Micheva Kristina D, Burden Jemima J, Schifferer Martina
Department of Neurosurgery, Stanford University, Stanford, CA, 94305, USA.
LMCB, University College London, London, UK.
Methods Microsc. 2024 Jul 17;1(1):9-17. doi: 10.1515/mim-2024-0001. eCollection 2024 Apr.
Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.
组织切片是许多研究生物结构方法的核心。在现代体积电子显微镜(vEM)方法中,阵列断层扫描(AT)基于连续超薄切片技术,将切片收集到固体支持物上,通过光学显微镜和/或扫描电子显微镜成像,并将连续图像重新组装成一个体积进行分析。虽然AT在很大程度上使用标准的电子显微镜设备,但它具有几个优点,包括样本的长期保存以及与多尺度和多模态成像的兼容性。此外,连续超薄切片的收集提高了轴向分辨率,并为分子标记提供了途径,这有利于光学显微镜和免疫标记,并便于与电子显微镜进行关联。尽管有这些优点,但由于人们认为AT技术难度较大且缺乏培训机会,因此在成像设施和实验室中的应用并不广泛。在这里,我们指出了连续切片和图像分析方面的新进展,这些进展促进了AT流程,并提出了克服限制的解决方案。由于没有一种单一的vEM技术能够满足所有关于视野和分辨率的需求,我们绘制了一个决策树,以帮助研究人员在众多可用选项中进行选择。最后,我们阐述了AT方法在不同生物领域中尚未被探索的潜力,以增加有价值的见解。