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

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.

Correlative light and electron microscopy (CLEM) allows the combination of optical microscopy images, which give molecular specificity through labelling with fluorescent probes, and electron microscopy, which provides high resolution ultrastructure. Ideally, CLEM would use super-resolution optical microscopy methods to bridge the resolution gap between light and electron imaging. We propose to develop a workflow for cryo-CLEM combining EM with STORM fluorescence microscopy on cryogenically vitrified samples.

We will purchase a cryo-focused ion beam (FIB)-scanning electron microscope (SEM) to prepare thin (100-300 nm) lamellas of frozen biological samples, suitable for imaging by cryo-electron microscopy (EM) and cryo-STORM. We will use two approaches to cyro-CLEM. Firstly, we will use a conventional cryo-STORM microscope capable of achieving resolutions in the region of 50 nm and correlate the data with cryo-electron microscopy. An ongoing collaboration with the Francis Crick Institute will underpin these efforts. Secondly, on the highest quality lamella (roughness <20 nm) we will also use Solid Immersion Lens (SIL) technology developed at our facility for cryo-STORM at high resolution (12 nm resolution already achieved). This method uses special lenses to couple the objective lens to the sample, achieving high numerical aperture and therefore high resolution using air objectives suitable for imaging under cryogenic conditions. Images obtained using these methods will also be correlated with cryo-EM images. Lamella milling strategies will be devised in collaboration with eBIC and Zeiss.

We will also use the cryo-FIB/SEM to implement at the RCaH 3D volume imaging. We are installing in the light sheet microscope an existing cryo-stage compatible with its epi-fluorescence imaging scheme we will aim at using to help to identify specific tissue types. Our ongoing collaboration with Lucy Collinson (Crick) will be crucial to the success of this part of the project.

The immediate beneficiaries of the partnership will be the academic user community of the new imaging capability. Collaborative programmes are outlined in this proposal, and in the long term, as access to the cryo-CLEM workflow becomes available through open access peer-review, we expect many beneficiaries in the academic community. These academics will be largely from the life-sciences research community, although other disciplines (e.g. biomedical materials research) will also benefit from the availability of a cluster of super-resolution imaging facilities. Users of the microscope will be trained in the use of the cryo-CLEM technique, and this expertise will be transferred to their home institutions, expanding the UK's base of experts in new imaging technologies. Ultimately, there will be societal benefits in the form of new medical treatments and diagnostic techniques, the collaborative nature of the Research Complex and Harwell Oxford campus speeding up the process of translating research findings into medical benefits.

The research that will be enabled by the availability of cryo-CLEM is expected to benefit other commercial sectors, such as pharmaceuticals and medical diagnostics. The applicants have a track record of working with these sectors (e.g. MMF's current and previous collaborations with Evotec, Illumina, and Astra Zeneca). There is a growing industrial user community on OCTOPUS and industrial users are expected to take advantage of the cryo-CLEM capability. There are a number of routes for industrial access to the facility, ranging from collaboration with academics for non-proprietary research, to fully paid commercial access. The facility also operates a number of schemes to assist industrial access, including the "B4I" and "A4I" programmes, providing proof-of-concept and longer term access, respectively, for industries to collaborate with the facility through supported access to equipment and expertise.

STFC provides a high level of support for identification and support of commercial opportunities, and this will be drawn upon to ensure maximum economic impact is derived from the work of the partnership. We have already established a collaboration with Zeiss, and it is expected that the proposed programme of work will ultimately lead to the development of a super-resolution cryo-CLEM microscope combining optical and electron imaging in the same instrument.

Finally, the research outputs will be of significant public interest because of the healthcare connections, and the high visual impact of microscopy work. There is already an extensive public engagement programme operated by STFC and RCaH, with regular organised visits from members of the public, schools, and undergraduates. The microscopy suite is a regular feature on these visits, and the new system will be seen and demonstrated. We actively encourage the dissemination of research outputs through additional routes to the "conventional" scientific literature, and regular press releases are issued when potentially high impact findings are published.