A multi-user light-sheet microscope for Bateson Centre researchers, University of Sheffield scientists, partners and collaborators

Research in the Bateson Centre is concerned with how the body develops across the life course, from the embryo to the adult. We wish to understand the genetic and cellular mechanisms that direct the development of a single cell - the fertilised egg - into a fully functioning mature body. We use a number of different animal model systems in our research. These include the zebrafish, which has a transparent embryo and is ideal for imaging; the fruit fly Drosophila, with its superb genetics; the chick, which has a large embryo that is easy to manipulate; and the mouse, which, as a mammal, has the closest parallels to our own development.

To follow dynamic processes, both during embryonic development and in the adult organism, we need to image samples in real time, using fluorescent proteins to light up different cells or tissues. Conventional microscopes, such as the laser scanning confocal microscope, are very good at detecting this fluorescence, but use a high intensity laser beam to illuminate the sample. This creates a number of problems, limiting the type of experiments that we can do. Firstly, the laser is focused onto a tiny area and scanned over the sample to build up a bigger image. This is time-consuming, and makes it difficult to image larger three-dimensional structures. The high intensity laser can also weaken or bleach the fluorescent signal, and can heat and damage cells. These factors limit the time for an experiment, and are likely to disrupt the very processes that we want to understand. Finally, because a conventional confocal microscope illuminates the sample from above, the image can be distorted by out-of-focus light.

The light-sheet microscope offers enormous improvements over existing confocal microscope systems for longer time-lapse experiments and imaging of larger objects: we are requesting funds to purchase one of these machines to support a whole variety of research projects in the Bateson Centre and elsewhere. In a light-sheet microscope, the sample is illuminated with a thin sheet of laser light from the side. No laser scanning is required to form a two-dimensional image, as information is captured instantaneously from the entire area that is illuminated. This vastly reduces the time required to capture three-dimensional information, meaning less bleaching and less damage to the sample. The amount of out-of-focus light is reduced to a minimum, giving a beautifully sharp and clear image.

A few specific examples will illustrate the range of different projects that we intend to pursue. Calcium signalling is one example: cells use calcium ions to respond to the external messages they receive, effecting changes in cell behaviour such as movement or adhesion to other cells. We use fluorescent reporters that flash on and off when calcium signalling is active. The light-sheet microscope will enable us to follow large numbers of cells over time, to identify these transient signalling events in the entire cell population. We will also use the light-sheet microscope to image complex and larger three-dimensional structures, including cultured tissues for the study of stem cells, and whole developing organ systems in the live embryo, such as the intricate labyrinth of the inner ear or the developing blood vessels.

The microscope will be housed within the University of Sheffield Light Microscopy Facility, and will be made available to all researchers at the University of Sheffield and further afield. Outside the Bateson Centre, other users are drawn from a number of different academic departments, including Molecular Biology and Biotechnology, Animal and Plant Sciences, and Mechanical Engineering. Some of our projects involve national or international collaboration (academic and industrial) and so these partners will also benefit. We therefore believe that the microscope will be very heavily and efficiently used, and that the proposal is excellent value for money.

This application is for funds to purchase a single plane illumination microscope (SPIM) for use by multiple research groups from the Bateson Centre, University of Sheffield science departments and beyond. The Centre is a cross-disciplinary grouping of basic and clinician scientists who use model organisms to study fundamental processes of embryonic development, adult tissue maintenance, and as models for disease. The equipment will be housed in the University's world-class Light Microscopy Facility, supported by excellent staff, computing and infrastructural facilities provided by the University of Sheffield.

Light-sheet microscopy offers faster acquisition, gentler illumination and deeper penetration than conventional laser scanning or spinning disk confocal microscopes, while retaining excellent (sub-cellular) resolution. It is thus especially useful for time-lapse studies of living material, capture of rare cellular events over a large field of view, and 3D imaging of large (mm-sized) objects. SPIM generates very large datasets (up to 1Tb/day), generating particular challenges for data storage and transfer, and so we are also requesting computing support in the application.

The Bateson Centre is home to one of the largest zebrafish groupings in the world. Light-sheet microscopy is a really exceptional technique for imaging this particular organism; adding this new technology to our imaging suite will open up experimental approaches that are currently not possible with our existing equipment. Groups working with different model systems in the University of Sheffield, together with our collaborators and partners, will benefit from shared access. We intend to put the microscope to use in a whole variety of basic science and applied projects, including the imaging of neural activity in the whole brain, analysis of stem cell behaviour in mammalian hypothalamic ex vivo cultures and whole organism imaging of the developing vasculature at cellular resolution.

Academics:
Light-sheet microscopy provides a unique niche in the expanding field of bioimaging. A wide range of researchers at Sheffield will benefit from the new experimental opportunities the SPIM provides through its ability to produce 4D images at the tissue, cellular and sub-cellular level at high resolution. Sheffield is a centre of excellence for Biological Sciences, ranked joint 3rd for research in the RAE 2008 (source: Times Higher Education). Advances in research through using the SPIM will be shared with academics in the UK and world-wide through collaborations, presentations at meetings and publication in high impact journals. A broad spectrum of research areas will benefit from using the SPIM and therefore the impact of the research generated will be wide-reaching.

Other SPIM users will benefit from new protocols for sample preparation and image capture that will be developed, optimized and shared through designated web pages and regular network meetings. Analysis of the data generated by the SPIM will facilitate interdisciplinary collaborations between U. Sheffield researchers, image analysis experts and modellers, and is likely to generate new software that will be widely applicable to this expanding field.

This project will also provide an excellent training opportunity for the RA involved, including specialist training in the SPIM microscope and associated software, general microscopy and image analysis, data management and storage, as well as developing project management skills. U. Sheffield has an excellent staff development and review programme to further career potential. In addition the SPIM microscope will enhance microscopy and imaging in the PhD training programme.

Commercial sector:
The SPIM microscope currently has only one commercial retailer (Zeiss); however, it is likely that the increased demand for such microscopes will lead to further advances in this technology and an expansion in the microscopes on the market. We will work with Zeiss to optimize and expand techniques using their SPIM. Big data generated from bioimaging projects will also benefit data storage manufacturers and data archiving companies such as Arkivum by increasing usage and bringing down costs. Individual PIs also have collaborative links with a number of different companies who will benefit from access to the research data generated by the SPIM.

Health sector/Charities:
A future goal for research is to impact on health and wellbeing. We envisage that research developed with this new technology will generate data that will feed in to a clinical setting from many of the different projects described in the Case for Support. The Bateson Centre focuses on biological research underpinning health and wellbeing. Many of the Co-Is work on basic biological areas that will impact on human health, including cardiovascular disease, cancer and neurodegenerative disease, and have links with relevant charities including the British Heart Foundation, CRUK and Action on Hearing Loss.

The public:
One of the goals of biological research is to further our understanding of how our bodies work: this is an area that the general public find fascinating, and we recognise the importance of passing on our knowledge. The Bateson Centre and University of Sheffield run a very well established outreach programme that engages with the public at many levels, including school visits and workshops, charity visits and the U. Sheffield programme of public engagement. We have had excellent feedback from all these activities. Our outreach events lead to a better informed public and can inspire the next generation of scientists, while providing us with an external viewpoint on the work we do.