High-resolution imaging of mammalian cells by confocal microscopy

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy


In animals, cells need to move during development and in response to infection or tissue damage. Cell movement is driven by the cytoskeleton, principally by filaments made of actin molecules, and also requires changes in the way cells attach to other cells or to molecules around them. We want to understand more about how proteins inside cells drive or inhibit cell movement, and therefore be able to more effectively use these proteins as targets for drug therapy against several diseases including chronic inflammatory disorders, heart disease, Alzheimer's and some cancers.

Technical Summary

The control of transmembrane proteins and associated cytoskeletal architecture at the plasma membrane is crucial to the regulation of numerous cellular functions including cell adhesion to the extracellular matrix, cell-cell adhesion in epithelia and endothelia, transendothelial migration of leukocytes, synaptogenesis and axon guidance. The spatial and temporal regulation of these events is tightly co-ordinated and can only be accurately studied using microscopy approaches, ideally in living specimens. Laser-scanning confocal microscopy allows the targeted analysis of events occurring at specific focal planes in the cell. This enables precise movements of molecules to be studied both spatially and temporally. Recent advances in technology have resulted in the development of laser scanning systems, that allow functional assays such as photo activation, FRAP (Fluorescence Recovery After Photobleaching), FRET and FLAP (Fluorescence Localisation After Photobleaching) using fluorophore-tagged proteins to analyse the dynamics of protein interactions and movement within cells. This application proposes the purchase of a state-of-the-art four-laser line confocal microscope to allow tracking of proteins in 3 dimensions over time in a living cell. We aim to employ this system to study a range of diverse membrane receptors and cytoskeletal proteins and their relative dynamics during cell adhesion, migration, synaptogenesis in brain slices and axon guidance of neuronal growth cones.
Description The confocal microscope (purchased from Nikon) has been and is being used to image live and fixed mammalian cells. In live cells, it is being used to track the movement of proteins tagged with fluorescent proteins such as green fluorescent protein (GFP). The advantage of this confocal microscope is that it is possible to track proteins that move rapidly, and also study protein localisation at very high resolution.
Exploitation Route It could be used for school groups to visit and learn about microscopy. Our success in working closely with Nikon to optimise the use of this confocal microscope has contributed to the set-up of a Nikon Imaging Centre at King's College London (http://www.kcl.ac.uk/innovation/research/corefacilities/smallrf/nikon/index.aspx), which provides multiple different microscopes available for all researchers at King's College London. It will also be available for visitors from other institutes/universities in the UK to see the latest Nikon microscopes and to use them.
Sectors Healthcare

Description Our findings using this microscope have been used in publications in journals.
First Year Of Impact 2009
Sector Healthcare