High throughput high resolution 4D imaging

A fundamental requirement for modern cell biology research is the ability to visualise, with high resolution and sensitivity, the localisation of proteins and structures within living cells. Over the past decade rapid advances in digital imaging and microscopy technologies, allied to the development of photoactivatable fluorescent proteins, has revolutionised the capacity to undertake such studies. These advances have led to new insights into the highly dynamic nature of molecular and biochemical processes occurring within living cells. Indeed, the visualisation and quantitation of proteins (often resolved to the level of a single molecule), in both a spatial and temporally resolved manner, is increasingly viewed as indispensable in cell biology studies. As a result of successful funding under two previous REI bids, we have created , a DeltaVision RT microscope facility within the Department of Biological Sciences at Lancaster University that provides essential bioimaging support to six research laboratories. The research projects undertaken in this facility are firmly within the BBSRC's remit and strategic priorities and many of the principal investigators involved have an established track record of attracting BBSRC funding. However, this DeltaVision RT imaging system is aging and so we are requesting funds to upgrade this microscope facility to a DeltaVision CORE imaging system (Applied Precision). Through a combination of improved software, optics and hardware this new system will deliver significant advances to our bioimaging capabilities and help to maintain our research competitiveness.

The technical improvements provided by DeltaVision CORE include a redesigned 10 position excitation filter wheel and 6 position emission filter wheel and long-life Xenon light source to provide over 1000 hours of continuous illumination. These features allow multi-channel image acquisition from an extended number of probes over increased time periods; important considerations for the increasingly demanding 4D live-imaging experiments undertaken in the applicants' laboratories. The EMCCD camera upgrade requested, allied to the improved control system and deconvolution algorithm integral to the DeltaVision CORE system, will considerably improve the sensitivity, spatial resolution and speed of image acquisition during live-cell imaging experiments. This increased sensitivity will considerably reduce the amount of excitation energy needed to visualise fluorescently labelled proteins within live cells, and so greatly reduce the phototoxic effects of fluorescence illumination and increase cell viability. In essence this offers far better temporal resolution meaning that it extends experimental lifetime either through the collection of more images over shorter time windows or alternatively running experiments over extended time periods. As well as considerably improving our existing DeltaVision imaging system, DeltaVision CORE delivers additional capabilities at present lacking in our bioimaging provision. These include a long travel stage and a provision of Total Internal Reflection Fluorescence Microscopy (TIRFM) capability. The extended stage will enable the use of multi-chambered slides in high-throughput fluorescence imaging screens while the TIRFM capability provides enhanced signal-to-background ratios and will be of considerable use in the detailed examination of cell surface phenomena including single molecule dynamics, protein-protein interactions, receptor-ligand behaviour, vesicle trafficking and cell surface structures such as focal adhesion contacts.