Bearing the context in mind: A cryo FIB-SEM based CLEM workflow to investigate relationships between molecular interactions and ultrastructure

21世纪生物学家的中心目标之一是在组织的宏观组织水平和细胞和组织的构建块的分子甚至原子水平的组织之间提供结构理解的无缝联系。对所有这些长度尺度的理解通常需要使用不止一种显微镜方法。目前正在使用的一种方法是“相关光学和电子显微镜”(Clem)，即用光学显微镜对标记有荧光标记的特定分子成像，这些图像与电子显微镜的图像相结合，可以非常高的分辨率描绘整个细胞结构。由于传统光学显微镜的分辨率远远低于电子显微镜的分辨率，因此CLEM一直受到限制，因此光学显微镜图像实际上只被用来识别感兴趣的区域。然而，近年来，所谓的“超分辨率”光学显微镜的发展使这项技术在分辨率上更接近于电子显微镜。因此，结合超分辨率光学显微镜和电子显微镜将提供显著更好的相关性，但一个限制是样品制备方法的不兼容性，特别是当样品通过冷冻保存用于电子显微镜时，这一过程有助于保持样品结构接近自然状态，但降低了可以用光学显微镜获得的分辨率。通过这项授权，我们建议开发一种结合超分辨率光学显微镜和电子显微镜的冷冻样品的CLEM方法。为此，我们将购买一台低温聚焦离子束扫描电子显微镜(FIB-SEM)，这将使我们能够制备既适合超分辨率又适合电子显微镜的非常薄的样品。Clem方法将被用来研究一些生物学相关的问题。高分辨率相关成像是理解基础细胞生物学的关键。我们已经形成了一个跨学科的合作伙伴关系，寻求利用这种Clem方法，并在多学科环境中的一系列相关样本中对其进行测试。在调试完显微镜后，我们的经验将帮助其他科学家和合作者应用这种方法来回答他们的科学问题。英国哈威尔校区的光学成像和电子显微镜设施与弗朗西斯·克里克研究所之间正在进行的合作，将支持英国在未来十年使用低温克莱姆进行基础性发现的努力。

One of the central goals of 21st century biologists is to provide a seamless link of structural understanding between the macroscopic level of tissue organization to the molecular and even atomic level organization of the building blocks of cells and tissues. Understanding across all these length scales often requires the use of more than one microscopy method. One approach being used is known as "Correlative Light and Electron Microscopy" (CLEM), in which light microscopy is used to image specific molecules labelled with fluorescent markers, and these images combined with images from electron microscopes that can picture the overall cellular structure with very high resolution.CLEM has been limited because the resolution of conventional optical microscopy is much lower than the resolution of electron microscopes, so that the light microscope images have only really been used to identify areas of interest. However, in recent years the development of so-called "super-resolution" light microscopy has brought the technique much closer in resolution to that of the electron microscope. Combining super-resolution light microscopy with electron microscopy would therefore provide significantly better correlation, but one limitation is an incompatibility of sample preparation methods, particularly when samples are preserved for electron microscopy by freezing, a process that helps to keep the sample structure close to the natural state but degrades the resolution that can be obtained with light microscopy.Through this grant we propose to develop a methodology for CLEM of frozen samples combining super-resolution optical and electron microscopy. To do this we will purchase a cryogenic Focussed Ion Beam Scanning Electron Microscope (FIB-SEM) that will allow us to prepare very thin samples suitable for both super-resolution and electron microscopy. The CLEM method will be used to investigate a number of problems of biological relevance.High resolution correlative imaging is critical to understand basic cell biology. We have formed an interdisciplinary partnership that seeks to exploit this CLEM methodology, and test it in a range of relevant samples within a multidisciplinary environment. After commissioning the microscope, our experience will help other scientists and collaborators to apply this method to answer their scientific questions. An ongoing collaboration between UK facilities for light imaging and electron microscopy at the Harwell Campus and the Francis Crick Institute, where significant experience in CLEM resides, will underpin efforts in the UK to use cryo-CLEM to make fundamental discoveries in the next decade.