A lattice lightsheet microscope for imaging highly dynamic processes in living cells and organisms.

Light microscopy lies at the heart of biological research. Ever since the light microscope was invented in the seventeenth century, it has been revealing profound insights into how cells function, grow, divide and die, and how individual cells work together to generate tissues and organisms. The development of fluorescent probes for studying protein function in living cells coupled with extraordinary progress in microscope design have signalled an era of unprecedented insight into cell function via light microscopy.

This has been marked by two recent Nobel Prizes. One was awarded in 2008 for the discovery of green fluorescent protein from jelly fish, which can be joined to a protein of interest by genetic engineering. The chimeric protein can then be expressed and observed in living cells. Since then, proteins that fluoresce in different colours have been identified, allowing researchers to follow multiple proteins in the same cell at the same time, providing vital information about cell behaviour. The second Nobel Prize was awarded in 2014 for the development of super-resolution microscopy. These methods gave a way of seeing structures with great level of detail than possible using diffraction-limited techniques, where the resolution is set by Ernst Abbe's nineteenth century equation.

One of the 2014 winners, Eric Betzig, has gone on to design a microscope called the lattice lightsheet microscope (LLSM). This technological breakthrough has many advantages. It captures images in 3D very rapidly, meaning that cellular structures that move very fast inside the cell can now be followed in 3D. For slower-moving structures, it offers the option of super-resolution imaging within living cells. Lastly, and perhaps most importantly, the lattice light-sheet illumination is very gentle on cells, tissues and organisms, because it causes minimal photo-damage. This means that cellular processes can be followed for longer times than previously possible, and at faster speeds and higher resolution.

This ground-breaking technology is now commercially available from Intelligent Imaging Innovations (3i), and here we propose to use the LLSM to image a wide range of different cellular structures within living cells over timescales ranging from minutes to days. We will be able to analyse the behaviour of fast-moving components such as endosomes and mRNA particles, and the cargoes transported by fast axonal transport in nerve cells. We will also image structural components of the cell's cytoskeleton - actin filaments and microtubules - as they work in processes as varied as cell division, migration and cell-cell communication. The LLSM's ability to image samples of different thickness will allow us to follow these processes in samples ranging from single cells through to cells in tissue samples or 3D cultures. In addition, we can use it to watch cell behaviour in developing embryos of fruit fly and zebrafish. The LLSM will benefit at least 24 groups of highly productive scientists holding significant BBSRC funding. This will also enhance the training of the next generation of researchers in a sophisticated light microscopic technique and the data analysis needed to interpret the results. In addition, the quantitative data generated will be used enhance collaborations between biologists, mathematicians and computer scientists, so promoting interdisciplinary research.

Lattice lightsheet microscopy (LLSM) is a recently developed advanced imaging technique that is revolutionising live cell imaging in 4D by virtue of its speed, resolution and the minimal photodamage it causes. It is ideal for many groups in the Faculty of Biology, Medicine and Health who use imaging to study a wide range of biological questions. We propose purchasing a system from the sole commercial supplier, 3i. The microscope will be located in our well-established Bioimaging Facility. LLSM will transform our ability to investigate a wide range of cell and developmental biological topics including: i) Function and dynamics of the endocytic pathway; ii) Neuronal development and function; iii) Mitotic spindle assembly and positioning; iv) Immune cell migration and function in tissues and in vivo; v) Signalling and gene expression in living and apoptotic cells.

These studies will benefit from the key advantages of LLSM.
1) The very rapid rate of acquisition of high resolution images in 3D allows visualisation of very small rapidly-moving sub-cellular structures such as endosomes and mRNA granules throughout the cell volume.
2) Structures that move more slowly, such as microtubules and actin filaments, can be imaged at enhanced resolution in SIM mode.
3) The lattice lightsheet illumination ensures very little out-of-focus illumination, and delivers a minimal total light dose, thereby minimising phototoxicity. This allows living samples to be imaged at high temporal and spatial resolution for far longer than previously possible.
4) The flexible sample mounting allows a wide range of samples to be imaged. These range from single cells (mammalian cultured cells; mouse and Drosophila neurons; yeast), through cells in complex 3D environments in vitro (organotypic cultures; human lung tissue samples; Drosophila larval and adult brains; chick brain slices), to cells in living organisms (Drosophila ovarian stem cells and macrophages; zebrafish kidney cells).

The requested lattice lightsheet microscope will support the research of a large number of users whose projects address biological and physical questions. Bioimaging is of key strategic importance to UK science, as clearly described in the BBSRC Strategic Review of Bioimaging. It falls under the remit of UKRI Technology Touching Life, and closely aligns with the drivers for BBSRC Exploiting New Ways of Working, by supporting the "strengthening of the skills base is required in order to embed the latest equipment in facilities, and enable multidisciplinary research". Extending the reach of this cutting edge technology to researchers in the UoM and across the North, will substantially enhance the delivery of BBSRC-funded world-class bioscience.
There are a wide range of direct and indirect beneficiaries:
(1) Biotechnology and biosciences. LLSM data contributes to research that will reach a broad audience across disciplines, including biomedical sciences, biophysics and tissue engineering. It will lead to the development of Biotechnological tools, ranging from cell lines stably expressing fluorophore-tagged proteins (that may be valuable for screening materials and drugs affecting cellular behaviour) to the development of new reagents, devices and sample preparation methods that promote imaging. We expect potential impact in the biotechnology area and will actively search for relevant systems/companies to share our knowledge. The impact will be direct and mid-term.
(2) Industry. We will develop a collaboration between Hamamatsu and 3i to test the applicability of new camera technology for LLSM (MW has a longstanding collaboration with Hamamatsu that has involved the loan of equipment and support for symposia and training courses). LLSM is also of direct relevance to an existing collaboration with Syngenta that is aimed at finding new ways of analysing the effects of pesticides on cells. Other projects in the Faculty relate to cell behaviour under changing mechanical and biochemical environments. Unravelling the mechanisms through imaging will provide a starting point for the development of pharmaceutical products influencing cellular responses to changing environments in healthy or diseased tissue. These impacts will be direct and short- to long-term.
(3) General public. Images generated from this project are colourful, intuitive, attractive and make science more accessible. They are useful for educating the public, and particularly capture the imagination of children, who will be encouraged to engage with science through the school sessions run by the co-applicants. The breadth of science covered by this proposal will show how disciplines can be integrated to deliver tangible benefits for society, in terms of finding new ways to understand and treat disease as well as to develop new materials. The general public of the UK and beyond will also benefit indirectly from improved quality of life, as discoveries pertaining to the processes maintaining health are translated into new healthcare solutions or policy guidance. These impacts are both direct and indirect, from short- to long-term.
(4) Researchers from various backgrounds. LLSM datasets will be used to drive new collaborative projects with computer scientists and mathematicians. The development of novel sample handling methodology and experimental approaches for LLSM is equally relevant for biosciences and engineering. Data analysis tools developed by users will be shared freely. The impact will be direct and immediate.
(5) Staff working on the project. Researchers will work in an interdisciplinary way, interact with scientists from different backgrounds and with companies. A direct outcome is training users in computational and mathematical approaches, so fulfilling a core BBSRC goal. They will further develop communication, problem solving and entrepreneurial skills, which will be useful in any later profession. The impact will be direct and evident in the short-term.