Life in 3D, expanding the structural length scales via Serial Block Face Scanning Electron Microscopy (SBF-SEM)

Bioimaging is one of the key technologies for life science research but the way life functions has however often been studied by reducing this to 2-dimensional projections for instance by light and / or electron microscopy. Although this approach has led and will also in the future lead to breakthrough discoveries there is a realisation that if possible "life" should be studied as much as possible in a 3D environment, ideally in the context of a tissue or organism. Some of the newer microscopy techniques therefore are focussing on this aspect and allow for imaging large volumes in all 3 dimensions, e.g. by using light sheet light microscopy. Electron Microscopy has generally focussed on higher resolution structural information but alongside the "resolution revolution" in structural cryo Electron Microscopy (EM) which is revealing ever greater molecular details, there has been a quieter but probably equally important revolution in volume EM. By sequentially scanning the block face of an embedded sample and removing the scanned top layer, a large high-resolution 3-dimensional volume can be acquired. Although at somewhat lower resolution this volume is much larger than possible by standard (serial thick section) Electron Tomography. This allows for the structural visualisation of complete cells and the interactions of these cells with other cells or the extracellular matrix inside a tissue. As all life is built in 3 Dimensions, Serial Block Face Scanning Electron Microscopy (SBF-SEM) has now become the method of choice for a growing number of life science studies.

We would like to apply for a Scanning Electron Microscope (SEM) with an integrated diamond knife based microtome. We will integrate the technology into the Wolfson Bioimaging Facility, the centralised microscopy facility of the Faculty of Biomedical Sciences at the University of Bristol which houses both light and electron microscopy and is well-known for its Correlative Microscopy technologies.

We will apply the tool to study a variety of research questions that can only be studied in 3D. These include but are certainly not limited to the formation of synapses in the brain, the infiltration of macrophages into tissue, and the formation and release of platelets in the blood stream. Importantly this tool will allow us to also study the interaction of the extracellular matrix with cells, an area where 3D imaging is critical.

Last but not least we will use the microscope to advance our understanding in the Synthetic Biology field, one of the highlight areas of the BBSRC. Some of the Synthetic Biology research in Bristol focusses on the development of artificial extracellular supports with the idea to grow cells on, and ultimately to use these for transplantation. In order to study the interaction of seeded cells with such matrix supports the SBFSEM will be of critical importance.

Training of the next generation of scientists is an important aspect within the Wolfson Bioimaging Facility. As home of an international EMBO course and other national training courses the technology would be of special interest to seamlessly integrate into some of those training courses, providing students with training in the latest state of the art technology.

By sequentially scanning the block face of a sample and removing the scanned top layer, a large high-resolution 3-dimensional volume can be acquired. This allows for the structural visualisation of complete cells and its interactions inside a tissue. As all life is built in 3 Dimensions, Serial Block Face Scanning Electron Microscopy (SBF-SEM) has now become the method of choice for a growing number of life science studies. We would like to apply for such a SBF-SEM.
There are a number of SBF-EM tools available, each with its unique advantages but also disadvantages. Focussed-Ion Beam SEM allows for very thin sections to be "milled" off the surface of the block and then subsequently scanned. Although of the SBF techniques it allows for the highest resolution it also has a very limited area that can be imaged. We are very strong in serial section Electron Tomography which would cover that need. Therefore a solution that allows for larger volumes with still good resolution in x-y (5-10 nm) best suits our needs.

We would like to apply for a Scanning Electron Microscope (SEM) with an integrated diamond knife based microtome. Available systems currently are the Gatan 3View and the Volumescope from Thermo Fisher. The best-established configuration of Gatan 3View is with a Zeiss Merlin SEM and this is currently our preferred instrument. The Volumescope is less established but only last month a new model has been released including a very interesting deconvolution approach allowing for better z-resolution.

We will apply SBFSEM to a wide variety of projects but most importantly we will, as far as we are aware, uniquely apply it to Synthetic Biology research. In the production of synthetic scaffolds to grow cells on and ultimately use as implants it is of critical importance to analyse the interaction of cells with the scaffold. We will pay particular attention to the technical challenges of charging in such samples because of the "open" nature of such samples.

This application does not directly seek to develop new imaging tools. However, with the need to specifically look at charging issues in "empty" samples such as cell scaffolds in the Synthetic Biology Area, the development or adaption of resins could lead to significant interest from industry and academic parties. It is therefore of great advantage that the University of Bristol Vice Chancellor fellow Dr. Sara Careira who will be working on this aspect has a Chemistry background, has previous experience with EM by having worked in Prof. Verkade's lab and is currently based in Dr. Perriman's lab.

In addition, within the individual research projects there is significant potential for impact in the longer term. Our work addresses the basic function of all mammalian cells which therefore underpins our understanding of the healthy organism and age-related changes. Furthermore, a higher spatial and through correlative microscopy approaches also temporal understanding of cancer cell biology, synaptic function, platelet formation, and skeletal development and dysfunction highlights the importance of a full understanding of these pathways to guide possible future clinical intervention. While outside of BBSRC remit, these more clinical possibilities must be considered in the context of long term impact. It is always very hard to define in which way and on what timescales such impacts might occur, we can develop such lines through our impact plan. Through informing our basic understanding of a critical cellular process, it is most likely our work will inform long term projects in other fields including the pharmaceutical industry.

Potential applications of this work are identified from within the labs involved as well as by continuing liaison with our Research and Enterprise Department (RED). Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by the highly experienced team within RED; who engage closely with funders such as BBSRC when appropriate. As with all of our projects, this one includes considerable opportunity to train the researcher involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration with public communication and outreach programmes, for instance through visits to the EM unit of the Wolfson Bioimaging Facility from schools in our Widening Participation programme and direct training of secondary school teachers for Ongoing Career Development. The extensive network of University schemes is designed to benefit the training and development of research staff (Bristol is at the forefront of research staff development).

Several applicants have good track records in facilitating the placement of staff in areas outside our core research activity. For example, a previous postdoc in the Stephens lab undertook a period of flexible working in order to shadow some of our Research and Enterprise team and subsequently undertook a part-time course in intellectual property management and now works full-time for a company in this role; similarly Martin has facilitated the movement of a graduate student to a career in science communication (now performing this role at Columbia University in New York). This demonstrates that the environment as a whole is highly conducive to career development of our staff beyond academic, basic science research alone and thus contributes to the economic development of the nation. Our projects are also very data intensive- notably from imaging work - and the management and analysis of such large (terabyte) datasets is applicable to many areas of professional life. This work will lead to significant image data that is readily used in both public understanding of science and artistic arenas.