Studying Single Molecules on living cells

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• Dr David Klenerman (Department of Chemistry, Cambridge)
• Friday 17 November 2006, 11:00-11:30
• Kaetsu Centre, New Hall.

If you have a question about this talk, please contact Duncan Simpson.

Watching Living Matter

One major challenge in biology is to understand how the individual molecules and complexes of the cell are organised and interact to form a functional living cell. To address this problem new biophysical tools are needed. One method that we have developed for functional nanoscale mapping of the cell surface is based on a scanned nanopipette. This allows high resolution, non-contact imaging of the soft and responsive cell surface using the ion current that flows between an electrode in the nanopipette and bath for distance feedback control.1 We have used the nanopipette to perform patch clamp at specific positions on the cell surface to study single ion channels.2 We have also combined high resolution topographic imaging with simultaneous recording of the fluorescence from the cell surface.3 The pipette can be used for controlled voltage driven delivery and deposition of biomolecules down to the single molecule level1 and this is being used to probe the structure of the cell membrane using single molecule fluorescence tracking. Recently we have made a major advance in the resolution of the topographic images, by scanning with fine quartz pipettes, so we can directly visualise protein complexes on the surface of live cells. To determine the oligomerisation state of proteins on the surface of living cells we have used two colour single molecule coincidence detection based on the excitation of two distinct fluorophore labels on proteins with two lasers focussed to the same spot.4 This method requires no prior knowledge of the structure of any complex formed or control of fluorophore position on the molecule. We show that this method can distinguish between protein monomers and dimers on the cell surface.

This talk is part of the Physics of Living Matter PLM6 series.

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