Probing the structure of the extracellular matrix using gold nanoparticle based single molecule microscopy

• Main Author: Nieves, Daniel
• Other Authors: Fernig, David Garth; Levy, Raphael; Yates, Ed
• Published: University of Liverpool 2013
• Subjects: 570; QP Physiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617452

The observation of single biomolecules via optical microscopy eliminates all the implicit averaging of ensemble techniques and thereby provides access to the heterogeneity of molecular systems that will be the key to at least some biological functions. The implementation of photothermal microscopy at the University of Liverpool to achieve the detection of single gold nanoparticles over long times at high signal-to-noise-ratio is presented here, along with the development of Photothermal Raster Image Correlation Spectroscopy, PhRICS. PhRICS was shown to be equally effective as Photothermal Absorption Correlation Spectroscopy, PhACS, in the determination of the hydrodynamic diameter of colloidal gold nanoparticles in solution. The use of gold nanoparticles as labels for biomolecules has been of great interest due to their favorable optical properties and surface chemistry. The development of a new strategy for the covalent biofunctionalisation of gold nanoparticles with a single maleimide group is described. Nanoparticles functionalised this way were used to label FGF-2 protein and heparin-derived oligosaccharides. Both the PhRICS and the new nanoparticles developed in this thesis are combined to investigate the heterogeneity of FGF binding to heparin-derived oligosaccharides and to HS in the pericellular matrix of Rama 27 fibroblasts. The cooperativity of the interaction of FGF-2 with a dodecasaccharide is investigated. Although oligomerization of FGF-2 on the dodecasaccharide is observed, it is not cooperative. The first photothermal imaging of FGF-1 in the pericellular matrix of Rama 27 fibroblasts reveals that its diffusion is quite different from FGF-2. Imaging of FGF-2 on live cells is also revisited and probed with PhRICS. In comparison to photothermal tracking, PhRICS indicates that FGF-2 diffuses faster than first thought, and that the pericellular matrix is remodeling at timescales much shorter than previously observed.