Dynamic Bioimaging Facility for Cells and Cell Surfaces

This research proposal would create a novel instrument which would combine Physics and Biology, and which would make it possible to simultaneously record several types of information from biological cells. The Atomic Force Microscope is a nanometre-resolution microscope, which physically 'touches' the surface of the cell. It is sensitive to the shape and structure of the outer layer of the cell, and produces a 3-dimensional map of the cell surface. Confocal microscopy on the other hand is a powerful light microscope technique, in which the focal point of a laser beam can be controllably moved throughout the 3-dimensional body of the cell, and therefore we can build up a picture of the inside of the cell by moving this focal point around inside the cell. If we are able to record information from these 2 microscopes at the same time, we can correlate directly features on the cell surface with internal cellular properties. One example of this would be to record fluorescence from membranes whilst obtaining a surface map. Another application would be to purposefully indent the cell with the tip of the AFM probe, and record the response of the cell, as its internal skeleton adapts to the stimulus on the outside surface. We will also combine these two microscopes with a technique called electrophysiology which will give information on the electrical signals crossing the surface of the cell. Ultimately the information we obtain will give us more insights into cell processes and specialised functions, how cells communicate with one another, and also how they interact and adapt to their environment.

This is a multi-user, multi-project interdisciplinary research consortium targeted on the imaging of cells and cell surfaces. A combined Confocal Microscope and Atomic Force Microscope (CM-AFM) will be developed and sited in the new Interdisciplinary Nanoscience centre in Bristol. Drawing extensively on the expertise of the Bristol Physics AFM group in instrumentation, the consortium also includes cell biologists from three University of Bristol Departments: Ophthalmology, Biochemistry and Physiology. It is planned that this initial user base will grow significantly. The initial research aims are targeted on a number of biophysical problems which will benefit from a combined approach. In Physiology, we will exploit CM-AFM and patch-clamp electrophysiology, to learn how cilium bending is coupled to ion transport; he results of our study will provide new insight into the molecular biophysics of the primary cilium and the regulation of transepithelial ion transport in the kidney. We will also use a modified AFM tip to actuate bundles of Outer Hair Cells (OHC), using high-resolution optical microscopy to locate and direct the AFM probe, and simultaneously record electrophysiological signals to reveal activation and adaptation kinetics of these cells. In Ophthalmology, a number of projects will include visualization of the cell-substrate cleft to understand better the cell movement and cytoskeletal changes, monitoring of membrane-bound thrombospondin, mapping of molecular adhesion if mucous epithelia, and conformation changes with temperature of gap junctions. In Biochemistry, we will study the specific anchoring mechanism of an integral membrane protein, BST-2, believed to be involved in stabilisation of lipid rafts.