Developing a label-free single molecule imaging technology: transitioning from material science to biological science

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Visualising single proteins in the absence of a label represents a vital step towards studying native protein function. In this application we propose to combine the technologies available at Danish Fundamental Metrology (DFM) with that of the Kad lab (University of Kent) to generate a new label-free imaging tool. This will be used to study DNA repair as a model biological system.The Kad lab presently uses single molecule fluorescence imaging to study nucleotide excision DNA repair (NER) in prokaryotes. This approach has led to considerable new insights into the physical search mechanisms employed by these proteins. To enable further insights we have constructed a high-speed dark-field imaging system that measures the light scattered from individual gold nanoparticle labelled proteins. One key drawback of this system is that the gold nanoparticles (~40 nm) increase the size and thus reduce the diffusion constant of the protein. Therefore we must use theoretical calculations to account for the expected contribution of the attached nanoparticle. Ideally, imaging proteins without a label would provide an accurate measure of how these proteins locate their target sites.We have teamed up with DFM who specialise in measuring the fundamental properties of materials. Such technologies are crucial for advanced optical component development (for example nanostructured gratings). One such system detects light scatter from single unlabelled molecules ordered on a surface (scatterometry). By using scatterometry we will dispense with gold nanoparticles and instead detect the scatter signal from single proteins directly. This technology requires very little hardware development; we use an open optical system that can easily be adapted to accommodate for scatterometry. A major component in this PhD project is the application of Fourier image analysis software, which is the subject of on-going development at DFM.