Multi-dimensional imaging of cellular dynamics and function

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A key requirement in modern biology is the ability to image multiple structures at high resolution in three and four dimensions. Confocal microscopy provides an excellent solution, since it provides a means of imaging multiple different fluorophores in thin optical sections, eliminating the contribution of out-of-focus light to the image, and allowing the generation of 3 dimensional stacks of images. For living samples, this process can be repeated over time to provide information on the dynamics of a multitude of biological processes. Using multiphoton illumination, such analysis can be performed on thick samples, considerably extending the use of this important approach. This application seeks funds for a multiphoton-ready Zeiss LSM 510 META confocal microscope, to form part of the bioimaging facility within the Faculty of Life Sciences (FLS). This multi-user environment provides researchers within the FLS, other faculties, and outside users with easy access to a range of state-of-the-art microscopes. The excellent technical support and training provided within the facility means that this equipment is accessible to first time users as well as experts. However, demand for single photon confocal microscopy can not be met by our existing Leica SP2 AOBS. We therefore have an urgent need for a new confocal system that is suited to a multi-user environment. Since there are a number of users whose research is dependent on the multiphoton confocal, we are requesting a system to which we will attach our existing Spectra Physics Tsunami Ti-Sapphire laser. The re-use of this laser, coupled with the 50% discount provided by Zeiss and the 14.4 per cent contribution from FLS, combine to make this bid excellent value for money. Together with our existing Leica confocal, this equipment will form the basis of an outstanding imaging centre that will meet current and future needs and, in addition, represents a significant upgrade to current state-of-the-art technology for the multi-photon users amongst the applicants. The research projects outlined in this application fall into three main areas of research, all of which require access to single or multi-photon confocal microscopy. 1. We will investigate organelle function and dynamics in health, disease and apoptosis. A major focus will be the endoplasmic reticulum, how its function relates to organisation, and how this is altered during apoptosis. 2. We will use the ability of two-photon confocal microscopy to image tissues in order to visualise cells in natural and artificial 3D environments. The goal of this work is to understand better how cells generate 3D organisation, how this influences cell behaviour, and how this can be harnessed for tissue engineering and stem cell research. 3. Fluorescence resonance energy transfer (FRET) will be used to analyse protein-protein interactions during yeast cell growth and gene transcription, and in the triggering of apoptosis in mammalian cells.