A Bessel beam light sheet microscope

Fluorescence microscopy has become a very important technique in the biosciences because it allows the distributions of specific proteins to be imaged. The resolution it provides is not as good as electron microscopy, but it has the major advantage that it can be used in live cells. However, the imaging process can still have a toxic effect on cells, and this is a major limitation in cell biology studies.

A popular way to minimise the effects of imaging on cells is called light sheet microscopy. This only illuminates the part of the sample that is in focus, meaning many more pictures can be taken without damaging the cell. Generally, this also makes the resolution worse - but an approach has recently been developed which not only illuminates using a light sheet, but also improves the resolution. This is achieved using Bessel beams, which use interference and are therefore not subject to the same limitations as most microscopy techniques. Using this lattice light sheet approach, the sample can be imaged quickly and with excellent resolution.

We will build a replica of the lattice light sheet system at Janelia Research Campus (where the technique was developed) in the Microscopy Innovation Centre (MIC) at King's College London, using blueprints and equipment lists provided to us by Janelia. The MIC has been created by KCL in recognition of the central role that microscopy plays in biological and biomedical innovation. It's purpose is to make advanced microscopy techniques available to users, and this is made possible by two experienced optical microscopy specialists. They will build the system, maintain it, and make it available to users. A particularly notable feature of this system is that it will have an extra module to allow specific areas of the samples to either be bleached or for fluorophores which can emit in two different colours to be switched from one to the other. We believe this would be the first system of its kind in the UK.

A wide range of different project applications have been proposed. These include three from groups who already travel to Janelia Research Campus to use the system there, so they are familiar with the equipment and sample preparation requirements. Projects will improve understanding of various basic biological processes: cell migration, cell division, how cancer cells invade, the characteristics of neurons which have been derived from stem cells, and the cytoskeletal structure and mitochondria in heart cells. The lattice light sheet microscope will let us image these live systems at very high resolution, and observe how they change over time, enabling new biological discoveries. After an initial evaluation period where the microscope is only available to users at King's, the microscope will be opened to users in other institutions, who would them be able to access this system with full technical support without travelling to the USA.

The ability to image cells at high resolution, without the the imaging process changing their behaviour, promises to vastly increase the amount of information available about cells. By making so much more information available, this microscope will help to maintain the UK's leading position in cell biology and biomedical research.

Fluorescence microscopy experiments face a number of limits: spatial resolution (in-plane and out-of-plane), time resolution, phototoxicity, and number of colours which can be imaged. Lattice light sheet imaging is an outstanding system for imaging small numbers of lives cells as it can image fast, in multiple colours, and with low phototoxicity. This is achieved using a Bessel beam for illumination, which uses interference to achieve a beam width smaller than that achievable by diffraction. However, this is at the cost of some experimental complexity and data analysis (the Bessel beams have side lobes and this must be taken into account when processing the data). The complexity of the technique has proved to be a limiting factor, with systems needing substantial technical support. Here we propose to create a clone of the system at the Janelia Research Campus (where the technique was developed), with two technical specialists to create and run the system. This will be used to investigate a number of different types of cells, with a focus on interactions between cells and other cells, or cells and their surrounding environment. The ability of the microscope to image the details of cells in 3D with almost isotropic resolution makes it particularly suited to detecting details of interactions between cells and their local environment which would not appear when using other techniques.

This project would create a lattice light sheet microscope with the capability to perform targeted photobleaching or photoactivation. The first beneficiaries of this system would be the fourteen co-Is whose projects are detailed in this proposal. They would all benefit from being able to use the resolution, speed and low phototoxicity of the lattice light sheet system to get new information about the biological systems they are interested in. For a substantial number of the proposed projects the information which will be obtained from lattice light sheet experiments is impossible to obtain using other techniques, and the researchers currently either travel to the US to carry out experiments or do not attempt to answer these particular biological questions. For these researchers, the availability of this microscope will be transformative, since they will be able to perform experiments and adapt their model or experimental approach on the timescale of a few weeks rather than six months as is currently the case when the facility at Janelia is used.

For all users, the microscope would enhance the quality of information available on the biological systems. This would enhance the robustness of the results and thus improve their impact. In general, the ability to perform measurements with low phototoxicity and photobleaching is very important to ensure that results are truly characteristic of cell behaviour. Similarly, the ability to image cells in 3D is important to the growing understanding that the local environment of cells can strongly influence their behaviour, and that therefore there is a substantial advantage to ensuring that the cells are in a 3D environment which approximates the environment they would experience in the body.

More broadly, the biological and biomedical community at KCL would benefit by having a new microscope capability available. The importance of microscopy has been recognised at King's through investment in a number of facilities including the Nikon Imaging Centre and the Microscopy Innovation Centre. Making a cutting-edge technique like lattice light sheet available in a facility setting will benefit those researchers with less microscopy experience. Participating in information sessions (which will be run by through the King's Imaging Network) will allow them to decide whether this particular technique will be of benefit to them, and set up communication channels which will allow them to plan their experiments effectively.

Lattice light sheet microscopes are still not commonly available, and the photobleaching/photoactivation option would be, to our knowledge, unique in the UK. When opened to the wider UK research community, this instrument would benefit those who want to obtain data about the 3D dynamics of cells, but for whom other techniques are either too low resolution or too phototoxic. From the enthusiastic response within KCL, we anticipate this would be a substantial number of researchers. This capability would benefit a broad range of the UK biological and biomedical community, with potential impacts in basic research, drug discover and personalised therapy.