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

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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.