Confocal Microscope for Centre for Cell Imaging

Live cell imaging has emerged as a very important technology for the study of the functions of genes and cells. In the Centre for Cell Imaging we have established a facility that specialises in timelapse microscopy looking at the same cells as they change, over time periods from seconds to days. This work involves the use of fluorescent labels to follow biological processes. Our existing microscopes are ageing but are extremely heavily used by a very large number of scientists from different disciplines, Departments and Institutions. We need a new system to take advantage of improvements that have occurred since the last confocal microscope was purchased 6 years ago. Since then, electronics and detection capabilities have been improved substantially. During this period, we have developed new methods that allow us to perform many experiments in a single dish containing a 'cell array' which consists of spots of around one tenth of a millimetre which are placed on a glass surface next to each other. Each spot has about 50 cells on it which have had different genes (or drugs) introduced into them. The new microscope will allow us to look at more spots every day . The improved speed is mainly because the new microscope will spend less time automatically setting the focus as it moves from spot to spot. We will also connect this microscope to a new computer controlled laser (purchased separately) that can excite fluorescence using intense pulses of light, where each photon carries half the energy normally used to excite fluorescence. Compared to our previous system, this microscope will be far more flexible and powerful, since the intensity of light that can be shone onto the cells will be far greater and there is new equipment for more efficient detection of the signal. The microscope will be used to study a variety of processes including: the timing of protein movement in cells, protein stability and protein interactions and gene expression. Protein interactions have been difficult to study in timelapse experiments and it is becoming very important that we understand which proteins bind to each other and when and where they do this inside cells. The research objectives lie in a broad area of applied biology. We are interested in how the timing of signals may change when genes (and which genes) are switched on in cells and how this affects cell division and cell death. We have discovered that NF-kappaB, one of the most important signals in the cell moves into the cell nucleus repeatedly suggesting that the timing of movement may control cell functions. We want to study this further and to relate this to other important signalling systems in the cell such as p53, the 'guardian of the genome' a tumour suppressor protein that is changed in most cancers. The information that we get will be fed into computer models of these systems because they are so complicated that it is difficult to understand in any other way, exactly what is going on. We will also study related projects in the areas of neurobiology, endocrinology and glycobiology. The research that this equipment will support is substantially funded by BBSRC and also by other organisations. There is a strong record of collaboration with industry and excellent strategic support for the Centre for Cell Imaging from Liverpool University.

The Centre for Cell Imaging has an established track record in pioneering the development and application of novel timelapse live cell imaging techniques. The facility at present consists of 3 widefield microscopes (mainly used for luminescence imaging) and 3 confocal microscopes. Our existing multiphoton microscope is housed in a darkened room for luminescence imaging, with the lasers outside the room to allow dual luminescence imaging. The current manually tunable multiphoton laser is therefore fibre-optically coupled to the microscope. This meant a compromise with incident light intensity compared to a direct laser connection. The microscope was purchased before direct descanned detectors became available. Therefore both illumination and detection efficiencies are lower than could now be achieved. Zeiss made available to us a Becker and Hickl fluorescence lifetime system. However, this ideally needs direct coupled multiphoton laser and direct detection. Since the last two confocal microscopes in the Centre were funded in 2000, we have concentrated on maximising the efficient use of time on the microscopes. The three confocal microscopes are used almost 100% for fluorescence and luminescence timelapse experiments, 7 days per week 24 h per day and time is allocated up to a month in advance. Through a DTI Beacon project we have developed live cell imaging of reverse transfected cell arrays as a method of increasing the number of parallel timelapse experiments that can be performed at the same time. We have substantially improved the computer facilities for data capture, handling and storage by establishing a local gigabit local area network and our own RAID storage. We have also written new automated timelape image analysis software. We request funds for a new LSM510 confocal microscope equipped for live cell imaging. Through an associated but independent purchase we will directly link the microscope to a computer controlled chameleon multiphoton laser.