Grant: Research Tools and Instruments

No technology has had a larger impact on our understanding of cells than the microscope. The ability to magnify cellular features to sizes viewable by humans has allowed researchers to unravel the structure, function, and behaviour of cells ranging from bacteria, to plants, to those which make up our bodies. Over the past 100 years there have been many advances in microscopy which have added to our ability to probe the behaviour of cells. Attaching fluorophores-molecules which emit specific colours of light-to a cell allow us to visualize how specific cellular features function. Specialized techniques allow us to visualize living cells and how they respond to their environment. But while these techniques are powerful, they all share one limitation: as light passes through lenses it becomes distorted, which places a firm limit on how small of an object we can observe. This "resolution limit" prevents us from seeing cellular structures smaller than 0.25 mm, but a lot of important biology occurs in regions of cells smaller than this. New techniques can overcome this resolution limitation, allowing for smaller cellular features to be observed-in some cases, even allowing individual molecules to be observed within cells. This proposal seeks to upgrade an existing microscope to add three new imaging approaches which will enable imaging these minute features. The first - SRRF microscopy - more than doubles the maximum possible magnification while imaging living cells, allowing for minute structures such as the interior of bacterial cells to be resolved. The second technology, dual-colour single particle tracking, allows for the diffusion (movement) of two different molecules to be directly observed as they make their way through living cells. This will allow us to determine where in cells specific metabolic and molecular processes take place, and to directly image molecules as they function within living cells. Finally, we will add smFRET imaging, which allows us to directly detect when two molecules interact by detecting the transfer of energy which occurs when two molecules collide. This will allow us to investigate inter-protein interactions and enzymatic activity-but instead of performing these experiments in a test-tube, we can instead make observations within the context of these molecules native environment - a living cell. To enable these abilities we propose to upgrade our existing microscope. This upgrade includes a new camera that can operate at the speeds required to image moving molecules (50-100 frames per second), a new fluorescent light source which will allow us to image a broader range of fluorescent molecules, better optics to enable the "breaking" of the resolution limit, and a dual-view system which allows for two different molecules to be imaged simultaneously. This technology will be housed in a core facility where it will be accessible to over a hundred researchers from labs across Western University and South-Western Ontario.