In the past, the analysis of the cellular events and molecular interactions of pathogens with their hosts was limited by the availability of suitable antibodies to detect particular proteins, or by inference from biochemical studies. These approaches, however, represent experimental 'autopsies' in which dynamic cellular processes must be deduced from a fixed sample. Developments in the imaging of molecular events in live cells, however, now mean that it is possible to follow, in real time, the location, movement and interactions of molecules within cells, and the interactions between cells- for example when a virus or bacterium infects cells of the body. These developments have been made possible by the engineering and expression within cells of proteins fused to naturally fluorescent proteins and by improvements in microscope technology. In particular, current confocal microscope systems enable both conventional high resolution positioning of proteins within fixed samples and the detection of fluorescent proteins in live samples under conditions that limit the cell damage that results from fluorescence illumination. The application of live cell imaging technology is particularly powerful for analysis of the interactions of a pathogen with its host, or host cell. Recently, it has become technically possible to engineer infectious agents such as viruses, bacteria or protozoan parasites to express one or more proteins conjugated to a fluorescent protein. When combined with existing technology for the expression of distinct fluorescent proteins within a mammalian host cell, it is possible to track molecular interactions between the pathogen and host, or track the infection pathway as a pathogen moves through distinct host cellular compartments, for example. These technologies have very wide ranging applications in infectious disease research, with researchers comprising this application being interested in such diverse processes as: - The complete interactions of viral proteins with other viral proteins and the proteins of the host cell. - The use of fluorescently labelled viruses to track the infection process in host cells and tissues. - The presentation of antigens by antibody producing cells to the immune system and the signalling events and protein interactions of immune effector and regulatory cells. - The infection process, developmental biology and evolutionary strategies of protozoan pathogens such as Leishmania, trypanosomes and malaria. The advantage of this technology- the ability to image live pathogens and their interactions with host cells- also presents a limitation- the need for safe containment of the pathogen. In consequence facilities available routinely in University departments are not available for use with infectious agents. This application proposes to establish a live cell imaging facility for infectious agents housed under suitable conditions for effective and safe containment. The equipment requested, a Leica TMC-SP5, has the capability for high resolution imaging of fixed and live samples and of high-speed capture, at excellent resolution, of dynamic cellular events. The equipment will be available to a large group of researchers contained within the Centre for Infectious Diseases at the University of Edinburgh, and provide a high-end facility with the potential to encourage resource, technology and knowledge sharing among a far wider group.

Researchers at the University of Bristol School of Biological Sciences are using confocal microscopy to obtain high quality images of processes in plants, animals, and yeast. These images provide crucial information that can help us to understand how biological systems work. We request funds for a new confocal microscope to investigate biological questions including: how cells specialise and grow in plant roots, how guard cells on leaves open and close to control the flow of water and gases, how wheat grains form, how pollen and stigmas interact during fertilisation in flowering plants, and how insect ears work. At the moment we use confocal microscopes in other buildings at the University or in other cities or countries to do this work, but it is difficult or impossible to transport delicate experimental material safely and without damage. We request a contribution towards a dedicated microscope for the Department. This bid is supported by funds from within our Department and a substantial discount from the manufacturer.

Laser microdissection (LMD) is a relatively new technique that is revolutionising the way scientists analyse biological processes. It allows researchers to examine a specimen under the microscope, cut out a precise area of interest (a single cell, perhaps, or a collection of cells) with a laser, and then collect the excised sample in a tube without contaminating it in any way. Once the sample has been captured, it can be subjected to a wide spectrum of chemical analyses as required. These can include analyses of DNA, RNA, proteins and the thousands of other cell chemicals that are the foundation of biological processes. In the case of many chemicals (DNA and RNA, for example) routine methods now allow researchers to work with the tiny amounts of these substances that can be isolated from single cells. Although all cell types share their basic chemistry, it is the subtle differences between them that give them their unique character and functions. They express different genes, build different proteins and contain different suites of active molecules. Understanding these differences and their purpose is central to many areas of the biological sciences. Without the LMD technique, researchers often have to work with samples containing many different types of cells. This means that the unique character of individual cell types is difficult or impossible to discern, because it is masked by the presence of several types of cell. As a result, it is often difficult to study specific localised events such as cell death, fungal infection or transport of proteins or sugars in or out of specific cells. Although single cells can be sampled with a micro needle if they are easily accessible, many cells are hidden within tissues and cannot be reached directly. LMD answers these problems by allowing easy access to any part of any material that can be cut into thin slices and mounted on a microscope slide. Because the slices are prepared from quickly frozen tissue (using a supplementary piece of equipment, a freezing microtome), cells suffer the absolute minimum of disturbance before examination and laser dissection. Access to these cells via LMD will help IGER researchers to understand key biological processes with greater precision. IGER carries out a wide range of research designed to underpin the targeted development of new plant varieties and agricultural methods, which are better for farming and better for the environment. Other things being equal, increased precision in research will accelerate the pace of discovery and development. Many research projects will benefit directly from the new equipment. These include studies aimed at understanding the roles of specific genes in plant growth and development, studies on plant responses to stress, and studies on the interactions taking place when plant material is broken down in the rumen of farm animals. These research programmes are central to key aims of UK science, including development of renewable energy for biofuels and the development of sustainable agriculture. LMD will be used to analyse gene expression in specific plant cells, focusing on areas such as plant senescence following stress. It will contribute to ongoing studies that are examining the way different types of cell process carbohydrate in the leaves of grasses. Studies on signalling between plant cells during fungal invasion will also benefit. LMD will help researchers study colonisation of plant material by microorganisms in the rumen, a key part of digestion in ruminant animals. Specifically, there are plans to follow gene expression during colonisation and to understand what microbial species are involved.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

The development of the first practical confocal laser scanning microscope in the 1980's was a major advance in optical microscopy. These microscopes allow researchers to obtain optical sections from intact specimens up to a depth of several hundred microns, and are now widely used in plant biology for the visualisation of biological processes and ultrastructure in intact and living plant tissues. A successful JREI application in 1998 led to the installation of an early generation confocal microscope in the Department of Plant Sciences in the University of Cambridge. As a result, plant researchers here have contributed heavily to the early development and application of confocal imaging techniques. Our existing facilities need upgrading in order to maintain our lead in the development of new fluorescent proteins, transgenic marker lines in Arabidopsis, algal and crop systems, new microtechniques for 3D imaging, software for image segmentation, visualisation and quantitation, and the application of advanced optical techniques to the analysis of cell, protein and metabolite dynamics in a wide variety of algal and plant systems.