Glen T;Smith JLR;Ho EML;Perdigão LMA;Pennington A;Klumpe S;Yee NBY;Farmer DA;Lai PYA;Bowles W;Kelley R;Plitzko JM;Wu L;Basham M;Clare DK;Siebert CA;Darrow MC;Naismith JH;Grange M
串行聚焦离子束扫描电子显微镜 (FIB/SEM) 能够对介观尺度（10 nm 至 10 µm）的亚细胞结构进行成像和评估。当应用于玻璃化样品时，系列 FIB/SEM 也是一种针对细胞和组织中特定结构的方法，同时保持下游原位结构生物学成分的水合壳。然而，由于低对比度、帘幕和充电伪影，未染色低温生物样品的连续 FIB/SEM 成像的应用受到限制。我们使用低温等离子体 FIB/SEM 来应对这些挑战。我们评估了等离子体离子源和成像方案的选择，以生成一系列不同生物样品的高质量 SEM 图像。使用自动化工作流程，我们生成了细菌、人体细胞和组织的三维体积，并计算了它们的分辨率估计值，通常达到 20-50 nm。此外，需要一种针对感兴趣区域的无标签定位工具来推动原位结构生物学在组织中的应用。系列 FIB/SEM 与基于等离子体的离子源的结合提供了一个框架，用于针对批量冷冻样品 (>100 µm) 中的特定特征，以生成用于低温电子断层扫描的薄片。
Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents’ hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20–50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.