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

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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.